isolation, characterization and genome sequencing of a

Isolation characterization and genome sequencing of a soil-borne

Citrobacter freundii strain capable of detoxifying trichothecene mycotoxins

by

Rafiqul Islam

A Thesis

Presented to

The University of Guelph

In Partial Fulfilment of Requirements

for the Degree of

Doctor of Philosophy

in

Plant Agriculture

Guelph Ontario Canada

copy Rafiqul Islam April 2012

ABSTRACT

ISOLATION CHARACTERIZATION AND GENOME SEQUENCING OF A SOIL-

BORNE CITROBACTER FREUNDII STRAIN CAPABLE OF DETOXIFIYING

TRICHOTHECENE MYCOTOXINS

Rafiqul Islam Advisors

University of Guelph 2012 Dr K Peter Pauls

Dr Ting Zhou

Cereals are frequently contaminated with tricthothecene mycotoxins like

deoxynivalenol (DON vomitoxin) which are toxic to humans animals and plants The goals

of the research were to discover and characterize microbes capable of detoxifying DON under

aerobic conditions and moderate temperatures To identify microbes capable of detoxifying

DON five soil samples collected from Southern Ontario crop fields were tested for the ability

to convert DON to a de-epoxidized derivative One soil sample showed DON de-epoxidation

activity under aerobic conditions at 22-24degC To isolate the microbes responsible for DON

detoxification (de-epoxidation) activity the mixed culture was grown with antibiotics at 50ordmC

for 15 h and high concentrations of DON The treatments resulted in the isolation of a pure

DON de-epoxidating bacterial strain ADS47 and phenotypic and molecular analyses

identified the bacterium as Citrobacter freundii The bacterium was also able to de-epoxidize

andor de-acetylate 10 other food-contaminating trichothecene mycotoxins

A fosmid genomic DNA library of strain ADS47 was prepared in E coli and screened

for DON detoxification activity However no library clone was found with DON

detoxification activity The whole genome of ADS47 was also sequenced and from

comparative genome analyses 10 genes (characterized as reductases oxidoreductases and

deacetylase) were identified as potential candidates for DONtrichothecene detoxifying

enzymes Bioinformatic analyses showed that the major animal pathogenic locus of

enterocyte effacement (LEE) operon genes absent from the ADS47 genome Strain ADS47

has the potential to be used for feed detoxification and the development of mycotoxin

resistance cereal cultivars

iii

DEDICATIONS

The PhD thesis is dedicated to the departed souls of my mother MOZIBUN NESSA and

father-in-law DR MA AHAD MIAH

iv

ACKNOWLEDGEMENTS

Foremost I am deeply indebted to my advisors Prof K Peter Pauls and Dr Ting Zhou

for the continuous support of my PhD study for their endurance inspiration enthusiasm and

mentoring Their continued guidance has helped me in all aspects of my research and in

writing of this dissertation I sincerely express my gratitude to them for their co-operation

from the beginning to conclusion of the thesis and many other areas This will guide me in my

future professional career

My thesis committee guided me through all these years I would like to express my

gratitude to Prof Paul Goodwin for his insightful comments and advice on my thesis research

Special thanks to Dr Chris Young for teaching me chemistry chemical structures and

interpreting liquid chromatography ultraviolet mass spectra data I am sincerely grateful to

Dion Lepp for helping in analyzing the bacterial genome and sharing his knowledge and

valuable suggestions on writing the genomic chapter

I would like to express my appreciation to staff members of Dr Ting Zhou and Prof

K Peter Pauls who supported me in many aspects during my PhD study I am thankful to

Jianwei He Xiu-Zhen Li Honghui Zhu for collections and preparation of soil samples and

LC-UV-MS analyses of numerous samples Special thanks to Dr David Morris Johnston

Monje for sharing his knowledge discussions and camaraderie at coffee breaks

I wish to acknowledge the Natural Sciences and Engineering Research Council of

Canada (NSERC) for awarding me a prestigious scholarship for my PhD program and

Deanrsquos Tri-Council Scholarship (University of Guelph) which made my life easier during this

study I also would like to convey my thanks to Agriculture and Agri-Food CanadaBinational

v

Agricultural Research and Development Fund (AAFCBARD) project for providing me

financial support

Mike Bryan a very supportive person helped provide solutions to my software issues

that improved my exposure and caliber at conference awards competitions Several student

competition awards honored and inspired me to work hard and lead to the successful

completion of my thesis research I would like to convey my appreciation to the judges of

different student award committees who recognized my work They are Society for Industrial

Microbiology and Biotechnology conference 2010 (USA) Food Safety Research Forum-

Ontario Ministry of Agriculture and Rural Affairs annual meeting 2010 (Canada)

International Mycological Conference 2009 (UK) and Southwestern Ontario Regional

Association of Canadian Phytopathological Society annual meeting 2009-2011 (Canada)

I would like to express my very special thanks to Prof Golam Hafeez Kennedy my

brother-in-law who is like my elder brother In many aspects I am indebt to him for his

cooperations which aided me to achieve my current status

Lastly and most importantly I wish to give my special thanks to my wife Mimi and

lovely daughter Racy whose endless love sacrifice support and encouragement enabled me

to cross the tough long road and made possible to achieve my goal and aspirations Racy

once you told me that one of your desires for me is to finish my PhD which is done now I

know you want to be a scientist which is a great ambition I wish you all the best in your

future aims Racy

vi

TABLE OF CONTENTS

DEDICATIONShelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellipiii

ACKNOWLEDGEMENTShelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellipiv

TABLE OF CONTENTShelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellipvi

LIST OF TABLEShelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellipix

LIST OF FIGUREShelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellipx

LIST OF ABBREVIATIONShelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellipxiii

10 GENERAL INTRODUCTION AND LITERATURE REVIEW helliphelliphelliphelliphelliphelliphelliphellip1

11 Backgroundhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip1

12 Plant pathogenesis and trichothecene productionhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip3

13 Biosynthesis of food contaminating TChelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip6

14 Occurrence of TC mycotoxins in food and feed helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip11

15 Significance of deoxynivalenol and trichotheceneshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip16

16 Toxic effects of deoxynivalenol and trichotheceneshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip19

17 Cellular and molecular mechanisms of DON toxicity in animals and plantshelliphelliphelliphellip22

18 Strategies to control Fusarium head blight and trichotheceneshelliphelliphelliphelliphelliphelliphelliphelliphellip26

19 Isolation of DONTC de-epoxidating micro-organisms and geneshelliphelliphelliphelliphelliphelliphelliphellip28

110 The bacterial species Citrobacter freundiihelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip30

111 Hypotheses and objectiveshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip31

vii

20 AEROBIC AND ANAEROBIC DE-EPOXIDATION OF MYCOTOXIN

DEOXYNIVALENOL BY BACTERIA ORIGINATING FROM AGRICULTURAL

SOIL33

21 Abstract helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip33

22 Introduction helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip34

23 Materials and methodshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip37

24 Results helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip45

25 Discussionhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip62

26 Acknowledgementshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip64

30 ISOALTION AND CHARACTERIZATION OF A NOVEL SOIL BACTERIUM

CAPABLE OF DETOXIFYING ELEVEN TRICHOTHECENE MYCOTOXINS65






36 Acknowledgementshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip100

40 CONSTRUCTION AND FUNCTIONAL SCREENING OF GENOMIC DNA

LIBRARY FOR ISOLATION OF DON DE-EPOXYDIZING GENE(S) FROM

BACTERIAL STRAIN ADS47helliphelliphelliphelliphelliphelliphelliphellip102

41 Abstract helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip102



viii




50 GENOME SEQUENCING CHARACTERIZATION AND COMPARATIVE

ANALYSIS OF A TRICHOTHECENE MYCOTOXIN DETOXIFYING

CITROBACTER FREUNDII STRAIN ADS47helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip122







60 CONCLUSIONhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip171

61 Summary helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip171

62 Research contributions helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip174

63 Future workhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip175

70 REFERENCEShelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip176

ix

LIST OF TABLES

Table 1-1 Fusaria produced type-A and type-B trichothecene mycotoxinshelliphelliphelliphelliphelliphelliphellip9

Table 1-2 Worldwide occurrence of deoxynivalenol (DON) in animal feedshelliphelliphelliphelliphellip12

Table 1-3 Occurrence of DON in Canadian grains grains foods and beershelliphelliphelliphelliphelliphelliphellip13

Table 1-4 Legislative maximum tolerated levels of trichothecene mycotoxins in Canadahellip14

Table 1-5 In vitro and in vivo synergistic effects of different food contaminating

mycotoxins helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip18

Table 3-1 Microbial transformation of type-A and type-B tricthothecene mycotoxins

after 72 h of incubation under aerobic conditions and at room temperaturehelliphelliphelliphelliphelliphellip95

Table 4-1 Analysis of Fosmid DNA library for deoxynivalenol (DON) de-epoxidation

capability helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip117

Table 5-1 General features of C freundii strain ADS47 genomehelliphelliphelliphelliphelliphelliphelliphelliphellip132

Table 5-2 Genome comparisons between strain ADS47 and five Citrobacter species141

Table 5-3 Functional categorized gene abundances among the six Citrobacter genomeshellip145

Table 5-4 Unique genes of C freundii strain ADS47 helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip156

Supplemental Table S5-1 List of unique genes of strain ADS47 gene IDs and their

functional roleshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip169

x

LIST OF FIGURES

Figure 1-1 Structures of trichothecene mycotoxins produced by Fusariahelliphelliphelliphelliphelliphelliphelliphellip7

Figure 1-2 Cellular and molecular mechanisms of deoxynivalenol toxicity in

eukaryotic organismshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip23

Figure 2-1 Mechanism of microbial DON de-epoxidationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip36

Figure 2-2 LC-UV-MS chromatographic analysis of DON and its de-epoxidated product

dE-DON obtained by culturing with selected soil microbeshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip46

Figure 2-3 Time course of relative biotransformation of DON to dE-DON by the initial

soil suspension culture in MSB mediumhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip49

Figure 2-4 Microbial de-epoxidation of DON after five days of incubationhelliphelliphelliphelliphelliphellip51

Figure 2-5 Analysis of microbial population reduction by terminal restriction fragment

length polymorphism (T-RFLP)helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip53

Figure 2-6 Microbial genera identified in the enriched DON de-epoxidizing microbial

culturehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip56

Figure 2-7 Effects of culture conditions on DON to dE-DON transformation by the

enriched microbial culturehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip58

Figure 2-8 Microbial DON to dE (de-epoxy)-DON transformation activity under aerobic

and anaerobic conditionshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip61

Figure 3-1 Colony color shape and size of the mycotoxin degrading bacterial strain

ADS47 helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip76

Figure 3-2 Dendrogram based on 16S-rDNA sequences for strain ADS47 and 11

reference Citrobacter specieshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip77

xi

Figure 3-3 Effects of DON concentration on bacterial growth and de-epoxidation

activitieshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip79

Figure 3-4 Bacterial growth and DON de-epoxidation capabilities under aerobic and

anaerobic conditions helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip82

Figure 3-5 Effects of media on bacterial growth and DON de-epoxidation activityhellip85

Figure3-6 Effects of temperature on microbial growth and DON de-epoxydation helliphelliphellip87

Figure 3-7 Influence of pH on microbial growth and DON de-epoxydation capabilityhellip89

Figure 3-8 Effects of sodium azide (SA) on bacterial DON de-epoxidation activityhelliphelliphellip91

Figure 3-9 Determination of DON to deepoxy-DON transformation by bacterial culture

filtrate and cell extracthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip92

Figure 3-10 Determination of microbial de-epoxidation and de-acetylation of a type-A

trichothecene mycotoxin (HT-2 toxin) by LC-UV-MShelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip94

Supplemental Figure S3-1 Flow chart depicted the procedures of enrichment and isolation

of DON de-epoxidation bacterial strain ADS47helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip101

Figure 4-1 Schematic overview of the construction and functional screening of Fosmid

DNA libraryhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip105

Figure 4-2 Genetic map of copy controlled Fosmid vector (pCC1FOS)helliphelliphelliphelliphelliphelliphellip108

Figure 4-3 Preparation of 40 kb size insert DNA from a DON de-epoxidizing bacterium113

Figure 4-4 Production of genomic DNA library in E coli EPI300-T1 host strainhelliphelliphelliphellip114

Figure 4-5 Confirmation of the Fosmid DNA library construction in E coli helliphelliphelliphelliphellip 116

Figure 5-1 Co-linearity between C freundii strain ADS47 genome and C koseri helliphelliphellip129

Figure 5-2 Circular genome map of C freundii strain ADS47 helliphelliphelliphelliphelliphelliphelliphelliphelliphellip133

xii

Figure 5-3 Size distribution of the predicted genes identified in C freundii strain

ADS47helliphelliphellip 135

Figure 5-4 Functional classification of the predicted coding sequences (CDS) of

C freundii strain ADS47 helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip137

Figure 5-5 Phylogeny of the trichothecene detoxifying C freundii strain ADS47 139

Figure 5-6 Comparisons of coding sequences between C freundii strain ADS47 and

five Citrobacter specieshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip143

Figure 5-7 Determination of the locus enterocyte effacement (LEE) operons in

Citrobacter species by comparing C rodentium helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip151

Figure 5-8 Locations of unique genes of C freundii strain ADS467helliphelliphelliphelliphelliphelliphelliphellip157

Figure 5-9 Unique genomic regions and associated genes in strain ADS47 compared

to five Citrobacter specieshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip159

xiii

LIST OF ABBREVIATIONS

APCI= atmospheric pressure chemical ionization

AE= attaching and effacing

A= aerobic conditions

ABC= ATP-binding cassette

ACT= artemis comparative tools

AN= anaerobic conditions

3-ADON= 3-acetyl-deoxynivalenol


ATA= alimentary toxic aleukia

bp= base pair

BGI= Beijing genomics institute

BLAST= basic local alignment search tool

BRIG= blast ring image generator

Bw= body weight

C= carbon

CDS= coding sequence

CFIA= Canadian food inspection agency

CHEF= clamped homogeneous electric fields

CMB= corn meal broth

CVTree= composition vector tree

DAS= diacetoxyscirpenol

dH2O= distilled water

DOGT1= UDP-glucosyltransferase

DON= deoxynivalenol

dsRNA= double stranded RNA

dE= de-epoxy

dE-DON= de-epoxy DON

E coli= Escherichia coli

ER=endoplasmic reticulum

FAO= Food amp Agriculture Organization

FM= full medium

FHB= Fusarium head blight

FUS= fusarenonX

gDNA= genomic DNA

GC= guanine cytosine

GER= Gebberella ear rot

GPI= glycosylphosphatidylinositol

h= hour

Hck= hematopoietic cell kinase

Kb= kilobase

xiv

Kg= kilogram

LC-UV-MS= liquid chromatography-ultraviolet-

mass spectrometry

LEE= locus of enterocyte effacement

LD50= lethal dose of 50 animal

LB= Luria Bertani

LMP= low melting point

MAPK= mitogen-activated protein kinase

microg= microgram

mg= milligram

mL= milliliter

microL= microliter

microM= micromole

min= minute

Mb= megabases

MCS=multiple cloning site

MSA= MSB+ agar

MSB= mineral salts broth

MM= minimal medium

MSC= mineral salts + 03 Bacto Peptone

MS-DON= mineral salts + 200 microgmL DON

mRNA= messenger RNA

MSY= mineral salts + yeast extract

mz= mass ion ratio

N= nitrogen

Na-Cl= sodiumndashclorine

NADH= (reduced) nicotinamide adenine

dinucleotide

NAD(P)= nicotinamide adenine

dinucleotide phosphate

NCBI= national center for biotechnology

information

NEO= neosolaniol

ng= nanogram

NIV= nivalenol

OD600nm= optical density at 600

nanometer wavelength

OMAFRA= Ontario Ministry of

Agriculture and Rural Affairs

Paa= phenylacetic acid

PCR= polymerase chain reaction

PDB= potato dextrose broth

ppm= parts per million

PFGE= pulsed field gel electrophoresis

PKR= protein kinase

xv

PNDO= pyridine nucleotide-disulfide

oxidoreductase

QTL= quantitative trait locus

RAST= rapid annotation subsystem technology

RDP= ribosomal database project

Rpl3= 60S ribosomal protein L3

rRNA= ribosomal RNA

s= second

SA= sodium azide

SDR= short chain dehydrogenasereductase

SE= soil extract

SIM= selected ion monitoring

SOAP= short oligonucleotide analysis package

sp= species

sRNA= small RNA

TC= trichothecene

TCA= tricarboxylic acid cycle

TSB= tryptic soy broth

TF= transcription factor

tRNA= transfer RNA

TIC= total ion count

TRAP= tripartite ATP-independent

periplasmic

T-RFLP=terminal restriction fragment

length polymorphism

TRI101= trichothecene 3-O-

acetyltransferase

YEB= yeast extract broth

VER= verrucarol

VKOR= vitamin K epoxide reductase

wv= weight by volume

1

CHAPTER 1

10 GENERAL INTRODUCTION AND LITERATURE REVIEW

11 Background

According to the Food and Agriculture Organization (FAO) of the United Nations

more than 25 of the worldrsquos food crops are contaminated with mycotoxins every year

causing serious adverse effects on livestock industry crop production and international trade

(Boutrif and Canet 1998 Desjardins 2006 Binder et al 2007 Surai et al 2010) The negative

effects of mycotoxins on the consumers are diverse including organ toxicity mutagenicity

carcinogenicity neurotoxicity teratogenicity modulation of the immune system reproductive

system effects and growth retardation Mycotoxins are toxic secondary metabolites produced

by various fungal species during crop colonization in field andor post harvest conditions

(FAO 1991 Barrett 2000) Aspergillus Penicillum and Fusarium are the predominant fungal

genera that accumulate economically important mycotoxins such as aflatoxins zearalenone

trichothecenes fumonisins ochratoxins and ergot alkaloids in agricultural commodities

Humans and animals are principally exposed to mycotoxins through consumption of

contaminated foodfeed and animal products such as milk meat and eggs (Fink-Gremmels

1989 Hollinger and Ekperigin 1999)

Trichothecenes (TCs) produced by a variety of toxigenic Fusarium species are

probably the most food and feed contaminating mycotoxins in the temperate regions of

America Europe and Asia (Creppy 2002 Yazar and Omurtag 2008) The fungal pathogen

accumulates various type-A (eg T-2 toxin HT-2 toxin) and type-B trichothecenes (eg

2

deoxynivalenol nivalenol and their derivatives) in cereals Among them type-B

trichothecenes particularly deoxynivalenol (DON) andor its derivatives appear to be the

most commonly found mycotoxins in grain and grain products worldwide impairing human

health and productivity of farm animals (Placinta et al 1999 Sherif et al 2009) A number of

human and animal disease outbreaks associated with TC contaminated grain food and feed

consumption have been reported in Asia (Japan India and central Asia) Europe New

Zealand and United States of America (USA) (Pestka and Smolinski 2005 Desjardins 2006)

A severe outbreak of Alimentary Toxic Aleukia (ATA) mycotoxicosis killed 100000 people

in the Orenburg region of Russia (Eriksen 2003) In 1996 losses of over $100 million were

estimated in Ontario because of costs related to managing DON (Schaafsma 2002)

Historically T-2 toxin DON and nivalenol (NIV) have likely been used as a biological

warfare in Laos Kampuchea and Afghanistan and there is concern because of the potential

use of these compounds for bioterrorism (Wannemacher et al 2000 Tucker 2001)

Despite the extensive efforts that have been made to prevent TC contamination with

the current varieties and production practices its presence in agricultural commodities is

likely unavoidable There are no crop cultivars currently available that are completely

resistant to Fusarium head blightGibberella ear rot (FHBGER) diseases Chemical or

physical methods cannot safely remove TC mycotoxins from grain completely or they are not

economically viable (Varga and Toacuteth 2005) Therefore an alternative strategy to address the

issue is of great importance (Awad et al 2010 Karlovsky 2011)

3

12 Plant pathogenesis and trichothecene production

Cereals infested by the ascomyceteous fungi may cause Gibberella ear rot (GER) in

maize (Zea mays L) and Fusarium head blight (FHB also known as lsquoscabrsquo) of small grain

cereals [wheat (Triticum aestivum L) barley (Hordeum vulgare L)] and other small grain

cereals (Goswami and Kistler 2004) As many as 17 fungal species have been reported to be

associated with FHBGER which include F graminearum Schwabe [telemorph Gibberella

zeae (Schwein) Petch] F culmorum (WG Smith) Sacc F sporotrichioides Scherb F

poae (Peck) Wollenw Microdochium nivale (Fr) Samuels amp IC Hallett and F avenaceum

(Fr) Sacc (Osborne and Stein 2007 Audenaert et al 2009) The two species F

graminearum and F culmorum are the principal species causing crop damage Among them

F graminearum is the widely adapted and most damaging species across the world including

North America (Dyer et al 2006) F culmorum tend to be the main species in the cooler parts

of North Central and Western Europe (Wagacha and Muthomi 2007)

The fungi overwinter mainly on crop residues as saprophytes where they produce

asexual (micro- and macro-conidia) andor sexual (ascospores) fruiting structures which act

as the initial source of inoculums in the subsequent crop season (Sutton 1982 Wagacha and

Muthomi 2007) Ascospores are primarily dispersed by air (Trail et al 2002) and conidia

move by rain-splash (Paul et al 2004) After they land on maize silks and florets of small

grain cereals infection occurs through natural openings (eg stomata) or occasionally by

direct penetration The pathogens can invade maize kernels through wounds caused in the

husks by insects or birds Infection may occur throughout the crop growing season but

disease incidence and severity is facilitated by warm temperatures (gt20degC) high humidity

(gt80) and frequent precipitation that coincides with anthesis (Osborne and Stein 2007

4

Wagacha and Muthomi 2007) The Fusarium pathogen initially infects as biotroph which

becomes a necrotroph as the disease progresses After penetration Fusaria produce different

extracellular cell wall degrading enzymes that trigger infection and hyphal progression

towards the developing earkernel where further colonization occurs and development of

FHBGER diseases (Voigt et al 2005 Miller et al 2007 Kikot et al 2009) FHB disease is

characterized by a bleached appearance around the middle of the head Severely infected

grains have a pinkish discoloration and can be shriveled In maize the disease produces a

reddishndashbrown discoloration of the kernels and progresses from the tip to the bottom of

affected ears

The fungi associated with FHB and GER contaminate cereals by accumulating various

host-nonspecific TC mycotoxins (Brennen et al 2007) TCs have typically been associated

with cereals grown in temperate climatic conditions including Canada (Doohan et al 2003)

DON production by F graminearum and F culmorum is maximal at warm temperatures (22-

28˚C) high grain moisture contents (gt17) and humid conditions (gt70 RH) (Martins and

Martins 2002 Hope et al 2005 Llorens et al 2006) Grain can be contaminated with TCs in

the field as well as in storage if conditions for fungal growth are optimal and grain contains

high moisture (gt17) (Larsen et al 2004 Birzele et al 2002)

Populations of FHBGER causing pathogens are dynamic Genetic variability between

and within Fusarium species appears to have occurred to allow for their adaptation to new

agro-climates (Desjardins 2006 Fernando et al 2011) Three strain specific Fusarium

chemotypes NIV 3-ADON (3-acetyl-deoxynivalenol) and 15-ADON (15- acetyl-

deoxynivalenol) have been identified which correlate with the geographical distributions

(Starkey et al 2007 Zhang et al 2007 Audenaert et al 2009) The NIV chemotype mainly

5

produced by F culmorum accumulates NIV and 4-NIV (Fusarinon X) is predominant in

Asia Africa and Europe (Lee et al 2001) The 3-ADON chemotype which synthesizes DON

and 3-ADON is the dominant strain in EuropeFar East (Larsen et al 2004) In North

America 15-ADON is the main chemotype which produces DON and 15-ADON (Goswami

and Kistler 2004) Significant Fusarium population shifting was observed in the past and in

recent years (Kant et al 2011) A dynamic change from F culmorum to F gramninearum

and DON to NIV producing chemotyes occurred in North West European countries The 3-

ADON chemotype which is uncommon in North America was found in North Dakota and

Minnesota (Goswami and Kistler 2004) F pseudograminearum which historically occurs in

Australia Africa and North West Pacific America has been found in Western Canada (Larsen

et al 2004 Kant et al 2011) In Manitoba a replacement of 15-ADON chemotype by the

more phytoxic 3-ADON type was observed in recent years (Fernando et al 2011) All

together the FHBGER associated pathogen complex is highly dynamic which not only

reduces crop yield but also decreases grain quality by accumulating various mycotoxins

6

13 Biosynthesis of food contaminating TC

TCs are fungal secondary metabolites that were initially identified in Trichotheceium

roseum Thus the name of this class of compounds follows the name of the genus

Trichotheceium (Freeman and Morrison 1949) There have been 180 TCs identified in various

fungal species belonging to the genera Fusarium Myrothecium Stachybotrys Trichoderma

and Thrichothecium (Eriksen 2003 Eriksen et al 2004) The FHBGER associated TCs are

mainly synthesized by different Fusaria and are characterized as tricyclic sesquiterpenoids

with a double bond at position C9-10 and a C12-13 epoxide ring

7

Figure 1-1 Structures of trichothecene mycotoxins produced by Fusaria (a) General

structure of trichothecenes R1 = H OH OiValeryl keto R2 = H OH R3= OH OAc R4 =

H OH OAc R5= OH OAc (b) Type-A trichothecene mycotoxin (c) Type-B trichothecene

mycotoxin which contain a carbonyl functional group at C8 position (Young et al 2007)

(a) (b)

(c)

8

The fungal metabolites are small in size (molecular weight 250-550) heat stable and

non-volatile molecules (Pestka 2007 Yazar and Omurtag 2008) Food contaminating TCs are

divided into two groups type-A and type-B (Figure 1-1b and 1c) Type-A is characterized by

the lack of a ketone function at C8 position and include T-2 toxin HT-2 toxin

diacetoxyscirpenol (DAS) and neosolaniol (NEO) which are mainly produced by F

sporotrichioides and F poae (Creppy 2002 Quarta et al 2006) The most frequently found

TC belongs to type-B which contains carbonyl functions at C8 positions DON NIV and

their acetylated derivatives The two types are further differentiated by the presence or

absence of functional hydroxyl (-OH) and acetyl (CH3-CO-) groups at C-3 C-4 C-7 C-8 and

C-15 positions (Table 1-1)

The syntheses of various TCs are speciesstrain specific and are genetically controlled

but influenced by environmental conditions (Desjardins 2006 Paterson and Lima 2010) Until

now 15 Fusaria catalytic genes and regulators involved in TC productions have been

identified (Merhej et al 2011) These biosynthetic genes are located at three loci on different

chromosomes The core trichothecene (TRI) gene cluster is comprised of 12 genes localized

in a 25-kb genomic region (Kimura et al 2001) The remaining three genes are encoded by

the TRI1-TRI16 and TRI101 loci (Gale et al 2005 Alexander et al 2009) Fusaria produce

TC following a number of oxygenation isomerization acetylation hydroxylation and

esterification reaction steps (Desjardins 2006 Desjardins and Proctor 2007 Kimura et al

2007 Merhej et al 2011) In brief farnesyl pyrophosphate is initially cyclized into

trichodiene by TRI5 which codes for trichodiene synthase In the next four steps

isotrichotriol is produced by TRI4 (a multifunctional cytochrome P450) via C-2 hydroxylation

and 1213 epoxidation followed by two hydroxylation reactions

9

Table 1-1 Fusaria produced type-A and type-B trichothecene mycotoxins Molecular weight

(MW) and acetyl (-R) hydroxyl (-OH) and ketone (=O) functions at different carbon (C)

position of the tricyclic rings A-types T-2 toxin HT-2 toxin DAS (diacetoxyscirpenol)

NEO (neosolaniol) VER (verrucarol) B-type DON (deoxynivalenol) 3-ADON (3-acetyl-

deoxynivalenol) 15-ADON (15-acetyl-deoxynivalenol) NIV (nivalenol) and FusX

(fusarenon X) (Young et al 2007)

Trichothecenes MW R1C3 R2C4 R4C7 R3C15 R5C8

Type-A

T-2 toxin 466 OH OCOCH3 H OCOCH3 OCOCH2CH(CH3)2

HT-2 toxin 424 OH OH H OCOCH3 OCOCH2CH(CH3)2

DAS 366 OH OCOCH3 H OCOCH3 H

NEO 382 OH OCOCH3 OH OCOCH3 H

VER 266 H OH H OH H

Type-B

DON 296 OH H OH OH =O

3-ADON 338 OCOCH3 H OH OH =O

15-ADON 338 OH H OH OCOCH3 =O

NIV 312 OH OH OH OH =O

FusX 354 OH OCOCH3 OH OH =O

10

The next two non-enzymatic reactions results in isotrichodemol Eventually the

trichothecene skeleton is produced via the formation of a C-O bond between the C-2 oxygen

and C-11 Subsequent acetylation at C-3 by TRI101 hydroxylation at C-15 by TRI1 and

catalytic activity of TRI3 leads to calonectrin production The pathway described above is

common for both type-A (eg T-2 toxin HT-2 toxin) and type-B (eg DON NIV and their

acetylated derivatives) TCs

The following steps are speciesstrain specific In DON producing F graminearum

strains calonectrin is converted to either 3-ADON or 15-ADON followed by DON production

by the catabolic activities of TRI1 and TRI8 The biosynthesis of 3-ADON or 15-ADON is

controlled by variation in the esterase coding sequences of TRI8 F graminearum produces

NIV from calonectrin via synthesis of 4-NIV followed by NIV by TRI1 and TRI8 gene

actions NIV can be produced either from DON by hydroxylation at C-4 or via

transformation of calonectrin to 315-diacetoxyscirpenol (315-DAS) followed by addition of

a ketone at C-8 position by the activity of the TRI7 and TRI13 genes 315-DAS is the

principal substrate for synthesis of type-A TCs by F sporotrichioides the main type-A

producer The biosynthetic pathway of T-2 toxin involves C-4 acetylation of 315-DAS by the

TRI7 gene product and TRI8 triggered by TRI1 315-DAS is also the substrate for HT-2

toxin synthesis In the absence of TRI7 expression HT-2 toxin is produced through

hydroxylation of 315-DAS and isovalerate addition to C-8 position (Foroud and Eudes 2009)

11

14 Occurrence of TC mycotoxins in food and feed

TC producing Fusaria are adapted to variable climatic conditions and agricultural

ecosystems As a consequence this class of mycotoxins particularly DON is frequently

detected in foods and feeds including breakfast cereals bakery products snack foods beer

and animal feed derived from cereals in most part of the world (Pittet 2001 Desjardins 2006

Lancova et al 2008) A two-year survey during 2003-2005 conducted by Romer Lab Inc

revealed unacceptable levels of DON in animal feedsfeed raw materials (such as maize

wheat wheat bran) in Asia and Oceania (1062 microgg) central Europe (802 microgg) Mid-West

USA (55 microgg) northern Europe (551 microgg) and southern Europe (303 microgg) (Binder et al

2007) (Table 1-2)

Quite high levels of DON contamination (19 microgg) were detected in north Asian and

Chinese feedfeed materials Recently five-year on-farm studies in Switzerland showed that

74 of the wheat grain and straw samples were contaminated with high levels of DON

(average 5 microgg) The amount is higher than the European maximum limit of 125 microgg

(Vogelgsang et al 2011)

The occurrences of TCs in Canadian agricultural commodities are indicated in Table

1-3 Seventeen surveys conducted during the 1980s and 1990s showed up to 100 DON

incidence in Ontario wheat and maize samples with an 18 average incidence (Scott 1997) A

three-year survey recently (2006-08) conducted by the Ontario Ministry of Agriculture and

Rural Affairs (OMAFRA) detected high levels of DON in spring (maximum 26 microgg) and

winter wheat (maximum 29 microgg) samples (Ana Matu personal communication) The DON

levels were much higher than Health Canadarsquos maximum limit of 2 microgg for uncleaned soft

wheat for staple foods (Table 1-4)

12

Table 1-2 Worldwide occurrence of DON in animal feed Feed and feed raw material

samples were collected from animal farmsfeed production sites during 2003-2005 The

survey was conducted by Romer Labs Inc (httpwwwromerlabscom)

Country

region

Sample

type

Incidence

()

Maximum

DON content

(microgg)

Mean DON

content

(microgg)

Survey

period

Source

North

AsiaChina

Animal

feed

071 1899 092

Binder et

al 2007

South East

Asia

014 152 016

South Asia 070 1062 049

Oceania 2003-05

Australia 041 186 030

Northern

Europe

070 551 059

Central

Europe

066 802 057

Southern

Europe

052 303 030

13

Table 1-3 Occurrence of DON in Canadian grains grain foods and beers Infant cereal foods

oat barley soy and multi-grain based foods

Province Sample

type

Incidence

()

Maximum

DON

contents

(microgg)

Multiple

mycotoxins

samples ()

Survey

period

Sources

Ontario Spring

Wheat

70 260 Yes 2006-08 OMAFRA1

Ontario Winter

wheat

49 290 Yes 2006-08 OMAFRA

Ontario Maize 78 140 Yes 2009-10 Limay-Rios

and

Schaafsma

2010

Ontario Maize - 1000 Yes 2011 AampL

Biologicals

Canada Breakfast

cereals

46 094 430 1999-01 Roscoe et al

2008

Canada Animal

feeds

73 425 Yes 1990-10 Zeibdawi et

al 2011

Canada Beers 58 0052 50 1990s Scott 1997

Canada Infant

Cereals

63 098 Yes 1997-99 Lombaert et

al 2003

1

Ontario Ministry of Agriculture and Rural Affairs

2 microgmL

14

Table 1-4 Legislative maximum tolerated levels of trichothecene mycotoxins in Canada

according to Canadian Food Inspection Fact Sheet April 23 2009 DON- deoxynivalenol

DAS- diacetoxyscirpenol

Products DON

(microgg)

T-2 toxin

(microgg)

HT-2 toxin

(microgg)

DAS

(microgg)

Uncleaned soft wheat for human

consumption

2 - - -

Cattlepoultry diets 5 lt1 01 lt1

Swinedairy cattle feeds 1 lt1 0025 lt2

15

High DON incidence (78) was observed in maize samples collected from

southwestern Ontario fields with moderate levels (lt1 microgg) of contamination during 2009 and

2010 (Limay-Rios and Schaafsma 2010) Some samples had high DON levels (14 microgg) In

the 2011 crop season extremely high levels of DON (gt100 microgg) were found in some

southern Ontario maize samples (AampL Biologicals London ON Personal communication)

In a Canadian Food Inspection Agency (CFIA) 20 year survey (1990-2010) 73 of

animal feed samples collected from feed mills farms and retail stores across Canada were

contaminated with DON and for more than 25 of the samples the mycotoxin levels were

above the maximum limit (ge 1 microgg) (Zeibdawi et al 2011) The highest level of DON (15

microgg) detected in the 2006-2007 samples were much lower than the levels found in the 1999-

2000 samples which contained a maximum of 425 microgg DON Importantly this study

reported a significant number of samples contaminated with multiple mycotoxins DON NIV

3-ADON 15-ADON T-2 toxin HT-2 toxin DAS and NEO

Earlier studies in the 1990s detected high levels of DON content in Eastern Canadian

cereals maize (maximum 175 microgg) wheat (maximum 916 microgg) barley (maximum 911

microgg) and oats (maximum 12 microgg) (Campbell et al 2002) The study identified a significant

number of samples (15-367) that contained multiple mycotoxins and up to 33 of the

wheat samples exceeded 1 microgg DON Low amounts but high incidences of TCs were found

in cereal-based foods collected from Canadian retail stores Sixty three percent of infant

cereal foods 46 of breakfast cereals and 58 of beers were contaminated with one or

multiple mycotoxins (Scott 1997 Lombaert et al 2003 Roscoe et al 2008) The maximum

amount of DON was found in infant foods (098 microgg) followed by breakfast cereals (094

microgg) and beers (005 microgmL)

16

15 Significance of deoxynivalenol and trichothecenes

For most cereals including wheat maize rice barley oats and rye the risk of

contamination with TCs resulted in grain quality reduction and FHBGER disease progression

(Bai et al 2002 Jansen et al 2005) The impacts of TC contamination in agricultural

commodities are diverse including reduced livestock production induced human illnesses

and enhanced FHB disease severity which may result in the reduction of crop yield Exposure

of farm animals (particularly pigs) to DON is a continuing threat to the livestock industries in

Canada USA and Europe (DrsquoMello et al 1999) Ingestion of TC-contaminated feeds

generally causes reduction of feed intake feed refusal poor feed conversion retardation of

weight gain increased disease susceptibility and reduced reproductive capacities (Binder et al

2007) High doses of DON result in acute toxic effects in pigs (eg emesis) while low doses

(ge1 microgmL) cause chronic effects (eg growth retardation in pigs) (Rotter et al 1996

Accensi et al 2006) Reduction of growth and reproduction ability was observed in

experimental rats exposured to much higher doses of TC (Sprando et al 2005 Collins et al

2006) Several animal disease outbreaks that occurred in different continents have been

attributed to consumption of feed contaminated with DON NIV and DAS (Desjardins 2006)

The consequences of human exposure to TCs include liver damage gastricintestinal

lesions and central nervous system toxicity resulting in anorexia nausea hypotensionshock

vomiting diarrhea and death (Pestka 2007) Human mycotoxicosis (illnesses and outbreaks)

by consumption of type-A and type-B TC mycotoxins in Europe New Zealand Russia

Japan South America India and Vietnam have been reported (Bhat et al 1989 Yoshizawa

1993 Luo 1994 Desjardins 2006) Severe outbreaks of Alimentary Toxic Aleukia (ATA)

associated with TC killed thousands of people in the Orenburg region of Russia during the

17

1930rsquos and 1940rsquos (Eriksen 2003) TCs (T-2 DON and NIV) were likely used for biological

warfare in Laos Kampuchea and Afghanistan and its potential application for bioterrorism is

an additional concern associated with TC (Tucker 2001 Bennett and Klich 2003)

TCs are phytotoxic and may contribute to FHB disease severity in cereals (Proctor et

al 1995 Maier et al 2006 Masuda et al 2007) Studies demonstrated that DON caused

wilting chlorosis inhibition of seedling growth and necrosis in cereals (Cossette and Miller

1995 Mitterbauer et al 2004 Rocha et al 2005) DON-mediated host cell damage and

subsequent necrotrophic fungal spread has been shown to lead to FHBGER disease

development and higher kernel damage (Procter et al 1995 Mitterbauer et al 2004 Maier et

al 2005) This suggests that DON contributes to yield losses as well as to reductions in the

market value of cereals (Osborne and Stein 2007) Severely infected grain may contain more

than 60 microgg DON which is unacceptable to the milling and brewing industries (Okubara et

al 2002) Significant crop losses due to FHB disease epidemics and mycotoxin contamination

have been reported in Ontario (Schaafsma 2002) FHB disease epidemics during 1998 to 2000

caused huge crop losses resulting in farm failures in the northern Great Plains in the USA

(Windels 2000) In that period FHB associated losses were estimated at US $ 27 billion in

the region (Nganje et al 2002)

Importantly Fusaria infested grains are often co-contaminated with multiple

mycotoxins which further increase health risks as interactions between the mycotoxins may

cause greater toxic effects than expected (Schollenberger et al 2006) This implies that an

apparently low level of contamination of a single mycotoxin can have severe effects on

consumers because of synergistic effects among multiple co-occuring mycotoxins (Surai et al

2010)

18

A number of studies demonstrated synergistic toxic effects among Fusarium

mycotoxins on pigs turkeys lamb chicks as well as in in vitro Caco-2 cell and yeast

bioassays (Table 1-5)

Table 1-5 In vitro and in vivo synergistic effects of different food contaminating mycotoxins

DON-deoxynivalenol DAS-diacetoxyscirpenol FB1-fumonisins 1 NIV-nivalenol ZEN-

zearelenone FA-fusaric acid Afl-aflatoxin

Mycotoxins source Interacting

mycotoxins

Experimental

animalcells

Effects Reference

Naturally contaminated

cereals

DON FA Pigs Inhibit growth Smith et al

1997

DON contaminated

wheat and FB1

DON FB1 Pigs Weight loss Harvey et al

1996

Culture materials

contained mycotoxins

T-2 FB1 Turkey poults Weight loss oral

lesion

Kubena et al

1997

Inoculated rice and

pure mycotoxin

DAS Afl Lamb Weight reduction Harvey et al

1995

Naturally DON

contaminated wheat

and pure T-2 toxin

DON T-2 Chicks Reduced body

weight significantly

Kubena et al

1989

Pure mycotoxins DAS Afl Chicks Reduced body

weight

Kubena et al

1993

Pure mycotoxins T-2 Afl Chicks Reduced weight

oral lesion

Kubena et al

1990

Pure mycotoxins DON

ZENFB1

Human intestinal

Caco-2 cells

Increased DNA

fragmentations

Kouadio et al

2007

Pure mycotoxins DON NIV Yeast bioassay Synergistic effects Madhyastha et

al 1994

Pure mycotoxins T-2 HT-2 Yeast bioassay Synergistic effects Madhyastha et

al 1994

19

16 Toxic effects of deoxynivalnol and trichothecenes

As small molecules TCs simply enter into cells and interact with different cell

components resulting in disruption of cellular functions and causing various toxic effects that

include inhibition of protein synthesis and ribosomal fragmentation (Eudes et al 2000

Minervini et al 2004 Pestka 2007) Simple absorption of DON into human intestinal Caco-2

cells and crossing human placenta barriers through passive diffusion has been reported

(Sergent et al 2006 Nielsen et al 2011)

TC may cause various toxicities in animals and plants including apoptosis

immunotoxicity chromatin condensation disruption of cellularmitochondrion membranes

disruption of gut tissues inhibition of protein and ribosome synthesis breakage of ribosome

and cell damage The toxicity of TC is influenced by structural variability (ie position

number and type of functional groups attached to the tricyclic nucleus and C-C double bond at

C9-10 position) (Desjardins 2006) Nevertheless epoxide function at the C12-13 position is

the key for TC cytotoxicity (Swanson et al 1988 Eriksen et al 2004)

Different in vitro and in vivo studies revealed that the method and period of exposure

amount of TC and types of celltissue and host species have significant effects on TCrsquos

cytotoxicity (Scientific Committee for Food 2002 Rocha et al 2005 Nielsen et al 2009)

Administration of 10 mgkg body weight (bw) NIV caused apoptosis in mice thymus Peyerrsquos

patch and spleen tissues (Poapolathep et al 2002) DON and NIV at 887 mgkg bw for 3 days

a week for four weeks induced immunotoxicity in experimental mice determined by

estimating increased immunoglobulins (IgA) and decreased IgM levels (Gouze et al 2007)

Exposure of mice to 1 mgkg bw of DON induced skeletal malformations and reduced mating

tendency while significant resorption of embryos observed at the dose of gt10 mgkg bw

20

(Khera et al 1982) At high acute doses (4972 mgkg bw) DONNIV and type-A

trichothecenes caused mice death because of intestinal haemorrhage necrosis of bone

marrowlymphoid tissues and kidneyheart lesions (Yoshizawa and Morooka 1973

Yoshizawa et al 1994 Forsell et al 1987 Ryu et al 1988) In a separate study 15-ADON

showed more toxicity than DON when the mycotoxins were orally administered to mice

(Forsell et al 1987)

Studies suggested that type-A TCs are more toxic in mammals than type-B which

means that functional variability at the C-8 position of TC has a significant role in toxicity

The toxicity of various TC mycotoxins in animals can be ranked in the order of T-2

toxingtHT-2 toxingtNIVgtDONge3-ADONgt15-ADON according to the results from human cell

lines and mice model systems bioassays (Ueno et al 1983 Pestka et al 1987 Nielsen et al

2009) Moreover animal feeding assays demonstrated that monogastric animals are most

sensitive to DON The order of sensitivity of animals to DON is

pigsgtrodentsgtdogsgtcatsgtpoultrygtruminants (Pestka 2005) The reasons for the variations in

DON toxicity species might be due to differences in metabolism absorption distribution and

elimination of the mycotoxin among animal species (Pestka 2007)

To my knowledge the mechanism of TC uptake by plant cells has not been studied

Nonetheless cereals are exposed to TC by toxigenic Fusaria infection which may cause

various toxicities such as cell damage wilting chlorosis necrosis program cell death and

inhibition of seedling growth in sensitive plants (eg wheat maize and barley) (Cossette and

Miller 1995 Rocha et al 2005 Boddu et al 2007) As animals TC inhibits protein synthesis

in non-host A thaliana (Nishiuchi et al 2006) Immunogold assays showed that the cellular

targets of DON in wheat and maize were cell membrane ribosome endoplasmic reticulum

21

vacuoles and mitochondria Modulation of cell membrane integrity and electrolyte losses

were observed by direct DON interference in maize and wheat cell assays (Cossette and

Miller 1995 Mitterbauer et al 2004) DON-mediated mitochondrion membrane alterations

disruption of host cells enzymatic activity apoptosis and necrosis have been observed in

cereals (Mitterbauer et al 2004 Rocha et al 2005) In contrast to animal type-B is more toxic

to plants than type-A DON showed 8- and 13-fold phytotoxicity to wheat coleoptiles than T-

2 and HT-2 toxins respectively (Eudes et al 2000) By compilation of different studies TC

phytoxicity can be ordered as DONge3-ADONgtT-2 toxingtDAS (Wakulinski 1989 McLean

1996 Eudes et al 2000)

22

17 Cellular and molecular mechanisms of DON toxicity in animals and plants

The underlying cellular and molecular mechanisms of DONTC toxicity are evolving

Exposure to DON proceeds with the up and down regulation of numerous critical genes of

eukaryotic organisms which causes various negative effects on animal and plant organisms

(Figure 1-2)

One of the early events of DON toxicity occurs when the mycotoxin binds to the

active ribosome resulting in blocking protein translation through different routes by

damaging the ribosome (Shifrin and Anderson 1999) degrading or altering conformational

changes of 28S-rRNA (Alisi et al 2008 Li and Pestka 2008) and inducing expression of

miRNAs This results in down regulation of the ribosome associated genes (He et al 2010a

Pestka 2010) Both animal and plant cell assays demonstrated that DON bound to the peptidyl

transferase (Rpl3) domain of the 60S subunit of ribosome and blocked protein chain

elongation and termination at high DON concentrations while peptide chain termination and

ribosome breakage occurred at a low level of DON exposure (Zhou et al 2003a)

23

Figure 1-2 Cellular and molecular mechanisms of deoxynivalenol toxicity in eukaryotic

organisms DON is simply absorbed by the cell and interacts with the cytoplasmic membrane

(CM) mitochondrion (MT) membrane and ribosome Disruption of the CM and alteration of

the MT membrane causes a loss of electrolytes and affects the function of the MT

respectively DON binds to the active ribosomes which leads to ribosome damage and

induces micro RNAs (miRNA) Expression of miRNA impairs the synthesis of ribosome

associated protein The subsequent transduction and phosphorylation of the RNA-activated

protein kinase (PKR) and hematopoietic cell kinase (Hck) activate the mitogen-activated

protein kinase (MAPK) cascade Phosphorylation of the MAPKs induces targeted

transcription factors (TF) which results in ribotoxis stress response upregulation of

proinflammatory cytokines and dysregulation of neuroendocrine signaling (derived from

Pestka 2007 and 2010)

24

The consequence of DON interaction with ribosomes leads to cellular and molecular

changes which are complex and not fully understood However it is well-demonstrated that

the activation of the mitogen-activated protein kinase (MAPK) and subsequent molecular

actions play key roles in the cytotoxicity of TC Upon binding to the ribosome DON rapidly

induced interactions between the protein kinases and the ribosomal 60S subunit as observed in

mononuclear phagocytes (Bae and Pestka 2008) Ribosomal binding to DON activated the

ribosome associated kinases for instances protein kinase R (PKR) hematopoietic cell kinase

(Hck) and P38

mitogen-activated protein kinase The protein kinases are induced by double-

stranded RNA (dsRNA) or possibly by the damaged ribosomal peptides (Iordanov et al 1997

Shifrin and Anderson 1999 Zhou et al 2003b Pestka 2007) Subsequent phosphorylation of

kinases (eg PKRHckP38

) activates the MAPK cascade resulting in ribotoxis stress

response upregulation of proinflammatory cytokines and dysregulation of neuroendocrine

signaling (Pestka and Smolinki 2005 Pestka 2010) Studies suggested that high doses of

DON induces ribotoxis stress response (apoptosis) preceded by immunosuppression and

disruption of intestinal permeabilitytissue functions (Yang et al 2000 Pestka 2008) Further

study observed stabilization of the mRNA and expression of transcription factors for critical

genes when cells were exposed to a low concentration of DON (Chung et al 2003)

The MAPK pathway proceeds with upregulation of a broad range of proinflammatory

cytokine genes and dysregulation of neuroendocrine signaling within the central and enteric

nervous systems Modulation of neuroendocrine signaling by DON increased the level of

serotonin resulted in vomiting reduced food intake and growth retardation in experimental

animalscell lines (Prelusky and Trenholm 1993 Li 2007) Upregulation of proinflammatory

cytokines and chemokines genes was determined in mice liver spleen kidney and lung

25

following in 1 h of exposure to DON A subsequent shock-like effect was observed in mice

because of a ldquocytokine stormrdquo (Pestka 2008) Induction of proinflammatory cytokines also led

to various cellular effects on the central nervous system interference of the innate immune

system and growth hormone signaling These contribute to anorexia growth retardation

aberrant immune response and tissue dysfunction as demonstrated in experimental animal

systems (Pestka 2010)

DON-mediated growth retardation in mice via suppression of growth hormones

(insulin-like growth factor 1 and insulin-like growth factor acid-labile subunit) has been

reported by Amuzie et al (2009) Disruption of gut tissues by DONNIV and subsequent

impairment of intestinal transporter activity was demonstrated in a human epithelial cell

model (Marseca et al 2002) The study proposed that alteration of gut tissue permeability

may cause gastroenteritis induction and growth retardation in livestock animals Growth

retardation of livestock may also occur via dysregulation of neuroendocrine signaling-

mediated induction of emesis and anorexia (Pestka et al 2010)

The molecular mechanism of TC toxicity in plant systems has not been well

documented A recent study showed the upregulation of numerous defense related genes in

wheat upon exposure to DON (Desmond et al 2008) The study further suggested that DON-

induced program cell death in wheat occurred through a hydrogen peroxide signaling

pathway This mechanism had not been observed in mammals

26

18 Strategies to control FHB and trichothecenes

During the past decades many pre- and post-harvest strategies have been applied to

protect cereal crops from Fusarium infestation and to eliminate TC from contaminated

agricultural commodities (FAO 2001 Munkvold 2003 Varga and Toth 2005 Kabak and

Dobson 2009) Some of the above crop protection techniques have been patented (He and

Zhou 2010) In spite of those efforts TC contamination of cereals appears to be unavoidable

using the currently available methods

Pre-harvest control strategies involving cultural practices (eg crop rotation tillage

early planting) and fungicide applications partially reduced Fusarium infection and DON

contamination in cereals (Dill-Macky and Jones 2000 Schaafsma et al 2001) However

repeated application of fungicides may lead to the evolution of Fusarium strains resistant to

fungicides with greater mycotoxin production ability (DrsquoMello et al 1998) At post-harvest

reduction of grain moisture by artificial drying storage at low temperatures (1-4degC) and

proper ventilation of the storage room have shown to be effective in inhibiting fungal growth

and mycotoxin production in infected grain (Midwest Plan Service 1987 Wilcke and Morey

1995) This technology has become less applicable particularly in developing countries

because of the frequent observation required to maintain proper storage conditions (Hell et al

2000)

Removal of Fusarium infected grains by physical methods (eg mechanical

separation density segregation colour sorting etc) was unable to eliminate TC completely

(Abbas et al 1985 He et al 2010b) This is partly because FHB symptomless kernels may

contain a considerable amount of DON which are not eliminated by physical separation

procedures (Seitz and Bechtel 1985) Several studies showed that milling and food processing

27

steps were unable to completely remove DON from grain-based foods (Jackson and

Bullerman 1999 Placinta et al 1999 Kushiro 2008) Detoxification of TC by chemical

treatments (eg ozone ammonia chlorine hydrogen peroxide and sodium metabisulfite) has

been demonstrated (Young et al 1986 Young et al 1987 Young et al 2006) The problem

with these techniques is that the application of such chemicals is not economically viable and

it also has a negative impact on grain quality (Karlovsky 2011) Although the technique has

been popular and showed effectiveness to certain mycotoxins utilization of absorbents to

inhibit mycotoxin absorption by the livestock gastrointestinal tract appeared to be ineffective

for TC (Huwig et al 2001)

The introduction of Fusarium resistant cultivars would be the best strategy to address

the FHB and associated mycotoxin problems in cereals However breeding for cultivars

resistant to FHBGER is difficult and time consuming as natural resistance against Fusarium

is quantitative in nature FHB resistant quantitative trait loci (QTLs) have been identified in

several cereal genotypes (eg Sumai 3 Ning 7840 Bussard) (Bernando et al 2007 Golkari

et al 2009 Haumlberle et al 2009 Yang et al 2010) The 3B 5A and 6B QTLs for type-I

(resistant to initial infection) and type-II (resistant to fungal spread in the kernel) resistances

have been identified on different wheat chromosomes (Buerstmayr et al 2009) Type-I

(Qfhsifa-5A) and type-II (Fhb1Fhb2-3B) QTLs from Sumai 3 are being used in many

breeding programs and pyramiding of the QTLs in spring wheat showed additional effects

Nonetheless the key genes involved in FHB resistance in cereals are yet to be identified This

makes it difficult to integrate the resistance genes into elite cultivars

28

Cultivar development by direct incorporation of TCDON resistance genes may be an

alternative way of protecting grains from these mycotoxins contaminations Many genes from

distantly related species have been integrated into crop cultivars using a transgenic approach

with great economic benefits (Brookes and Barfoot 2006) DON transforming trichothecene

3-O-acetyltransferase (TRI101 from Fusarium sporotrichioides) and UDP-

glucosyltransferase (from Arabidopsis thaliana) genes expressed in cereal cultivars showed

some DONDAS detoxifying ability under in vitro and controlled conditions (Kimura et al

1998 Okubara et al 2002 Poppenberger et al 2003 Ohsato et al 2007) By contrast no

reduction of mycotoxins in either of the above cases was observed under field conditions

(Manoharan et al 2006) Importantly the transformed acetylated and glycosylated

compounds could revert to the original substances or convert to even more toxic products

(Gareis et al 1990 Alexander 2008)

19 Isolation of DONTC de-epoxidating micro-organisms and genes

An enzyme with the capability of de-epoxidizing DONTC appears to be a potential

source for completely removing TC from contaminated cereals (Karlovsky 2011) However

such trichothecene detoxifying genes do not exist in cereals and other plants (Boutigny et al

2008) Because of this several research groups have attempted to obtain microorganisms that

encode DONTC de-epoxidation genes Mixed microbial culture capable of de-epoxydizing

trichothecenes have been isolated from chicken gut (He et al 1992) cow ruminal fluid (King

et al 1984 Swanson et al 1987 Fuchs et al 2002) pig gut (Kollarczik et al 1994) and

digesta of chicken (Young et al 2007) rats (Yoshizawa et al 1983) sheep (Westlake et al

1989) and fish (Guan et al 2009) All of the microbial cultures cited above showed de-

29

epoxydation capabilities only under anaerobic conditions realtively high temperatures and at

lowhigh pH except microbes from fish intestine that showed efficient de-epoxidation at low

temperatures and wide range of temperatures (Guan et al 2009) Such enzymes active under

anaerobic conditions high temperatues and low pH may have potential applications as feed

additives and the eubacterial strain BBSH 797 is an animal feed additive product with the

brand name Mycofixreg by BIOMIN (He et al 2010b) Biotechnological applications of

anaerobically-active enzymes from the above microbial sources seem to be inappropriate

under the aerobic and moderate conditions at which cereals plants are grown

Most of these efforts have failed to isolate single DONTC degrading microbes

effective under aerobic conditions because of inappropriate microbial growth conditions

under laboratory conditions Earlier attempts were made to obtain TC de-epoxidizing aerobic

bacteria but the culture did not retain DONNIV de-epoxidation activity after reculturing

(Ueno 1983 He et al 1992) In addition the co-metabolic nature of the microbial TC de-

epoxidation reaction has limited the use of mycotoxins as the sole nutrient source for selective

isolation of the target microorganisms Several DONTC de-epoxidation microbial strains

including Bacillus strain LS100 and Eubacterial strain BBSH 797 have been isolated from

chicken gut and rumen fluids through multi-step enrichment procedures (Fuchs et al 2002

Yu et al 2010) A DON to 3-keto DON transforming bactial strain was islated from soil by

prolonged incubation with high concentration DON (Shima et al 1997)

Microbial genesenzymes with TC de-epoxidation activity are not known Based on

the microbial TC de-epoxidation activity of a mixed microbial culture it has been proposed

that ceratin reductases or oxidoreductases may catalyze such a biochemical reaction (Islam et

al 2012) With the advent of molecular techniques many novel genes with

30

industrialagricultural value have been cloned by reverse genetics (genomic DNA library

preparation and functional screening of the library clones) forward genetics (transposon

mutagenesis) and comparative genomics methods (Frey et al 1998 Koonin et al 2001

Suenaga et al 2007 Chung et al 2008 Ansorge 2009) The tremendous advancement of next

generation sequencing technology and bioinformatics tools may facilitate the identification of

the microbial genes responsible for DONTC detoxification (Ansorge 2009) Isolation of TC

de-epoxidation genes may play a major role in producing safe foods and feeds derived from

grains

110 The bacterial species Citrobacter freundii

Citrobacter freundii is a bacterial species belonging to the family Enterobacteriace It

is a facultatively anaerobic and Gram-negative bacillus (Wang et al 2000) The bacterium

survives almost everywhere including animalhuman intestines blood water and soils

(Wang et al 2000 Whalen et al 2007) C freundii is known as an opportunistic human

pathogen and causes a variety of respiratory tract urinary tract and blood infections and

responsible for serious neonatal meningistis disease (Badger et al 1999 Whalen et al 2007)

Interestingly this bacterium plays a positive environmental role through nitrogen recycling

(Puchenkova 1996) Strains of C freundii also contribute to the environment by sequenstering

heavy metals and degrading xenobiotic compounds (Kumar et al 1999 Sharma and Fulekar

2009)

31

111 Hypotheses and objectives

The study was based on the following hypotheses

i Certain microorganisms in DON contaminated agricultural soils are able to transform DON

to de-epoxy DON (de-epoxidation) under aerobic conditions and moderate temperatures

ii DON de-epoxidation may be achieved by a single microbial strain

iii Microbial DON detoxifying genes and associated regulators are able to express and

function in a closely related host organism

iv DON detoxifying microbial strains carry genes for de-epoxidation activity that are absent

from related species that have not evolved under DON selection pressure

The objectives of this research were to

i Select microbial cultures from soil having DON de-epoxidation activity under aerobic

conditions and moderate temperatures by analyzing potential DON contaminated soils in

various growth conditions namely aerobicanaerobic conditions different growth media

temperature and pH

ii Select for organisms with DON de-epoxidation activity from the mixed microbial culture

by treating with antibiotics heating subculturing on agar medium and incubating for a

prolonged period in media with a high DON concentration

iii Identify microbial de-epoxidation genes by producing a microbial genomic DNA library

from the DON de-epoxidizing strain followed by screening of the clones for DON de-

epoxidation

32

iv Determine microbial de-epoxidation genes by sequencing of the genome and comparative

analysis of the genome with closely related species and identification of putative DON

detoxification genes

33

CHAPTER 2



SOIL

Published in World Journal of Microbiology and Biotechnology 2012 287-13

21 Abstract

Soil samples collected from different cereal crop fields in southern Ontario Canada

were screened to obtain microorganisms capable of transforming deoxynivalenol (DON) to

de-epoxy DON (dE-DON) Microbial DON to dE-DON transformation (ie de-epoxidation)

was monitored by using liquid chromatography-ultraviolet-mass spectrometry (LC-UV-MS)

The effects of growth substrates temperature pH incubation time and aerobic versus

anaerobic conditions on the ability of the microbes to de-epoxidize DON were evaluated A

soil microbial culture showed 100 DON to dE-DON biotransformation in mineral salts

broth (MSB) after 144 h of incubation Treatments of the culture with selective antibiotics

followed an elevated temperature (50ordmC) for 15 h and subculturing on agar medium

considerably reduced the microbial diversity Partial 16S-rRNA gene sequence analysis of the

bacteria in the enriched culture indicated the presence of at least Serratia Citrobacter

Clostridium Enterococcus Stenotrophomonas and Streptomyces The enriched culture

completely de-epoxidized DON after 60 h of incubation Bacterial de-epoxidation of DON

occurred at pH 60 to 75 and a wide array of temperatures (12 to 40ordmC) The culture showed

34

rapid de-epoxidation activity under aerobic conditions compared to anaerobic conditions This

is the first report on microbial DON to dE-DON transformation under aerobic conditions and

moderate temperatures The culture could be used to detoxify DON contaminated feed and

might be a potential source for gene(s) for DON de-epoxidation

Keywords biotransformation de-epoxidation deoxynivalenol soil bacteria vomitoxin

22 Introduction

Deoxynivalenol (DON commonly known as lsquovomitoxinrsquo) 3α 7α 15-trihydroxy-

1213-epoxytrichothec-9-en-8-one is a trichothecene produced by toxigenic Fusarium species

causing Fusarium head blight (FHB) or Gibberella ear rot (GER) disease on small grain

cereals and maize respectively (Starkey et al 2007 Foroud and Eudes 2009) DON

contamination in cereals and maize is widespread and can lead to tremendous losses to the

growers It has also detrimental effects on human and livestock health (Pestka and Smolinski

2005 Rocha et al 2005) The toxin interferes with the eukaryotic ribosomal peptidyl

transferase (Rpl3) activity resulting in human and animal mycotoxicosis (Ueno 1985 Bae

and Pestka 2008) Common effects of DON toxicity in animals include diarrhea vomiting

feed refusal growth retardation immunosuppression reduced ovarian function and

reproductive disorders (Pestka and Smolinski 2005 Pestka 2007) In plants DON causes a

variety of effects viz inhibition of seed germination seedling growth green plant

regeneration root and coleoptile growth (cited in Rocha et al 2005) In addition DON

enhances disease severity in sensitive host plants (Maier et al 2006)

35

Avoidance of DON contamination or reduction of toxin levels in contaminated grains

remains a challenging task Physical and chemical processes are either inappropriate or

prohibitively expensive to use to decrease the DON content of agricultural commodities

(Kushiro 2008 He et al 2010b) The use of Fusarium resistant crop varieties might be the

best method to reduce DON contamination However breeding for FHB or GER resistant

cultivars appears to be very difficult and time consuming as natural sources of resistance

against Fusarium-caused diseases is quantitative in nature (Ali et al 2005 Buerstmayr et al

2009) As an alternative expressing genes for enzymatic detoxification of DON in elite

cultivars may be an effective strategy for protecting the value of feeds and foods (Karlovsky

1999) Heterologous expression of eukaryotic trichothecene 3-O-acetyltransferase and UDP-

glucosyltransferase genes in cereals and Arabidopsis have resulted in reduced toxicity via

acetylation and glycosylation of the DON (Poppenberger et al 2003 Ohsato et al 2007)

It has been proposed that enzymatic de-epoxidation of trichothecene toxins could be

used to limit DON contamination (Zhou et al 2008 Foroud and Eudes 2009) DON belongs

to type B-trichothecenes which contain an epoxide ring at C12-13 and the epoxide is highly

reactive and mainly responsible for toxicity (Figure 2-1) (Swanson et al 1988 Eriksen et al

2004)

36

Figure 2-1 Mechanism of microbial DON de-epoxidation Removal of the oxygen atom from

the epoxide ring results in formation of a C=C double bond at C-12 position which reduces

cytotoxicity (Swanson et al 1988 Eriksen et al 2004)

Microbial DON de-epoxidation by single bacterial strains and by mixed cultures has

been reported (King et al 1984 Swanson et al 1987 He et al 1992 Kollarczik et al 1994

Fuchs et al 2002 Young et al 2007 Guan et al 2009 Yu et al 2010) However all of these

microbes originated from fish and other animals and were strictly anaerobic The recovered

microbial cultures and single strains only showed de-epoxidation of trichothecenes at low

(le15ordmC) and high (gt30degC) temperatures Such enzymes might have limited applications for

toxin decontamination in cereal grains The aim of the current study was to isolate the

microbes from soil capable of transforming DON to a de-epoxy metabolite under aerobic

conditions and moderate temperatures

De-epoxydation

Deoxynivalenol (DON)

De-epoxyDeoxynivalenol (dE-DON)

37

23 Materials and methods

231 Soil samples

The upper layer (0-30 cm) of soils along with crop debris were randomly collected

from cereal (eg maize wheat barley) crop fields during 2004-2006 in Southern Ontario

Canada Detailed of the crops and locations of the fields have been described in Jianwei Hersquos

Master Degree thesis (He 2007) Briefly soil samples were collected in sterile plastic bags

and transported to the laboratory Samples were sieved to remove large roots stones and

stored at 4ordmC until used The samples were mixed with grounded mouldy corn (containing 95

microgg DON) + F graminearum and incubated at 22-24degC for six weeks Five samples were

prepared for DON de-epoxidation activity analysis by dispersing 5 g soil in 50 mL distilled

water and storing at4degC until further use

232 Culture media

Media used in the study were nutrient broth (NB) Luria Bertani (LB) minimal

medium (MM) (He 2007) six mineral salts + 05 bacto peptone (MSB) (He 2007) tryptic

soy broth (TSB) corn meal broth (CMB) (Guan et al 2009) potato dextrose broth (PDB) six

mineral salts + 05 yeast extract broth (MSY) and soil extract (SE) MM was composed of

sucrose-100 g K2HPO4-25 g KH2PO4-25 g (NH4)2HPO4-10 g MgSO47H2O-02 g

FeSO4-001 g MnSO47H2O- 0007 g water- 985 mL and MSB was composed of the same

salts as MM but was supplemented with Bactotrade Peptone (05 wv) as the sole C source

instead of sucrose To prepare soil extract 100 g of the composite soil was homogenized in 1

L water The soil particles and plant debris were removed by passing the soil suspension

through Whatmanreg grade 3 filter paper (Sigma-Aldrich Oakville Ontario Canada) The

38

extract was autoclaved at 121degC for 30 min Media and ingredients were purchased either

from Fisher Scientific Inc (Fair Lawn New Jersey USA) Sigma-Aldrich (Oakvile Ontario

Canada) or Difco (Sparks Maryland USA) Prior to autoclaving pH of the broth media was

adjusted to 70 Solid media were prepared by adding 15 agar

233 Screening of soil samples

Frozen soil suspensions were thawed and vortexed to homogenize the microbial

population An aliquot (100 microL) of the suspension was immediately transferred to MSB PDB

and MM broth media DON at 50 microgmL was added to the microbial suspension cultures The

cultures were incubated at 22-24degC with continuous shaking at 180 revolutions per minute

(rpm) The extent of DON de-epoxidation was determined at 24 h 48 h 72 h 96 h 120 h and

144 h of incubation

234 Determination of de-epoxidation product

Microbial DON de-epoxidation was measured according to Guan et al (2009)

Samples were prepared by mixing equal volume of microbial culture and methanol and

allowed to stand for 30 min at room temperature Microorganisms in the mixture were

removed by passing them through a 045 microm Millipore filter (Whatmanreg Florham Park New

Jersey USA) The filtrates were used for de-epoxidation analysis by a liquid chromatography-

ultraviolet-mass spectrometry (LC-UV-MS) Identification and quantification of DON and its

transformation product was achieved using a Finnigan LCQ DECA ion trap LC-MS

instrument (ThermoFinnigan San Jose CA USA) The equipment detects 30-2000 mz

(molecular mass ion ratio) compounds and able to determine 001 micromL DON The HPLC

39

system was equipped with a Finnigan Spectra System UV6000LP ultraviolet (UV) detector

and an Agilent Zorbax Eclipse XDB C18 column (46times150 mm 35 μm) The binary mobile

phase consisted of solvent A (methanol) and solvent B (water) The system was run with a

gradient program 25 A at 0 min 25ndash41 A over 5 min isocratically at 41 A for 2 min

and 41ndash25 A over 1 min There was a 3 min post-run at its starting condition to allow for

the equilibration of the column The flow-rate was set at 10 mLmin UV absorbance was

measured at 218 nm Atmospheric pressure chemical ionization (APCI) positive ion mode

was used for MS detection A DON standard was used to adjust the instrument to its

maximum response The system was operated as follows shear gas and auxiliary flow-rates

were set at 95 and 43 (arbitrary units) voltages on the capillary tube lens offset multipole 1

offset multipole 2 offset and entrance lens were set at 3000 minus1700 minus370 minus850 minus3500

and minus4200 V respectively capillary and vaporizer temperatures were set at 200 and 450degC

respectively and the discharge needle current was set at 7 μA Identification of mycotoxin

was achieved by comparing their retention times UV spectra and mass spectrum with DON

and de-epoxy DON (Biopure Union MO USA) as standards Quantification of DON and

product was based on peak areas related to their UV and MS calibration curves

40

235 Biological DON de-epoxidation

To help identify the types of microorganisms causing the DON de-epoxidation MSB

medium was supplemented with either 100 microgmL penicillin G or streptomycin sulfate for

suppressing gram positive or negative bacterial growth and activities respectively Three

replications of antibiotic-treated media were inoculated with 100 microL of microbial culture

capable of de-epoxidizing DON The antibiotic-treated cultures were incubated at 27degC

spiked with 50 microgmL DON Controls were prepared without adding antibiotics in the

microbial culture Inhibition of DON de-epoxidation was analyzed after five day of

incubation

236 Reduction of microbial diversity by antibiotic treatments heating and

subculturing on agar medium

In order to reduce non-DON degrading microbial populations the DON de-

epoxidation capable cultures were sequentially treated with antibiotics having diverse modes

of action including i) dichloramphenicol that kills various gram positive Gram-negative and

anaerobic bacteria ii) trimethoprim that kills Gram-positive and negative bacteria iii)

penicillin that is lethal to Gram-positive bacteria and iv) cyclohexamide to kill filamentous

fungi and yeasts Antibiotics were purchased from Sigma-Aldrich Antibiotic stock solutions

(10 mgmL) were prepared with sterile water or methanol Microbial cultures in MSB

medium were treated with 100 microgmL of each antibiotic followed by incubation of the culture

at 50ordmC for 15 hours to kill undesirded microbial species After heating cultures were treated

with 50 microgmL DON and incubated at 27ordmC for five days DON de-epoxidation activity of the

microbial cultures was determined following the procedure described above Cultures that

41

showed gt99 de-epoxidation were considered for subsequent antibiotic and heat treatments

Aliquots of the achieved culture were spread on MSB agar plates and grown at room

temperature for few days Colonies appeared on the plates were collected and incubated in

MSB medium supplemented with 50 microgmL DON De-epoxidation ability of the subcultured

microbial cultures was determined after five days of incubation at 27degC by LC-UV-MS

method

237 Analysis of microbial population diversity

Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis

Initially microbial diversity was determined by terminal restriction fragment length

polymorphism (T-RFLP) fingerprint analysis (Bruce and Hughes 2000) Genomic DNA from

microbial cultures with and without enrichment was extracted using DNeasy Plant DNA

Extraction Mini Kit (Qiagen Inc Mississauga Ontario Canada) 16S-rDNA was amplified

by PCR using the HEX-labeled forward primer Bac8f

(5-agagtttgatcctggctcag-3) and

unlabeled reverse primer Univ1492r (5-ggttaccttgttacgactt-3) (Fierer and Jackson 2006)

Fifty microlitter PCR mixture for each sample was prepared having 1x HotStarTaq Master

Mix (Qiagen) 05 microM of each primer 50 microg of BSA and 50 ng of DNA The 35 PCR cycles

followed 60 s at 94degC 30 s at 50degC and 60 s at 72degC After size verification by agarose-gel

electrophoresis 50 ng of purified PCR products was digested for 3 h in a 20 microL reaction

mixture with 5 U of HhaI restriction enzyme (New England Biolabs Ipswich MA USA)

The size and relative abundance of the labeled T-RFLP fragments were separated by capillary

electrophoresis in an automated DNA sequencer equipped with fluorescence detector

(Applied Biosystem 3730 DNA Analyzer) The DNA fragments were analyzed with the Peak

42

Scannertrade Software v10 to create an electropherogram with the genetic fingerprints of the

microbial community

Analysis of V3-V5 region of the 16S-rDNA

To define the bacterial genera present in the enrichment culture the 16S-rDNA was

amplified using two sets of bacterial universal primers Primarily 16S-rDNA was PCR

amplified using the primers 46f-5rsquogcytaacacatgcaagtcga3rsquo (Kaplan and Kitts 2004) and

1371r-5rsquogtgtgtacaagncccgggaa3rsquo (Zhao et al 2007) PCR was performed in a total volume of

25 μL which contained 06 μM of each primer 1x of HotStarTaqreg Master Mix (Qiagen Inc)

and 20-40 ng of DNA The PCR cycles included the following steps 95ordmC (15 min) 35 cycles

of 94ordmC (20 s) 52ordmC (15 s) 72ordmC (1 min 30 s) and a 72ordmC (7 min) extension step The second

pair of universal bacterial primers were 27f-5rsquoagagtttgatcmtggctcag3rsquoand 1492r-

5rsquoggttaccttgttacgactt3rsquo (Lane 1991) PCR was conducted with 25 cycles following the

procedures of Tian et al (2009) Amplification was carried out in a GeneAmpreg PCR System

9700 (Applied Biosystems) Furthermore the PCR products were used as templates to

amplify the hypervariable V3-V5 region of the 16S-rDNA using primers 357f-

5rsquocctacgggaggcagcag3rsquo and 907r7 5rsquoccgtcaattcctttgagttt3rsquo (Yu and Morrison 2004)

After purification of the PCR products by QIAquick PCR purification kit two

amplicon libraries were prepared by ligating to pCR-TOPO vector (Invitrogen) and the library

clones were transformed into TOPO10 competent cells according to Invitrogenrsquos TOPO

cloning manual The clone libraries were plated on LB plates containing 25 μgmL

kanamycin Fifty colonies from each library were randomly picked up from the overnight

plates and inserts were sequenced as described by Geng et al (2006) PCR products were

43

sequenced with an automated ABI 3730 DNA analyzer (Applied Biosystems) The sequence

similarities of the amplified V3-V5 region were compared with the ribosomal database project

(RDP) (httprdpcmemsuedu) Sequences with greater than 95 identity were assigned to

the same bacterial genus

238 Factors influencing DON de-epoxidation

DON de-epoxidation ability of the enriched microbial culture was investigated under

different conditions as described below

Maintaince of active culture

To retain microbial de-epoxidation activity the active culture has been maintained in

MSB medium containing 50 microgmL DON

Media growth temperature and pH

Overnight microbial cultures (100 μL) were added to 2 mL of NB LB MM TSB

MSB CMB MSY PDB or SE broths The pH of the media was adjusted to 70 and the

experiments were conducted at 27degC temperature The effect of temperature was studied by

incubating the culture in MSB medium (pH 7) at 12 20 25 30 and 40ordmC To determine the

effects of pH MSB medium pH was adjusted to 55 60 65 70 75 and 80 Media with

different pHs were inoculated with aliquots of the overnight active culture and then incubated

at 27ordmC The cultures were supplemented with 50 μgmL DON before incubation Three

replications for each medium temperature and pH condition were evaluated

44

De-epoxidation under aerobic and anaerobic conditions

Three replicate microbial cultures of five mL each were grown in sterile 15 mL

centrifuge tubes under aerobic conditions and in an anaerobic chamber filled with 95 CO2

and 5 H2 (COY Laboratory Products Inc Ann Arbor MI USA) Three days after initiation

half (25 mL) of the cultures from anaerobic conditions was transferred to new centrifuge

tubes and brought to aerobic conditions Similarly half of the aerobically grown cultures were

placed into an anaerobic chamber Cultures moved from anaerobic to aerobic conditions were

shaken after opening the lids of the tubes in a laminar flow every eight hours intervals to

maintain aerobic conditions in the culture Lids of the culture tubes transferred to anaerobic

conditions were kept loose to allow oxygen to escape from the culture Two days after

transferring to the new conditions the cultures in both conditions were treated with 50 microgmL

DON and incubated at 27degC Every 12 h 500 microL were removed from each tube and analyzed

for microbial DON de-epoxidation activity by LC-UV-MS

239 Data analysis

Data were processed using Microsoft Excel Program 2007 Statistical analysis was

done using General Linear Model procedures in SAS version 91 (SAS Institute Inc Cary

NC USA 2002-03) The results were the average of three replications stated as mean plusmn

standard error Differences between means were considered significant at plt 005 according

to Tukeyrsquos-HSD test

45

24 Results

241 Analyses of DON to dE-DON biotransformation

A typical LC-UV-MS analysis where there is incomplete de-epoxidation of DON by a

particular soil suspension culture is shown in Figure 2-2 Analysis by LC with UV detection

and integration of peak areas showed 662 conversion of DON to dE-DON (Figure 2-2a)

Analysis LC with MS detection (expressed as total ion counts) indicated 643 conversion

(Figure 2-2b) LC analyses with UV and TIC (total ion count) detection are subject to

interferences due to co-eluting contaminants and therefore integration of small peaks was

very difficult making quantification inaccurate MS detection by SIM (selected ion

monitoring) can eliminate the influence of co-eluting contaminants For example the SIM LC

chromatogram for DON (Figure 2-2c) was generated by using the major ions resulting from

the APCI positive ion MS of DON (Figure 2-2e)

Similarly the SIM LC chromatogram for dE-DON (Figure 2-2d) was derived from the

MS ions of dE-DON (Figure 2-2f) The signal to noise ratios (SN) of peaks observed by SIM

were at least 20 times greater than those detected by TIC because there is reduced likelihood

of co-eluting peaks having the same ions Integration of the SIM peaks revealed a 616

conversion The three methods gave similar results but the SIM LC results were considered

to be the most accurate Using that method the conversion of DON to dE-DON by the mixed

culture occurred most quickly at 48 h and was completed by 144 h (Figure 2-3)

46

47

Figure 2-2 LC-UV-MS chromatographic analysis of DON and its de-epoxidated product dE-

DON obtained by culturing with selected soil microbes (a) LC-UV chromatogram of product

mixture monitored at 218 nm (b) LC-MS total ion chromatogram (TIC) of product mixture

(c) selected ion monitoring (SIM) chromatogram of cultured DON at mz 231 249 267 279

and 297 (d) SIM chromatogram of cultured dE-DON at mz 215 233 245 251 263 and 281

(e) atmospheric pressure chemical ionization (APCI) positive ion mass spectrum of DON (f)

APCI positive ion mass spectrum of dE-DON

48


Initially samples originating from soils were analyzed in three types of culture media

to support the growth of fungi yeast and bacteria However cultures growing in PDB which

is a suitable substrate for many fungi and yeasts did not permit transform DON to dE-DON

(data not shown by microorganisms) A define medium (ie MM) was also used in the

sample screening assay None of the soil samples showed de-epoxidation activity in MM

(data not shown) From five soil samples one soil sample culture showed de-epoxidation of

DON in MSB The culture in MSB converted gt99 DON to dE-DON within 144 h (Figure 2-

3) Conversion began after a lag of 48 h and was linear thereafter with a rate of 1 per hour

49

Figure 2-3 Time course of relative biotransformation of DON to dE-DON by the initial soil

suspension culture in MSB medium The culture contained 50 μgmL DON at time 0 Culture

with three replications was incubated at room temperature with continuous shaking at 180

rpm The conversion of DON to dE-DON was analysed by LC-UV-MS at 24 h intervals Blue

and red lines are the percent conversion of DON and recovery of dE-DON respectively

50

243 Evidence of biological DON de-epoxidation

Treatment of active microbial culture with 100 microgmL of streptomycin completely

inhibited DON de-epoxidation In contrast the same concentration of penicillin G did not

affect activity of the culture (Figure 2-4) Streptomycin is effective against Gram-negative

bacteria while penicillin G is lethal to Gram-positive bacteria These experiments provided

evidence that DON de-epoxidation may be caused by certain Gram-negative bacteria

51

Figure 2-4 Microbial de-epoxidation of DON after five days of incubation The active DON

de-epoxidation culture was treated with either 100 microgmL penicillin G or streptomycin

(Strepto) in MSB Cultures having three replications were added 50 microgmL DON and

incubated at room temperatures with continuous shaking at 180 rpm For controls cultures

were without antibiotics DON to dE-DON transformation was monitored after five day of

incubation by LC-UV-MS method

0

20

40

60

80

100

120

Penicillin Strepto Control

DO

N d

e-e

po

xyd

ati

on

(

)

Antibiotics

52

244 Reduction of the microbial diversity

Plating the suspension cultures onto MSB agar revealed colonies with a variety of

sizes morphologies and colours reflecting a mixed culture (data not shown) To reduce the

complexity of the culture three steps of selection pressures were applied namely treatment

with antibiotics heat shocks and subculturing on solid medium The above indicated

treatments did not significantly affect DON de-epoxidation of the microbial cultures (data not

shown) T-RFLP profiles of the mixed culture before treatments showed six distinct peaks of

about 110 150 200 220 340 and 420 bp restriction fragments with the 110 bp peak being

the predominant one (Figure 2-5a)

After treatment with antibiotics and heat only three peaks at 110 150 and 325 bp

remained (Figure 2-5b) suggesting that there was a reduction in the microbial diversity

without affecting DON de-epoxidation After enrichments the bacteria species in the culture

changed their growth patterns as the electropherogram produced different sizes of pick

lengths compared to untreated culture

53

(b)

(a)

Rela

tive flu

ore

scen

ce

32000

0

0 500

16S-rDNA fragment size (bp)

100 200 300 400

Rela

tive flu

ore

scen

ce

32000

0

500


0 300200100 400

54


length polymorphism (T-RFLP) (a) Restriction fragments of the 16S-rDNA of the untreated

culture and (b) fragmentation patterns of the enriched culture Picks along the X-axis showed

the fragmentation patterns of the 16S-rDNA which is related to the bacterial species diversity

The length of picks (along the Y-axis) showed abundance of terminal restriction fragments

related to the abundance of particular bacterial speciesgenera

55

245 Microbial population structures in the enriched culture

Microbial diversity in the enriched culture was determined by analyzing the sequences

of the V3-V5 region (~586 bp) amplified from the 16S-rDNAs of the microorganisms it

contained The sequence analysis showed the presence of Serratia Citrobacter Clostridium

Enterococcus Stenotrophomonas and Streptomyces in the enriched culture (Figure 2-6)

Out of 100 V3-V5 sequences 54 sequences were found to be belonging to Serratia

the predominant species of the culture The remaining sequences matched with 12

Citrobacter 8 Clostridium 14 Enterococcus 8 Stenotrophomonas and 4 Streptomyces

species

56


culture The bacterial genera were determined by comparing the hypervariable regions (V3-

V5) of the 16S-rDNA using ribosomal database project (httprdpcmemsuedu) X-axis

showed the identified bacterial genera and Y-axis demonstrated the abundance of the V3-V5

sequences belonging to different bacterial genera

54

12 8

14

8 4

0

20

40

60 S

eq

uen

ce

fre

qu

en

cy

Microbial genera

57

246 Effects of media growth temperature pH and oxygen requirements on DON de-

epoxidation

Of the nine media that were tested DON de-epoxidation by the enriched microbial

culture only occurred in NB and MSB (Figure 2-7a) It appeared that C andor N

concentrations in the media were crucial for the enzymatic activity because less or more than

05 Bacto Peptone in MSB reduced the microbial DON transformation ability

DON de-epoxidation activity occurred over a wide range of temperatures from 12 to

40ordmC (Figure 2-7b) The fastest rates of DON de-epoxidation occurred between 25 to 40ordmC

De-epoxidation was observed within a relatively narrow range of pH (60 to 75) with

the most rapid DON biotransformation activity observed at pH 70 (Figure 2-7c) At pH 70

the enriched culture showed DON de-epoxidation activity 125 times faster than at pH 60 or

75 At pH 55 and 80 there was no observable DON de-epoxidation

58

0

20

40

60

80

100

120

12degC

20degC

25degC

30degC

40degC

DO

N d

e-e

po

xyd

ati

on

(

)

Temperature

0

20

40

60

80

100

120

DO

N d

e-e

pxyd

ati

on

(

)

Media

(a)

(b)

59

Figure 2-7 Effects of culture conditions on DON to de-epoxy DON transformation by the

enriched microbial culture For the media experiment (a) all the media were adjusted to pH 7

and the incubation temperature was 27degC For the temperature experiment (b) MSB pH 7 was

used For the pH experiment (c) MSB at 27degC was used Microbial cultures were inoculated

with 50 μgmL DON De-epoxidation was determined after 72 h of incubation Media MSY

(mineral salts + 05 yeast extract) LB (Luria Bertani) TSB (tryptic soy broth) MSB

(mineral salts + 05 bacto peptone) SE (soil extract) CMB (corn meal broth) PDB (potato

dextrose broth) and MM (minimal medium)

0

20

40

60

80

100

120

55

60

65

70

75

80

DO

N d

e-e

po

xyd

ati

on

(

)

pH

(c)

60

The enriched culture showed DON de-epoxidation activity under both aerobic and

anaerobic conditions (Figure 2-8) The fastest transformation (100 de-epoxidation after 60 h

of incubation) occurred in cultures (A) that were grown under aerobic conditions throughout

the culture growth period A medium rate of de-epoxidation (100 de-epoxidation after 72 h)

was observed in cultures that were initially grown in either aerobic or anaerobic conditions

followed by transferring to anaerobic or aerobic conditions (A+AN and AN+A) respectively

The lowest rate occurred (100 de-epoxidation after 96 h) in cultures grown in continual

anaerobic conditions (AN) The means comparison of microbial DON de-epoxidation

efficiencies at 60 h of incubation showed that cultures continually grown in conditions A and

AN were statistically different Biotransformation abilities of the cultures that were switched

from one conditions to another (A+AN and AN+A) were statistically similar among each

other but different from cultures that were only grown in conditions A or AN (plt005)

61

Figure 2-8 Microbial DON to dE (de-epoxy DON)-DON transformation activity under

aerobic and anaerobic conditions A = culture incubated and examined the activity only under

aerobic conditions A+AN = culture initially was grown in aerobic conditions and then

transferred to anaerobic conditions AN = cultures grown throughout the assay under

anaerobic conditions AN+A = culture initially grown under anaerobic conditions and then

transferred to aerobic conditions Differences of lower case letters are significantly different

in de-epoxidation capability (after 60 h) among incubation conditions according to Tukeyrsquos

HSD test (p lt 005) The experiment was conducted with three replications

62

25 Discussion

The current study describes a novel bacterial culture isolated from agricultural soil that

was capable of transforming DON to de-epoxy DON under aerobic and anaerobic conditions

at moderate temperatures Because it has been speculated that DON translocates to soil from

contaminated crops and DON that enters into the soil could be metabolized by microbes

(Voumllkl et al 2004) soil samples for the current study were mainly collected from fields where

cereal crops contaminated with DON were grown

In our initial sample screening steps upwards of 144 h of incubation were required to

achieve gt99 DON de-epoxidation Such a long period may have been due to the presence of

fast growing microorganisms that had suppressive effects on the growth or activity of the

DON transforming bacteria (Davis et al 2005) The sequential use of different antibiotics

with different modes of actions followed by thermal treatments and subculturing on solid

medium resulted in a reduction in the number of bacterial species as determined by T-RFLP

analysis Analysis of V3-V5 region of the 16S-rDNA showed that five bacterial genera were

present in the enriched culture The culture showed higher DON de-epoxidation activity (only

60 h required for gt99 biotransformation) compared to initial soil suspension culture that

required 144 h to complete the de-epoxidation This microbial culture could be used to isolate

the DON de-epoxidizing gene(s) applying metagenomics approaches (Daniel 2005)

The culture showed complete de-epoxidation activity both in MSB and NB media

However we have found that the use of nutritionally rich NB medium enhanced non-target

bacterial (eg Serratia lequifaciences) growth but this did not occur in MSB which is a

comparably poor medium To avoid the possible suppressive and detrimental effects of the

fast growing bacteria on the target bacteria MSB was used for enrichment and

63

characterization of the culture An important characteristic of the DON de-epoxidation

activity observed for the microbial culture in the current study is its activity at moderate

temperature (25-30degC) In contrast previously isolated cultures showed optimal activities at

low (le15degC) or high temperatures (37degC) (Guan et al 2009) Also the culture showed DON

de-epoxidation activity at neutral pH range (pH 60-75) in contrast to previous reports which

found that microbial trichothecene de-epoxidation occurred at acidic and basic pHs (Swanson

et al 1987 He et al 1992 Kollarczik et al 1994 Young et al 2007) However a microbial

culture from fish gut showed de-epoxidation under wide range of pHs (Guan et al 2009)

Probably the pH range reflects the environment in which the bacteria naturally grow which in

this case is similar to the soil pH of a typical crop field in Southern Ontario of Canada The

advantage of the present finding is that DON de-epoxidation activity of the culture was

optimal in a temperature range that matches the temperature range of activity for most arable

plants Therefore it is reasonable to suggest that the gene(s)enzyme(s) responsible for the

activity have potential application for enzymatic detoxification of DON in cereals (Karlovsky

1999)

The current observation that bacterial DON de-epoxidation activity occurred under

both aerobic and anaerobic conditions was interesting because DON de-epoxidation is a

reductive chemical reaction that might be expected to be promoted by anaerobic conditions

However there are previous reports of aerobic de-epoxidation of eldrindieldrin by soil

bacteria (Matsumoto et al 2008) and Goodstadt and Ponting (2004) described aerobic

reductive de-epoxidation by vitamin K epoxide reductasephylloquinone reductase A

definitive determination of the oxygen sensitivity of the enzyme responsible for DON de-

epoxidation will ultimately come from experiments with the purified protein

64

In conclusion this study reports on a novel bacterial source from soil that efficiently

de-epoxidizes DON at moderate temperatures under both aerobic and anaerobic conditions

This research may lead to the isolation of microbes and gene(s) encoding enzyme(s) capable

of de-epoxidizing DON that could create transgenic plants with reduced DON contamination

thus improving food and feed safety and reducing negative effects of the toxin on public

health

26 Acknowledgements

The authors gratefully acknowledged the Natural Sciences and Engineering Research

Council of Canada (NSERC) for awarding a scholarship to Rafiqul Islam for PhD program

The research was supported by the Agriculture and Agri-Food Canada Binational

Agricultural Research and Development Fund (AAFCBARD) project We are thankful for

the contributions of Jianwei He Xiu-Zhen Li and Honghui Zhu

65

CHAPTER 3

30 ISOLATION AND CHARACTERIZATION OF A NOVEL SOIL BACTERIUM

CAPABLE OF DETOXIFYING ELEVEN TRICHOTHECENE MYCOTOXINS

31 Abstract

Deoxynivalenol (DON vomitoxin) is one of the most commonly detected food and

feed contaminating mycotoxins worldwide In order to develop a control method effective

under aerobic conditions and moderate temperatures a bacterial strain ADS47 was isolated

capable of transforming DON to the less toxic de-epoxy DON The bacterial strain is

facultatively anaerobic Gram-negative rod shaped with an average cell size of 150 X 072

microm and has peritrichous (multiple) flagella The 16S-rRNA gene sequence had gt99

nucleotide identity to Citrobacter freundii a bacterium that belongs to the gamma subclass of

proteobacteria Strain ADS47 showed near 100 de-epoxidation of DON after 42 h of

incubation in nutrient broth (NB) under aerobic conditions moderate temperature (28˚C) and

neutral pH (70) However de-epoxidation could occur at 10-45˚C and pH 60-75 Under

anaerobic conditions near 100 de-epoxidation was observed after 72 h mainly during the

stationary growth phase Microbial DON de-epoxidation ability increased with increasing

DON concentrations in the culture media Inhibition of bacterial de-epoxidation by sodium

azide suggested that an active electron transport chain was required In addition to DON

strain ADS47 was able to de-epoxidize andor de-acetylate five type-A and five type-B

trichothecene mycotoxins This is the first report of a bacterial isolate capable of de-

epoxidizing DON and trichothecene mycotoxins under aerobic conditions and moderate

66

temperatures Strain ADS47 could potentially be used in bioremediation to eliminate DON

and other trichothecene mycotoxins from contaminated food and feed

Keywords aerobic de-epoxidation Citrobacter freundii isolation soil bacterium

trichothecenes

32 Introduction

Cereals are often contaminated with a variety of type-A and type-B trichothecene

mycotoxins produced by a number of toxigenic Fusarium species (Placinta et al 1999

Binder et al 2007 Foroud and Eudes 2009) Trichothecenes are tricyclic sesquiterpenes with

a double bond at the C9 10 position and a C12 13 epoxy group (Desjardins 2006) The two

types are distinguished by the presence of a ketone at position C8 in type-B which is absent

in type-A (Yazar and Omurtag 2008) The type-B toxin deoxynivalenol (DON) is the most

frequently found mycotoxin in food and feed around the world (Larsen et al 2004 Desjardins

2006 Starkey et al 2007) Different Fusarium species associated with Fusarium head blight

(FHB) or Gibberella ear rot (GER) disease of small grain cereals and maize are mainly

responsible for contaminating grains with DON and other type-A and type-B trichothecenes

These can cause serious mycotoxicosis in humans animals and plants (Ueno 1985 DrsquoMello

et al 1999 Bae and Pestka 2008) Common effects of DONTC toxicity in animals include

diarrhea vomiting feed refusal growth retardation immunosuppression reduced ovarian

functionsreproductive disorders and even death (Pestka and Smolinski 2005 Pestka 2007)

DON enhances FHB disease severity in wheat and maize (Proctor et al 1995 Desjardins

1996 Maier et al 2006)

67

Reduction of mycotoxin levels in contaminated agricultural products remains a

challenging task Current physical and chemical processes to decrease DON content are either

ineffective or prohibitively expensive (Kushiro 2008 He et al 2010b) However the use of

detoxifying microbes to biodegrade mycotoxins is an alternative (Awad et al 2010

Karlovsky 2011) The toxicity of DON is mainly due to the epoxide and there is a significant

reduction of DON toxicity (54) when it is reduced to produce de-epoxy DON Related

mycotoxins also show reductions in toxicity when they are de-epoxidized such as NIV (55)

and T-2 toxin (400) (Swanson et al 1988 Eriksen et al 2004) Bacillus arbutinivorans

strain LS100 and Eubacterial strain BBSH 797 originating from chicken intestine and rumen

fluid respectively are capable of cleaving the epoxide ring of DON under anaerobic

conditions and high temperature (37degC) (Fuchs et al 2002 Yu et al 2010) Strain BBSH 797

which can de-epoxidize DON scirpentriol and T-2 triol trichothecene mycotoxins has been

used as an additive for poultry and swine diets (Karlovsky 2011) However there are no

reports of microbes able to de-epoxidze DON and other trichothecenes under aerobic

conditions and at moderate temperatures

To develop an effective method for eliminating DON and trichothecene mycotoxins

from contaminated grains soils from fields with FHBGER infected crops were screened for

DON de-epoxidizing microbes (refer to chapter 2 Islam et al 2012) One soil sample yielded

a mixed microbial culture was partially enriched for bacteria with DON de-epoxidizing

activity (chapter 2) The current study describes the purification and characterization of a

novel DON de-epoxidizing bacterium from this culture

68


331 Microbial source

A mixed microbial culture originating from a screen of more than 150 separate soil

samples collected from maize and wheat fields near Guelph Ontario Canada (Islam et al

2012) was used for enrichment purification and characterization of the DON de-epoxidizing

bacterium

332 Media and chemicals

The media used were nutrient broth (NB) 110 strength NB (110 NB) minimal

medium (MM) consisting of six mineral salts (MS) + 100g per liter sucrose (Islam et al

2012) MS + 05 Bacto Peptone (MSB) MS + 03 Bacto Peptone (MSC) MS + 05

yeast extract (MSY) MS + 200 microgmL DON (MS-DON) sterile water-soluble soil extract

(SE) (Islam et al 2012) potato dextrose broth (PDB) and Luria Bertani (LB) Prepared media

and media components were obtained from Fisher Scientific (Fair Lawn New Jersey USA)

Sigma-Aldrich (Oakville ON Canada) andor Difco (Sparks Maryland USA) Media pH

was adjusted as required prior to autoclaving For solid media preparation 15 agar was

added DON and all trichothecene mycotoxins were purchased from Biomin (Romer Lab

Union MO USA)

333 Isolation of the DON de-epoxidizing bacterial strain

Aliquots (20 microL) of the microbial culture partially enriched for DON de-epoxidizing

ability (Islam et al 2012) was transferred to 05 mL MSB (pH 70) in sterile 15 mL

Eppendorf tubes After addition of 200 microgmL pure DON the culture was incubated at room

69

temperature with continuous shaking at 220 revolutions per minute (rpm) Seven to ten days

later 20 microL of the culture was transferred to fresh MSB supplemented with 200 microgmL DON

The culture was then incubated for 7 to 10 days under the above conditions This procedure

was repeated 12 times

After three months of repeated transfers in broth with 200 microgmL DON 100 microL of the

culture was serially diluted in 09 mL MSB containing 05 Tweenreg20 (Sigma Aldrich) to

reduce bacterial cell aggregation (Mabagala 1997) The dilutions were vigorously shaken for

ten minutes A 100 microL aliquot from each dilution was spread on MSB + agar (MSA) The

MSA plates were incubated at room temperature until single colonies appeared

Single colonies from plates were transferred to 1 mL MSB+ 50 microgmL DON and

incubated at room temperature with continuous shaking at 180 rpm under aerobic conditions

After three days the DON de-epoxidation ability was examined by mixing an equal volume

of the bacterial culture and methanol and incubating for 30 min at room temperature to lyse

the cells Insoluble materials in the mixture were removed by passing it through a 045 microm

Millipore filter (Whatmanreg Florham Park New Jersey USA) The level of de-epoxy DON

(dE-DON also known as DOM-1) in the filtrate was determined by liquid chromatography-

ultraviolet-mass spectrometry (LC-UV-MS) (Guan et al 2009)

Individual colonies that showed de-epoxidation activity were serially diluted in

MSB+05 Tweenreg20 and plated onto MSA Single colonies that appeared on the agar plate

were incubated in MSB as described above and retested for DON de-epoxidation ability To

ensure that pure cultures were obtained this was repeated 10 times and individual colonies

were selected on MSA each time

70

334 Identification of a DON de-epoxidizing bacterium

Strain ADS47 with DON de-epoxidation ability was obtained from a single colony

after 10 serial transfers and was grown on MSA at room temperature under aerobic

conditions for 72 h Colony color shape edge and elevation were observed The cell

morphology of the bacterium was examined with a transmission electron microscope at the

University of Guelph Guelph Ontario Canada Whole bacterial cells were placed directly on

a formvar and carbon-coated 200-mesh copper grid They were washed two times by touching

the grids to a drop of clean dH2O and the excess water was blotted off The samples were

negatively stained using 1 Uranyl Acetate (aq) for five seconds Electron microscopy was

performed on a Philips CM10 transmission electron microscope at 60kV under standard

operating conditions (Philips Optics Eindhoven Netherlands) Gram staining was performed

using the procedure described in the commercial gram stain kit from BD Canada

(Mississauga Ontario Canada)

Total bacterial DNA was extracted using a DNeasy Plant DNA Extraction Mini Kit

(Qiagen Inc) The 16S-rDNA was PCR amplified using the primers 46f-

5rsquogcytaacacatgcaagtcga3rsquo (Kaplan and Kitts 2004) and 1371r-5rsquogtgtgtacaagncccgggaa3rsquo

(Zhao et al 2007) PCR was performed in 25 microL containing 06 microM of each primer 1x of

HotStarTaqreg Master Mix (Qiagen Inc) and ~40 ng of DNA The PCR cycles were 95ordmC (15

min) 35 cycles of 94ordmC (20 s) 52ordmC (15 s) 72ordmC (1 min 30 s) and a 72ordmC (7 min) extension

step Amplification was carried out in a GeneAmpreg PCR System 9700 (Applied Biosystems)

PCR products were sequenced with an automated ABI 3730 DNA analyzer (Applied

Biosystems) The 16S-rRNA gene sequence similarity was performed by BlastN using the

Ribosomal Database Project (RDP) (httprdpcmemsueduseqmatch)

71

A phylogenetic tree was constructed by comparing the partial 16S-rDNA sequence of

strain ADS47 with the 11 known Citrobacter species (Bornstein and Schafer 2006 Brenner et

al 1993) The Citrobacter strains were C freundii [FN997616] C koseri [AF025372] C

amalonaticus [AF025370] C farmeri [AF025371] C youngae [AB273741] C braakii

[AF025368] C werkmanii [AF025373] C sedlakii [AF025364] C rodentium [AF025363]

C gillenii [AF025367] and C murliniae [AF025369] Escherichia coli O157H7 [AE005174]

was used as an out group strain The analysis was performed using distance matrix with a gap

opening of 100 and a gap extension of 01 by a server based ClustalW2 program

(httpwwwebiacukToolsmsaclustalw2) A dissimilarity tree was created and viewed

using a TreeView X software (996)

335 Effects of DON concentration aeration substrate temperature pH and antibiotic

To determine if DON concentration can affect the growth of the isolated bacteria

tubes containing 5 mL of NB or MSB with 20 200 or 500 microgmL of DON were inoculated

with strain ADS47 that had been grown for 16 h with continuous shaking at 220 rpm and then

adjusted to cell density at 025 OD600nm (107 cellsmL) The cultures were incubated at 28degC

with continuous shaking at 180 rpm and 05 mL was removed every 12 h to measure

OD600nm The culture (05 mL) was then analysed for DON de-epoxidation activity by LC-

UV-MS (Guan et al 2009) as described above Three replicate cultures were tested

To examine the effect of aeration five mL of NB or MSB containing 50 microgmL DON

were inoculated with an overnight culture of strain ADS47 prepared as described above and

incubated at 28˚C with continuous shaking at 180 rpm One mL of culture was removed every

six hours and bacterial growth was determined by measuring cell density at OD600nm At the

72

same time points culture samples were analysed for DON to dE-DON transformation ability

(Guan et al 2009) The experiment was repeated with MSB medium containing 50 microgmL

DON under anaerobic conditions as described by Islam et al (2012) with DON to dE-DON

transformation ability being determined every 6 h Three replicate cultures were prepared for

each assay

The effect of substrate on DON de-epoxidation ability was tested after growing

ADS47 for 72 h in NB 110 NB MM MS-DON MSB MSC MSY SE PDB or LB

containing 50 microgmL DON For this assay the media were maintained at pH70plusmn02 and 28degC

for 72 h The effect of temperature was examined with MSB containing 50 microgmL DON at 10

15 20 25 30 35 40 45 or 50degC The effect of pH on growth of strain ADS47 was examined

with MSB adjusted to pH 55 60 65 70 75 or 80 and the cultures were incubated at 28degC

for 72 h Three replicate cultures were used for each assay

Overnight cultures of ADS47 in MSB were treated with 001 and 0001 (wv) of

an antibiotic sodium azide (SA) (Sigma-Aldrich) Controls were prepared in MSB without

SA Three replicate cultures were spiked with 50 microgmL DON DON to dE-DON

transformation ability of the bacterial culture was analysed (Guan et al 2009) after 72 h at

28degC with continuous shaking at 180 rpm Three replicate cultures were tested

336 Localization of DON to dE-DON transformation ability in the bacterium

Bacterial cell extracts were prepared using B-PER II Bacterial Protein Extraction

Reagent following the manufacturerrsquos instructions (PI 78260 Thermo Scientific Rockford

IL USA) ADS47 was grown in 15 mL MSB for 48 h under the growth conditions described

above A culture (05 mL) was centrifuged at 5000 x g for 10 minutes and the pellet was

73

collected B-PER II reagent (2 mL) was added to the pellet and the cells were lysed by

pipetting the suspension up and down several times The cell lysate was treated with 20 microL

Halt Protease Inhibitor Cocktail (Thermo Scientific Rockford IL USA) Culture filtrate was

prepared by passing 05 mL of the culture through a 022 microm Millipore filter (Fisher

Scientific) The remaining 05 mL culture was used as control Cell lysate culture filtrate and

control were incubated with 50 microgmL DON and incubated at 28degC DON to dE-DON

transformation ability of the cell lysate culture filtrate and control were analysed after 72 h

incubation (Guan et al 2009) Three replicate cultures were tested

337 Detoxification of additional trichothecene mycotoxins

Strain ADS47 was evaluated for its ability to degrade five type-A trichothecene

mycotoxins (HT-2 toxin T-2 toxin T2-triol diacetoxyscirpenol and neosolaniol) and five

type-B trichothecene mycotoxins (nivalenol verrucarol fusarenon X 3-acetyl-

deoxynivalenol and 15-acetyl-deoxynivalenol) One and a half mL NB was added with 100

microgmL of each mycotoxin and then inoculated with 100 microL of ADS47 culture grown

overnight as previously described The culture of NB + mycotoxin + ADS47 was incubated at

28degC with continuous shaking at 180 rpm and DON to dE-DON transformation ability of the

culture was analysed by LC-UV-MS (Guan et al 2009) Five replicate cultures were tested for

each trichothecene mycotoxin

74

338 Statistical analysis


performed using General Linear Model procedure in SAS version 91 (SAS Institute Cary

NC USA 2002-03) Replications were averaged and stated as mean plusmn standard error The

mean differences were considered significant at plt 005 according to Tukeyrsquos-HSD test

75

34 Results

341 Isolation and identification of a DON de-epoxidizing bacterium

A DON de-epoxidizing bacterium designated strain ADS47 was isolated after 12

transfers over three months from cultures growing in MSB containing 200 microgmL DON

Strain ADS47 produced small to medium size circular convex and white colonies on MSA at

room temperature under aerobic conditions (Figure 3-1a) The bacterium is rod shaped and

150 x 072 microm in size (Figure 3-1b) It has numerous flagella that cover the surface of the cell

(peritrichous flagella) and is Gram-negative

BlastN analysis of the 16S-rDNA sequence of strain ADS47 showed the most

similarity to the eleven known Citrobacter species in the gamma proteobacteria of the

Enterobacteriaceae In particular the 16S-rDNA nucleotide sequence of ADS47 was more

than 99 identical to C fruendii (Figure 3-2) The next most similar sequences were a cluster

of C werkmanii and C murliniae followed by more distantly related C braakii C gilleni

and C youngae A separate cluster was formed by C rodentium C farmeri C sedlakii C

koseri and C amalonaticus

76

Figure 3-1 (a) Colony color shape and size of the mycotoxin degrading bacterial strain

ADS47 on mineral salts agar (MSA) medium (b) Transmission electron microscopic (TEM)

image of the Citrobacter freundii strain ADS47 The bacterium showed multiple flagella

(peritrichous flagella) and numerous fimbriae around the cell surface

(a) (b)

77

Figure 3-2 Dendrogram based on 16S-rDNA sequences for strain ADS47 and 11 reference

Citrobacter species An E coli O157H7 strain was considered as an out group species The

tree was constructed using a genetic distance matrix by ClustalW 2 and viewed by

TreeViewX software The distance was converted to scale and shown by branch lengths (scale

bar 001 which indicated the 16S-rDNA nucleotide sequence variability between the bacterial

species) GenBank accession numbers for 16S-rDNA sequences of the species used for

comparison are shown in materials and methods section (334)

78

342 Effects of DON concentrations on bacterial DON de-epoxidation

Growth of ADS47 aerobically in MSB with 20 200 and 500 microgmL DON was not

significantly different from that observed in control cultures without DON (Figure 3-3a)

Similarly increasing DON concentrations did not affect bacterial growth in NB medium

(Figure 3-3b)

DON de-epoxidation activity determined as microgmL DON convertedcell density

(OD600) was measured lowest in MSB+20 microgmL DON and highest in MSB+ 500 microgmL

DON containing cultures (Figure 3-3c) The highest rate of dE-DON formation was 115 microgh

in ADS47 cultures in MSB with 500 microgmL DON between 48 to 72 h All of the DON was

converted in the cultures containing 20 microgmL DON and 200 microgmL DON between 48-60 h

and 60-72 h respectively In 200 microgmL and 500 microgmL of DON cultures the rate of de-

epoxidation increased significantly after 36 h

79

00

04

08

12

16

0 24 36 48 60 72 96

OD

600n

m

Incubation time (h)

Control

20 microgmL

200 microgmL

500 microgmL

(b)

(a)

80

Figure 3-3 Effects of DON concentration on bacterial growth and de-epoxidation activities

Strain ADS47 was grown in MSB and NB media with three different DON concentrations 20

microgmL 200 microgmL and 500 microgmL Control cultures were prepared without DON Cultures

were grown at 28degC with continuous shaking at 180 rpm (a) Microbial density (OD600 nm) in

MSB with different DON concentrations was measured at 24 36 48 60 72 and 96 h (b)

Microbial growth (OD600 nm) in NB having different DON concentration was measured at 24

48 72 and 96 h (c) Bacterial DON de-epoxidation activity in 20 200 and 500 microgmL DON

containing cultures at different time points DON de-epoxidation was measured in microgmL per

OD600 nm Three replicate cultures for each assay were analysed

(C)

81

343 Influence of growth conditions and antibiotic on microbial DON de-epoxydation

Aerobic and anaerobic conditions

ADS47 grew more quickly uner aerobic conditions under MSB+50 microgmL DON than

in MSB+50 microgmL DON under anaerobic conditions (Figure 3-4a) Aerobic growth was

faster in NB+50 microgmL DON compared to MSB+50 microgmL DON

However the DON de-epoxidation activities on a cell density basis were very similar

in NB+50 microgmL DON compared to MSB+50 microgmL DON (Figure 3-4b) Also DON de-

epoxidation activity of ADS47 under aerobic conditions in MSB+50 microgmL DON was higher

than in the same medium under anaerobic conditions (Figure 3-4b)

82

(b)

(a)

83


anaerobic conditions (a) Microbial growth under aerobic conditions in MSB and NB medium

and anaerobic conditions in MSB medium (b) Microbial DON de-epoxidation ability under

aerobic conditions in MSB and NB media and anaerobic conditions in MSB medium

Cultures media having 50 microgmL DON were inoculated with overnight culture of strain

ADS47 Initial microbial concentration in the cultures was adjusted to 025 OD600nm Cultures

were incubated at 28degC having three replications Bacterial growth at OD600nm and DON de-

epoxidation in microgmL per OD600nm were measured at six hour interval

84

Media temperature and pH

Growth of ADS47 in media containing 50 microgmL DON under aerobic conditions at 72

h was three-fold higher in NB and LB than in SE two-fold higher than in 110 NB and MSC

and did not occur in MS-DON (Figure 3-5a) Growth in MM MSB and MSY was

intermediate

Over the 72 h DON de-epoxidation per cell density was highest in MSB and MSY and

approximately 25 lower in NB and LB and lower by 75 and 87 in 110 NB and MSC

respectively (Figure 3-5b) No DON de-epoxidation was observed in MS-DON in which

DON was used as a carbon source Bacteria grown in MM broth which contains sucrose as a

sole carbon source were unable to de-epoxidize DON No DON de-epoxidation was observed

in SE and PDB grown cultures

Growth of ADS47 under aerobic conditions for 72 h in MSB+50 microgmL DON was

greatest between 25-40degC but no growth occurred at 50degC (Figure 3-6a) DON de-

epoxidation activity was greatest and equivalent between 20-40degC and lower at 10 15 and

45degC temperatures (Figure 3-6b)

ADS47 growth under aerobic conditions for 72 h in MSB+50 microgmL DON was

limited to neutral pHs (ie 60 - 75) (Figure 3-7a) The transformation of DON to dE-DON

occurred at the same pH range and DON de-epoxidation activity was not significantly

different between pH 60 to 75 (Figure 3-7b)

85

b

c

a

b

a

d c

b b

(a)

(b)

b

c

b

a a

d

86

Figure 3-5 Effects of media on bacterial growth and DON de-epoxidation activity (a)

Microbial growth (at OD600nm) on different culture media Media pHs were adjusted to

70plusmn02 (b) Bacterial DON to dE-DON transformation in microgmL by OD600nm in different

media after 72 h The cultures having three replications were incubated for 72 h at 28degC and

DON de-epoxidation was measured The Media NB = nutrient broth 1NB = 110 strength

NB MM = minimal medium MS-DON = six mineral salts + 200 microgmL DON as sole C

source MSB = six mineral salts + 05 bacto peptone MSC = six mineral salts broth + 03

bacto peptone MSY = six mineral salts with 05 yeast extract SE = soil extract PDB =

potato dextrose broth and LB = Luria Bertani Means with the same label are not significantly

different according to Tukeyrsquos HSD test (p lt 005)

87

(a)

(b)

88

Figure 3-6 Effects of temperature on microbial growth and DON de-epoxydation capability

(a) Microbial growth (at OD600nm) at different temperatures after 72 h of incubation (b)

Bacteria DON de-epoxidation in microgmL by OD600nm in different temperatures The most

efficient temperature range (ge40 microgmL DON de-epoxidation) is marked by dot lines The

experiment was conducted in MSB medium at pH 70plusmn02 with three replications

89

(a)

(b)

90

Figure 3-7 Influence of pH on microbial growth and DON de-epoxydation (a) Microbial

growth (OD600nm) in MSB at different pH (b) Bacterial DON to de-epoxy-DON

transformation in microgmL by OD600nm in different pH Dot lines indicated the efficient de-

epoxidation (ge40 microgmL DON conversion) pH range Experiment was conducted at 28degC

with three replications

91

Antibiotic

Treatment of ADS47 at 72 h in MSB+50 microgmL DON with sodium azide (SA) at

0001 and 001 reduced DON de-epoxidation activity to 5 (25 microgmL) and 0

respectively The control with no SA added was able to de-epoxidize DON completely

(100) (Figure 3-8)

Figure 3-8 Effects of sodium azide (SA) on bacterial DON de-epoxidation activity

Microbial culture in MSB+50 microgmL DON was treated with 001 (SA-1) and 0001 (SA-

2) sodium azide In control (C) no SA was added The experiments with three replications

were incubated for 72 h at 28degC and DON de-epoxidation was measured by LC-UV-MS

method

0

20

40

60

80

100

120

SA1 SA2 C

D

ON

de

-ep

oxyd

ize

d

92

344 Localization of bacterial DON de-epoxidation

A cell lysate prepared from ADS47 grown for 48 h in MSB transformed 20 (10

microgmL) of DON added at 50 microgmL to dE-DON within 72 h (Figure 3-9) In contrast a cell-

free culture filtrate did not show DON de-epoxidation activity

Figure 3-9 Determination of DON to de-epoxy DON transformation by bacterial culture

filtrate and cell extract The treatments having three replications were spiked with 50 microgmL

DON and incubated at 28degC for 72 h In control strain ADS47 was incubated in MSB broth

with 50 microgmL DON DON de-epoxidation activity in percentage was examined

0

20

40

60

80

100

120

Filtrate Extract Control

D

ON

de

-ep

oxyd

ize

d

93

345 Bacterial detoxification (de-epoxidtaion and de-acetylation) of other trichothecene

mycotoxins

Aerobic cultures of ADS47 grown for 72 h in NB+100 microgmL HT-2 converted 74 of

the toxin to de-epoxy HT-2 toxin and 26 to a de-acetylated product (Figure 3-10 and Table

3-1)

In a culture treated with T2-toxin 46 was converted to de-epoxy T-2 16 was

converted to an unknown compound and 38 was not converted In ADS47 culture with

diacetoxyscirpenol (DAS) 61 was transformed into de-epoxy DAS and 39 was de-

acetylated DAS (39) Neosolaniol (NEO) was transformed to de-epoxy NEO (55) and

de-acetylated NEO (45) T2-triol was transformed in to de-epoxy T2-triol (47) and de-

acetylated T2-triol (53)

Aerobic cultures of ADS47 were also grown for 72 h in NB+100 microgmL of one of five

type-B trichothecenes For nivalenol (NIV) 92 was de-epoxydized while the remaining 8

NIV was transformed to DON 3-acetyl-deoxynivalenol (3-ADON) was transformed into

three products de-epoxy NIV (5) de-epoxy DON (54) and DON (41) For 15-

acetyldeoxynivalenol (15-ADON) 8 was transformed to de-epoxy 15-ADON and 92 to

de-epoxy DON (dE-DON) FusarenonX (FUS) had 95 transformed into de-epoxy FUS

while 97 of verrucarol (VER) was transformed to de-epoxy VER The remaining 5 FUS

and 3 VER were not transformed

94

Figure 3-10 Determination of microbial de-epoxidation and de-acetylation of HT-2 toxin by

LC-UV-MS Bacterial culture in NB was added with 100 microgmL HT-2 toxin Bacterial HT-2

toxin transformation ability was examined after 72 h of incubation at 28degC The upper panel

illustrated mass spectrometry chromatograph of the de-acetylated (red peak) and de-

epoxydized (blue peak) products Arrows indicated the targeted acetyl (red) and epoxy (blue)

functional groups of HT-toxin The middle and lower panels showed the mass spectra of the

de-acetylated (dA) and de-epoxidized (dE) compounds Arrows on the upper panel indicated

the targeted acetyl (red) and epoxy (blue) functional groups of HT-2 toxin Red and blue

arrows on the middle and lower panels pointed at the degradation sites of HT-2 toxin

95

Table 3-1 Microbial transformation of type-A and type-B tricthothecene mycotoxins after 72

h of incubation under aerobic conditions at room temperature DAS=diacetoxyscirpenol

DON=deoxynivalenol FUS=FusarenonX 3AON=3-acetyl-deoxynivalenol and

15ADON=15-acetyl-deoxynivalenol

1 Convertion of trichothecene mycotoxins to either DON or an unknown compound

Trichothecene

mycotoxin

Untransformed

trichothecenes ()

Transformed products of

trichothecenes ()

De-

epoxy

De-

acetylate

Other1

Type-A

HT-2 toxin 0 74 26 -

T-2 toxin 38 46 - 16

(unknown)

DAS 0 61 39 -

Neosolanol 0 55 45 -

T2-triol 0 47 53 -

Type-B

DON 0 100 - -

Nivalenol 0 92 - 8 (DON)

3-ADON 0 59 41 (DON)

15-ADON 0 100 - -

FUS 5 95 - -

Varrucarol 3 97 - -

96

35 Discussion

The ADS47 strain of C freundii was isolated in the present study by a prolonged

incubation of a mixed microbial culture with a high concentration DON since the bacterium

efficiently de-epoxidized (detoxified) DON and number of other trichothecene mycotoxins

under aerobic and anaerobic conditions at moderate temperatures and neutral pH The total

procedures for enrichment and isolation of the strain ADS47 is depicted in supplemental

Figure S3-1 The reductive de-epoxidation activity may be due to a cytoplasmic enzyme

which would be in agreement with a previous study that showed de-epoxidation of

epoxyalkanes was catalyzed by a bacterial cytoplasmic flavoprotein (Westphal et al 1998)

The ability of the currently isolated strain of C freundii to biodegrade environmental

pollutants such as aminoaromatic acids and heavy metals (copper) is also likely mediated by

cytoplasmic enzymes since these compounds need to be taken up by the cells before they are

metabolized (Savelieva et al 2004 Sharma and Fulekar 2009) In contrast oxidative

transformation of DON to 3-keto DON by microbial extracellular enzyme has been reported

(Shima et al 1997)

Isolation of a trichothecene de-epoxidizing microorganism from a mixed microbial

community is very challenging Although trichothecenes are toxic to eukaryotes prokaryotes

appear to be resistant to these mycotoxins (He et al 1992 Rocha et al 2005) The present

study showed that the bacteria grew well in media with high concentrations of DON (500

microgmL) Bacterial de-epoxidation of DON involves the removal of an oxygen atom from the

functional epoxy ring of the tricyclic DON structure (Yoshizawa et al 1983) Such co-

metabolic degradation does not provide the bacteria with any nutrients from the reacting

compounds (Horvath 1972 Fedorak and Grabic-Galic 1991) This is supported by the results

97

of the current study which showed that strain ADS47 was unable to grow in media with DON

as a sole carbon source and increasing the concentration of DON did not increase ADS47

growth in MSB Hence DON could not be used as sole nutrient source in the mixed culture

enrichment process

Many previous attempts to obtain DON de-epoxidizing microbes have failed

particularly under aerobic conditions because media or laboratory conditions were

inappropriate to support growth or initially active cultures lost de-epoxidizing function after

serial transfer (Karlovsky 2011)

Media composition appears to be a crucial factor for microbial trichothecene de-

epoxidation activity (Voumllkl et al 2004) Effective biotransformation of trichothecene

mycotoxins by the fish digesta microbial culture C133 occurred in full media (FM) but a low

level of de-epoxidation occurred in media having a single nitrogen source and no

transformation activity was observed in the common bacterial medium LB (Guan et al

2009) Zhou et al (2010) also showed that chitin de-acetylation by soil bacterial strains

occurred more rapidly in yeast extract compared to common bacterial media Therefore a

screening strategy for microbial DON de-epoxidation ability should use different media in

parallel as was done in this study

This study observed efficient de-epoxidation in common bacterial media (NB and LB)

as well as in mineral salts medium containing yeast extract (MSY) or 05 Bacto Peptone

(MSB) However reduction of Bacto-Peptone to 03 (MSC) significantly reduced the

bacterial de-epoxidation activity suggesting that the activity is sensitive to the availability of

protein and amino acids The study found that medium containing sucrose as carbon source

(MM) and a commonly used medium PDB blocked microbial DON de-epoxidation even

98

though bacteria grew in those growth media These suggest that media containing high sugar

contents had inhibitory effects on microbial de-epoxidation enzymatic activity Previously

sugars have been found to have negative effects on the production andor activity of bacterial

enzymes (Saier and Roseman 1971)

Losses of microbial functions under laboratory conditions limited researchers to obtain

economically important microbial genetic sources The soil borne Curtobacterium sp strain

114-2 efficiently de-epoxydized nivalenol but unfortunately the strain lost its activity in

latter generations (Ueno et al 1983) Instability of de-epoxidation activity of ADS47

observed in cultures grown on agar media without DON resulted in the loss of DON de-

epoxidation activity by more than 80 of the colonies after one generation However strain

ADS47 retained de-epoxidation activity after ten subcultures in MSB (data not shown)

The non-specific de-epoxidation of DON made it impossible to use DON as a sole

substrate C source for selective isolation of ADS47 Thus a number of techniques were

utilized to select ADS47 and suppress other bacteria including the use of a variety of

antibiotics and culture at elevated temperatures (Islam et al 2012) Selection of the isolate

ADS47 required repeated subculturing and prolonged exposure to media containing 200

microgmL DON After three months of selection with DON culture that was obtained had a

DON de-epoxidation activity that was up to 30-fold higher than the mixed culture As growth

of ADS47 was not affected by 200 microgmL DON the prolonged incubation may have reduced

populations of other bacteria that are more sensitive to DON Enrichment and isolation of a

DON to 3-keto DON transforming bacterial strain E3-39 by prolonged incubation of a soil

microbial culture with 200 microgmL DON was reported by Shima et al (1997)

99

This study demonstrated that microbial DON de-epoxidation occurred mainly at the

stationary growth phase A DON degrading strain of Citrobacter may have gained energy

from DON by co-metabolic cleavage of the epoxy ring under less nutrient conditions

Microbial use of energy from co-metabolic reaction has been suggested by Chiu et al (2005)

The rate of de-epoxidation activity increased when ADS47 grew at higher concentrations of

DON Taken together a prolonged exposure to high concentration DON may have resulted in

non-DON metabolizing microbial populations declining by DON toxicity while facilitated

the population of DON metabolizing bacterium by providing energy

DON is a small molecule and is simply absorbed by cells (Sergent et al 2006)

Microbial DON de-epoxidation occurred in intact and lysed bacterial cells In addition

treating the ADS47 culture with SA which blocks gram negative bacterial electron transport

and activity inhibited DON de-epoxidation activity (Lichstein and Malcolm 1943 Erecinska

and Wilson 1981) implying that an active electron transport system required for microbial de-

epoxidation reaction (He et al 1992) The data suggest that a cytoplasmic epoxy reductase is

involved in the DON de-epoxidation reaction in the ADS47 microbial strain that was isolated

in the present study

Since agricultural commodities are often contaminated with multiple fungal

mycotoxins the ability of bacteria like ADS47 to degrade mycotoxins by several

mechanisms may be an advantage De-acetylation might be another mechanism of microbial

mycotoxin detoxification since toxicity of trichothecenes also relies on the acetyl functional

groups (Desjardins 2006) Strain ADS47 showed efficient de-epoxidation and de-acetylation

of several type-A and type-B trichothecene mycotoxins that are commonly detected in cereals

in association with DON In conclusion the soil-borne gram-negative bacterium C freundii

100

strain ADS47 is capable of detoxifying trichothecene mycotoxins by both de-epoxidation and

de-acetylation under aerobic and anaerobic conditions neutral pH and a range of

temperatures ADS47 has great potential to detoxify DON contaminated animal feed

Furthermore isolation of novel bacterial de-epoxidation genes might be used to generate

transgenic plants that can detoxify DON in contaminated seed Alternatively contaminated

seed might be treated with manufacture de-epoxidation enzyme(s) These applications can

improve the quality and safety of agricultural commodities that are prone to trichothecene

mycotoxin contamination

36 Acknowledgements

Thank to the Natural Sciences and Engineering Research Council of Canada (NSERC) for a

scholarship to Rafiqul Islam The work was funded by the Agriculture and Agri-Food Canada

Binational Agricultural Research and Development Fund (AAFCBARD) project We

appreciate Honghui Zhu for technical assistance

101

Supplemental Figure S3-1 Flow chart depicts the procedures of enrichment and isolation of

DON de-epoxidation bacterial strain ADS47 Steps 1 to 4 describe the optimization of

microbial DON to dE-DON transformation activity and removal of undesired microbial

species Step 5 to 7 shows the culture enrichment by incubation with high concentration DON

and isolation of the single strain ADS47

Step 1 Screening of soil samples

Step 2 Optimization of cultural conditions

Step 3 Elimination of undesired microbial species

Step 4 Removal of untargeted microbial species

Step 5 High concentration DON enrichment

Step 6 Separation of bacterial cells and dilution plating

Step 7 Single colony analysis

Active culture was sequentially treated with different mode of actions antibiotics heating cultures at 50degC and subculturing on agar media

Colonies were tested for their DON de-epoxidation ability Purified the single colony via several times subculturing on agar medium

Optimized the culture conditions ie growth substrates temperatures pH and oxygen requirements

Obtained DON de-epoxidation capable microbial culture obtained by analyzing soil samples collected from cereal fields

Serially diluted active culture was grown on agar media Collected washed colonies having DON de-epoxidation activity

The partially enriched culture that retained DON de-epoxidation activity was incubated with 200 microgmL DON in MSB media with continuous shaking at 220 microgmL for 7 d The procedure was repeated 12 times

The enriched culture was serially diluted and treated dilutes with 05 Tween 20 After vigorously vortex for ten min aliquot cultures were spread on different agar media plates to produce single colonies

102

CHAPTER 4



CITROBACTER FREUNDII STRAIN ADS47

41 Abstract

Enzymatic activity-based screening of a genomic DNA library is a robust strategy for

isolating novel microbial genes A genomic DNA library of a DON detoxifying bacterium

(Citrobacter freundii strain ADS47) was constructed using a copy control Fosmid vector

(pCC1FOSTM

) and E coli EPI300-T1R as host strain A total of 1250 E coli clones containing

large DNA inserts (~40 kb) were generated which resulted in about 10-fold theoretical

coverage of the ADS47 genome The efficiency of E coli transformation with the

recombinant Fosmid vector was examined by restriction digestion of Fosmids extracted from

15 randomly selected library clones The fragmentation patterns showed that all of the clones

contained an 81 kb Fosmid backbone and ~40 kb of insert DNA except one that had a small

insert This suggests that a DNA library was prepared with expected DNA inserts The de-

epoxidation ability of the library was analysed by incubating clones in Luria Bertani (LB)

broth medium supplemented with 20 microgmL DON with induction (by addition of L-arabinose)

or without induction None of the 1000 clones showed DON de-epoxidation activity The

potential DON de-epoxidation gene(s) of ADS47 may not have been expressed in E coli or

might have been expressed but lacked additional factors required for DON de-epoxidation

103

Keywords DNA library fosmid vector functional library screening DON de-epoxidation

activity

42 Introduction

Microorganisms contain numerous valuable genes and biocatalysts which may have

significant roles in agricultural productivity (Jube and Borthakur 2007) Various techniques

have been used to isolate novel genes from different organisms including transposon

mutagenesis (Frey et al 1998 Tsuge et al 2001) gene knockout (Winzeler et al 1999)

whole genome sequencing (Krause et al 2006) comparative genomics (Koonin et al 2001)

and suppression subtractive hybridization (Dai et al 2010)

The genomic DNA library preparation and activity-based screening of the library is a

relatively new technique which has been efficiently used for isolation of microbial novel

enzymes and compounds such as lipases proteases oxidoreductases antibiotic resistance

genes membrane proteins and antibiotics (Henne et al 2000 Gupta et al 2002 Knietsch et

al 2003 Gabor 2004 Suenaga et al 2007 van Elsas et al 2008) The strength of the

technique is that genes from microbial sources could be obtained without prior sequence

information and not requiring culturing the organisms under laboratory conditions Using this

technique an unknown gene or a set of genes could be obtained by determining particular

phenotypes of the library clones Eventually the gene of interest from the positive clones is

isolated by using a forward (eg mutation) or reverse genetics method (eg subcloning the

positive clones into a microbial expression vector) followed by functional analysis of the

subclones

104

Although this strategy has been used effectively for isolating novel microbial genes

several factors need to be considered in order for the method to be successful These factors

include DNA quality vector type (eg plasmid cosmid fosmid bacterial artificial

chromosome etc) vector copy number and host strains (Daniel 2005 Singh et al 2009) The

Fosmid vector-based technique has been used successfully to clone bacterial genes including

xenobiotics degradation genes antifungal gene clusters and antibiotic resistance genes from

soil metagenomes (Hall 2004 Suenaga et al 2007 Chung et al 2008) This study reports the

construction and activity-based screening of a Fosmid DNA library in an E coli host with the

aim of isolating a novel DON de-epoxidation gene from Citrobacter freundii strain ADS47

(Chapter 3)


431 Genomic DNA library construction

The genomic DNA library was prepared using a Copy Control Fosmid Library

Production Kit (Epicentre Biotechnologies Madison Wisconsin USA) following the

manufacturerrsquos instructions as depicted in Figure 4-1

105

Figure 4-1 Schematic overview of the construction and functional screening of a Fosmid

DNA library

106

Citrobacter freundii strain ADS47 was grown in NB medium at 28degC with continuous

shaking at 220 rpm The high molecular weight bacterial genomic DNA was extracted from

the overnight culture using the Gentra Puregene YeastBacteria Kit (Qiagen Inc Mississauga

Ontario Canada) The purity of the DNA was confirmed by determining OD260280 at ~190 by

a NanoDrop spectrophotometer A 5 microL sample having ~25 microg high molecular weight DNA

was sheared by passing through a 200-microL pipette tip and achieved a sufficient amount of

DNA about 40 kb in size Blunt ended 5rsquo-phosphorylated insert DNA was generated by

thoroughly mixing it with End-Repair Enzyme reagents on ice The 20 microL mixture was

incubated for 45 min at room temperature followed by incubation at 70degC for 10 min to

inactivate the End-Repair Enzyme activity

The treated DNA was size fractionated by a Low Melting Point-Pulsed Field Gel

Electrophoresis (LMP-PFGE) on the clamped homogeneous electric fields (CHEF-DR III)

system (Bio-RAD Laboratories) using 1 UltraPure Low Melting Point Agarose (Invitrogen)

The PFGE was conditioned according to manufacturerrsquos instructions as follows initial switch

time-42 s final switch time-761 s run time 16 h 50 min angle-120deg gradient- 6 Vcm

temperature-14degC and running buffer-05 TBE DNA size markers for the 42 kb Fosmid

control DNA (Epicentre Biotechnologies) and low range Pulse Field Gel (PFG) markers (New

England Biolabs Inc) were loaded adjacent to the DNA sample slots After electrophoresis a

portion of the gel that contained DNA size markers was cut off The gel was stained in SYBR

Safe DNA gel stain (Invitrogen) for 30 min visualized and marked on the 23 42 and 485 kb

marker bands A slice of PFGE gel containing 40 plusmn 10 kb size DNA was cut off with a sterile

razor blade by comparing the marker sizes DNA from the gel was recovered by digesting it

with GELase enzyme ( 1 U100 microL melted agarose) in 1X GELase Buffer followed by a

107

sodium acetate-ethanol precipitation The DNA with the desired size was stored at 4degC for

next dayrsquos ligation to a cloning-ready Fosmid vector (pCC1FOSTM

)

Ligation was performed in a 10 microL reaction mixture by thoroughly mixing 10X Fast-

Link Ligation Buffer 10mM ATP 025 microg of ~40 kb insert DNA 05 microg Fosmid Copy

Control Vector (pCC1FOS) (Figure 4-2) and 2 U Fast-Link DNA lipase The mixture was

incubated for 4 h at room temperature preceded by inactivating the ligation reaction by

heating at 70degC for 15 min The recombinant Fosmid vector was stored at -20degC for later use

Ten microL of the ligated DNA was thawed on ice and transferred to a pre-thawed

MaxPlax Lambda Packaging Extract tube containing 25 microL extract By pipetting several

times the mixture was incubated at 30degC for lamda packaging After 2 h of packaging an

additional 25 microL packaging extract was added into the mixture and incubated for a further 2 h

at 30degC The packaged phage particles were volumed to 05 mL by adding ~400 microL Phage

Dilution Buffer and 25 microL chloramphenicol

Transformation was performed via transduction by mixing the packaged particles with

the overnight cultured E coli EPI300-T1R cells (1 10 vv ratio) in LB supplemented with 10

mM MgSO4 and 02 Maltose Transformation was completed by incubation at 37degC for 1 h

The transformed E coli cells were spread on an LB agar plate containing 125 microgmL

chloramphenicol The plates were incubated overnight at 37degC Colonies that appeared on the

plates were individually transferred to 96-well microtiter plates having 200 microL LB medium

supplemented with 10 glycerol and 125 microgmL chloramphenicol The microtiter plates

were incubated at 37degC for 24 h with continuous shaking at 180 rpm The prepared library

clones were stored at -80degC for further analysis and functional screening

108

109

Figure 4-2 Genetic map of copy controlled Fosmid vector (pCC1FOSTM

) The 813 kb

Fosmid vector backbone shows a multiple cloning site (MCS) chloramphenicol resistance

selectable marker (CmR) replication initiation fragment (redF) inducible high copy number

origin (oriV) replication initiation control (repE) and F-factor-based partitioning genes (ParA

ParB ParC) The vector contains a cos site for lamda packaging (not shown) and lacZ gene

for bluewhite colony selection The vector map was provided by the Epicentre

Biotechnologies (httpwwwepibiocomitemaspid=385)

110

432 Confirmation of library construction

The quality of the DNA library prepared was determined by examining 15 E coli

clones randomly selected A 15 mL LB broth supplemented with 125 microgmL

chloramphenicol was inoculated by each of the transformed clones To obtain higher Fosmid

DNA yield 1 microL autoinduction solution (L-arabinose) to increase the copy number of the

Fosmid vector (Epicentre) was added to the cultures After an overnight incubation of the

cultures at 37degC with continuous shaking at 220 rpm Fosmid DNA was extracted using a

Qiagen Large-Construct Kit followed by restriction digestion with Not I enzyme A 20 microL

digestion mixture containing 05 microg Fosmid DNA 1X digestion buffer and 05 U Not I was

incubated at 37degC for 15 h The partially digested DNA and a 42 kb DNA size marker

(Fosmid control DNA) were ran on 08 agrose gel for 15 h at 100 V The linear Fosmid

vector backbone (813 kb) and insert DNA fragments were visualized on the gel and the

image was recorded

433 Functional screening of the clones

Eppendorf tubes (15 mL) containing 500 microL LB supplemented with 125 microgmL

chloramphenicol were inoculated with a single clone (400 clones tested) or a combination of

12 library clones (600 tested) from microtiter plates stored at -80degC One half of the cultures

in each group (single or combined clones) were induced to increase the Fosmid copy number

by adding a 1 microL (500X) autoinduction solution (contained L-arabinose) to the culture tubes

L-arabinose induces the expression of a mutant trfA gene of the E coli host strain which

leads to the initiation of Fosmid replication from a low-copy to a high-copy number origin of

replication (oriV) The cultures were spiked with 20 microgmL DON and incubated at 37degC for

111

72 h with continuous shaking at 180 rpm The DON to de-epoxy DON transformation ability

of the clone cultures was examined by a liquid chromatography-ultraviolet-mass spectrometry

(LC-UV-MS) method as described by Islam et al (2012)

112

44 Results

441 Production of the DNA library

High molecular weight intact (non-degraded) and good quality (OD600280 ~190)

genomic DNA was obtained from the DON de-epoxidizing bacterial strain ADS47 (data not

shown) Pipetting of the DNA extract resulted in an adequate amount of insert DNA (gt025

microg) of approximately 40 kb size as showed on a LMP-PFGE (Figure 4-3)

A DNA library in E coli host was constructed using LMP-PFGE purified DNA The

library was comprised of 1250 E coli transformants which were selected on LB agar plates

supplemented with chloramphenicol (Figure 4-4)

113

Figure 4-3 Preparation of a 40 kb size insert DNA from a DON de-epoxidizing bacterium

Genomic DNA was extracted from the overnight culture of a DON de-epoxidizing

Citrobacter freundii strain ADS47 After fragmentation the DNA was size fractionated by

Low Melting Point- Pulse Field Gel Electrophoresis (LMP-PFGE) with markers for the PFGE

(M1) and 42 kb Fosmid control DNA (M2) S1 S2 S3= different DNA samples extracted

from bacterial strain ADS47 The red dots area indicates the section of the gel containing

genomic DNA of the expected size (~40 kb)

114

Figure 4-4 Production of the genomic DNA library in E coli EPI300-T1 host strain

Approximately 40 kb long blunt ended genomic DNA from a DON de-epoxidizing bacterial

strain ADS47 was ligated to a Copy Control Fosmid vector pCC1FOS The construct was

packaged into lamda bacteriophage followed by a transduction-mediated transformation of E

coli host cells The clones which appeared on the LB agar plate supplemented with 125

microgmL chloramphenicol are expected to be transformed by the recombinant Fosmid vector

containing insert DNA

115

442 Conformation of the library construction

All of the clones tested acquired the Fosmid vector Out of 15 clones examined 14

received the recombinant Fosmid vector that contain the expected size insert DNA as

determined by an 81 kb vector backbone and at least one high molecular weight insert DNA

band (~40 kb) on agarose gel (Figure 4-5) In contrast one clone (lane 4) showed an 81 kb

vector backbone and a low molecular weight band which is also a fragment of the Fosmid

vector The result indicated that approximately 94 of the library clones were transformed

with the recombinant vector

443 Activity-based screening of the library

Out of 500 single copy number clones (200 individual and 300 combined clones)

none showed DON to de-epoxy DON transformation activity (Table 4-1) An additional 500

high copy number clones (200 single and 300 combined clones) were unable to de-epoxidize

DON as well

116

Figure 4-5 Confirmation of the Fosmid DNA library production in E coli Fosmid DNA

collected from 15 clones was digested with Not I and run on 08 agarose gel at 100 V for 15

h The presence of the vector backbone (81 kb) and the insert DNA fragment (~40 kb) in the

clones was determined by comparing a 42 kb molecular size marker (M) Lanes (1-15) denote

the recombinant Fosmid DNA extracted from 15 E coli clones

117

Table 4-1 Analysis of Fosmid DNA library clones for deoxynivalenol (DON) de-epoxidation

capability

Type of clones Analysis of

singlecombined

clones

Number of

clones

examined

Number of clones

showed DON de-

epoxidation activity

Without induction

(single copy number clones)

Singly 200 0

Combination of 12 300 0

With induction (multi-copy

number clones)

Singly 200 0


De-epoxidation activity of the clones was examined alone or in combination of 12 clones

without or with induction by adding L-arabinose The clones grown in LB supplemented with

125 microgmL and 20 microgmL DON were analysed for DON to de-epoxy DON transformation

ability by a liquid chromatography-ultra violet- mass spectrum (LC-UV-MS)

118

45 Discussion

While a Fosmid DNA library with the expected genome coverage of a DON de-

epoxidizing bacterial genome was produced the function-derived screening of the library

clones did not identify any clones that showed DON de-epoxidation enzymatic activity This

is unlikely due to lack of coverage of the bacterial genome as 1000 clones analyzed in the

study should have provided eight fold coverage of the genome Therefore it was likely due to

a lack of expression of the enzyme(s) for DON de-epoxidizing activity under the conditions

used

Thus far there is only one report of heterologous expression of bacterial epoxide

reductase gene in another bacterium A high level of heterologous expression of a

Synechococcus sp vitamin K epoxide reductase gene was achieved in E coli with codon

optimization and placing the gene under the control of a prokaryotic T7 promoter in an

expression vector (Li et al 2010) Therefore heterologous expression of bacterial epoxide

reducatse gene using its own promoter has not yet been demonstrated The success of the

method used in this chapter relies on an intact copy of the gene or operon being cloned the

selection of an appropriate cloning vector a host organism that can recognize the cloned C

fruendii promoter and a suitable library screening method for DON degradation by E coli

(Mocali and Benedetti 2010)

The unbiased cloning efficiency of the Fosmid vector its ability to contain large insert

DNA high stability in E coli as single copy number and autoinduction to increase copy

number in host cells made it appear to be a suitable method for the present work (Kim et al

1992 Lee et al 2004) Library production with large insertions is a major advantage as gene

cluster involved in particular pathway could be trapped into single clone and their functions

119

could be analysed The pathway of microbial DON de-epoxidation activity and the genes

encoding the activity are not known Therefore it was reasonable to prepare a library with

large inserts to ensure the possibility of cloning single as well as whole pathway genes of

interest for functional screening A further advantage of the method is that Fosmid clones are

inducible which allows detecting poorly expressed genes in a heterologous host by inducing

high copy numbers (Daniel 2005)

One of the limitations of the method is that the functions of the target genes rely

mostly on the host organismrsquos transcription and translation machineries Therefore selection

of the host organism is critical for activity-based screening of library clones in heterologous

hosts A number of host strains including E coli Streptomyces lividans Rhizobium

leguminosarum and Pseudomonas aeruginosa have been used for functional screening

activities from diverse organisms (Daniel 2005) The present work attempted to isolate C

freundii strain ADS47 genes encoding DON de-epoxidation enzymes Both strain ADS47 and

E coli belong to gamma proteobacteria which suggest that E coli would be an appropriate

host organism for heterologous expression of ADS47 genes Poor expression of heterologous

genes could be another limitation of the method which may be overcome by overexpression

to increase the catalytic activity of less expressed genes However clones with DON de-

epoxidation activity were not identified in this study even the Fosmid copy number was

increased by induction

In general a large number of clones must be analysed to obtain the gene of interest

using an activity-based screening technique (Lee et al 2004 van Elsas et al 2008) In this

study the constructed DNA library comprised of 1160 clones having approximately ~40 kb

insert DNA suggesting that the prepared library achieved ~10 fold coverage of the ADS47

120

genome (48 Mb) which ensured gt99 probability of the presence of all DNA sequence in

the library In the produced library three clones (lanes 3 10 and 12) showed one intact band

greater than 42 kb in size on the agarose gel It is presumed that the Fosmid DNA of the three

samples was not digested because of insufficient amount of restriction enzyme added to the

digestion mixture About half of the samples demonstrated multiple bands suggesting that the

insert DNA might have contained multiple Not I restriction sites

However not every heterologous gene may necessarily express or function in the E

coli host (Gabor et al 2004) The functionality of the gene requires expression of the genes

and proper folding of the protein which may be influenced by environmental conditions (eg

substrate temperature and pH) and genetic factors In this study library clones were screened

in growth conditions at which strain ADS47 performed the best de-epoxidation activity The

expression of genes may be controlled by the promoter suitable ribosome binding site and

transndashacting factors (eg special transcription factors inducers chaperones cofactors proper

modifying enzymes or an appropriate secretion system) (Gabor et al 2004) Those factors

have to be supplied by the cloned DNA fragments or host cells to allow any heterologous

gene to be functional Since DON de-epoxidation genes of strain ADS47 evolved in a distinct

environmental condition the expression and activity of the gene(s) might have required

specific factorsregulators that were absent in E coli Even though the Fosmid based library

screening technique facilitated isolation of many microbial genes clones having de-

epoxidation activity were not identified using such technique in this study

121

46 Acknowledgements

We appreciate the Agriculture and Agri-Food CanadaBinational Agricultural

Research and Development Fund (AAFCBARD) project for providing the financial support

for this work The authors thank Honghui Zhu for analyzing numerous samples by liquid

chromatography ultraviolet mass spectrum method

122

CHAPTER 5


ANALYSIS OF CITROBACTER FREUNDII STRAIN ADS47

51 Abstract

The genome of a trichothecene mycotoxin detoxifying C freundii strain ADS47

originating from soil was sequenced and analyzed Strain ADS47 contains a circular

chromosome of 4878242 bp that encodes 4610 predicted protein coding sequences (CDSs)

with an average gene size of 893 bp Rapid Annotation Subsystems Technology (RAST)

analysis assigned functions to 2821 (62) of the CDSs while 1781 (38) CDSs were

assigned as uncharacterized or hypothetical proteins Comparative bioinformatics analyses

were done for strain ADS47 C freundii Ballerup 785139 Citrobacter sp 30_2 C youngae

ATCC 29220 C koseri ATCC BAA-895 and C rodentium ICC168 There were a significant

number of common orthologous genes (2821) were found in the six Citrobacter genomes

implying a common evolutionary relationship ADS47 is most closely related to the

opportunistic human pathogen C freundii Ballerup The human and mouse pathogenic

species C rodentium and C koseri are the most distantly related to ADS47 and non-

humananimal pathogens C youngae and Citrobacter sp 30_2 were closely related ADS47

C koseri C youngae and Citrobacter sp 30_2 lacked the clinically important locus for

enterocyte effacement (LEE)-encoded type-III secretion systems which are present in C

rodentium and C freundii Ballerup Strain ADS47 had 270 unique CDSs many of which are

located in the large unique genomic regions potentially indicating horizontal gene transfer

123

Among the unique CDSs nine were reductasesoxidoreductases and a de-acetylase that may

be involved in trichothecene mycotoxin de-epoxidation and de-acetylation reactions

Keywords Citrobacter freundii comparative genomics genome sequencing unique genes

reductases

52 Introduction

According to the Food and Agriculture Organization (FAO) at least 25 of the

worldrsquos food crops are contaminated with mycotoxins causing serious negative impacts on

livestock and international trade (Boutrif and Canet 1998 Desjardins 2006 Binder et al

2007) Trichothecenes (TCs) produced by a variety of toxigenic Fusarium species are

probably the most prevalent food and feed contaminating mycotoxins It is estimated that

infections by the fungus caused more than $1 billion in crop yield losses in USA and Canada

alone (Windels 2000 Schaafsma et al 2002 Yazar and Omurtag 2008) TC mycotoxins can

have adverse affects on human health including organ toxicity mutagenicity carcinogenicity

neurotoxicity modulation of the immune system negative reproductive system affects and

growth retardation (DrsquoMello et al 1999 Pestka et al 2007) A strain of Citrobacter freundii

ADS47 was isolated from soil in southern Ontario that is capable of detoxifying (ie de-

epoxidation and de-acetylation) several TC mycotoxins including deoxynivalenol nivalenol

and T2-toxin (Chapters 2-3)

Citrobacter freundii is a gram negative motile rod shaped facultatively anaerobic

bacterium belonging to the family of Enterobacteriaceae (Wang et al 2000 Whalen et al

2007) The opportunistic human pathogen is responsible for a number of infections including

124

nosocomial infections of respiratory tract urinay tract blood and causes neonatal meningitis

(Badger et al 1999 Wang et al 2000) C freundii exists almost ubiquitously in soil water

wastewater feces food and the human intestine Biodegradations of tannery effluents copper

aromatic amino acids by C freundii strains isolated from water mine tailings and soil

respectively have been previously reported (Kumar et al 1999 Savelieva et al 2004 Sharma

and Fulekar 2009) Because of their biochemical capabilities bacteria have played important

roles in industrial biotechnology including de-epoxidation and de-acetylation activities

(Demain and Adrio 2008 Plank et al 2009 Zhao et al 2010) However the identification of

genes for de-epoxidation activity against trichothecene mycotoxins is limited (Boutigny et al

2008)

The advent of molecular techniques such as next generation sequencing technologies

and the related bioinformatics tools has enabled the discovery of novel genes and the

enzymes they encode in pathogenic and beneficial bacteria (van Lanen and Shen 2006

Ansorge 2009 Zhao 2011) In the present study the genome of mycotoxin-detoxifying strain

C freundii ADS47 was sequenced and subjected to comparative genomic analyses with other

Citrobacter species to identify putative mycotoxin-detoxifying genes

125


531 Sequencing quality control and assemble of ADS47 genome

Genomic DNA of the strain ADS47 was extracted using Gentra Puregene

YeastBacteria Kit (Qiagen Inc Mississauga Ontario Canada) and purity of the DNA was

confirmed by determining OD 260280 at ~190 using a Nanodrop spectrophotometer

The genomic DNA was sent to be sequenced by the Illumina HiSeq 2000 sequencing

analyzer at Beijing Genomics Institute (BGI) Hong Kong

(httpwwwbgisequencecomhomeservicessequencing-servicesde-novo-sequencing) The

sequencing workflow involved short read library preparation cluster generation sequencing

and data analysis Briefly 3-5 ug of bacterial gDNA was fragmented to produce a 500 bp

insert library by preparative electrophoresis through a 08 agarose gel The fragmented

DNAs were blunt-ended by treating with a mixture of enzymes (T4 DNA polymerase klenow

fragment) and subsequently adapters were ligated to the blunt ends Each of the DNA

fragments was amplified to a cluster which was performed in the flow cell by template

hybridization and bridge amplification Millions of clusters were then sequenced by

sequencing-by-synthesis technology leading to base calls The raw data was filtered to remove

low abundant sequences from the assembly according to 15-mer frequencies The low

complexity reads low quality (leQ20) base calls and adapter contaminations (24 bp overlap

between adapter and reads) were eliminated The quality of the sequences was analysed by

GC content and depth correlative analysis (for GC biasness) K-mer analysis was performed

using 15 bp of the short reads for heterologous sequence identification

126

The cleaned short reads (approximately 90 bp) were self-assembled using the Short

Oligonucleotide Analysis Package (SOAP) denovo (httpsoapgenomicsorgcn) (Li et al

2008) The assembly errors were corrected by filling gaps and proofreading single base pairs

using mapping information The assembly produced 441 contigs with an N50 contig size of

29222 bp (the largest contig size 114839 bp) and 80 scaffolds with an N50 scaffold size of

325726 bp (largest scaffold 662452 bp) with an total sequence length of 4878242 bp which

constitutes 33-fold genome coverage The scaffolds were ordered and oriented according to a

reference genome C koseri ATCC BAA-895 the closest completely sequenced species of

the newly sequenced bacterial genome by alignment with the MUMmer program v322

(httpmummersourceforgenet) (Delcher et al 2002) Genome coverage was determined by

mapping the reads to the assembled genome with Bowtie software (Langmead et al 2009) A

preliminary genome annotation was performed to define the origin of replication by

identifying colocalization of multiple genes ie gyrB recF dnaN and dnaA (usually found

near the origin of replication) and comparison with the C koseri reference genome Synteny

analysis between the reference bacterium and assembled scaffolds was performed with the

MUMmer program

532 Gene function annotation and classification

The genome of ADS47 was annotated by Rapid Annotation using Subsystems

Technology v40 (RAST) a server-based genome analysis system (httprastnmpdrorg)

(Aziz et al 2008) A circular genome map was constructed using CGView program

(httpstothardafnsualbertacacgview_server) (Stothard and Wishart 2005)

127

533 Molecular phylogenetic analysis

A robust phylogenetic tree was produced using Composition Vector Tree (CVTree)

web-based program v20 (httpcvtreecbipkueducn) (Qi and Hao 2004) by comparing

whole genome amino acid sequences of strain ADS47 two completely sequenced Citrobacter

genomes [C rodentium ICC168 (NC_013716) and C koseri ATCC BAA-8959 NC_009792)]

and three partially sequenced Citrobacter genomes [C freundii strain Ballerup 785139

(CACD00000000) Citrobacter sp 30-2 (NZ_ACDJ00000000) and C youngae ATCC 29220

(NZ_ABWL00000000)] E coli O157H7 strain EDL933 (NC_002655) was used as an

outgroup species The tree was constructed from the generated pairwise distance matrix data

using Neighbor-Joining Program in PHYLIP v369 package (Saitou and Nei 1987 Felsenstein

1989)

534 Comparative genomic analysis

Genome-wide comparative analyses between C freundii ADS47 C freundii strain

Ballerup 785139 Citrobacter sp 30-2 C youngae ATCC 29220 C Koseri ATCC BAA-895

and C rodentium ICC168 was carried out The nucleotide sequence idendities were

determined by pairwise alignments of the ADS47 genome with the five other Citrobacter

genomes using MUMmer software (Delcher et al 2002) and GC content total CDSs genome

coverage by CDSs and number of stable RNAs were determined by RAST program

Orthologous CDS groups among the total set of 29079 CDSs derived from the six

Citrobacter genomes were determined using OrthoMCL software v202

(httporthomclsourceforgenet) based on all-against-all reciprocal BLASTP alignment of

amino acid sequences (Li et al 2003) Genes unique to each strain were identified as those

128

CDSs not included in any orthologous group of the five other Citrobacter genomes BLASTP

comparisons were visualized with Blast Ring Image Generator (BRIG) software v095

(httpsourceforgenetprojectsbrig) (Alikhan et al 2011)

Protein sequences of the five Citrobacter genomes pooled from the GenBank data

bases were compared with ADS47 A circular map was developed by BRIG software that

showed the unique genomic regionsgenes of ADS47 The unique genes were grouped

according to their functional similarities previously identified by RAST software and

genomic regions possibly acquired by horizontal transfer were identified using RAST

software

129

54 Results

541 Assembly and organization of the ADS47 bacterial genome

Scaffolds derived from Citrobacter freundii strain ADS47 were ordered and oriented

by alignment to Citrobacter koseri ATCC BAA-895 the most closely related genome that has

been completely sequenced The genomes of ADS47 and C koseri displayed close synteny

(Figure 5-1)

Figure 5-1 Co-linearity between C freundii strain ADS47 genome and C koseri Scaffolds

derived from ADS47 genome sequences (Y- axis) were aligned and assembled by comparison

with the closest completely sequenced genome C koseri ATCC BAA-875 (X- axis)

130

542 An overview of the ADS47 genome

General features of the C freundii strain ADS47 are presented in Table 5-1 Some

notable features are that strain ADS47 contains 4881122 bp circular chromosome with an

average GC content of 5202 The genome contains 4610 CDSs which comprise 8784 of

the whole genome Annotation by RAST assigned 2821 (62) CDSs to known biological

functions and remaining 1789 (38) genes were classified to unknown or hypothetical

proteins

The GC content in the coding regions is significantly higher (5318) than in the non-

coding intergenic region (4379) which covers 1215 (592781 bp) of the genome The

intergenic region is comprised of 018 repeat sequences (total 8999 bp) 5 (009)

transposons (4806 bp) 025 stable RNA (12091 bp) and the remaining ~115 is

classified as regulatory and other functions The ADS47 genome contains 62 tRNAs 3

rRNAs and 34 copies of sRNAs The average lengths of the tRNAs rRNAs and sRNAs are

785 885 and 13441 bp respectively There are also 42 phageprophage-related genes

identified in the genome

Figure 5-2 shows the distribution of the 26 categorized CDSs rRNA tRNA GC

content and GC skew It appears that ADS47 genome contains equal quantity of genes in

leading and lagging strands and showed occasional clustering (likely operons) of genes in the

two strands The genome contains three rRNAs (two large subunit and a small subunit) and 62

tRNAs which are distributed across the genome A consistent GC content was observed in

the ADS47 genome however 7-10 low GC content (high black color peaks) genomic regions

were found at locations origin of replication 600 2000 3000 3300 3550 3850 4300 and

4800 kbp This is indicative of possible horizontally transferred genes or insertions in those

131

genomic locations GC skew denotes the excess of C over G in certain genomic locations

Positive GC skew (+) (green color) indicates the skew value in the leading strand while

negative skew (-) (purple color) in the lagging strand As in many bacterial genomes the

ADS47 genome map shows the separation of positive and negative GC skew at the origin of

replication (base pair position 1) The GC skew in the leading strand is highly skewed (high

green color peak) indicating that possible horizontal gene transfer occurred into the ADS47

genome

The CDSs ranged from ~100 bp to ~2000 bp and have an average size of 893 bp

(Figure 5-3) The highest numbers of CDS (770) were 600-800 bp long A considerable

number of genes (220) were gt2000 bp in size Some of the biological functions for the

smallest genes (100-200 bp) are sulfur carrier protein methionine sulfoxide reductase

ribonuclease P protein component while the largest genes (gt2000 bp) are ferrous iron

transport protein B translation elongation factor G pyruvate flavodoxin oxidoreductase and

tetrathionate reductase

132

Table 5-1 General features of the C freundii strain ADS47 genome

Feature Chromosome

Genome size 4881122 bp

Overall GC content 5202

Number of genes 4610

Genes with assigned function (number and ) 2821 (62 )

Genes without assigned function (number and ) 1789 (38 )

Total length of protein coding sequences 4285461 bp

Genome coverage by coding sequences () 8784

Average length of coding sequences 893 bp

GC content in the coding region 5318

GC content in the non-coding region 4379

Length and percentage of intergenic region 592781 bp and 1215

Total repeat sequences and genome coverage 8999 bp and 018

Total transposons sequences and genome coverage 4806 and 009

Number average length and genome coverage of tRNA 62 copies 785 bp and 0099

Number average length and genome coverage of rRNA 3 copies 885 bp and 0067

Number average length and genome coverage of sRNA 34 copies 13441 bp and 0093

133

134

Figure 5-2 Circular genome map of C freundii strain ADS47 Map was created with the

CGView software From outside to inside the first circle indicates the genomic position in kb

Different functional categories of predicted coding sequences (CDS) are distinguished by

colours with clockwise transcribed CDSs in the second circle and anticlockwise transcribed

CDSs in the third circle Bars on the fourth and fifth circles indicate the rRNAs and tRNAs

respectively Sixth and seventh circles show the coverage and GC content respectively The

innermost circle demonstrates the GC skew + (green) and GC skew- (purple)

135

Figure 5-3 Size distribution of the predicted genes identified in C freundii strain ADS47

The X axis identifies the gene size categories (bp) and Y axis the abundance of genes

136

543 Annotation and functional classification of ADS47 genome

RAST analysis identified predicted CDSs in the genome of Citrobacter strain ADS47

assigned functions and grouped the annotated genes into 26 functional subsystem categories

out of 27 No CDS was found belonging to the photosynthesis category A graphical

presentation of the distribution of the CDSs into each of the 26 categories is given in

Figure 5-4

A large portion (38) of the CDSs belong to hypothetical or unknown functions

Among the 62 known or predicted proteins the highest numbers (835) belong to the

carbohydrate category followed by amino acids and derivatives (468) and

cofactorsvitaminsprosthetic groups (324) A noticeable number of stress response (236) and

respiration (198) categories genes were identified in the bacterial genome The ADS47

genome encoded by 171 membrane transport genes 113 virulencedefense genes and 126

genes involved in regulationcell signaling categories Two hundred and one CDSs were

grouped into the miscellaneous category whose functions are different than the indicated

RAST categories The RAST annotations further divided the categorized CDSs into

subcategories based on similar functional roles which is discussed in section 545

137

Figure 5-4 Functional classification of the predicted coding sequences (CDS) of C freundii

strain ADS47 The annotation was performed by Rapid Annotation using Subsystems

Technology (RAST) program

138

544 Molecular phylogenetic relationships

Genome-wide phylogenetic analysis based on comparisons of whole genome amino

acid sequences of two completely sequenced Citrobacter genomes (C koseri and C

rodentium) and three partially sequenced Citrobacter genome sequences (C freundii

Citrobacter sp and C youngae) illustrates the relationships of ADS47 to five Citrobacter

species (Figure 5-5)

C youngae ATCC 29220 originating from human feces and two pathogenic species

(C koseri ATCC BAA-895 and C rodentium strain ICC168) are progressively more distantly

related to ADS47 The present analysis is consistent with the 16S-rRNA sequence based

phylogenetic analysis (Chapter 3) which showed that the closest bacterial strain to ADS47 is

C freundii (Figure 3-2) This confirmed the identification of strain ADS47 as the gamma

proteobacterial species C freundii made in Chapter 3

139

Figure 5-5 Phylogeny of the trichothecene detoxifying C freundii strain ADS47 The

dendrogram was constructed by comparing the ADS47 genome (amino acid sequences) with

five Citrobacter genomes and a E coli strain (out group species) using the CVTree software

The calculated pairwise distances matrix by Protdist program was transformed into a tree by

neighbor-joining program The dissimilarity tree was drawn to scale with branch lengths

(scale bar 002 indicated the amino acid substitutions among the genomes) The C koseri C

rodentium C freundii Citrobacter sp C youngae and E coli protein sequences were

uploaded from GenBank (Benson et al 2009)

140

545 Comparative genomics

Genome-wide comparative analyses between the genome of strain ADS47 and the five

previously sequenced Citrobacter genomes showed that 872 of the total nucleotides

between ADS47 and C freundii Ballerup were identical The values for the comparisons

between C freundii strain ADS47 were 8212 for Citrobacter sp 30-2 7637 for C

youngae 5981 for C koseri and 4213 for C rodentium (Table 5-2)

The two human and mouse pathogenic species namely C rodentium and C koseri

and the bacteria originally isolated from human feces (C youngae) have higher GC contents

(546 and 532 respectively) compared to C freundii strain ADS47 C freundii strain

Ballerup and Citrobacter sp 30_2 which have slightly lower GC contents (~52) The two

pathogenic species contain 22 copies of rRNA which is significantly higher than the 9 found

in C freundii strain Ballerup 6 in Citrobacter sp 30_2 and 3 in C freundii strain ADS47 but

less than the 25 copies in C youngae The genome of C freundii strain ADS47 C freundii

strain Ballerup C youngae and Citrobacter sp 30-2 had a similar percentage of the genome

comprising coding sequences (865-88) C koseri and C rodentium genomes contained the

highest (90) and lowest (84) genome coverage by coding sequences respectively

141

Table 5-2 Genome comparisons between strain ADS47 and five Citrobacter species The

base nucleotide sequence identities were performed using MUMmer DNAdff script program

(Delcher et al 2002) The features (GC content total CDS percentage CDS and number of

ribosomal RNA) were determined using RAST program (Aziz et al 2008)

Genome feature Bacterial species

C

freundii

ADS47

C freundii

Ballerup

Citrobacter

sp 30_2

C

youngae

C koseri C

rodentium

Genome length

(bp)

4878242

4904641

5125150

5154159

4720462

5346659

Base similarity to

ADS47 ()

NA 8715 8212 7637 5981 4213

GC () 5202 5218 516 5265 530 546

Number of coding

sequences

4610 4685 4728 5278 4978 4800

Genome coverage

with coding

sequences ()

880 865 866 872 900 840

rRNA copies 3 9 6 25 22 22

Total stable RNAs 99 81 78 105 120 142

142

The number of orthologs shared between ADS47 and other Citrobacter species is

illustrated in Figure 5-6 ADS47 shares the smallest number of orthologous CDSs with C

rodentium and C koseri 3250 and 3341 respectively The highest number of shared CDS

were found with C freundii Ballerup (3706) followed by C youngae (3693) and Citrobacter

sp 30_2 (3676) A total of 2821 CDSs were found to be common to all of the six Citrobacter

genomes analysed (data not shown) Nonetheless significant diversity exists between C

freundii ADS47 and the other species as a considerable number of unshared genes were

identified from the comparisons with C freundii Ballerup [979 (20)] Citrobacter sp 30_2

[1064 (23)] C youngae [1654 (31)] C koseri [1693 (34)] and C rodentium [1634

(34)]

143

Figure 5-6 Comparisons of coding sequences between strain C freundii ADS47 and five

Citrobacter species Coding sequences of ADS47 were compared with C freundii Ballerup

Citrobacter sp 30_2 C youngae C koseri and C rodentium genomes based on all-versus-all

reciprocal FASTA comparison of at least 30 amino acid identity and gt70 coverage of the

total length using OrthoMCL program A Venn diagram demonstrates the shared and

unshared (indicated in the brackets) coding sequences

144

Table 5-3 shows differences in the 26 RAST categorized genes between the six

Citrobacter genomes Even though a low abundance of virulencediseasedefense category

genes were found in the animalhuman pathogenic bacteria of C rodentium (85) and C koseri

(92) significantly higher mycobacterium virulence genes (13) were identified in this two

species The genomes of the two closely related strains C freundii ADS47 and C freundii

Ballerup lack this type of virulence gene Instead elevated numbers of genes for resistance to

antibiotic and toxic compounds were found in C freundii ADS47 (94) C freundii Ballerup

(88) and Citrobacter sp (110) compared to other three species Copper homeostasis and

cobalt-zinc-cadmium resistance genes were identified in strain C freundii ADS47 (13 and 21

respectively) and closely related C freundii Ballerup (12 and 22) and Citobacter sp (18 and

22) Significantly lower numbers of these genes were present in C rodentium (6 and 4) and C

koseri (7 and 8) C rodentium and C koseri lack mercuric reductase mercuric resistance and

arsenic resistance genes perhaps because being animalhuman pathogens but these genes

were found in the remaining four genomes The C koseri genome is richer in betandashlactamase

genes (5) compared to the remaining genomes which encodes 1-3 genes

145

Table 5-3 Functional categorized gene abundances among the genomes of C freundii

ADS47 C freundii Ballerup Citrobacter sp 30_2 C youngae C koseri and C rodentium

The genomes were classified into different functional categories with the RAST program

which classified the total CDSs

RAST subsystem category Citrobacter species

C

freundii

ADS47

C

freundii

Ballerup

C

sp

C

youngae

C

koseri

C

rodentium

Cofactorsvitaminsprosthetic

groupspigments

323

317

310

311

325

304

Cell wall and capsule 279 272 248 262 247 262

Virulence disease and defense 113 107 134 100 92 85

Potassium metabolism 50 53 33 32 30 28

Miscellaneous 210 228 200 185 245 185

Phageprophagetransposable

element plasmid

24 75 25 4 6 49

Membrane transport 170 156 171 195 171 172

Iron acquisition and metabolism 80 81 86 62 113 27

RNA metabolism 220 222 217 215 238 212

Nucleosides and nucleotides 119 112 110 111 120 114

Protein metabolism 249 280 269 298 287 254

Cell division and cell cycle 30 29 32 29 38 29

Motility and chemotaxis 133 124 90 147 143 133

Regulation and cell signaling 126 130 124 135 132 123

Secondary metabolism 21 22 20 21 0 0

DNA metabolism 145 167 165 155 147 152

Fatty acids lipids and isoprenoids 121 149 122 121 134 133

Nitrogen metabolism 50 52 53 49 56 58

Dormancy and sporulation 3 3 3 4 3 3

146

Table 5-3 (continued)

RAST subsystem category

Citrobacter species

C

freundii

ADS47

C

freundii

Ballerup

C

sp

C

youngae

C

koseri

C

rodentium

Respiration 198 184 180 191 166 162

Stress response 236 235 170 177 179 174

Metabolism of aromatic

compounds

20 20 28 25 17 48

Amino acids and derivatives 482 476 450 466 428 416

Sulfur metabolism 48 50 52 57 56 56

Phosphorus metabolism 50 49 43 42 43 28

Carbohydrates 837 803 708 731 733 737

147

Strains C freundii ASD47 and C freundii Ballerup have much more potassium

metabolism category genes (50 and 53 respectively) than the other bacterial species which

code for 28-33 proteins Larger numbers of genes associated with the glutathione regulated

potassium efflux system were also identified in C freundii ASD47 with 23 and C freundii

Ballerup with 26 compared to the remaining four species that encode only five genes All of

the six genomes contained phageprophage-related genes with the maximum identified in C

freundii Ballerup (75) followed by C rodentium (49) Citrobacter sp (25) and C freundii

ADS47 (24) Lower numbers of phageprophage-related genes were found in C koseri (6) and

C youngae (4)

The six Citrobacter genomes showed remarkable variability in their secretion systems

The type-III secretion operon genes are only found in C rodentium and C freundii Ballerup

Type-II secretion genes are predominant in C rodentium (12) followed by ADS47 (9)

Type-IV secretion system is uncommon in Citrobacter species since it was only

identified in Citrobacter sp where it is represented by 19 genes Variable numbers of type-VI

secretion system genes are present in the five genomes except C koseri which lacks such

genes C youngae has the highest number (43) of type-VI secretion proteins The other strains

have a range of type-VI secretion proteins in particular 27 were identified in C freundii

Ballerup 15 in C freundii ADS47 and 8 in Citrobacter sp Type-VII (chaperoneusher)

secretion system genes were found in all of the six Citrobacter species with the largest (42)

and smallest (13) numbers of these genes occurring in C koseri and C freundii Ballerup

respectively

Although a relatively similar number of ABC transporters (37-42) were identified in

the six genomes C freundii ADS47 contain the largest number of tripartite ATP-independent

148

periplasmic (TRAP) transporter genes (7) By contrast the closely related C freundii

Ballerup lacks such transporter system An identical number of iron acquisition and

metabolism category genes were found in C freundii ADS47 (80) and C freundii Ballerup

(81) while the two pathogenic species C rodentium and C koseri code for the lowest (27)

and highest (113) numbers of such genes C koseri is rich in siderophore related genes (64)

but the remaining five genomes have smaller numbers of siderophore-associated genes (20-

22)

Variable numbers of motility and chemotaxis related genes were found in the

Citrobacter genomes with the largest number occurring in C youngae (147) followed by C

koseri (143) C freundii ADS47 (133) C rodentium (133) C freundii Ballerup (124) and

Citrobacter sp (90) The two pathogenic species (C rodentium and C koseri) encode a

remarkably low number of quorum sensing genes (2) In contrast C youngae codes for the

highest number of quorum sensing genes (19) A common and distinctive feature of the two

pathogenic species is the lack of genes required for secondary metabolism In contrast the

non-pathogenic species contain a common set of genes for degrading a range of

phenylpropanoids and cinnamic acid (20-22)

The lowest number of genes related to phospholipidsfatty acids biosynthesis and

metabolism were found in C freundii ADS47 (66) compared to C freundii Balllerup (94) and

C koseri (98) The smallest numbers of nitratenitrite ammonification genes were found in

ADS47 (31) and its closest genome C freundii Ballerup (32) In contrast the two pathogens

encode the highest number of such proteins (39-40)

C rodentium and C koseri code for significantly low numbers of respiratory category

genes (162-165) while C freundii ADS47 contains the highest number of such genes (198)

149

Further analysis of the subcategory genes determined that the two gut species (C youngae and

Citrobacter sp 30_2) contain the largest numbers of genes encoding anaerobic reductases

(22) and identical numbers of formate dehydrogenases (17) The largest numbers of formate

dehydrogenase genes were identified in C freundii Ballerup (32) and C freundii ADS47 (30)

but the two pathogenic species differed from the other species by encoding the lowest number

(13) of c-type cytochrome biogenesis genes

A higher number of aromatic compound metabolism category genes were found in the

mouse pathogen C rodentium (48) compared to the human pathogen (C koseri) which

contain 17 genes A significantly higher number of genes involved in central aromatic

intermediate metabolism (26) were found in C rodentium compared to the mouse pathogen

(C koseri) and other genomes which only contain 6-8 such genes The two C freundii strains

ADS47 and Ballerup lacked the anaerobic degradation of aromatic compound

(hydroxyaromatic decarboxylase family) genes

In spite of the abundance of stress response category genes strain ADS47 has only a

small number of desiccation stress genes (5) compared to the other species which ranged

from 9 to10 However ADS47 and Ballerup code for higher number of genes related to

oxidative stress (94 and 91 respectively) and cold shock (8 and 7 respectively) than the other

species

The smallest numbers of amino acids and its derivatives category genes were found in

the two pathogens C rodentium and C koseri (416 and 428 respectively) while the largest

number of these genes were identified in strain ADS47 (482) and C freundii Ballerup (476)

Despite the similar number of sulfur metabolism category genes determined across the six

species only ADS47 lacks taurine utilization genes Strains ADS47 and Ballerup were found

150

to be rich in the phosphorous metabolism gene category (49-50) while C rodentium contains

a significantly decreased number of these category genes (28)

The bioinformatics analyses found the largest number of carbohydrate category genes

in ADS47 (837) followed by Ballerup (803) The gut bacteria (Citrobacter sp 30_2) codes for

the least number of genes assigned to the carbohydrate category (708) while C koseri C

rodentium and C youngae contained 731-737 in this category genes The analyses also

determined that there are relatively small numbers of TCA and Entner Doudoroff pathway

genes in C rodentium and C youngae compared to the other four species and relatively large

numbers of fermentation-related genes in ADS47 (81) and C freundii (85) compared to the

other four species

A comparison of the pathogenicity operons [locus of enterocyte effacement (LEE)] of

C rodentium with other five Citrobacter genomes revealed the absence of this virulence

determinant gene region in the other four Citrobacter species including ADS47 (Figure 5-7)

The analysis showed the presence of LEE operon genes in C freundii strain Ballerup as no

gap was observed in that genomic location while C freundii ADS47 C koseri C youngae

and Citrobacter sp 30_2 lacked that loci (7a) Further ananlysis showed alignments between

C rodentium and E coli LEE operon genes (Figure 7b) In contrast no alignment of such

genes was observed in ADS47 genome

151

(a)

152

Figure 5-7 (a) Blast Ring Image Generator (BRIG) output based of Citrobacter rodentium

(NC_013716) compared against ADS47 C freundii C koseri C youngae E coli and

Citrobacter sp genomes The targeted comparative genome analysis was performed based on

locus of enterocyte effacement (LEE) operons of C rodentium (Petty et al 2011) The

innermost rings show GC content (black) and GC skew (purple and green) The remaining

rings show BLAST comparisons of the other Citrobacter and E coli genomes against C

rodentium The region shown in red is LEE operon genes (b) The absence of LEE operons in

the ADS47 genome comparing to C rodentium and E coli Analysis was performed and

visualized by Artemis Comparison Tool software v132

(httpwwwsangeracukresourcessoftwareact) (Tatusov et al 2000 Craver et al 2005)

LEE operons

(b)

153

546 Analysis of the unique genes in C freundii ADS47

Two hundred and seventy genes unique to ADS47 were identified from a comparison

of its genome to the other five Citrobacter species In general the unique genes are

distributed throughout the ADS47 genome but some clusters of unique genes occurred

including eight unique genomic regions (Figure 5-8) Six large unique regions are located at

767204-775090 (~8 kb) 1025572-1042510 (17 kb) 2798289-2807217 (~9 kb)

3045220-3055912 (~11 kb) 3930366-3943075 (12 kb) and 4022195-4032444 (10 kb)

Out of 270 unique genes 147 (54 ) are hypothetical proteins The remaining 123

(46) genes are grouped into reductasesoxidoreductases (9 genes) de-acetylase (1)

hydrolases (2) transferases (4) sulfatasesarylsulfatases (3) bacteriocins (3) type VI

secretion proteins (2) kinases (4) transporters (11) membraneexported proteins (8)

regulators (12) integraserecombinases (5) phagesprophagestransposaes related genes (27)

and miscellaneous (32) that includes DNA helicases ligases fimbrial proteins and

chromosome partitioning genes (Table 5-4)

The unique regions encode various functional genes that may cause trichothecene de-

epoxidation or de-acetylation reactions The 8 kb unique regions contains six genes encoding

glycosyltransferases and two copies of oxidoreductase-like coding sequences (CMD_0711

and CMD_0712) that showed 39 identity to the Marinobacter aquaeolei F420

hydrogenasedehydrogenase beta subunit The largest (17 kb) unique genomic region codes

for 16 genes including dehydrogenase hydrolase bacteriocin (pyocin) integrase and a short

chain dehydrogenasereductase-like (SDR) (CMD_0979) gene (Figure 5-9a)

154

The SDR like gene is 49 amino acid similarity to an oxidoreductase of the

phytopathogenic Dickeya dadantii and the enzyme contains a domain with 77 homology to

the Dickeya zeae NAD(P) binding site The 9 kb unique genomic section contains 8 genes

including a NaCl symporter a dehydratase (CMD_2679) with 86 homology to

Enterobacteriaceae bacterium dihydroxy-acid dehydratase and a putative oxidoreductase

(CMD_2683) having 37 similarity with oxopropionate reductase of Bacillus sp The unique

region also encodes a short chain dehydrogenasereductase (SDR) (CMD_2684) that shows

85 identity to Enterobacteriaceae bacterium (Figure 5-9b) A unique thiol-disulfide and

thioredoxin like reductase (CMD_3721) was identified in a 10 kb unique region that contains

13 genes (Figure 5-9c) This gene is 59 identical to Oligotropha carboxidovorans thiol-

disulfide isomerase and thioredoxin In addition several reductasesoxidoreductase-like

unique genes were identified in the ADS47 genome including an oxidoreductase

(CMD_1024) with 27 identity to Canadidatus nitrospira NADH-quinone oxidoreductase

membrane L Another oxidoreductase-like gene identified in a different location of the

ADS47 genome (CMD_1393) shows 87 homology to Klebsiella pneumoniae

deoxygluconate dehydrogenase contains a SDR active site A putative nitrate reductase gene

(CMD_3184) has a low level of identity (27) with an uncultured bacterial respiratory nitrate

reductase The ~11 kb unique region encodes sulfatasesarylsulfatses

(CMD_2898CMD_2905) and related genes Furthermore ADS47 contains a unique

glycosylphosphatidylinositol (GPI) anchor biosynthesis operon comprised of four genes

including a N-acetylglucosaminyl-phosphatidylinositol de-acetylase (CMD_2431) which

showed 78 similarity to E coli de-acetylase (Figure 5-9e)

155

Other unique genespredicted genes in the ADS47 genome include

polysaccharidephenazine biosynthesis genes (CMD_0710CMD_1406) heavy metal kinase

(CMD_0989) two component heavy metal regulator (CMD_0990) nitrate reductase regulator

(CMD_3477) putative potassiumzinc transporters (CMD_0973CMD_4600) sodium

symporter (CMD_2678) (tripartite ATP-independent periplasmic transporters) TRAP

transporters (CMD_3693 CMD_3694 CMD_3695 CMD_4337) glutamineribose ABC

transporters (CMD_3176CMD_3544) toxin transcription regulator (CMD_3464) fimbrial

proteinchaperone (CMD_1695 CMD_2434 CMD_3277 CMD_3278) and ParB

chromosomeplasmid partitioning proteins (CMD_4033CMD_4034)

Majority of the unique genes and their distributions are shown in comparative circular

genome maps (Figure 5-8)

156

Table 5-4 Unique genes of C freundii strain ADS47 Genes were categorized according to

the functional similarity The unique genes were determined by comparing ADS47 with five

sequenced Citrobacter species using OrthoMCL software

Unique gene classes Number

Total unique genes

270

Reductases oxidoreductases 9

De-acetylase 1

Hydrolase 2

Transferase 4

Sulfatase 3

Bacteriocin 2

Type VI secretion 2

Kinase 4

Transporter 9

Membraneexported protein 8

Regulator 12

Integraserecombinase 5

Phageprophagetransposae 27

Miscellaneous 34

Hypothetical protein 147

157

158

Figure 5-8 Locations of the unique genes of C freundii strain ADS47 The unique genes

were determined by comparing the total CDS of strain C freundii ADS47 with C freundii

Ballerup Citrobacter sp C youngae C koseri and C rodentium CDSs by OrthoMCL

program The map was developed by Blast Ring Generator (BRIG) software First second

and third circles from inside to out side show the genome size GC and GC skew of strain

ADS47 Forward (Fwd) and reversed (Rev) transcribed CDSs of ADS47 are shown in green

(forth circle) and red (fifth circle) respectively The next five rings represent the circular

genome maps of five Citrobacter species distinguished by different ring colours (C

rodentium as sky blue C koseri as dark blue C youngae as light blue C freundii Ballerup as

yellow and Citrobacter sp as green) The degree of orthologous gene identity between

ADS47 and other five genomes are shown by color intensity (light color= 75 gene identity

ash color= 50 gene identity) Gaps across five genome rings indicate the absence of

genesregions compared to ADS47 Unique genes are shown surrounding the map rings and

the identities of some of the genes are given Arrows indicate putative

reductasesoxidoreductases and de-acetylase

159

(a)

(b)

160

(c)

(d)

161

Figure 5-9 Unique genomic regions and associated genes in C freundii ADS47 compared to

five Citrobacter species RAST server based analysis compared the unique genesregions of

ADS47 with the publicly available databases The upper panels demonstrate the large unique

regions defined by yellow boxes and genes located in the genomic regions are indicated by

arrows The lower panels are closely related genesoperons found in distantly related species

The unique regions that contain potential trichothecene de-epoxidation genes are shown in (a)

short chain dehydrogenasereductase (SDR) and dehydrogenase (b) putative oxidoreductase

dehydratase and SDR (c) thiol-disulfide isomerase and thioredoxin oxidoreductase and (d)

unique glycosylphosphatidylinositol (GPI) biosynthesis operon having N-acetylglucosaminyl-

phosphatidylinositol de-acetylase gene

162

55 Discussion

The de novo sequenced ADS47 genome was oriented and assembled using a

completely sequenced C koseri genome as reference strain The synteny analysis between

ADS47 and C koseri showed very similar overall organization of the two genomes The high

preservation of genes among five Citrobacter species and ADS47 were emphasized by the

close alignments observed by Blast Ring Image Generator (BRIG) outputs obtained from

these genomes

Genome-wide phylogenetic analyses based on a large number of common CDSs

(2821) observed within the six Citrobacter genomes confirmed the species identification

based on 16S-rDNA sequencing of the trichothecene mycotoxin-detoxifying C freundii strain

ADS47 The base sequence idendity was 8715 between these two genomes They shared

~80 CDS and had a similar GC content (52) Citrobacter are Gram-negative rod shaped

and facultatively anaerobic bacteria belonging to the family of Enterobacteriaceae (Wang et

al 2000 Whalen et al 2007) Strains ADS47 and Ballerup of C freundii are both aerobic

gram-negative bacilli that have numerous flagella and inhabit in soil (Cong et al 2011 Islam

et al 2012) C freundii is considered to have a positive environmental role by reducing nitrate

to nitrite (Rehr and Klemme 1989) which is an important step in the nitrogen cycle

C freundii C rodentium and C koseri are animalhuman pathogenic species C

rodentium is a natural mice pathogen causing hemorrhagic colitis and hemolytic uremic

syndrome (Gareau et al 2010) C koseri causes human disease (Badger et al 1999 Kariholu

et al 2009) particularly neonatal meningitis and C freundii is an opportunistic human

pathogen responsible for a various types of infections like respiratory tract urinary tract and

blood (Whalen et al 2007) C freundii is very common representing approximately 29 of

163

all opportunistic human infections (Hodges et al 1978) Outside of animals they can be found

in soil water sewages and different organs of diseased animals (Wang et al 2000) These

pathogens code for large numbers of mycobacterium virulence operonsgenes which were

absent in the three other genomes However they have fewer genes involved in environmental

stress such as copper homeostasis genes and no mercuricarsenic resistance and secondary

metabolism category genes unlike the other Citrobacter genomes Nonetheless considerable

variability exists between the two pathogens correlates with C rodentium causing disease in

mice C koseri causing meningistis in human and C freundii causing infections in different

human organsThere were attaching and effacing (AE) lesion forming (LEE) operons in C

rodentium and C freundii but not in C koseri Those genes have shown to be acquired by

lateral gene transfer playing major roles in virulence of enteropathogenic and

enterohemorrhagic E coli and C rodentium (Mundy et al 2005 Petty et al 2010 Petty et al

2011 The ~356 kb pathogenic LEE gene island is comprised of five operons (LEE1 to

LEE5) that code for ~40 virulence genes that include type-III secretion genes and associated

effectors (Berdichevsky et al 2005) In addition an elevated number of type-II and -VI

secretion proteins identified in C rodentium which may act as additional virulence factors

(Petty et al 2010) Because it lacks type-III secretion apparatus genes it is likely that C

koseri uses a different strategy to cause human neonatal meningitis (Badger et al 1999 Ong

et al 2010)

To adapt to different environmental niches or hosts bacteria like Citrobacter species

frequently gainlose genes increase repeat sequences and recombine genomic regions using

different mechanisms (Parkhill et al 2001 Petty et al 2011) The existence of large numbers

of phageprophagetransposae related genes in the Citrobacter genomes particularly in C

164

freundii Ballerup suggests that their genomes are unstable For example C freundii Ballerup

and C koseri might have acquired LEE operons by horizontal transfer via phage mediated

transpositions Interestingly although ADS47 is closely related to C freundii Ballerup it

lacks the pathogen-related LEE gene island and mycobacterium virulence operons required

for invasion and intracellular survival in macrophages (Gao et al 2006) Instead ADS47 has a

larger number and variety of stress-responsive genes such as efflux genes and bacteriocins

(pyocin hemolysin) genes that provide protection against heavy metals and antibiotics and

increase their competitiveness against other bacteria These findings suggest that the strain

ADS47 is not an opportunistic animal pathogen but has evolved to be highly competitive in

the soil micro-environment (Arnold 1998 Bengoechea and Skurnik 2000) Perhaps ADS47

evolved trichothecene degrading genes for adaptation to soils containing trichothecene

mycotoxins contaminated soils (Gadd and Griffiths 1978 Sharma and Fulekar 2009 Han et

al 2011) Conversely the absence or reduced number of stress-responsive genes in C

rodentium and C koseri may imply that those two pathogenic species are not exposed to such

stressful and xenobiotic-contaminated environments as ADS47

This interpretation is supported by the observation that the two of the three

animalhuman pathogenic species contained high of rRNA copy number but lower numbers

were found in ADS47 C freundii Ballerup and Citrobacter sp 30_2 Bacteria maintain large

number of rRNA for higher production of ribosome during high growth rates (Stevenson et al

1997 Klappenbach et al 2000) such as might occur during the establishment of an infection

However a large copy number of rRNAs may not be metabolically favourable for bacteria

such as ADS47 that evolved in a highly competitive environment as that costs higher cellular

energy (Hsu et al 1994)

165

C freundii Ballerup seems to be an exception in that it is similar to the other animal

pathogens for containing virulent factors (ie LEE-operon genes) but more like the

environmental species for encoding high number genes for stress defence heavy metal and

toxic compounds resistances

The comparison of the genes in C freundii ADS47 with the unique ability to detoxify

trichothecenes with genes in other Citrobacter species allowed approximately 270 unique

genes to be identified in ADS47 Some of those genes may be involved in trichothecene de-

epoxidation or de-acetylation Currently the pathways and microbial enzymes leading to

trichothecene de-epoxidation and de-acetylation are unknown but based on the required

reaction ie the elimination of an oxygen atom from epoxide moiety it is likely that epoxide

reductases or epoxide oxidoreductases are responsible for such biodegradation activity (He et

al 1992)

From database searches three enzymes having epoxide reductase activity were found

These are a vitamin K epoxide reductase (VKOR) a monooxygenase PaaABCE and pyridine

nucleotide-disulfide oxidoreductase (PNDO) which reduce vitamin K epoxide phenylacetyl-

CoA and epoxyalkanes respectively (Swaving et al 1996 Li et al 2004 Teufel et al 2012)

In mammals VKOR plays important roles in vitamin K cycle by reducing vitamin K epoxide

to vitamin K and its homologues that are found in plants bacteria and archaea (Goodstadt

2004 Li et al 2010) The microbial multi-component PaaABCE monooxygenase catalyzes

degradation of variety of aromatic compounds or environmental contaminants via epoxidation

of phenylacetic acid (Paa) and NADPH-dependant de-epoxidation of epoxy-Paa (Luengo et

al 2001 Teufel et al 2012) PNDO is a unique type of NADP-dependent oxidoreductase

identified in Xanthobacter strain Py2 that was obtained by propane enrichment procedure

166

(van Ginkel and de Bont 1986) However no orthologs of these genes were found in ADS47

genome

Nine ADS47-unique reductaseoxidoreductase-like genes are potential candidates for

the trichothecene de-epoxidation enzyme Genes belong to short chain

dehydrogenasereductase (SDR) family are structurally and functionally diverse and cause

different enzymatic activities including xenobiotic degradation (Kavanagh et al 2008) This is

a promising candidate for the trichothecene de-epoxidation gene Genes belong to dehydratase

family catalyze reduction reactions by cleaving carbon-oxygen bonds of organic compounds

that result in removal of oxygen in various biosynthetic pathways (Flint et al 1993) This

mechanism may be relevant to microbial trichothecene de-epoxidation reaction since the latter

involves removal of the oxygen atom forming the epoxide in trichothecene mycotoxins

(Young et al 2007)

Another ADS47-unique gene was for a nitrate reductase that may be a candidate de-

epoxidation gene Under stress conditions such an enzyme causes reductive removal of an

oxygen molecule (Morozkina and Zvyagilskaya 2007)

A fourth type of ADS47-unique gene is for a thiol-disulfide isomerasethioredoxin like

(Trx) oxidoreductase The thiol-disulfide isomerase and thioredoxin oxidoreductases contain a

thioredoxin like domain with a CXXC catalytic motif which catalyzes various biological

processes that allow the organism to adapt to environmental stress and develop competence to

sporulate (Scharf et al 1998 Petersohn et al 2001 Smits et al 2005) The membrane-

anchored Trxndashlike domain initiates the vitamin K de-epoxidation reaction by reducing the

active motif (CXXC) of VKOR (Schulman et al 2010)

167

The present study did not find any homologues of VKOR in ADS47 genome but we

identified two copies of structurally similar quinone reductase protein disulfide bond

formation (DsbB) gene (CMD_1044 and CMD_2641) Both VKOR and DsbB contain four

transmembrane segments an active site CXXC motif and cofactor quinone and use a

common electron transfer pathway for reducing substrates (Li et al 2010 Malojcic et al

2008) As VKOR bacterial DsbB requires a Trx-like partner (eg DsbA) for activity Further

experiments are required to examine if DON de-epoxidation occurs by the catalytic activity of

DsbB coupled with a unique thiol-disulfide isomerase and thioredoxin oxidoreductase present

in ADS47 genome

The analysis also identified an ADS47-unique glycosylphosphatidylinositol (GPI)

biosynthetic operon that is comprised of four genes including an N-

acetylglucosaminylphosphatidylinositol de-acetylase The GPI membrane anchor protein

synthesis is initiated by transferring N-acetylglucosamine from uridine diphosphate N-

acetylglucosamine to phosphatidylinositol by N-acetylglucosaminyltransferase an

endoplasmic reticulum (ER) membrane-bound protein complex (Hong and Kinoshita 2009)

In the second step of the pathway N-acetylglucosamine-phosphatidylinositol is de-acetylated

to produce glucosaminyl-phosphatidylinositol by the catalytic activity of a zinc

metalloenzyme N-acetylglucosaminylphosphatidylinositol de-acetylase which is localized on

the cytoplasmic side of the ER (Nakamura et al 1997 Hong and Kinoshita 2009) It is

possible that the identified unique N-acetylglucosaminylphosphatidylinositol de-acetylase

gene of ADS47 may be involved in trichothecene de-acetylation activity

168

Further analysis is needed to determine which of these candidate gene or genes may be

involved in tricthothecene mycotoxin de-epoxidation and de-acetylation activities

Identification of gene responsible for the trichothecene detoxification activities of C freundii

ADS47 opens many potential applications for its use in agri-based industries to contribute to

cropanimal production and food safety (Karlovsky 2011)

56 Acknowledgement

We gratefully acknowledge Agriculture and Agri-Food CanadaBinational

Agricultural Research and Development Fund (AAFCBARD) project for providing the

financial supports of the work

169


functional roles The unique genes were determined by comparing the ADS47 genome with

five sequenced Citrobacter species (C freundii Citrobacter sp C youngae C koseri and C

rodentium) using OrthoMCL (Li et al 2003)

Unique gene

classes

Number Gene ID and functions

Total unique

genes

270 123 known functions 147 hypothetical proteins

Reductases

oxidoreductases

9 CMD_0711 coenzyme F420 hydrogenasedehydrogenase beta subunit

CMD_0979 Short chain dehydrogenase reductase (SDR) Oxidoreductase

SDR dehydrogenase CMD_1024 NADH-quinone oxidoreductase

membrane L CMD_1393 oxidoreductase SDR putative deoxygluconate

dehydrogenase (NAD+ 3-oxidoreductase) CMD_2679

Dehydratasedihydroxy acid dehydratase CMD_2683 oxidoreductase

oxopropionate reductase CMD_2684 short-chain dehydrogenasereductase

SDR CMD_3184 nitrate reductase alfa subunit CMD_3721 thiol-disulfide

isomerase and thioredoxin oxidoreductase

Deacetylase 1 CMD_2431 N-acetylglucosaminylphosphatidylinositol deacetylase

superfamily

Hydrolase 2 CMD_0991 Dienelactone hydrolase CMD_2895 Putative glycosyl

hydrolase

Transferase 4 CMD_1640 D12 class N6 adenine-specific DNA methyltransferase

CMD_2432 Glycosyltransferase CMD_2433 putative acetyltransferase

CMD_3023 adenine-specific DNA modification methyltransferase

Sulfatase 3 CMD_2898 Possible sulfatase arylsulfatasearylsulfatransferase

CMD_2902 Arylsulfatasesulfatase CMD_2905 Putative

sulfatasearylsulfatase

Bacteriocin 3 CMD_0982 Thermosatble hemolysin superfamily CMD_0993 Pyocin S2

superfamily (bacteriocin that kills other bacterial strain) CMD_3705 S-type

pyocin containing domain protein

Type VI secretion 2 CMD_1802 EvpB family type VI secretion protein CMD_2364 type VI

effector Hcp1

Kinase 4 CMD_0989 Heavy metal Sensor kinase CMD_1794 Polo kinase kinase

(PKK) superfamily CMD_3124 predicted diacylglycerol kinase CMD_4266

putative signal transduction kinase

Transporter 11 CMD_0973 TrkA-N domain-containing protein (K uptake transporter)

CMD_1407 Branched-chain amino acid transport system carrier protein

CMD_2678 Sodium Symporter CMD_3176 glutamine ABC transporter

CMD_3544 Ribose ABC transport system CMD_3693 TRAP-type C4-

dicarboxylate transport system binding protein CMD_3694 TRAP-type C4-

dicarboxylate transport system binding protein small permease component

(predicted N-acetylneuraminate transporter) CMD_3695 TRAP-type C4-

dicarboxylate transport system large permease CMD_4337 23-diketo-L-

gulonate TRAP transporter small permease protein yiaM component

CMD_4338 23-diketo-L-gulonate TRAP transporter large permease protein

yiaN CMD_4600 putative transporter involved in Zinc uptake (YciA

protein)

170

Supplmental Table S5-1 (continued)

Unique gene

classes


Membrane

exported protein

8 CMD_0713 putative membrane protein wzy CMD_0983 Putative exported

protein precursor CMD_0986 Putative transmembrane protein

CMD_0987 putative exported protein precursor CMD_1458 putative

exported protein CMD_2903 Putative exported protein CMD_2904

putative secreted 5-nucleotidase CMD_4291 (S) putative integral

membrane export protein

Regulator 12 CMD_0977 probable transcriptional regulator protein CMD_0980 Putative

transcription activator TenA family CMD_0990 Two component heavy

metal response transcription regulator CMD_1766 Leucine responsive

regulatory protein CMD_2677 transcriptional regulator GntR CMD_2909

putative arylsulfatase regulator CMD_3279 Uncharacterized outer

membrane usher protein yqiG precursor CMD_3464 toxin transcription

regulatory protein CMD_3465 EAL regulator protein (signaling)

CMD_3475 putative transcription regulator CMD_3477 Fumarate and

nitrate reduction regulatory protein (for anaerobic adaptation) CMD_3812

putative regulator from phage

Integrase

recombinase

5 CMD_0785 CMD_0992 CMD_0994 CMD_4032 CMD_4424

Phage prophage

transposae related

genes

27 Phages CMD_1608 CMD_1609 CMD_1610 CMD_1611 CMD_1619

CMD_1631 CMD_1913 CMD_1914 CMD_1915 CMD_1917



CMD_4610 Transposae CMD_0252 CMD_0398 Prophage CMD_1645

CMD_3806 CMD_4425 CMD_4597

Miscellaneous 32 CMD_0095 ATP-dependent DNA helicase Rep CMD_0656 LysA protein

CMD_0710 Polysaccharide biosynthesis family protein CMD_0978 TPR

repeat containing protein CMD_0981 AMP dependant synthatase and

ligase CMD_0985 Cupin superfamily CMD_0987 flavodoxin

CMD_1404 Putative endoribonuclease CMD_1405 Putative PAC domain

protein CMD_1406 Phenazine (PhzF) biosynthesis protein CMD_1695

Putative fimbrial protein CMD_1793 HPChromosome segregation ATPase

domain protein CMD_1922 FRG uncharacterized protein domain

CMD_2206 L-fucose isomerase like protein CMD_2434 CS12 fimbria

outer membrane usher protein CMD_2680 HpcHHpaI aldosecitrate lyase

protein family CMD_2681 Aldose -1 epimerase CMD_2682

Gluconolaconase LRE like region CMD_2900 Putative cytoplasmic

protein CMD_3009 putative restriction endonuclease CMD_3277 Putative

yqi fimbrial adhesion CMD_3278 Hypothetical fimbrial chaperone yqiH

CMD_3340 Pea-VEAacid family CMD_3518 3-hydroxy-3-methylglutaryl

CoA synthase CMD_3801 putative reverse transcriptase CMD_3840 beta-

D-galactosidase alpha subunit CMD_4033 ParB family chromosome

partitioning protein CMD_4034 ParBrepB chromosomeplasmid

partitioning protein CMD_4035 RelASpoT protein CMD_4532 putative

DNA ligase -like protein CMD_4541 Antitermination protein Q and

CMD_4585 putative ATP dependent helicase

171

CHAPTER 6

60 CONCLUSION

61 Summary

Contamination of grain with mycotoxins including deoxynivalenol (DON) and other

trichothecenes is becoming a limiting factor for cereal and animal production (Binder et al

2007) To address the issue this study obtained a novel microbial source for DON

detoxification and determined candidate genes for the function which may have potential

applications to detoxify grain and grain-derived foodfeed contaminated with DON and TC

mycotoxins

From a screen of soil samples from crop field microorganism was isolated that is able

to de-epoxidize the most frequently detected foodfeed contaminating mycotoxin DON The

effects of media compositions temperature pH and aerobicanaerobic conditions on

microbial detoxification activity were assessed From 150 analyzed soil samples a mixed soil

microbial culture that showed DON de-epoxidation activity under aerobic conditions and

moderate temperature (~24degC) was obtained This culture exhibited a slow rate of microbial

DON to de-epoxyDON transformation (de-epoxidation) possibly because of competition

between de-epoxidizing and other microorganisms in the culture

The mixed microbial culture was enriched for DON detoxifying bacteria by reducing

the non-targeted microbial species through antibiotic treatments heating and subculturing on

agar medium and selecting colonies The enriched culture had higher DON de-epoxidation

activity than the initial culture Because of low abundance of DON detoxifying

microorganisms further culture enrichments were required to increase the target microbial

172

population Reculture of the partially enriched microbial community in nutritonally poor MSB

medium with high level of DON increased the DON de-epoxidizing bacterial population

Serial dilution plating and selecting single colonies eventually led to isolation of a DON-

detoxifying bacterial strain named ADS47

Phenotypic and 16S-rDNA sequence analyses provided evidence that the bacterium is

a strain of C freundii Strain ADS47 is a gram negative rod-shaped facultative anaerobe and

has peritrichous flagella Strain ADS47 was unable to use DON as sole carbon source

however increasing DON concentrations in growth media enhanced its DON metabolism

efficiency The pure bacterial culture was shown to be able to rapidly de-epoxidize DON

under aerobic conditions moderate temperatures and neutral pH Strain ADS47 more quickly

de-epoxidizes DON in aerobic conditions than the anaerobic conditions In addition to DON

strain ADS47 showed capability to de-epoxidize andor de-acetylate 10 different type-A and

type-B TC mycotoxins which often co-contaminant grain with DON

To clone the DON detoxifying genes a DON detoxifying bacterial DNA library in E

coli host was constructed followed by screening of the library clones for DON detoxification

activity The study produced a library giving approximately 10 fold coverage of the genome

of strain ADS47 Restriction digestion analysis determined that most of the clones contained

ADS47 DNA inserts This indicates that a good DNA library was produced which had gt99

probability of containing all the DNA of the ADS47 genome However activity-based

screening of the library could not identify any clones with DON de-epoxidation ability It is

assumed that bacterial DON de-epoxidation genes evolved in a certain environmental niche

which may be required for the DON de-epoxidation to be expressed and active but was not

supplied by the host E coli cells

173

An attempt was made to identify the bacterial genes encoding DON de-epoxidation

and de-acetylation enzymes by sequencing the genome of strain ADS47 and comparing its

sequence to the genomes of related bacteria that are not known to posses DON de-epoxidation

activity A RAST server based bioinformatics analysis determined that the 48 Mb circular

genome of ADS47 codes for 4610 predicted CDS A genome wide phylogenetic analysis

determined that strain ADS47 was gt87 identical to the pathogen C freundii strain Ballerup

The relationship to two other animalhuman pathogenic species C rodentium and C koseri

was more distant This result is consistent with the 16S-rDNA based phylogenetic analysis

It is assumed that the microbial DON detoxifying genes were acquired by horizontal

gene transfer or other means during the adaptation of ADS47 in mycotoxin contaminated soil

If it is correct then this means that those newly acquired genes are not common in other

related microbial species and no other Citrobacter species is known to degrade DON Hence

the study identified 270 unique genes to ASDS47 with a subset of 10 genes for reductases

oxidoreductases and a de-acetylase which were considered to be candidate genes for the

enzymes that have DON detoxifying activity Furthermore a targeted comparative genome

analysis showed that the mycotoxin detoxifying ADS47 genome does not harbour the

clinically important LEE encoded typendashIII secretion system and mycobacterium virulence

genes This suggests that strain ADS47 is not likely to be an animal pathogenic bacterium

unlike C freundii Ballerup which encodes the pathogenic relevance LEE operons

174

62 Research contributions

The research made four major contributions which may aid in reducing the risks of

animals and humans from DONTC exposure Firstly the study identified a novel bacterial

strain ADS47 that is able to detoxify DON and 10 related mycotoxins over a wide range of

temperatures under aerobic and anaerobic conditions The discovery has led to a patent

application for strain ADS47 supporting the novelty and importance of the finding This

bacterial strain could be used to detoxify trichothecene mycotoxins in contaminated animal

feed thus reducing food safety risks

Secondly the study resulted in the development of a method for isolating DON de-

epoxidizing microbes from soil as well as optimizing the conditions for bacterial DONTC

detoxification activity to increase the effectiveness usefulness of the strain

Thirdly the thesis work resulted in availability of the genome sequence of a

trichothecene detoxifying microbe which may ultimately lead to an understanding of the

cellularmolecular mechanisms of DONTC de-epoxidation and de-acetylation

genespathways The genome analysis resulted in identification of several candidate genes for

DONTC de-epoxidation and de-acetylation activities Genes which show de-epoxidation

activity could be used to develop DONTC resistant cereal cultivars Fourthly strain ADS47

lacks major animal pathogenicity genes (in particular the LEE-operons) as well as does not

encodes mycobacterium virulence genes Thus strain ADS47 has a great potential to be

utilized to detoxify cereal grain and may be used as a feed additive for livestockfishery All

together the reported work may have significant applications to produce and supply

DONTC-free food and feed

175

63 Further research

This research provides the basis for developing biological detoxification of DON and

related mycotoxins This goal might be achieved by using DON de-epoxidizing gene to

develop resistant plants enzymatic detoxification of contaminated feed and use of the bacteria

as a feed additive Follow-up studies may reach the above indicated goals Because the

bacterium showed de-epoxidation activity under aerobicanaerobic conditions and under wide

range of temperatures it is likely to have an application for enzymatic detoxification of

contaminated animal feed Therefore additional studies on bacterial efficiency to detoxify

contaminated cereal and grain-derived feed need to be conducted Although strain ADS47

does not encode major animal pathogenicity genes the bacterium must be directly tested on

animals to see if it could negatively affect animal digestive systems If not then the potential

for ADS47 is considerable for its use as a feed additive for pig poultry and fish Animal

feeding trials will also need to be performed to determine if DON detoxification is sufficient

to be cost effective as a feed additive In order to develop DONTC resistant cereal cultivars

the gene responsible to DON degrading activity among the presently identified candidate

genes (reductases oxidoreductases and de-acetylase) will need to be expressed in other

organisms like E coli and a codon modified version adapted for expression in plants like

corn and wheat

176

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Desjardins AE Proctor RH Bai G McCormick SP Shaner G Beuchley G Hohn TM 1996

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Canadian Workshop on Fusarium Head Blight edited by

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Kouadio JH Dano SD Moukha S Mobio TA Creppy EE 2007 Effects of combinations of

Fusarium mycotoxins on the inhibition of macromolecular synthesis malondialdehyde

levels DNA methylation and fragmentation and viability in Caco-2 cells Toxicon

49306ndash317

Krause L Diaz NN Bartels D Edwards RA Puumlhler A et al 2006 Finding novel genes in

bacterial communities isolated from the environment Bioinformatics 22281ndash289

Kubena LF Huff WE Harvey RB Phillips TD Rottinghaus GE 1989 Individual and

combined toxicity of deoxynivalenol and T-2 toxin in broiler chicks Poultry Science

68622ndash626

Kubena LF Harvey RB Huff WE Corrier DE Phillips TD Rottinghaus GE 1990 Efficacy

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toxin Poultry Science 691078ndash1086

Kubena LF Harvey RB Huff WE Elissalde MH Yersin AG Phillips TD Rottinghaus GE

1993 Efficacy of hydrated sodium calcium aluminosilicate to reduce the toxicity of

aflatoxin and diacetoxyscirpenol Poultry Science 7251ndash59

Kubena LF Edrington TS Harvey RB Buckley SA Phillips TD Rottinghaus GE Casper

HH 1997 Individual and combined effects of fumonisin B1 present in Fusarium

moniliforme culture material and T-2 toxin or deoxynivalenol in broiler chicks Poultry

Science 761239ndash1247

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Kumar RA Gunasekaran P Lakshmanan M 1999 Biodegradation of tannic acid by

Citrobacter freundii isolated from a tannery effluent Journal of Basic Microbiology

39161-168

Kushiro M 2008 Effects of milling and cooking processes on the deoxynivalenol content in

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Lane DJ 1991 16S23S rRNA sequencing In Nucleic acid techniques in bacterial

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Lancova K Hajslova J Poustka J Krplova A Zachariasova M Dostalek P Saschambula L

2008 Transfer of Fusarium mycotoxins and ldquomaskedrdquo deoxynivalenol (deoxynivalenol-3-

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25732ndash744

Langmead B Trapnell C Pop M Salzberg SL 2009 Ultrafast and memory-efficient

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Larsen JC Hunt J Perrin I Ruckenbauer P 2004 Workshop on trichothecenes with a focus

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deoxynivalenol- and nivalenol-producing chemotypes of Gibberella zeae by using PCR

Applied and Environmental Microbiology 672966ndash2972

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65720-726

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Li L Christian J Stoeckert J David SR 2003 OrthoMCL Identification of Ortholog Groups

for Eukaryotic Genomes Genome Research 132178-2189

Li T Chang CY Jin DY Lin PJ Khvorova A Stafford DW 2004 Identification of the gene

for vitamin K epoxide reductase Nature 427541ndash544

Li Y 2007 Sensory signal transduction in the vagal primary afferent neurons Current

Medicinal Chemistry 142554ndash2563

Li MX Pestka JJ 2008 Comparative induction of 28S ribosomal RNA cleavage by ricin and

the trichothecenes deoxynivalenol and T-2 toxin in the macrophage Toxicological


Li R Li Y Kristiansen K Wang J 2008 SOAP short oligonucleotide alignment program


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Lichstein HC Malcolm HS 1943 Studies of the effect of sodium azide on microbic growth

and respiration Journal of Bacteriology 47221ndash230

Limay-Rios V Schaafsma AW 2011 2010 Update on the development of an integrated

mycotoxin management system in Ontario corn 30th

Centralia Swine Research Update

Kirkton Ontario Canada

Llorens A Hinojo MJ Mateo R Gonzalez-Jaen MT Valle- Algarra FM Logrieco A Jimenez

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195

Lombaert GA Pellaers P Roscoe V Mankotia M Neil R Scott PM 2003 Mycotoxins in

infant cereal foods from the Canadian retail market Food Additives and Contaminants

20494ndash504

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catabolic unit with broad biotechnological applications Molecular Microbiology

391434ndash1442

Luo X 1994 Food poisoning caused by Fusarium toxins In Proceedings of the Second

Asian Conference on Food Safety ILSI Thailand pp 129ndash136

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bean reproductive tissues on seed infection of resistant and susceptible bean genotypes


Madhyastha MS Marquardt RR Abramson D 1994 Structurendashactivity relationships and

interactions among trichothecene mycotoxins as assessed by yeast bioassay Toxicon

321147ndash1152

Maier FJ Miedaner T Hadeler B Felk A Salomon S Lemmens M Kassner H Schaumlfer W

2006 Involvement of trichothecenes in fusarioses of wheat barley and maize evaluated

by gene disruption of the trichodiene synthase (Tri5) gene in three field isolates of

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5823301ndash3307

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Manoharan M Dahleen LS Hohn TM Neate SM Yu XH Alexander NJ et al 2006

Expression of 3-OH trichothecene acetyltransferase in barley (Hordeum vulgare L) and

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Maresca M Mahfoud R Garmy N Fantini J 2002 The mycotoxin deoxynivalenol affects

nutrient absorption in human intestinal epithelial cells The Journal of Nutrition

1322723ndash2731

Martins ML Martins HM 2002 Influence of water activity temperature and incubation time

on the simultaneous production of deoxynivalenol and zearalenone in corn (Zea mays) by

Fusarium graminearum Food Chemistry 79315ndash318

Masuda D Ishida M Yamaguchi K Yamaguchi I Kimura M Nishiuchi T 2007 Phytotoxic

effects of trichothecenes on the growth and morphology of Arabidopsis thaliana Journal

of Experimental Botany 581617ndash1626

Matsumoto E Kawanaka Y Yun SJ Oyaizu H 2008 Isolation of dieldrin- and endrin-

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Applied Microbiology and Biotechnology 801095-1103

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Merhej J Richard-Forget F Barreau C 2011 Regulation of trichothecene biosynthesis in

Fusarium recent advances and new insights Applied Microbiology and Biotechnology

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Midwest Plan Service 1987 Grain Drying Handling Storage Handbook Ames Iowa Iowa

State University 2nd edition

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Miller SS Reid LM Harris LJ 2007 Colonization of maize silks by Fusarium graminearum

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Minervini F Fornelli F Flynn KM 2004 Toxicity and apoptosis induced by mycotoxins

nivalenol deoxynivalenol and fumonisin B1 in a human erythroleukemia cell line

Toxicology In Vitro 1821ndash28

Mitterbauer R Poppenberger B RaditschnigA Lucyshyn D Lemmens M Glossl J Adam G

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Mocali S Benedetti A 2010 Exploring research frontiers in microbiology the challenge of

metagenomics in soil microbiology Research Microbiology 161497-505

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stress factors Biochemistry 721151ndash1160

Munkvold GP 2003 Cultural and genetic approaches to managing mycotoxins in maize

Annual Review of Phytopathology 4199-116

Mundy R MacDonald TT Dougan G Frankel G Wiles S 2005 Citrobacter rodentium of

mice and man Cellular Microbiology 71697ndash1706

Nakamura N Inoue N Watanabe R Takahashi M Takeda J Stevens VL Kinoshita T 1997

Expression cloning of PIG-L a candidate N-acetylglucosaminyl-phosphatidylinositol

deacetylase The Journal of Biological Chemistry 27215834ndash15840

Nganje WE Bangsund DA Leistritz FL Wilson WW Tiapo NM 2002 Estimating the

economic impact of a crop disease the case of Fusarium head blight in US wheat and

barley National Fusarium Head Blight Forum Proceedings East Lansing Michigan State

University pp 275ndash281

198

Nielsen C Lippke H Didier A Dietrich R Martlbauer E 2009 Potential of deoxynivalenol

to induce transcription factors in humanhepatoma cells Molecular Nutrition and Food

Research 53479ndash491

Nielsen JKS Vikstroumlm AC Turner P Knudsen LE 2011 Deoxynivalenol transport across

the human placental barrier Food and Chemical Toxicology 492046-2052

Nishiuchi T Masuda D Nakashita H Ichimura K Shinozaki K Yoshida S et al 2006

Fusarium phytotoxin trichothecenes have an elicitor-like activity in Arabidopsis thaliana

but the activity differed significantly among their molecular species Molecular Plantndash

Microbe Interactions 19512ndash520

Ohsato S Ochiai-Fukuda T Nishiuchi T Takahashi-Ando N Koizumi S Hamamoto H

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3-O-acetyltransferase show resistance to the Fusarium phytotoxin deoxynivalenol Plant

Cell Reports 26531-538

Okubara PA Blechl AE McCormick SP Alexander NJ Dill-Macky R Hohn TM 2002

Engineering deoxynivalenol metabolism in wheat through the expression of a fungal

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analysis of type 3 fimbrial genes from Escherichia coli Klebsiella and Citrobacter

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Osborne LE Stein JM 2007 Epidemiology of Fusarium head blight on small-grain cereals

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Parkhill J Dougan G James KD Thomson NR Pickard D Wain J Churcher C Mungall KL

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Pestka JJ Smolinski AT 2005 Deoxynivalenol Toxicology and potential effects on humans

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Pestka J2010 Toxicological mechanisms and potential health effects of deoxynivalenol and

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Poppenberger B Berthiller F Lucyshyn D Sieberer T Schuhmacher R Krska R Kuchler K

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Qi BW Hao BL 2004 Whole-proteome prokaryote phylogeny without sequence alignment

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of Toxicology and Environment Health 481ndash34

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Sherif SO Salama EE Abdel-Wahhab MA 2009 Mycotoxins and child health the need for

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Sprando RL Collins TFX Black TN Olejnik N Rorie JI Eppley RM Ruggles DI 2005

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Canadian Workshop on Fusarium Head Blight edited by J Gilbert A Tekauz N

Sweetland and K Slusarenko Winnipeg Manitoba Canada p 41

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Yoshizawa T 1993 Red-mold diseases and natural occurrence in Japan In Trichothecenes-

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hydroxy HT-2 toxins from T-2 by liver homogenates from mice and monkeys Applied


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Agricultural and Food Chemistry 34461ndash465

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with sodium bisulfite and evaluation of the effects when pure mycotoxin or contaminated

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Young JC Zhu H Zhou T 2006 Degradation of trichothecene mycotoxins by aqueous

ozone Food Chemistry and Toxicology 44417ndash424

Young JC Zhou T Yu H Zhu H Gong J 2007 Degradation of trichothecene mycotoxins by

chicken intestinal microbes Food Chemistry and Toxicology 45136-143

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in fingerprinting of microbial communities by PCR-denaturing gradient gel

electrophoresis Applied and Environmental Microbiology 704800-4806

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deoxynivalenol-transforming bacteria from the chicken intestines using the approach of

PCR-DGGE guided microbial selection BMC Microbiology 10182

Zeibdawi A Steele M Savoie A 2011 An overview of CFIAs mycotoxin monitoring

program in livestock feeds In Proc 7th

Canadian Workshop on Fusarium Head Blight

edited by J Gilbert A Tekauz N Sweetland and K Slusarenko Winnipeg Manitoba

Canada p 25

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trichothecene mycotoxins chemotypes and associated geographical distribution and

phylogenetic species of the Fusarium graminearum clade from China Mycological


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nov and Shewanella atlantica sp nov manganese dioxide- and hexahydro-135-trinitro-

135-triazine-reducing psychrophilic marine bacteria International Journal of Systemic

Evolution and Microbiology 572155ndash2162

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Zhao X-Q 2011 Genome-based studies of marine microorganisms to maximize the diversity

of natural products discovery for medical treatments Evidence-Based Complementary

and Alternative Medicine Article ID 384572 doi1011552011384572

ZhaoY Park RD Muzzarelli RA 2010 Chitin deacetylases properties and applications

Marine Drugs 824ndash46

Zhou HR Lau AS Pestka JJ 2003a Role of double-stranded RNA-activated protein kinase R

(PKR) in deoxynivalenol-induced ribotoxic stress response Toxicological Science

74335ndash344

Zhou HR Islam Z Pestka JJ 2003b Rapid sequential activation of mitogen-activated protein

kinases and transcription factors precedes proinflammatory cytokine mRNA expression in

spleen of mice exposed to the trichothecene vomitoxin Toxicological Sciences 72130ndash

142

Zhou T He J Gong J 2008 Microbial transformation of trichothecene mycotoxins World

Mycotoxin Journal 123-30

Zhou G Zhang H He Y He L 2010 Identification of a chitin deacetylase producing bacteria

isolated from soil and its fermentation optimization African Journal of Microbiology

Research 42597-2603

ABSTRACT

ISOLATION CHARACTERIZATION AND GENOME SEQUENCING OF A SOIL-

BORNE CITROBACTER FREUNDII STRAIN CAPABLE OF DETOXIFIYING

TRICHOTHECENE MYCOTOXINS

Rafiqul Islam Advisors

University of Guelph 2012 Dr K Peter Pauls

Dr Ting Zhou

Cereals are frequently contaminated with tricthothecene mycotoxins like

deoxynivalenol (DON vomitoxin) which are toxic to humans animals and plants The goals

of the research were to discover and characterize microbes capable of detoxifying DON under

aerobic conditions and moderate temperatures To identify microbes capable of detoxifying

DON five soil samples collected from Southern Ontario crop fields were tested for the ability

to convert DON to a de-epoxidized derivative One soil sample showed DON de-epoxidation

activity under aerobic conditions at 22-24degC To isolate the microbes responsible for DON

detoxification (de-epoxidation) activity the mixed culture was grown with antibiotics at 50ordmC

for 15 h and high concentrations of DON The treatments resulted in the isolation of a pure

DON de-epoxidating bacterial strain ADS47 and phenotypic and molecular analyses

identified the bacterium as Citrobacter freundii The bacterium was also able to de-epoxidize

andor de-acetylate 10 other food-contaminating trichothecene mycotoxins










iii

DEDICATIONS



iv

ACKNOWLEDGEMENTS






















v


financial support


















future aims Racy

vi

TABLE OF CONTENTS



















vii



SOIL33





















viii


















ix

LIST OF TABLES

















x

LIST OF FIGURES






















xi























xii













xiii











bp= base pair




Bw= body weight

C= carbon









DON= deoxynivalenol


dE= de-epoxy





FM= full medium


FUS= fusarenonX

gDNA= genomic DNA




h= hour


Kb= kilobase

xiv

Kg= kilogram


mass spectrometry



LB= Luria Bertani



microg= microgram

mg= milligram

mL= milliliter

microL= microliter

microM= micromole

min= minute

Mb= megabases


MSA= MSB+ agar


MM= minimal medium



mRNA= messenger RNA


mz= mass ion ratio

N= nitrogen



dinucleotide




information

NEO= neosolaniol

ng= nanogram

NIV= nivalenol










PKR= protein kinase

xv


oxidoreductase





rRNA= ribosomal RNA

s= second

SA= sodium azide


SE= soil extract



sp= species

sRNA= small RNA

TC= trichothecene




tRNA= transfer RNA



periplasmic


length polymorphism


acetyltransferase


VER= verrucarol



1

CHAPTER 1


11 Background



















2





















3
























4









affected ears















5















6









7





(a) (b)

(c)

8
























9








Type-A





VER 266 H OH H OH H

Type-B






10




















11










2007) (Table 1-2)














12




Country

region

Sample

type

Incidence

()

Maximum

DON content

(microgg)

Mean DON

content

(microgg)

Survey

period

Source

North

AsiaChina

Animal

feed

071 1899 092

Binder et

al 2007

South East

Asia

014 152 016

South Asia 070 1062 049

Oceania 2003-05


Northern

Europe

070 551 059

Central

Europe

066 802 057

Southern

Europe

052 303 030

13



Province Sample

type

Incidence

()

Maximum

DON

contents

(microgg)

Multiple

mycotoxins

samples ()

Survey

period

Sources

Ontario Spring

Wheat

70 260 Yes 2006-08 OMAFRA1

Ontario Winter

wheat

49 290 Yes 2006-08 OMAFRA


and

Schaafsma

2010


Biologicals

Canada Breakfast

cereals

46 094 430 1999-01 Roscoe et al

2008

Canada Animal

feeds


al 2011


Canada Infant

Cereals


al 2003

1


2 microgmL

14




Products DON

(microgg)

T-2 toxin

(microgg)

HT-2 toxin

(microgg)

DAS

(microgg)


consumption

2 - - -



15
























16
























17























2010)

18








mycotoxins

Experimental

animalcells

Effects Reference


cereals


1997

DON contaminated

wheat and FB1


1996

Culture materials



lesion

Kubena et al

1997

Inoculated rice and

pure mycotoxin


1995

Naturally DON

contaminated wheat

and pure T-2 toxin



Kubena et al

1989


weight

Kubena et al

1993


oral lesion

Kubena et al

1990

Pure mycotoxins DON

ZENFB1

Human intestinal

Caco-2 cells

Increased DNA

fragmentations

Kouadio et al

2007


al 1994


al 1994

19
























20
























21










22





(Figure 1-2)










23













24








(Hck) and P38


















25




















26


















2000)






27






















28























29
























30








grains











2009)

31














temperature and pH






epoxidation

32




33

CHAPTER 2



SOIL


21 Abstract















34






22 Introduction















35

















2004)

36













De-epoxydation



37


231 Soil samples










232 Culture media











38











144 h of incubation











39

















40









incubation














41






method


















42


microbial community





















43




















44














239 Data analysis






45

24 Results



















46

47








48










49






50







51







0

20

40

60

80

100

120


DO

N d

e-e

po

xyd

ati

on

(

)

Antibiotics

52















53

(b)

(a)

Rela

tive flu

ore

scen

ce

32000

0

0 500


100 200 300 400

Rela

tive flu

ore

scen

ce

32000

0

500


0 300200100 400

54







55









species

56






54

12 8

14

8 4

0

20

40

60 S

eq

uen

ce

fre

qu

en

cy

Microbial genera

57


epoxidation











58

0

20

40

60

80

100

120

12degC

20degC

25degC

30degC

40degC

DO

N d

e-e

po

xyd

ati

on

(

)

Temperature

0

20

40

60

80

100

120

DO

N d

e-e

pxyd

ati

on

(

)

Media

(a)

(b)

59









0

20

40

60

80

100

120

55

60

65

70

75

80

DO

N d

e-e

po

xyd

ati

on

(

)

pH

(c)

60













61









62

25 Discussion























63















1999)









64






health

26 Acknowledgements






65

CHAPTER 3



31 Abstract


















66




trichothecenes

32 Introduction

















67






















68






bacterium











Union MO USA)





69























70
























71










using a TreeView X software (Page 1996)













72





each assay


















73



















74






75

34 Results















76





(a) (b)

77








78





(Figure 3-3b)








79

00

04

08

12

16

0 24 36 48 60 72 96

OD

600n

m

Incubation time (h)

Control

20 microgmL

200 microgmL

500 microgmL

(b)

(a)

80










(C)

81










82

(b)

(a)

83









84





intermediate















85

b

c

a

b

a

d c

b b

(a)

(b)

b

c

b

a a

d

86











87

(a)

(b)

88






89

(a)

(b)

90






91

Antibiotic




(100) (Figure 3-8)





method

0

20

40

60

80

100

120

SA1 SA2 C

D

ON

de

-ep

oxyd

ize

d

92









0

20

40

60

80

100

120


D

ON

de

-ep

oxyd

ize

d

93


mycotoxins



3-1)















94










95






Trichothecene

mycotoxin

Untransformed

trichothecenes ()


trichothecenes ()

De-

epoxy

De-

acetylate

Other1

Type-A

HT-2 toxin 0 74 26 -

T-2 toxin 38 46 - 16

(unknown)

DAS 0 61 39 -


T2-triol 0 47 53 -

Type-B

DON 0 100 - -


3-ADON 0 59 41 (DON)

15-ADON 0 100 - -

FUS 5 95 - -

Varrucarol 3 97 - -

96

35 Discussion














(Shima et al 1997)









97




enrichment process




















98























99
























100









36 Acknowledgements





101




















102

CHAPTER 4




41 Abstract




(pCC1FOSTM













103


activity

42 Introduction


















subclones

104










(Chapter 3)






105


DNA library

106
























107



)





















108

109


) The 813 kb







110













image was recorded










111




112

44 Results









113








114








115













DON as well

116






117


capability


singlecombined

clones

Number of

clones

examined

Number of clones

showed DON de-


Without induction


Singly 200 0



number clones)

Singly 200 0






118

45 Discussion







used
















119
























120





















121

46 Acknowledgements





122

CHAPTER 5



51 Abstract


















123




reductases

52 Introduction

















124










2008)







125






















126
















MUMmer program






127











1989)












128









software

129

54 Results





(Figure 5-1)




130







proteins

















131







genome








132


Feature Chromosome

















133

134








135



136






Figure 5-4











137




138













139









140

















141






C

freundii

ADS47

C freundii

Ballerup

Citrobacter

sp 30_2

C

youngae

C koseri C

rodentium

Genome length

(bp)

4878242

4904641

5125150

5154159

4720462

5346659

Base similarity to

ADS47 ()

NA 8715 8212 7637 5981 4213

GC () 5202 5218 516 5265 530 546

Number of coding

sequences

4610 4685 4728 5278 4978 4800

Genome coverage

with coding

sequences ()

880 865 866 872 900 840

rRNA copies 3 9 6 25 22 22


142










(34)]

143







144
















145






C

freundii

ADS47

C

freundii

Ballerup

C

sp

C

youngae

C

koseri

C

rodentium


groupspigments

323

317

310

311

325

304




Miscellaneous 210 228 200 185 245 185


element plasmid

24 75 25 4 6 49



RNA metabolism 220 222 217 215 238 212







DNA metabolism 145 167 165 155 147 152




146



Citrobacter species

C

freundii

ADS47

C

freundii

Ballerup

C

sp

C

youngae

C

koseri

C

rodentium

Respiration 198 184 180 191 166 162

Stress response 236 235 170 177 179 174


compounds

20 20 28 25 17 48




Carbohydrates 837 803 708 731 733 737

147









C youngae (4)












respectively



148







22)

















149


















species






150










other four species









151

(a)

152











LEE operons

(b)

153






767204-775090 (~8 kb) 1025572-1042510 (17 kb) 2798289-2807217 (~9 kb)

3045220-3055912 (~11 kb) 3930366-3943075 (12 kb) and 4022195-4032444 (10 kb)















154























155












156





Total unique genes

270


De-acetylase 1

Hydrolase 2

Transferase 4

Sulfatase 3

Bacteriocin 2

Type VI secretion 2

Kinase 4

Transporter 9


Regulator 12



Miscellaneous 34


157

158
















159

(a)

(b)

160

(c)

(d)

161











162

55 Discussion






these genomes

















163



















et al 2010)





164
























165












al 1992)












166


genome










(Young et al 2007)











167









in ADS47 genome













168






56 Acknowledgement




169





Unique gene

classes


Total unique

genes


Reductases

oxidoreductases











superfamily


hydrolase











effector Hcp1















protein)

170


Unique gene

classes


Membrane

exported protein

















Integrase

recombinase


Phage prophage

transposae related

genes






CMD_3806 CMD_4425 CMD_4597






















171

CHAPTER 6

60 CONCLUSION

61 Summary






mycotoxins














172
























173




















174























175

63 Further research

















corn and wheat

176

70 REFERENCES



486


















177




25195-203




















178





108A20ndashA23



















179









121411ndash423














180
























181










Microbiology 10236













182











Pathology 9435-445





149ndash18






183






Oxford pp 145-160












73 FAO Rome




166

184























185






6879ndash886


4303ndash317














186






















1190

187
























188
























189









10216892ndash16897












190




















231ndash241



191













32181ndash1183



1333







2105ndash110

192







49306ndash317





68622ndash626











193



39161-168





York pp 115-175




25732ndash744










65720-726

194
























195



20494ndash504



391434ndash1442








321147ndash1152







5823301ndash3307

196






1322723ndash2731














91519ndash528



197
























198
























199




















5631

200





















201
























202











186








285



504271ndash276

203






Research 50543-551

















204





212347ndash368









494








205






















206















94181-189









207














7th







885



208
























209







2934



2135













261

210















Canada p 25









211








74335ndash344




142





Research 42597-2603










iii

DEDICATIONS



iv

ACKNOWLEDGEMENTS






















v


financial support


















future aims Racy

vi

TABLE OF CONTENTS



















vii



SOIL33





















viii


















ix

LIST OF TABLES

















x

LIST OF FIGURES






















xi























xii













xiii











bp= base pair




Bw= body weight

C= carbon









DON= deoxynivalenol


dE= de-epoxy





FM= full medium


FUS= fusarenonX

gDNA= genomic DNA




h= hour


Kb= kilobase

xiv

Kg= kilogram


mass spectrometry



LB= Luria Bertani



microg= microgram

mg= milligram

mL= milliliter

microL= microliter

microM= micromole

min= minute

Mb= megabases


MSA= MSB+ agar


MM= minimal medium



mRNA= messenger RNA


mz= mass ion ratio

N= nitrogen



dinucleotide




information

NEO= neosolaniol

ng= nanogram

NIV= nivalenol










PKR= protein kinase

xv


oxidoreductase





rRNA= ribosomal RNA

s= second

SA= sodium azide


SE= soil extract



sp= species

sRNA= small RNA

TC= trichothecene




tRNA= transfer RNA



periplasmic


length polymorphism


acetyltransferase


VER= verrucarol



1

CHAPTER 1


11 Background



















2





















3
























4









affected ears















5















6









7





(a) (b)

(c)

8
























9








Type-A





VER 266 H OH H OH H

Type-B






10




















11










2007) (Table 1-2)














12




Country

region

Sample

type

Incidence

()

Maximum

DON content

(microgg)

Mean DON

content

(microgg)

Survey

period

Source

North

AsiaChina

Animal

feed

071 1899 092

Binder et

al 2007

South East

Asia

014 152 016

South Asia 070 1062 049

Oceania 2003-05


Northern

Europe

070 551 059

Central

Europe

066 802 057

Southern

Europe

052 303 030

13



Province Sample

type

Incidence

()

Maximum

DON

contents

(microgg)

Multiple

mycotoxins

samples ()

Survey

period

Sources

Ontario Spring

Wheat

70 260 Yes 2006-08 OMAFRA1

Ontario Winter

wheat

49 290 Yes 2006-08 OMAFRA


and

Schaafsma

2010


Biologicals

Canada Breakfast

cereals

46 094 430 1999-01 Roscoe et al

2008

Canada Animal

feeds


al 2011


Canada Infant

Cereals


al 2003

1


2 microgmL

14




Products DON

(microgg)

T-2 toxin

(microgg)

HT-2 toxin

(microgg)

DAS

(microgg)


consumption

2 - - -



15
























16
























17























2010)

18








mycotoxins

Experimental

animalcells

Effects Reference


cereals


1997

DON contaminated

wheat and FB1


1996

Culture materials



lesion

Kubena et al

1997

Inoculated rice and

pure mycotoxin


1995

Naturally DON

contaminated wheat

and pure T-2 toxin



Kubena et al

1989


weight

Kubena et al

1993


oral lesion

Kubena et al

1990

Pure mycotoxins DON

ZENFB1

Human intestinal

Caco-2 cells

Increased DNA

fragmentations

Kouadio et al

2007


al 1994


al 1994

19
























20
























21










22





(Figure 1-2)










23













24








(Hck) and P38


















25




















26


















2000)






27






















28























29
























30








grains











2009)

31














temperature and pH






epoxidation

32




33

CHAPTER 2



SOIL


21 Abstract















34






22 Introduction















35

















2004)

36













De-epoxydation



37


231 Soil samples










232 Culture media











38











144 h of incubation











39

















40









incubation














41






method


















42


microbial community





















43




















44














239 Data analysis






45

24 Results



















46

47








48










49






50







51







0

20

40

60

80

100

120


DO

N d

e-e

po

xyd

ati

on

(

)

Antibiotics

52















53

(b)

(a)

Rela

tive flu

ore

scen

ce

32000

0

0 500


100 200 300 400

Rela

tive flu

ore

scen

ce

32000

0

500


0 300200100 400

54







55









species

56






54

12 8

14

8 4

0

20

40

60 S

eq

uen

ce

fre

qu

en

cy

Microbial genera

57


epoxidation











58

0

20

40

60

80

100

120

12degC

20degC

25degC

30degC

40degC

DO

N d

e-e

po

xyd

ati

on

(

)

Temperature

0

20

40

60

80

100

120

DO

N d

e-e

pxyd

ati

on

(

)

Media

(a)

(b)

59









0

20

40

60

80

100

120

55

60

65

70

75

80

DO

N d

e-e

po

xyd

ati

on

(

)

pH

(c)

60













61









62

25 Discussion























63















1999)









64






health

26 Acknowledgements






65

CHAPTER 3



31 Abstract


















66




trichothecenes

32 Introduction

















67






















68






bacterium











Union MO USA)





69























70
























71























72





each assay


















73



















74






75

34 Results















76





(a) (b)

77








78





(Figure 3-3b)








79

00

04

08

12

16

0 24 36 48 60 72 96

OD

600n

m

Incubation time (h)

Control

20 microgmL

200 microgmL

500 microgmL

(b)

(a)

80










(C)

81










82

(b)

(a)

83









84





intermediate















85

b

c

a

b

a

d c

b b

(a)

(b)

b

c

b

a a

d

86











87

(a)

(b)

88






89

(a)

(b)

90






91

Antibiotic




(100) (Figure 3-8)





method

0

20

40

60

80

100

120

SA1 SA2 C

D

ON

de

-ep

oxyd

ize

d

92









0

20

40

60

80

100

120


D

ON

de

-ep

oxyd

ize

d

93


mycotoxins



3-1)















94










95






Trichothecene

mycotoxin

Untransformed

trichothecenes ()


trichothecenes ()

De-

epoxy

De-

acetylate

Other1

Type-A

HT-2 toxin 0 74 26 -

T-2 toxin 38 46 - 16

(unknown)

DAS 0 61 39 -


T2-triol 0 47 53 -

Type-B

DON 0 100 - -


3-ADON 0 59 41 (DON)

15-ADON 0 100 - -

FUS 5 95 - -

Varrucarol 3 97 - -

96

35 Discussion














(Shima et al 1997)









97




enrichment process




















98























99
























100









36 Acknowledgements





101




















102

CHAPTER 4




41 Abstract




(pCC1FOSTM













103


activity

42 Introduction


















subclones

104










(Chapter 3)






105


DNA library

106
























107



)





















108

109


) The 813 kb







110













image was recorded










111




112

44 Results









113








114








115













DON as well

116






117


capability


singlecombined

clones

Number of

clones

examined

Number of clones

showed DON de-


Without induction


Singly 200 0



number clones)

Singly 200 0






118

45 Discussion







used
















119
























120





















121

46 Acknowledgements





122

CHAPTER 5



51 Abstract


















123




reductases

52 Introduction

















124










2008)







125






















126
















MUMmer program






127











1989)












128









software

129

54 Results





(Figure 5-1)




130







proteins

















131







genome








132


Feature Chromosome

















133

134








135



136






Figure 5-4











137




138













139









140

















141






C

freundii

ADS47

C freundii

Ballerup

Citrobacter

sp 30_2

C

youngae

C koseri C

rodentium

Genome length

(bp)

4878242

4904641

5125150

5154159

4720462

5346659

Base similarity to

ADS47 ()

NA 8715 8212 7637 5981 4213

GC () 5202 5218 516 5265 530 546

Number of coding

sequences

4610 4685 4728 5278 4978 4800

Genome coverage

with coding

sequences ()

880 865 866 872 900 840

rRNA copies 3 9 6 25 22 22


142










(34)]

143







144
















145






C

freundii

ADS47

C

freundii

Ballerup

C

sp

C

youngae

C

koseri

C

rodentium


groupspigments

323

317

310

311

325

304




Miscellaneous 210 228 200 185 245 185


element plasmid

24 75 25 4 6 49



RNA metabolism 220 222 217 215 238 212







DNA metabolism 145 167 165 155 147 152




146



Citrobacter species

C

freundii

ADS47

C

freundii

Ballerup

C

sp

C

youngae

C

koseri

C

rodentium

Respiration 198 184 180 191 166 162

Stress response 236 235 170 177 179 174


compounds

20 20 28 25 17 48




Carbohydrates 837 803 708 731 733 737

147









C youngae (4)












respectively



148







22)

















149


















species






150










other four species









151

(a)

152











LEE operons

(b)

153






767204-775090 (~8 kb) 1025572-1042510 (17 kb) 2798289-2807217 (~9 kb)

3045220-3055912 (~11 kb) 3930366-3943075 (12 kb) and 4022195-4032444 (10 kb)















154























155












156





Total unique genes

270


De-acetylase 1

Hydrolase 2

Transferase 4

Sulfatase 3

Bacteriocin 2

Type VI secretion 2

Kinase 4

Transporter 9


Regulator 12



Miscellaneous 34


157

158
















159

(a)

(b)

160

(c)

(d)

161











162

55 Discussion






these genomes

















163



















et al 2010)





164
























165












al 1992)












166


genome










(Young et al 2007)











167









in ADS47 genome













168






56 Acknowledgement




169





Unique gene

classes


Total unique

genes


Reductases

oxidoreductases











superfamily


hydrolase











effector Hcp1















protein)

170


Unique gene

classes


Membrane

exported protein

















Integrase

recombinase


Phage prophage

transposae related

genes






CMD_3806 CMD_4425 CMD_4597






















171

CHAPTER 6

60 CONCLUSION

61 Summary






mycotoxins














172
























173




















174























175

63 Further research

















corn and wheat

176

70 REFERENCES



486


















177




25195-203




















178





108A20ndashA23



















179









121411ndash423














180
























181










Microbiology 10236













182











Pathology 9435-445





149ndash18






183






Oxford pp 145-160












73 FAO Rome




166

184























185






6879ndash886


4303ndash317














186






















1190

187
























188
























189









10216892ndash16897












190




















231ndash241



191













32181ndash1183



1333







2105ndash110

192







49306ndash317





68622ndash626











193



39161-168





York pp 115-175




25732ndash744










65720-726

194
























195



20494ndash504



391434ndash1442








321147ndash1152







5823301ndash3307

196






1322723ndash2731














91519ndash528



197
























198
























199




















5631

200





















201
























202











186








285



504271ndash276

203






Research 50543-551

















204





212347ndash368









494








205






















206















94181-189









207














7th







885



208
























209







2934



2135













261

210















Canada p 25









211








74335ndash344




142





Research 42597-2603

iv

ACKNOWLEDGEMENTS






















v


financial support


















future aims Racy

vi

TABLE OF CONTENTS



















vii



SOIL33





















viii


















ix

LIST OF TABLES

















x

LIST OF FIGURES






















xi























xii













xiii











bp= base pair




Bw= body weight

C= carbon









DON= deoxynivalenol


dE= de-epoxy





FM= full medium


FUS= fusarenonX

gDNA= genomic DNA




h= hour


Kb= kilobase

xiv

Kg= kilogram


mass spectrometry



LB= Luria Bertani



microg= microgram

mg= milligram

mL= milliliter

microL= microliter

microM= micromole

min= minute

Mb= megabases


MSA= MSB+ agar


MM= minimal medium



mRNA= messenger RNA


mz= mass ion ratio

N= nitrogen



dinucleotide




information

NEO= neosolaniol

ng= nanogram

NIV= nivalenol










PKR= protein kinase

xv


oxidoreductase





rRNA= ribosomal RNA

s= second

SA= sodium azide


SE= soil extract



sp= species

sRNA= small RNA

TC= trichothecene




tRNA= transfer RNA



periplasmic


length polymorphism


acetyltransferase


VER= verrucarol



1

CHAPTER 1


11 Background



















2





















3
























4









affected ears















5















6









7





(a) (b)

(c)

8
























9








Type-A





VER 266 H OH H OH H

Type-B






10




















11










2007) (Table 1-2)














12




Country

region

Sample

type

Incidence

()

Maximum

DON content

(microgg)

Mean DON

content

(microgg)

Survey

period

Source

North

AsiaChina

Animal

feed

071 1899 092

Binder et

al 2007

South East

Asia

014 152 016

South Asia 070 1062 049

Oceania 2003-05


Northern

Europe

070 551 059

Central

Europe

066 802 057

Southern

Europe

052 303 030

13



Province Sample

type

Incidence

()

Maximum

DON

contents

(microgg)

Multiple

mycotoxins

samples ()

Survey

period

Sources

Ontario Spring

Wheat

70 260 Yes 2006-08 OMAFRA1

Ontario Winter

wheat

49 290 Yes 2006-08 OMAFRA


and

Schaafsma

2010


Biologicals

Canada Breakfast

cereals

46 094 430 1999-01 Roscoe et al

2008

Canada Animal

feeds


al 2011


Canada Infant

Cereals


al 2003

1


2 microgmL

14




Products DON

(microgg)

T-2 toxin

(microgg)

HT-2 toxin

(microgg)

DAS

(microgg)


consumption

2 - - -



15
























16
























17























2010)

18








mycotoxins

Experimental

animalcells

Effects Reference


cereals


1997

DON contaminated

wheat and FB1


1996

Culture materials



lesion

Kubena et al

1997

Inoculated rice and

pure mycotoxin


1995

Naturally DON

contaminated wheat

and pure T-2 toxin



Kubena et al

1989


weight

Kubena et al

1993


oral lesion

Kubena et al

1990

Pure mycotoxins DON

ZENFB1

Human intestinal

Caco-2 cells

Increased DNA

fragmentations

Kouadio et al

2007


al 1994


al 1994

19
























20
























21










22





(Figure 1-2)










23













24








(Hck) and P38


















25




















26


















2000)






27






















28























29
























30








grains











2009)

31














temperature and pH






epoxidation

32




33

CHAPTER 2



SOIL


21 Abstract















34






22 Introduction















35

















2004)

36













De-epoxydation



37


231 Soil samples










232 Culture media











38











144 h of incubation











39

















40









incubation














41






method


















42


microbial community





















43




















44














239 Data analysis






45

24 Results



















46

47








48










49






50







51







0

20

40

60

80

100

120


DO

N d

e-e

po

xyd

ati

on

(

)

Antibiotics

52















53

(b)

(a)

Rela

tive flu

ore

scen

ce

32000

0

0 500


100 200 300 400

Rela

tive flu

ore

scen

ce

32000

0

500


0 300200100 400

54







55









species

56






54

12 8

14

8 4

0

20

40

60 S

eq

uen

ce

fre

qu

en

cy

Microbial genera

57


epoxidation











58

0

20

40

60

80

100

120

12degC

20degC

25degC

30degC

40degC

DO

N d

e-e

po

xyd

ati

on

(

)

Temperature

0

20

40

60

80

100

120

DO

N d

e-e

pxyd

ati

on

(

)

Media

(a)

(b)

59









0

20

40

60

80

100

120

55

60

65

70

75

80

DO

N d

e-e

po

xyd

ati

on

(

)

pH

(c)

60













61









62

25 Discussion























63















1999)









64






health

26 Acknowledgements






65

CHAPTER 3



31 Abstract


















66




trichothecenes

32 Introduction

















67






















68






bacterium











Union MO USA)





69























70
























71























72





each assay


















73



















74






75

34 Results















76





(a) (b)

77








78





(Figure 3-3b)








79

00

04

08

12

16

0 24 36 48 60 72 96

OD

600n

m

Incubation time (h)

Control

20 microgmL

200 microgmL

500 microgmL

(b)

(a)

80










(C)

81










82

(b)

(a)

83









84





intermediate















85

b

c

a

b

a

d c

b b

(a)

(b)

b

c

b

a a

d

86











87

(a)

(b)

88






89

(a)

(b)

90






91

Antibiotic




(100) (Figure 3-8)





method

0

20

40

60

80

100

120

SA1 SA2 C

D

ON

de

-ep

oxyd

ize

d

92









0

20

40

60

80

100

120


D

ON

de

-ep

oxyd

ize

d

93


mycotoxins



3-1)















94










95






Trichothecene

mycotoxin

Untransformed

trichothecenes ()


trichothecenes ()

De-

epoxy

De-

acetylate

Other1

Type-A

HT-2 toxin 0 74 26 -

T-2 toxin 38 46 - 16

(unknown)

DAS 0 61 39 -


T2-triol 0 47 53 -

Type-B

DON 0 100 - -


3-ADON 0 59 41 (DON)

15-ADON 0 100 - -

FUS 5 95 - -

Varrucarol 3 97 - -

96

35 Discussion














(Shima et al 1997)









97




enrichment process




















98























99
























100









36 Acknowledgements





101




















102

CHAPTER 4




41 Abstract




(pCC1FOSTM













103


activity

42 Introduction


















subclones

104










(Chapter 3)






105


DNA library

106
























107



)





















108

109


) The 813 kb







110













image was recorded










111




112

44 Results









113








114








115













DON as well

116






117


capability


singlecombined

clones

Number of

clones

examined

Number of clones

showed DON de-


Without induction


Singly 200 0



number clones)

Singly 200 0






118

45 Discussion







used
















119
























120





















121

46 Acknowledgements





122

CHAPTER 5



51 Abstract


















123




reductases

52 Introduction

















124










2008)







125






















126
















MUMmer program






127











1989)












128









software

129

54 Results





(Figure 5-1)




130







proteins

















131







genome








132


Feature Chromosome

















133

134








135



136






Figure 5-4











137




138













139









140

















141






C

freundii

ADS47

C freundii

Ballerup

Citrobacter

sp 30_2

C

youngae

C koseri C

rodentium

Genome length

(bp)

4878242

4904641

5125150

5154159

4720462

5346659

Base similarity to

ADS47 ()

NA 8715 8212 7637 5981 4213

GC () 5202 5218 516 5265 530 546

Number of coding

sequences

4610 4685 4728 5278 4978 4800

Genome coverage

with coding

sequences ()

880 865 866 872 900 840

rRNA copies 3 9 6 25 22 22


142










(34)]

143







144
















145






C

freundii

ADS47

C

freundii

Ballerup

C

sp

C

youngae

C

koseri

C

rodentium


groupspigments

323

317

310

311

325

304




Miscellaneous 210 228 200 185 245 185


element plasmid

24 75 25 4 6 49



RNA metabolism 220 222 217 215 238 212







DNA metabolism 145 167 165 155 147 152




146



Citrobacter species

C

freundii

ADS47

C

freundii

Ballerup

C

sp

C

youngae

C

koseri

C

rodentium

Respiration 198 184 180 191 166 162

Stress response 236 235 170 177 179 174


compounds

20 20 28 25 17 48




Carbohydrates 837 803 708 731 733 737

147









C youngae (4)












respectively



148







22)

















149


















species






150










other four species









151

(a)

152











LEE operons

(b)

153






767204-775090 (~8 kb) 1025572-1042510 (17 kb) 2798289-2807217 (~9 kb)

3045220-3055912 (~11 kb) 3930366-3943075 (12 kb) and 4022195-4032444 (10 kb)















154























155












156





Total unique genes

270


De-acetylase 1

Hydrolase 2

Transferase 4

Sulfatase 3

Bacteriocin 2

Type VI secretion 2

Kinase 4

Transporter 9


Regulator 12



Miscellaneous 34


157

158
















159

(a)

(b)

160

(c)

(d)

161











162

55 Discussion






these genomes

















163



















et al 2010)





164
























165












al 1992)












166


genome










(Young et al 2007)











167









in ADS47 genome













168






56 Acknowledgement




169





Unique gene

classes


Total unique

genes


Reductases

oxidoreductases











superfamily


hydrolase











effector Hcp1















protein)

170


Unique gene

classes


Membrane

exported protein

















Integrase

recombinase


Phage prophage

transposae related

genes






CMD_3806 CMD_4425 CMD_4597






















171

CHAPTER 6

60 CONCLUSION

61 Summary






mycotoxins














172
























173




















174























175

63 Further research

















corn and wheat

176

70 REFERENCES



486


















177




25195-203




















178





108A20ndashA23



















179









121411ndash423














180
























181










Microbiology 10236













182











Pathology 9435-445





149ndash18






183






Oxford pp 145-160












73 FAO Rome




166

184























185






6879ndash886


4303ndash317














186






















1190

187
























188
























189









10216892ndash16897












190




















231ndash241



191













32181ndash1183



1333







2105ndash110

192







49306ndash317





68622ndash626











193



39161-168





York pp 115-175




25732ndash744










65720-726

194
























195



20494ndash504



391434ndash1442








321147ndash1152







5823301ndash3307

196






1322723ndash2731














91519ndash528



197
























198
























199




















5631

200





















201
























202











186








285



504271ndash276

203






Research 50543-551

















204





212347ndash368









494








205






















206















94181-189









207














7th







885



208
























209







2934



2135













261

210















Canada p 25









211








74335ndash344




142





Research 42597-2603

v


financial support


















future aims Racy

vi

TABLE OF CONTENTS



















vii



SOIL33





















viii


















ix

LIST OF TABLES

















x

LIST OF FIGURES






















xi























xii













xiii











bp= base pair




Bw= body weight

C= carbon









DON= deoxynivalenol


dE= de-epoxy





FM= full medium


FUS= fusarenonX

gDNA= genomic DNA




h= hour


Kb= kilobase

xiv

Kg= kilogram


mass spectrometry



LB= Luria Bertani



microg= microgram

mg= milligram

mL= milliliter

microL= microliter

microM= micromole

min= minute

Mb= megabases


MSA= MSB+ agar


MM= minimal medium



mRNA= messenger RNA


mz= mass ion ratio

N= nitrogen



dinucleotide




information

NEO= neosolaniol

ng= nanogram

NIV= nivalenol










PKR= protein kinase

xv


oxidoreductase





rRNA= ribosomal RNA

s= second

SA= sodium azide


SE= soil extract



sp= species

sRNA= small RNA

TC= trichothecene




tRNA= transfer RNA



periplasmic


length polymorphism


acetyltransferase


VER= verrucarol



1

CHAPTER 1


11 Background



















2





















3
























4









affected ears















5















6









7





(a) (b)

(c)

8
























9








Type-A





VER 266 H OH H OH H

Type-B






10




















11










2007) (Table 1-2)














12




Country

region

Sample

type

Incidence

()

Maximum

DON content

(microgg)

Mean DON

content

(microgg)

Survey

period

Source

North

AsiaChina

Animal

feed

071 1899 092

Binder et

al 2007

South East

Asia

014 152 016

South Asia 070 1062 049

Oceania 2003-05


Northern

Europe

070 551 059

Central

Europe

066 802 057

Southern

Europe

052 303 030

13



Province Sample

type

Incidence

()

Maximum

DON

contents

(microgg)

Multiple

mycotoxins

samples ()

Survey

period

Sources

Ontario Spring

Wheat

70 260 Yes 2006-08 OMAFRA1

Ontario Winter

wheat

49 290 Yes 2006-08 OMAFRA


and

Schaafsma

2010


Biologicals

Canada Breakfast

cereals

46 094 430 1999-01 Roscoe et al

2008

Canada Animal

feeds


al 2011


Canada Infant

Cereals


al 2003

1


2 microgmL

14




Products DON

(microgg)

T-2 toxin

(microgg)

HT-2 toxin

(microgg)

DAS

(microgg)


consumption

2 - - -



15
























16
























17























2010)

18








mycotoxins

Experimental

animalcells

Effects Reference


cereals


1997

DON contaminated

wheat and FB1


1996

Culture materials



lesion

Kubena et al

1997

Inoculated rice and

pure mycotoxin


1995

Naturally DON

contaminated wheat

and pure T-2 toxin



Kubena et al

1989


weight

Kubena et al

1993


oral lesion

Kubena et al

1990

Pure mycotoxins DON

ZENFB1

Human intestinal

Caco-2 cells

Increased DNA

fragmentations

Kouadio et al

2007


al 1994


al 1994

19
























20
























21










22





(Figure 1-2)










23













24








(Hck) and P38


















25




















26


















2000)






27






















28























29
























30








grains











2009)

31














temperature and pH






epoxidation

32




33

CHAPTER 2



SOIL


21 Abstract















34






22 Introduction















35

















2004)

36













De-epoxydation



37


231 Soil samples










232 Culture media











38











144 h of incubation











39

















40









incubation














41






method


















42


microbial community





















43




















44














239 Data analysis






45

24 Results



















46

47








48










49






50







51







0

20

40

60

80

100

120


DO

N d

e-e

po

xyd

ati

on

(

)

Antibiotics

52















53

(b)

(a)

Rela

tive flu

ore

scen

ce

32000

0

0 500


100 200 300 400

Rela

tive flu

ore

scen

ce

32000

0

500


0 300200100 400

54







55









species

56






54

12 8

14

8 4

0

20

40

60 S

eq

uen

ce

fre

qu

en

cy

Microbial genera

57


epoxidation











58

0

20

40

60

80

100

120

12degC

20degC

25degC

30degC

40degC

DO

N d

e-e

po

xyd

ati

on

(

)

Temperature

0

20

40

60

80

100

120

DO

N d

e-e

pxyd

ati

on

(

)

Media

(a)

(b)

59









0

20

40

60

80

100

120

55

60

65

70

75

80

DO

N d

e-e

po

xyd

ati

on

(

)

pH

(c)

60













61









62

25 Discussion























63















1999)









64






health

26 Acknowledgements






65

CHAPTER 3



31 Abstract


















66




trichothecenes

32 Introduction

















67






















68






bacterium











Union MO USA)





69























70
























71























72





each assay


















73



















74






75

34 Results















76





(a) (b)

77








78





(Figure 3-3b)








79

00

04

08

12

16

0 24 36 48 60 72 96

OD

600n

m

Incubation time (h)

Control

20 microgmL

200 microgmL

500 microgmL

(b)

(a)

80










(C)

81










82

(b)

(a)

83









84





intermediate















85

b

c

a

b

a

d c

b b

(a)

(b)

b

c

b

a a

d

86











87

(a)

(b)

88






89

(a)

(b)

90






91

Antibiotic




(100) (Figure 3-8)





method

0

20

40

60

80

100

120

SA1 SA2 C

D

ON

de

-ep

oxyd

ize

d

92









0

20

40

60

80

100

120


D

ON

de

-ep

oxyd

ize

d

93


mycotoxins



3-1)















94










95






Trichothecene

mycotoxin

Untransformed

trichothecenes ()


trichothecenes ()

De-

epoxy

De-

acetylate

Other1

Type-A

HT-2 toxin 0 74 26 -

T-2 toxin 38 46 - 16

(unknown)

DAS 0 61 39 -


T2-triol 0 47 53 -

Type-B

DON 0 100 - -


3-ADON 0 59 41 (DON)

15-ADON 0 100 - -

FUS 5 95 - -

Varrucarol 3 97 - -

96

35 Discussion














(Shima et al 1997)









97




enrichment process




















98























99
























100









36 Acknowledgements





101




















102

CHAPTER 4




41 Abstract




(pCC1FOSTM













103


activity

42 Introduction


















subclones

104










(Chapter 3)






105


DNA library

106
























107



)





















108

109


) The 813 kb







110













image was recorded










111




112

44 Results









113








114








115













DON as well

116






117


capability


singlecombined

clones

Number of

clones

examined

Number of clones

showed DON de-


Without induction


Singly 200 0



number clones)

Singly 200 0






118

45 Discussion







used
















119
























120





















121

46 Acknowledgements





122

CHAPTER 5



51 Abstract


















123




reductases

52 Introduction

















124










2008)







125






















126
















MUMmer program






127











1989)












128









software

129

54 Results





(Figure 5-1)




130







proteins

















131







genome








132


Feature Chromosome

















133

134








135



136






Figure 5-4











137




138













139









140

















141






C

freundii

ADS47

C freundii

Ballerup

Citrobacter

sp 30_2

C

youngae

C koseri C

rodentium

Genome length

(bp)

4878242

4904641

5125150

5154159

4720462

5346659

Base similarity to

ADS47 ()

NA 8715 8212 7637 5981 4213

GC () 5202 5218 516 5265 530 546

Number of coding

sequences

4610 4685 4728 5278 4978 4800

Genome coverage

with coding

sequences ()

880 865 866 872 900 840

rRNA copies 3 9 6 25 22 22


142










(34)]

143







144
















145






C

freundii

ADS47

C

freundii

Ballerup

C

sp

C

youngae

C

koseri

C

rodentium


groupspigments

323

317

310

311

325

304




Miscellaneous 210 228 200 185 245 185


element plasmid

24 75 25 4 6 49



RNA metabolism 220 222 217 215 238 212







DNA metabolism 145 167 165 155 147 152




146



Citrobacter species

C

freundii

ADS47

C

freundii

Ballerup

C

sp

C

youngae

C

koseri

C

rodentium

Respiration 198 184 180 191 166 162

Stress response 236 235 170 177 179 174


compounds

20 20 28 25 17 48




Carbohydrates 837 803 708 731 733 737

147









C youngae (4)












respectively



148







22)

















149


















species






150










other four species









151

(a)

152











LEE operons

(b)

153






767204-775090 (~8 kb) 1025572-1042510 (17 kb) 2798289-2807217 (~9 kb)

3045220-3055912 (~11 kb) 3930366-3943075 (12 kb) and 4022195-4032444 (10 kb)















154























155












156





Total unique genes

270


De-acetylase 1

Hydrolase 2

Transferase 4

Sulfatase 3

Bacteriocin 2

Type VI secretion 2

Kinase 4

Transporter 9


Regulator 12



Miscellaneous 34


157

158
















159

(a)

(b)

160

(c)

(d)

161











162

55 Discussion






these genomes

















163



















et al 2010)





164
























165












al 1992)












166


genome










(Young et al 2007)











167









in ADS47 genome













168






56 Acknowledgement




169





Unique gene

classes


Total unique

genes


Reductases

oxidoreductases











superfamily


hydrolase











effector Hcp1















protein)

170


Unique gene

classes


Membrane

exported protein

















Integrase

recombinase


Phage prophage

transposae related

genes






CMD_3806 CMD_4425 CMD_4597






















171

CHAPTER 6

60 CONCLUSION

61 Summary






mycotoxins














172
























173




















174























175

63 Further research

















corn and wheat

176

70 REFERENCES



486


















177




25195-203




















178





108A20ndashA23



















179









121411ndash423














180
























181










Microbiology 10236













182











Pathology 9435-445





149ndash18






183






Oxford pp 145-160












73 FAO Rome




166

184























185






6879ndash886


4303ndash317














186






















1190

187
























188
























189









10216892ndash16897












190




















231ndash241



191













32181ndash1183



1333







2105ndash110

192







49306ndash317





68622ndash626











193



39161-168





York pp 115-175




25732ndash744










65720-726

194
























195



20494ndash504



391434ndash1442








321147ndash1152







5823301ndash3307

196






1322723ndash2731














91519ndash528



197
























198
























199




















5631

200





















201
























202











186








285



504271ndash276

203






Research 50543-551

















204





212347ndash368









494








205






















206















94181-189









207














7th







885



208
























209







2934



2135













261

210















Canada p 25









211








74335ndash344




142





Research 42597-2603

vi

TABLE OF CONTENTS



















vii



SOIL33





















viii


















ix

LIST OF TABLES

















x

LIST OF FIGURES






















xi























xii













xiii











bp= base pair




Bw= body weight

C= carbon









DON= deoxynivalenol


dE= de-epoxy





FM= full medium


FUS= fusarenonX

gDNA= genomic DNA




h= hour


Kb= kilobase

xiv

Kg= kilogram


mass spectrometry



LB= Luria Bertani



microg= microgram

mg= milligram

mL= milliliter

microL= microliter

microM= micromole

min= minute

Mb= megabases


MSA= MSB+ agar


MM= minimal medium



mRNA= messenger RNA


mz= mass ion ratio

N= nitrogen



dinucleotide




information

NEO= neosolaniol

ng= nanogram

NIV= nivalenol










PKR= protein kinase

xv


oxidoreductase





rRNA= ribosomal RNA

s= second

SA= sodium azide


SE= soil extract



sp= species

sRNA= small RNA

TC= trichothecene




tRNA= transfer RNA



periplasmic


length polymorphism


acetyltransferase


VER= verrucarol



1

CHAPTER 1


11 Background



















2





















3
























4









affected ears















5















6









7





(a) (b)

(c)

8
























9








Type-A





VER 266 H OH H OH H

Type-B






10




















11










2007) (Table 1-2)














12




Country

region

Sample

type

Incidence

()

Maximum

DON content

(microgg)

Mean DON

content

(microgg)

Survey

period

Source

North

AsiaChina

Animal

feed

071 1899 092

Binder et

al 2007

South East

Asia

014 152 016

South Asia 070 1062 049

Oceania 2003-05


Northern

Europe

070 551 059

Central

Europe

066 802 057

Southern

Europe

052 303 030

13



Province Sample

type

Incidence

()

Maximum

DON

contents

(microgg)

Multiple

mycotoxins

samples ()

Survey

period

Sources

Ontario Spring

Wheat

70 260 Yes 2006-08 OMAFRA1

Ontario Winter

wheat

49 290 Yes 2006-08 OMAFRA


and

Schaafsma

2010


Biologicals

Canada Breakfast

cereals

46 094 430 1999-01 Roscoe et al

2008

Canada Animal

feeds


al 2011


Canada Infant

Cereals


al 2003

1


2 microgmL

14




Products DON

(microgg)

T-2 toxin

(microgg)

HT-2 toxin

(microgg)

DAS

(microgg)


consumption

2 - - -



15
























16
























17























2010)

18








mycotoxins

Experimental

animalcells

Effects Reference


cereals


1997

DON contaminated

wheat and FB1


1996

Culture materials



lesion

Kubena et al

1997

Inoculated rice and

pure mycotoxin


1995

Naturally DON

contaminated wheat

and pure T-2 toxin



Kubena et al

1989


weight

Kubena et al

1993


oral lesion

Kubena et al

1990

Pure mycotoxins DON

ZENFB1

Human intestinal

Caco-2 cells

Increased DNA

fragmentations

Kouadio et al

2007


al 1994


al 1994

19
























20
























21










22





(Figure 1-2)










23













24








(Hck) and P38


















25




















26


















2000)






27






















28























29
























30








grains











2009)

31














temperature and pH






epoxidation

32




33

CHAPTER 2



SOIL


21 Abstract















34






22 Introduction















35

















2004)

36













De-epoxydation



37


231 Soil samples










232 Culture media











38











144 h of incubation











39

















40









incubation














41






method


















42


microbial community





















43




















44














239 Data analysis






45

24 Results



















46

47








48










49






50







51







0

20

40

60

80

100

120


DO

N d

e-e

po

xyd

ati

on

(

)

Antibiotics

52















53

(b)

(a)

Rela

tive flu

ore

scen

ce

32000

0

0 500


100 200 300 400

Rela

tive flu

ore

scen

ce

32000

0

500


0 300200100 400

54







55









species

56






54

12 8

14

8 4

0

20

40

60 S

eq

uen

ce

fre

qu

en

cy

Microbial genera

57


epoxidation











58

0

20

40

60

80

100

120

12degC

20degC

25degC

30degC

40degC

DO

N d

e-e

po

xyd

ati

on

(

)

Temperature

0

20

40

60

80

100

120

DO

N d

e-e

pxyd

ati

on

(

)

Media

(a)

(b)

59









0

20

40

60

80

100

120

55

60

65

70

75

80

DO

N d

e-e

po

xyd

ati

on

(

)

pH

(c)

60













61









62

25 Discussion























63















1999)









64






health

26 Acknowledgements






65

CHAPTER 3



31 Abstract


















66




trichothecenes

32 Introduction

















67






















68






bacterium











Union MO USA)





69























70
























71























72





each assay


















73



















74






75

34 Results















76





(a) (b)

77








78





(Figure 3-3b)








79

00

04

08

12

16

0 24 36 48 60 72 96

OD

600n

m

Incubation time (h)

Control

20 microgmL

200 microgmL

500 microgmL

(b)

(a)

80










(C)

81










82

(b)

(a)

83









84





intermediate















85

b

c

a

b

a

d c

b b

(a)

(b)

b

c

b

a a

d

86











87

(a)

(b)

88






89

(a)

(b)

90






91

Antibiotic




(100) (Figure 3-8)





method

0

20

40

60

80

100

120

SA1 SA2 C

D

ON

de

-ep

oxyd

ize

d

92









0

20

40

60

80

100

120


D

ON

de

-ep

oxyd

ize

d

93


mycotoxins



3-1)















94










95






Trichothecene

mycotoxin

Untransformed

trichothecenes ()


trichothecenes ()

De-

epoxy

De-

acetylate

Other1

Type-A

HT-2 toxin 0 74 26 -

T-2 toxin 38 46 - 16

(unknown)

DAS 0 61 39 -


T2-triol 0 47 53 -

Type-B

DON 0 100 - -


3-ADON 0 59 41 (DON)

15-ADON 0 100 - -

FUS 5 95 - -

Varrucarol 3 97 - -

96

35 Discussion














(Shima et al 1997)









97




enrichment process




















98























99
























100









36 Acknowledgements





101




















102

CHAPTER 4




41 Abstract




(pCC1FOSTM













103


activity

42 Introduction


















subclones

104










(Chapter 3)






105


DNA library

106
























107



)





















108

109


) The 813 kb







110













image was recorded










111




112

44 Results









113








114








115













DON as well

116






117


capability


singlecombined

clones

Number of

clones

examined

Number of clones

showed DON de-


Without induction


Singly 200 0



number clones)

Singly 200 0






118

45 Discussion







used
















119
























120





















121

46 Acknowledgements





122

CHAPTER 5



51 Abstract


















123




reductases

52 Introduction

















124










2008)







125






















126
















MUMmer program






127











1989)












128









software

129

54 Results





(Figure 5-1)




130







proteins

















131







genome








132


Feature Chromosome

















133

134








135



136






Figure 5-4











137




138













139









140

















141






C

freundii

ADS47

C freundii

Ballerup

Citrobacter

sp 30_2

C

youngae

C koseri C

rodentium

Genome length

(bp)

4878242

4904641

5125150

5154159

4720462

5346659

Base similarity to

ADS47 ()

NA 8715 8212 7637 5981 4213

GC () 5202 5218 516 5265 530 546

Number of coding

sequences

4610 4685 4728 5278 4978 4800

Genome coverage

with coding

sequences ()

880 865 866 872 900 840

rRNA copies 3 9 6 25 22 22


142










(34)]

143







144
















145






C

freundii

ADS47

C

freundii

Ballerup

C

sp

C

youngae

C

koseri

C

rodentium


groupspigments

323

317

310

311

325

304




Miscellaneous 210 228 200 185 245 185


element plasmid

24 75 25 4 6 49



RNA metabolism 220 222 217 215 238 212







DNA metabolism 145 167 165 155 147 152




146



Citrobacter species

C

freundii

ADS47

C

freundii

Ballerup

C

sp

C

youngae

C

koseri

C

rodentium

Respiration 198 184 180 191 166 162

Stress response 236 235 170 177 179 174


compounds

20 20 28 25 17 48




Carbohydrates 837 803 708 731 733 737

147









C youngae (4)












respectively



148







22)

















149


















species






150










other four species









151

(a)

152











LEE operons

(b)

153






767204-775090 (~8 kb) 1025572-1042510 (17 kb) 2798289-2807217 (~9 kb)

3045220-3055912 (~11 kb) 3930366-3943075 (12 kb) and 4022195-4032444 (10 kb)















154























155












156





Total unique genes

270


De-acetylase 1

Hydrolase 2

Transferase 4

Sulfatase 3

Bacteriocin 2

Type VI secretion 2

Kinase 4

Transporter 9


Regulator 12



Miscellaneous 34


157

158
















159

(a)

(b)

160

(c)

(d)

161











162

55 Discussion






these genomes

















163



















et al 2010)





164
























165












al 1992)












166


genome










(Young et al 2007)











167









in ADS47 genome













168






56 Acknowledgement




169





Unique gene

classes


Total unique

genes


Reductases

oxidoreductases











superfamily


hydrolase











effector Hcp1















protein)

170


Unique gene

classes


Membrane

exported protein

















Integrase

recombinase


Phage prophage

transposae related

genes






CMD_3806 CMD_4425 CMD_4597






















171

CHAPTER 6

60 CONCLUSION

61 Summary






mycotoxins














172
























173




















174























175

63 Further research

















corn and wheat

176

70 REFERENCES



486


















177




25195-203




















178





108A20ndashA23



















179









121411ndash423














180
























181










Microbiology 10236













182











Pathology 9435-445





149ndash18






183






Oxford pp 145-160












73 FAO Rome




166

184























185






6879ndash886


4303ndash317














186






















1190

187
























188
























189









10216892ndash16897












190




















231ndash241



191













32181ndash1183



1333







2105ndash110

192







49306ndash317





68622ndash626











193



39161-168





York pp 115-175




25732ndash744










65720-726

194
























195



20494ndash504



391434ndash1442








321147ndash1152







5823301ndash3307

196






1322723ndash2731














91519ndash528



197
























198
























199




















5631

200





















201
























202











186








285



504271ndash276

203






Research 50543-551

















204





212347ndash368









494








205






















206















94181-189









207














7th







885



208
























209







2934



2135













261

210















Canada p 25









211








74335ndash344




142





Research 42597-2603

isolation, characterization and genome sequencing of a

Documents