Duška VujaklijaLaboratory for Molecular Genetics

Zagreb, May 10, 2013

INSTITUT RUĐER BOŠKOVIĆ

Molecular study of dominant soil bacteria: streptomycetes in nature and application to biotechnology

The 2nd International Symposium “Vera Johanides”

Actinobacteria - one of the major communities of the microbial population present in soil

responsible for the peculiar soil - smell after rain

• inhabit a wide range of environmental niches; soil, freshwater, marine sediments

• Gram-positive bacteria• produce a number of enzymes that help degrade organic plant material, lignin, and chitin…

•the best known known as secondary metabolite producers; Streptomyces as antibiotic producers

Most Actinobacteria of medical significance belong to order of Actinomycetales

Antibiotics Other Total

Actinomycetes* 7900 1220 9120

Other bacteria 1400 240 1640

Fungi 2600 1540 4140

Total 11,900 3000 14,900

*70% from StreptomycesCourtesy of D.A.

Hopwood

F. Marinelli: isolation of novel species A. Mikoč: isolation of novel speciesA. Mikoč: cave Tounjčica

Sporulating colonies(Courtesy of D. Hopwood)

S. coelicolor

S. rimosus colonies(Zagreb group)

from liquid media

Model systems

The best studied model systemAntibiotic producer

LH arm = 1.5 Mb

RH arm = 2.3 Mb

Core = 4.9 Mb

7825 ORFs(55 pseudogenes)

63 tRNA genes6 rRNA operons

72.12% G+C

Genetic adaptability to a wide range of environments is evident in the genome of S. coelicolor

Sporulating colonies

Spiral aerial hyphae

whiE sigF whiD

whiI

whiH

whiA whiB

whiG whiJ

bldA,B,C,D,G,H,I,K...

Spore formation

Spores

Substrate

mycelium

Complex life cycle

phylum Actinobacteria

order Actinomycetales

family Streptomycetaceae

S. coelicolor

Elliot MA et al. Multicellular Development in Streptomyces

Reproductive stage of S. coelicolor growth

repoductive stage

Courtesy of D.Hopwood

SSB

http://www.pdbj.org/eprots/index_en.cgi?PDB%3A3BEP

Molecular study of streptomycetes: Implication of SSB in chromosome segregation

S coelicolor possesses two ssb genes

SSBs- primary structures

What is the biological role of SSBs ?

ssbA is essential

ssbB exibits Whi phenotype

“knock out” eksperiments

Tina Paradzik, et al. Structure-function relationships of two paralogous single-stranded DNA binding proteins from Streptomyces coelicolor: implication of SSB-B in chromosome segregation during sporulation Nucleic Acids Res. 2013.

▪ Statistical analyses of spore length and number in S. coelicolor M145 and ssbB mutant showes slightly increased spore length and number of spores in spore chain ssbB

ssbB mutant has defect in chromosome segregation

▪ Abberant distribution od chromosome in ssbB mutant , 30% of spores lacked DNA(n=2200)

A

B

C

1 2

18h 24h 48h 96h - 18h 24h 48h 96h -

18h 24h 48h 96h -

18h 24h 48h 96h -

16s-RT

16s-RT

1

2

3

1

2

3

RMMM

18h 24h 48h 96h -

Expression profiles of ssb genes

Manteca A et al. J. Proteome Res. 2011

T. Paradzik, et al. Nucleic Acids Res. 2013.

▪Two transcription start of ssbA gene is75 bp and 163 bp upstream of rpsF gene

the transcription start of ssbB gene to be 73 bp upstream of start codon

▪Promoter region of ssbB (79 % GC, a palindromic sequence, DnaA box two long

imperfect direct repeats)

Promoter regions of two ssb genes

Binding of SSB proteins to ФX174 DNA (EMSA)

- NaCl 100 mM NaCl

Tryptophane fluorescent quenching of SSB-A (1) and SSB-B (2) while binding to (dT)35

Stefanic et al (2009) Acta Crystallogr D Biol Crystallogr. 2009

T. Paradzik, et al. Nucleic Acids Res. 2013.

Detection of disulphide bonds in SsbB

(A) S. coelicolor SsbB isolated from E. coli . (B) Western blot analysis: SsbB isolated from S. coelicolor(C) Binding of SSB proteins to ФX174 DNA in a presence of DTT

Fluorescence microscopy after in vivo staining by DAPI (A)the strain lacking ssbB (TSB01) or (C) only C-terminus of ssbB (ssbB∆C ,TSB03), (B) M145, wild type strain, and (D) TSB02, ssbB mutant complemented with ssbB.

TSB01 TSB03

M145 TSB02

A - Arial hyphae grow by tip extension; FtsZ helical filaments which are remodelled into Z rings After septation, MreB localizes to closing septa and spread around developing spore.

B- Chromosome segregation, ParA / ParB binds near oriC, its distribution is driven by ParA.

Flardth K and Buttner M, Nature Reviews/Microbiology 2009

Cell processes during sporulation; role of SSB-B?

DivIVA

ParA

ParB

FtsZ

FtsK

Collaboration with D. Jakimowicz from Wroclav, Poland started.

Streptomyces: still represent an excellent source for genome mining

John Innes Center

Some Genes that Adapt for Life in the Soil

01112Chitinase,

cellulase

92122135Secreted hydrolase

807732141ABC transporter

881344Ser/Thr PK

323411852-comp sensor

7 (2)17 (7)14 (11)65 (45)Sigma (ECF)

E. coliB. subt.M. tub.S. coel.

Courtesy of D.A. Hopwood

Lipolytic activity of various Streptomyces isolated from soil

A.Mikočtricaprylin/TSB medium

Number of retrieved

sequences

Species/strain of genus Streptomyces

17S. clavuligerus ATCC 27064S. scabiei 87.22

16S. hygroscopicus ATCC 53653S. violaceusniger Tu 4113

15S. roseosporusS. sp. AA4

14S. ghanaensis ATCC 14672S. pristinaespiralis ATCC 25486

13S. albus J1074S. avermitilis MA-4680S. viridochromogenes DSM 40736

12S. sp. ACTES. sp. C

11S. flavogriseus ATCC 33331S. sviceus ATCC 29083

10S. bingchenggensis BCW-1S. griseoflavus Tu4000

9

S. griseus subsp. griseus NBRC 13350S. sp. ACT-1S. sp. Mg1S. sp. SPB78

8 S. sp. SA3_actG

7

S. coelicolor A3(2)S. lividans TK24S. sp. e14S. sp. SPB74

6 S. sp. SA3_actF

3 S. ambofaciens ATCC 23877

2 S. rochei

1S. diastatochromogenesS. fradiaeS. rimosus R6

organism lipase esterase

Streptomyces coelicolor 20 55

Streptomyces avermitilis 29 76

Streptomyces griseus 12 39

Streptomyces scabies 22 69

Genome mining

GDSL lipolytic family

► Multifunctionality

EC number Activity

2.3.1.43phosphatidylcholine:sterol O-acyltransferase

3.1.1.1 esterase

3.1.1.2 arilesterase

3.1.1.3 lipase

3.1.1.4 phospholipase A(2)

3.1.1.5 lysophospholipase

3.1.1.6 acetylesterase

3.1.1.471-alkyl-2-acetylglycerophosphocholine esterase

3.1.1.53 sialate O-acetylesterase

3.1.1.72 acetylxylan esterase

3.1.1.77 acyloxyacyl hydrolase

3.1.2.2 palmitoyl-CoA hydrolase

► Activity and Stability (Temp., pH, and organic solvents)

► Potential for application in biotechnology/bioremediation

Prediction of SrL 3D structure

• Abramić et al, Enzyme Microb Technol, 1999 • Vujaklija et al, Arch Microbiol , 2002 • Vujaklija et al, Food Technol Biotechnol, 2003 • Leščić et al, Biological Chemistry, 2004 • Zehl et al, J Mass Spectrom, 2004 • Leščić Ašler et al, BBA, 2006• Bielen et al, Biochimie, 2009

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0,12%

0,14%

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GDSL lipolytic enzymes are abundant in Actinobacteria

Scanning for genes encoding GDS(L) hydrolases in Actinobacteria from wide diversity of ecological niches

AnaAna BielenBielen

The 2The 2ndnd International Symposium “VERA JOHANIDES”, 2013International Symposium “VERA JOHANIDES”, 2013 Zagreb, May 11, 10,40 am

Taxonomic distribution...

IDENTIFICATION

Metagenomics

•

Babu A. ManjasettyEMBL, Grenoble

Ivo Piantanida, IRB

Paul Herron

Meri Luic & Zoran Stefanic

Nives Ivic

University of Strathclyde, Glasgow

Tina Paradžik, Želimira Filić i Ana Bielen

CIM-IRB

Pau University

Christine Cagnon, Robert Duran

Emina Durmiši

Bojan Hamer

Marija AbramićJ.Pigac

Adris Group - donation

Senka Džidić