Complete Genome of the Starch-Degrading Myxobacteria

Sandaracinus amylolyticus DSM 53668T

Gaurav Sharma Indu Khatri and Srikrishna Subramanian

Protein Science and Engineering CSIR-Institute of Microbial Technology Sector-39A Chandigarh India

Corresponding author E-mail krishnaimtechresin

Accepted June 14 2016

Data deposition The complete genome sequence of Sandaracinus amylolyticus DSM 53668T and its annotations are deposited at DDBJENA

GenBank under accession number CP011125

Abstract

Myxobacteria are members of d-proteobacteria and are typified by large genomes well-coordinated social behavior gliding motility

and starvation-induced fruiting body formation Here we report the 1033 Mb whole genome ofa starch-degrading myxobacterium

Sandaracinus amylolyticus DSM 53668T that encodes 8962 proteins 56 tRNA and two rRNA operons Phylogenetic analysis in

silico DNA-DNA hybridization and average nucleotide identity reveal its divergence from other myxobacterial species and support its

taxonomic characterization into a separate family Sandaracinaceae within the suborder Sorangiineae Sequence similarity searches

using the Carbohydrate-active enzymes (CAZyme) database help identify the enzyme repertoire of S amylolyticus involved in starch

agar chitin andcellulosedegradation We identified16a-amylasesand twog-amylases in the S amylolyticusgenomethat likelyplay

a role in starch degradation While many of the amylases are seen conserved in other d-proteobacteria we notice several novel

amylases acquired via horizontal transfer from members belonging to phylum Deinococcus-Thermus Acidobacteria and

Cyanobacteria No agar degrading enzyme(s) were identified in the S amylolyticus genome Interestingly several putative b-gluco-

sidases and endoglucanases proteins involved in cellulose degradation were identified However the absence of cellobiohydrolases

exoglucanases corroborates with the lack of cellulose degradation by this bacteria

Key words CAZyme amylase agarase cellulase methylome phylogeny

Introduction

Myxobacteria [order Myxococcales] are Gram-negative and

aerobic (except Anaeromyxobacter) d-proteobacteria These

bacteria possess large genomes and a complex life cycle

wherein they coordinate within swarms and hunt other mi-

crobes (Shimkets 1990Whitworth 2008) Myxobacterial spe-

cies have been of industrial interest due to their significant role

in secondary metabolites production (Weissman and Muller

2010 Steinmetz et al 2012) Order Myxococcales at present

comprises of more than 27 taxonomically classified genera

and 60 species This order has been classified in three subor-

ders Cystobacterineae (four families) Nannocystineae (two

families) and Sorangiineae (four families) The members of

suborder Sorangiineae are cellulolytic bacteriolytic and pro-

teolytic as compared with other family members and form a

separate clade in myxobacterial 16S rRNA-based phylogeny

(Reichenbach 2005 Garcia and Muller 2014) Till date the

largest complete bacterial genomes Sorangium cellulosum

strain So0157-2 (1478 Mb) (Han et al 2013) and S cellulo-

sum strain Soce56 (1303 Mb) (Schneiker et al 2007) have

been reported from the suborder Sorangiineae These mem-

bers are known to degrade diverse complex polysaccharides

such as cellulose starch casein agar chitin gelatin dextrin

xylan agarose etc (Garcia and Muller 2014) They have also

been reported as microbial predators to organisms such as

Escherichia coli Bacillus subtilis Mycobacterium chitae

Cyanobacteria yeast etc (Garcia and Muller 2014) Among

the four characterized families of the suborder Sorangiineae

genomic data are available only for Sorangium and

Chondromyces that belong to Polyangiaceae family which

are the largest source of myxobacterial secondary metabolites

(Schneiker et al 2007Weissman and Muller 2010Han et al

2013)

Within suborder Sorangiineae Sandaracinus amylolyticus

strain DSM 53668T is the type strain of S amylolyticus gen

nov sp nov proposed as a novel species in the family

GBE

The Author 2016 Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (httpcreativecommonsorglicensesby40) which permits unrestricted reuse

distribution and reproduction in any medium provided the original work is properly cited

2520 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016

Sandaracinaceae within order Myxococcales (Mohr et al

2012) Morphologically and biochemically the strain is a

Gram-negative aerobic rod-shaped during vegetative

states catalase-negative oxidase-positive and pigment form-

ing myxobacteria (Mohr et al 2012) This strain was isolated in

1996 from a gray-brown soil sample (having plant residues)

collection by Helmholtz Zentrums fur Infektionsforschung

[HZI] during 1995 from a botanical garden in Lucknow

Uttar Pradesh India (Reichenbach and Dworkin 1992 Mohr

et al 2012) Sandaracinus amylolyticus possesses typical myx-

obacterial characteristics including gliding motility secondary

metabolite production (Steinmetz et al 2012) and spore gen-

eration during harsh environmental conditions Here we

report the complete genome of S amylolyticus DSM 53668T

with special emphasis on the starch degrading properties

along with the protein repertoire involved in starch agarose

chitin and cellulose degradation

Materials and Methods

Culturing and Genomic DNA Isolation

Actively growing plate cultures of S amylolyticus was pro-

cured from Deutsche Sammlung von Mikroorganismen und

Zellkulturen (DSMZ) as strain number DSM 53668T [NOSO-4T]

Sandaracinus amylolyticus DSM 53668T was grown aerobi-

cally on CY agar plate culture (DSM Medium 67 as prescribed

by DSMZ culture collection) at 28ndash30 C for a minimum of 5

days The culture was yellow-orange in color attributed to the

presence of carotenoids (Mohr et al 2012) DNA for sequenc-

ing was obtained using both the Zymogen Research Bacterial

fungal DNA isolation kit and PhenolndashChloroformndashIsoamyl

Alcohol-based manual method The quantity and quality of

the extraction were checked by gel electrophoresis along with

Nanodrop method and followed by Qubit quantification

Genome Sequencing Assembly and Annotation

Sandaracinus amylolyticus was sequenced on four single mol-

ecule real-time (SMRT) cells using P6 polymerase and C4

chemistry (P6C4) on a Pacific Biosciences RSII instrument at

McGill University and Genome Quebec Innovation Center

Montreal (Quebec) Canada DNA sample was sheared and

concentrated using AMPure magnetic beads and treated by

ExoVII to remove single stranded ends SMRTbell long libraries

were constructed with 10 mg whole genomic DNA using

ldquoProcedure and Checklistndash20 kb Template Preparation Using

BluePippinTM Size Selection System protocolrdquo (httpwww

pacbcomwp-contentuploads201509Shared-Protocol-20-

kb-Template-Preparation-Using-BluePippin-Size -Selection-

System-15-kb-Size-Cutoffpdf last accessed July 5 2016)

Blunt ligation reactions were prepared and SMRTbell tem-

plates were purified using AMPure magnetic beads

BluePippinTM size selection was performed to retain long

reads (gt20000) for sequencing The size-selected SMRTbell

templates were annealed and then loaded onto four SMRT

cells and sequenced using P6C4 chemistry with 180-min

movie times Sequencing yielded a total of 241674 reads

with a mean read length of 467 kb totaling

1129177817 bp (~85 coverage) De novo assembly was

carried out using the hierarchical genome assembly process

(HGAP) protocol in SMRT Analysis v20 including consensus

polishing with Quiver (Chin et al 2013) Since the

BluePippinTM Size Selection (gt20000) protocol filters out

small DNA fragments such as plasmids Illumina paired-end

(PE) data were also obtained using the Illumina HiSeq1000

sequencing platform at the C-CAMP Bangalore India

Sequencing resulted in 13103763 PE reads (insert size 350

bp and read length 101 bp) out of which 11663870 PE

reads were selected after quality filtering [PHRED quality

score 20 minimum read length 70] using NGSQC Toolkit

v223 (Patel and Jain 2012) To trace the presence of any

plasmid the filtered Illumina reads were mapped using

CLCbio wb80 (wwwclcbiocom last accessed July 8 2016)

to the bacterial plasmid database (httpwwwebiacukge-

nomesplasmidhtml last accessed July 8 2016)

Gene prediction and functional annotation were per-

formed by Rapid Annotation using Subsystem Technology

(RAST) (Aziz et al 2008) RNAmmer 12 (Lagesen et al

2007) and tRNAscan-SE-123 (Lowe and Eddy 1997) were

used to predict rRNA and tRNA genes respectively

Phylogenetic Analysis Genome-Genome Distance andAverage Nucleotide Identity

16S rRNA genes from various strains of suborder Sorangiineae

along with neighboring families were extracted from the

National Centre for Biotechnology Information (NCBI) and

were aligned using ClustalW module of BIOEDIT sequence

alignment tool (version 7130 Hall 1999) The resulting align-

ment was used as an input in Molecular Evolutionary Genetics

Analysis (MEGA) v606 (Tamura et al 2013) to generate a max-

imum likelihood (ML) tree [model Tamura 3-param bootstrap

100] Initial tree(s) for the heuristic search were obtained by

applying the Neighbor-Joining method to a matrix of pairwise

distances estimated using the Maximum Composite Likelihood

approach In silico DNA-DNA hybridization (DDH) values among

the members of suborder Sorangiineae and other species mem-

bers of order Myxococcales were calculated using Genome-To-

Genome Distance Calculator (GGDC) server (Auch et al 2010)

Average Nucleotide Identity (ANI) matrix was also generated

among the genomes studied (Richter and Rossello-Mora 2009)

Genome and Proteome Analysis

Sandaracinus amylolyticus proteome was subjected to

hmmscan (Eddy 2011) against Hidden Markov Model

(HMM) profiles of Carbohydrate-Active enZYmes (CAZY)

database (dbCAN HMMs 30 Yin et al 2012Lombard et al

2014) The functional domains and other known sequence

Complete Genome of a Starch-Degrading Myxobacteria GBE

Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2521

motifs involved in starch cellulose chitin and agar degrada-

tion were retrieved on the basis of EC number for each

enzyme category Also the proteins of S amylolyticus were

mapped against the CAZY protein database using Basic-Local

Alignment Search Tool (BLASTp) (E-value cutoff of 1e5 min-

imum query coverage of 50 and minimum percent identity

of 35) (Altschul et al 1990) NCBI BLAST + (v 2226+) and

nonredundant protein (NR) database downloaded on

September 6 2014 was used throughout the study

Clustered regularly interspaced short palindromic repeat

(CRISPR) elements and insertion elements were identified

using CRISPRfinder (Grissa et al 2007) and ISfinder (Siguier

et al 2006) respectively

Sandaracinus amylolyticus Amylases

The a-amylases and g-amylases from S amylolyticus were sub-

jected to the BLASTp against amylase sequences from the

CAZY database (clustered at 70 sequence identity) and

top hits were retrieved The amylases from S amylolyticus

were cut into the domains and were aligned using MUltiple

Sequence Comparison by Log-Expectation (MUSCLE) (Edgar

2004) incorporated in MEGA v606 Further the evolutionary

tree was generated by ML method [model Jones Taylor and

Thorton bootstrap 100] using MEGA v606 Initial tree(s) for

the heuristic search were obtained by applying the Maximum

Parsimony method to a matrix of pairwise distances estimated

using the Maximum Composite Likelihood approach Further

the S amylolyticus GH13 amylases along with the closely re-

lated members of each phylogenetic clade were aligned using

PROfile Multiple Alignment with predicted Local Structures

and 3D constraints (PROMALS3D) with Taka-Amylase A of

Aspergillus oryzae [2TAA] (Matsuura et al 1984) as a struc-

tural template (Pei et al 2008)

Results and Discussion

Sandaracinus amylolyticus Genome Properties

Sandaracinus amylolyticus genome was assembled de novo by

HGAP v20 (Chin et al 2013) of the SMRT portal as a single

contig of 1033 Mb with no plasmid and representing the

complete genome with approximately 85 coverage and

72 G+C content (fig 1) No complete plasmid could be

retrieved from the mapping of Illumina PE reads against the

bacterial plasmid database Plasmids are rare in the order

Myxococcales and till date only one plasmid pMF1 from

Myxococcus fulvus 124B02 has been reported (Zhao et al

2008) RAST-based annotation predicted 8962 coding

genes out of which 4939 proteins (5516) were function-

ally annotated while the remaining (4484) were hypothet-

ical proteins (supplementary table S1 Supplementary Material

online) Sandaracinus amylolyticus genome has two rRNA op-

erons and 56 aminoacyl-tRNA synthetase genes

This genome encodes two sequentially identical 16S rRNA

which shows 100 identity with previously reported 16S rRNA

sequence of S amylolyticus strain NOSO-4T [NCBI Reference

Sequence NR_1180011] followed by 8798 identity with

Chondromyces apiculatus strain Cm a14 [NR_0253441]

Among the myxobacteria with sequenced genomes it shows

the maximum identity (8789) to the 16S rRNA gene of C

apiculatus DSM 436 (strain Cm a2) [GenBank M942742] fol-

lowed by 875 identity to S cellulosum 16S rRNA (Soce0157-

2 and Soce56) The 16S rRNA-based phylogeny of

Sandaracinus and related myxobacteria suggest its classification

as a separate family within the suborder Sorangiineae (supple-

mentary fig S1 Supplementary Material online) Sandaracinus

amylolyticus 16S rRNA forms a separate branch distinct from

other families in the suborder Sorangiineae phylogenetic tree

representing family Sandaracinaceae We also calculated in

silico DDH values for all available myxobacterial genomes

using the GGDC server (Auch et al 2010) Sandaracinus

amylolyticus is not closely related to other Myxococcales as

the maximum DDH value was identified to be 181 with S

cellulosum followed by 174 with C apiculatus DSM 436 sug-

gesting a distant relation to Polyangiaceae members which

further confirms its placement in a separate family

Sandaracinaceae ANI calculations likewise support the above

conclusions (supplementary table S2 Supplementary Material

online)

The members of suborder Sorangiineae such as the S cel-

lulosum species have been well characterized for degrading

cellulose and other complex polysaccharides (Schneiker et al

2007 Han et al 2013) Similarly S amylolyticus has been

reported as the only myxobacteria capable of degrading

starch (Mohr et al 2012) Thus in order to gain insights into

the genes involved in the degradation of complex polysaccha-

rides especially starch the complete genome of S amylolyticus

was determined and investigated for the presence of carbo-

hydrate active enzymes

Sandaracinus amylolyticus CAZome

Two-hundred and forty-three proteins (~27 of total prote-

ome) of S amylolyticus were annotated with diverse combi-

nations of CAZY domains that consist of 72 GT domains 72

CE domains 58 GH domains 19 AA domains 4 PL domains

and 26 CBM domains (fig 2A) Fourteen out of these 243

proteins contained more than one CAZY domain whereas

the rest had only a single CAZY domain In multiple CAZY-

domain containing proteins various carbohydrate binding

modules such as CBM48 CBM50 and CBM21 are pre-

sent along with other CAZY category proteins for assisting in

their functions The CAZyme distribution observed in

S amylolyticus indicates its ability to degrade various

types of biomass Here we examine the S amylolyticus pro-

teome involved in starch agar chitin and cellulose

degradation

Sharma et al GBE

2522 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016

Evolutionary History of S amylolyticus Amylases

Amylases belonging to glycoside hydrolase (GH) enzymatic

category degrade starch into sugar moieties by the

hydrolysis of a-14- and a-16-glycosidic bond (Van der

Maarel et al 2002) Based on the site of action they are di-

vided into three categories a-amylase (EC3211 GH13

GH57 GH119 GH126) b-amylase (EC3212 GH14) and

-amylase (EC3213 GH15 GH97) Sandaracinus

amylolyticus genome contains both a- and -amylases

whereas no b-amylases could be found Among the 4 a-am-

ylases GH families 15 GH13 proteins and 1 GH57 protein

were found We could identify two -amylases belonging to

Sandaracinus amylolyticusTDSM 53668

CP01112510328378 bp

FIG 1mdashCircular representation of the Sandaracinus amylolyticus DSM 53668T complete genome Circles (from inside to outside) 1 and 2 (GC content

black line and GC skew magenta and green lines) circle 3 (CAZome Proteins annotated as carbohydrate active enzymes Red) circle 4 5 and 6 (Amylases

Cellulases and Chitinases Black) circle 7 (encoded RNA Magenta) circle 8 (CDS present on negative strand Blue) circle 9 (CDS present on positive strand

Green) circle 10 (S amylolyticus DSM 53668T complete genome Red) BRIG 095 was used to build the circular representation (Alikhan et al 2011)

Complete Genome of a Starch-Degrading Myxobacteria GBE

Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2523

GH15 category (fig 2B) The starch degrading proteins have

been reported to have CBM20 CBM21 CBM25 CBM26

CBM34 CBM41 CBM45 CBM48 CBM53 and CBM58

modules that help in glycogenstarch binding truncation of

which reduces its enzymatic activity (Noguchi et al 2011

Janecek et al 2014) GH15 and GH57 domains are present

as single domain proteins in S amylolyticus Interestingly in 5

out of 15 GH13 S amylolyticus proteins (AKF03796

AKF03860 AKF06952 AKF07413 and AKF11765) the a-

amylase GH13 domain was present along with a CBM48

module at the N-termini The CBM48 module assists in the

GH13 function by serving as starch-binding domains

The GH13 family include the a-amylase domains with an (b

a)8 barrel catalytic domain These proteins have 4ndash7 conserved

sequence regions (CSRs) and a specific catalytic triad consisting

of a catalytic nucleophile (aspartic acid) on strand b4 proton

donor (glutamic acid) on strand b5 and a transition state sta-

bilizer (aspartic acid) on strand b7 (Janecek et al 2014) GH13

members show extremely low sequence identity with their ho-

mologs but the catalytic triad is highly conserved GH57 family

a-amylases have a (ba)7 barrel structure and have five CSRs as

compared with the GH13 family However only two of the

catalytic residues viz the nucleophile on strand b4 and the

proton donor on strand b7 are conserved (Blesak and Janecek

2012) Irrespective of these differences GH13 and GH57 both

employ the same retaining mechanism of action (Janecek and

Kuchtova 2012) Sandaracinus amylolyticus GH13 proteins

were retrieved and the structure of Taka amylase of A

oryzae (PDBid 2TAA) was used to perform a structure-based

alignment using PROMALS3D (Matsuura et al 1984 Pei et al

2008) All the seven CSR regions and the three catalytic

residues were conserved in all S amylolyticus GH13 amylases

(supplementary figs S2 and S3 Supplementary Material

online) Similarly S amylolyticus GH57 (AKF06951) was

aligned with its homologs using PROMALS3D based on the

4-a-glucanotransferase structure of Thermococcus litoralis

(PDBid 1K1W) In the GH57 alignment all five CSRs and

both catalytic residues were conserved (supplementary figS4

Supplementary Material online)

In order to understand the provenance of these a- and g-

amylases they were subjected to BLASTp against the NR data-

base To include amylases across the entire bacterial phylum

the CAZY-NR database for GH13 and GH57 domains were

retrieved and subjected to BLASTClust at 70 identity and

70 length coverage that reduced the domain sequence

space from 19590 to 4038 domains Further amylase do-

mains from S amylolyticus were subjected to BLASTp against

these 4038 domains and their top hits were retrieved and

included in the phylogenetic analysis All these GH13 and

GH57 domains along with S amylolyticus amylase domains

were aligned using MUSCLE as incorporated in MEGAv606

and ML-based phylogeny with 100 bootstrap values was

drawn (fig 3) The ML tree obtained was divided into three

major clades where the clade at root composed of all the GH57

domains and the other two major clades further composed of

all the GH13 sequences The representative bacterial amylases

from various clades show close relations with amylases of di-

verse bacterial groups suggesting the multiple gain and loss of

these domains As all the amylase domains of S amylolyticus

are scattered across the tree the phylogenetic position of these

domains could provide us insights about their provenance and

reveal possible cases of horizontal transfers

19

26

72

58

72

4

0

10

20

30

40

50

60

70

80

AA CBM CE GH GT PL

A B

Num

ber o

f pro

tein

s

GH13

GH13

CBM48 GH13

GH13CBM48

GH13

GH57

GH13CBM48

GH13GH13CBM48

GH13

GH13

GH13

GH15

GH13CBM48

GH13

AKF03605AKF03642AKF03796

AKF03860AKF03861AKF03862AKF04017AKF06951AKF06952

AKF07406AKF07413AKF07527AKF09634AKF09643AKF10340AKF10487AKF11765AKF11772

GH13

GH13

GH15

FIG 2mdashSandaracinus amylolyticus DSM 53668T CAZome distribution (A) Sandaracinus amylolyticus DSM 53668T CAZY families presented in a bar

graph x axis represents the CAZY categories and y axis represents the number of proteins (B) a- and g-amylases along with their CAZome domain

architecture The domains have been colored as GH13 sky blue GH15 dark red GH57 green and CBM48 orange

Sharma et al GBE

2524 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016

Taking into account the GH57 clade the sole GH57

domain of S amylolyticus (GenBank ID AKF06951) shares

the clade with anaerobic Deltaproteobacteria Among the

GH13 domains of S amylolyticus AKF03862 AKF07527

AKF09643 AKF11765 and AKF11772 shares clade with

other myxobacteria Among these AKF11772 and

AKF07527 share sister branches and form sister clades with

other myxobacterial species such as S cellulosum Soce56 and

Plesiocystis pacifica SIR-1 and are further related to the clade

containing Gammaproteobacteria Apart from these two am-

ylases AKF03860 and AKF06952 are similar to each other and

group with Candidatus accumulibacter phosphtis amylase

(YP_0031672861) from the phylum Acidobacteria

Interestingly AKF03860 and AKF06952 group with members

GH13 ZP 021632211 Kordia algicida OT-1

GH13 ZP 067516041 Parascardovia denticolens F0305

GH13 YP 0035709041 Salinibacter ruber M8

GH13 YP 0032500041 Fibrobacter succinogenes subsp succinogenes S85

GH13 AKF03862 Sandaracinus amylolyticus DSM 53668

GH13 YP 0039537801 Stigmatella aurantiaca

GH13 CAJ738231 Candidatus Kuenenia stuttgartiensis

GH13 YP 0228461 Picrophilus torridus DSM 9790GH13 ZP 029306261 Verrucomicrobium spinosum DSM 4136

GH13 CAX841261 uncultured bacterium

GH13 YP 0031678771 Candidatus Accumulibacter phosphatis clade IIA str UW-1GH13 ZP 031321321 Chthoniobacter flavus Ellin428

GH13 ZP 018759791 Lentisphaera araneosa HTCC2155GH13 YP 0033894351 Spirosoma linguale DSM 74GH13 NP 7734061 Bradyrhizobium diazoefficiens USDA 110

GH13 YP 0037757321 Herbaspirillum seropedicae SmR1GH13 YP 4266911 Rhodospirillum rubrum ATCC 11170GH13 ZP 035079501 Rhizobium etli Brasil 5

GH13 ZP 057930691 Butyrivibrio crossotus DSM 2876

GH13 ZP 066231321 Turicibacter sanguinis PC909

GH13 ZP 044499741 Catonella morbi ATCC 51271

GH13 EGE534241 Streptococcus parauberis NCFD 2020

GH13 196049865 Lactobacillus Plantarum

GH13 YP 0042196261 Granulicella tundricola MP5ACTX9

GH13 YP 0011935611 Flavobacterium johnsoniae UW101

GH13 YP 0032910851 Rhodothermus marinus DSM 4252GH13 YP 0040967591 Bacillus cellulosilyticus DSM 2522GH13 ZP 037030351 Flavobacteria bacterium MS024-2A

GH13 XP 0025352531 Ricinus communis

GH13 YP 7579561 Maricaulis maris MCS10GH13 ZP 010417351 Erythrobacter sp NAP1GH13 YP 0018177571 Opitutus terrae PB90-1GH13 EFR587141 Alistipes sp HGB5

GH13 CBK627851 Alistipes shahii WAL 8301GH13 EFR582631 Alistipes sp HGB5

GH13 YP 0034042011 Haloterrigena turkmenica DSM 5511

GH13 YP 0035357541 Haloferax volcanii DS2

GH13 YP 0035357541 Haloferax volcanii DS2

GH13 YP 0025661621 Halorubrum lacusprofundi ATCC 49239

GH13 YP 1368761 Haloarcula marismortui ATCC 43049

GH13 ZP 080436741 Haladaptatus paucihalophilus DX253

GH13 YP 0025652161 Halorubrum lacusprofundi ATCC 49239

GH13 YP 0035346301 Haloferax volcanii DS2

GH13 YP 0033225661 Thermobaculum terrenum ATCC BAA-798

GH13 YP 0021353421 Anaeromyxobacter sp K

GH13 AKF09643 Sandaracinus amylolyticus DSM 53668

GH13 YP 0016137031 Sorangium cellulosum So ce56

GH1

3 99

0316

21 H

alot

herm

othr

ix or

enii

GH1

3 AA

D051

991

Pae

niba

cillu

s po

lymyx

a

GH1

3 YP

003

6999

601

Bac

illus

sele

nitir

educ

ens

MLS

10

GH1

3 ZP

022

0799

81

Copr

ococ

cus

euta

ctus

ATC

C 27

759

GH

13 B

AA07

401

1 Xa

ntho

mon

as c

ampe

stris

GH

13 Y

P 00

4052

835

1 M

ariv

irga

tract

uosa

DSM

412

6

GH1

3 ZP

037

0954

51

Cory

neba

cter

ium

mat

ruch

otii A

TCC

3380

6

GH1

3 NP

267

649

1 La

ctoc

occu

s la

ctis

subs

p la

ctis

Il140

3

GH1

3 YP

001

3023

201

Par

abac

tero

ides

dist

ason

is AT

CC 8

503

GH1

3 YP

001

2276

361

Syn

echo

cocc

us s

p R

CC30

7

GH1

3 YP

002

5652

591

Hal

orub

rum

lacu

spro

fund

i ATC

C 49

239

GH13

ZP

0709

0550

1 C

oryn

ebac

teriu

m g

enita

lium

ATC

C 33

030

GH13

YP 0

0283

4100

1 C

oryn

ebac

teriu

m a

urim

ucos

um A

TCC

7009

75

GH13 A

KF03

861

Sand

arac

inus a

mylo

lyticu

s DSM

536

68

GH13

ZP 0

0050

865

1 M

agne

tosp

irillum

mag

neto

tacti

cum

MS-

1

GH13 Y

P 00

1568

811

1 Pe

troto

ga m

obilis

SJ9

5

GH13 Y

P 02

2847

1 P

icrop

hilus

torri

dus D

SM 9

790

GH13 E

FT25

5011

Pro

pionib

acter

ium ac

nes H

L110

PA3

GH13 E

FA80

1951

Poly

spho

ndyli

um pa

llidum

PN50

0

GH13 A

BD4655

81 V

ermam

oeba

verm

iform

is

GH13 Z

P 0350

5050

1 Rhiz

obium

etli B

rasil

5

GH13 YP 00

1348

4651

Pseud

omon

as ae

rugino

sa PA7

GH13 YP 00

2238

3511

Klebsie

lla pn

eumon

iae 34

2

GH13 YP 00

1437

9781

Cron

obac

ter sa

kaza

kii ATCC B

AA-894

GH13 Y

P 0011

7103

71 P

seud

omon

as st

utzeri

A1501

GH13 Y

P 0019

7240

81 S

tenotr

opho

monas

malt

ophil

ia K27

9a

GH13 Y

P 0043

1901

11 S

phing

obac

terium

sp 2

1

GH13 YP 0013127791 Sinorhizobium m

edicae W

SM419

GH13 ZP 035028921 Rhizobium etli K

im 5

GH13 YP 67

6219

1 Che

lativo

rans s

p BNC1

GH13 YP 00

2376

8861

Cya

nothe

ce sp

PCC 74

24

GH13 YP 0036383061 Cellulomonas flavig

ena DSM 20109

GH13 YP 0040977291 Intrasporangium ca

lvum DSM 43043

GH13 YP 0011691691 Rhodobacter s

phaeroides ATCC 17025

GH13 ZP 011580901 Oceanicola granulosus H

TCC2516

GH13 YP 0038382651 Micromonospora aurantiaca ATCC 27029

GH13 YP 0040977251 Intrasporangium calvu

m DSM 43043

GH13 YP 0036954761 Starkeya novella DSM 506

GH13 ZP 035166441 Rhizobium etli IE4771

GH13 ZP 031273131 Chthoniobacter flavus Ellin

428

GH13 AKF03642 Sandaracinus amylolyticus DSM 53668

GH13 YP 0031252671 Chitinophaga pinensis DSM 2588

GH13 YP 0030850521 Dyadobacter fermentans DSM 18053

GH13 YP 0033879241 Spirosoma linguale DSM 74

GH13 YP 0034301801 Streptococcus gallolyticus UCN34

GH13 ZP 078184041 Eremococcus coleocola ACS-139-V-Col8

GH13 YP 0034774501 Thermoanaerobacter italicus Ab9

GH13 ZP 048517861 Paenibacillus sp oral taxon 786 str D14

GH13 YP 0038414241 Caldicellulosiruptor obsidiansis OB47

GH13 YP 0024279661 Desulfurococcus kamchatkensis 1221n

GH13 YP 0034262831 Bacillus pseudofirmus OF4

GH13 ZP 076854311 Oscillochloris trichoides DG-6GH13 YP 0016174961 Sorangium cellulosum So ce56

GH13 ZP 019113371 Plesiocystis pacifica SIR-1

GH13 YP 6318811 Myxococcus xanthus DK 1622

GH13 AKF11765 Sandaracinus amylolyticus DSM 53668

GH13 ZP 036771121 Bacteroides cellulosilyticus DSM 14838

GH13 YP 0037157841 Croceibacter atlanticus HTCC2559

GH13 ZP 012015681 Flavobacteria bacterium BBFL7

GH13 ZP 019113321 Plesiocystis pacifica SIR-1GH13 YP 0031962511 Robiginitalea biformata HTCC2501

GH13 YP 0040531921 Marivirga tractuosa DSM 4126

GH13 ZP 021817881 Flavobacteriales bacterium ALC-1

GH13 CBL150401 Ruminococcus bromii L2-63

GH13 ZP 080051001 Bacillus sp 2 A 57 CT2

GH13 CBK911271 Eubacterium rectale DSM 17629

GH13 YP 0029369771 Eubacterium rectale ATCC 33656

GH13 YP 0021366011 Anaeromyxobacter sp K

GH13 ZP 014604541 Stigmatella aurantiaca

GH

13 728851 Streptomyces lividans

GH

13 BAJ281541 Kitasatospora setae KM-6054

GH

13 YP 0033390201 Streptosporangium roseum

DSM

43021

GH

13 YP 9214291 Nocardioides sp JS614

GH

13 AEB462451 Verrucosispora maris AB-18-032

GH

13 CAJ810301 Actinoplanes sp

GH

13 NP 9387401 C

orynebacterium diphtheriae N

CTC

13129

GH

13 YP 0036976141 Arcanobacterium haem

olyticum D

SM 20595

GH

13 ZP 039259261 Actinomyces coleocanis D

SM 15436

GH

13 YP 0040999421 Intrasporangium calvum

DSM

43043

GH

13 YP 0030989701 Actinosynnema m

irum D

SM 43827

GH

13 YP 0036376081 Cellulom

onas flavigena DSM

20109

GH

13 ZP 009938561 Janibacter sp HTC

C2649

GH13 YP 0030608421 Hirschia baltica ATCC 49814

GH13 YP 7579631 M

aricaulis maris M

CS10

GH13 YP 0016857121 Caulobacter sp K31

GH13 ZP 013016731 Sphingom

onas sp SKA58

GH13 YP 0038198341 Brevundim

onas subvibrioides ATCC 15264

GH

13 YP 4971641 Novosphingobium

aromaticivorans D

SM 12444

GH13 YP 6609551 Pseudoalterom

onas atlantica T6c

GH13 ZP 011126451 Reinekea blandensis M

ED297

GH13 YP 2677341 Colwellia psychrerythraea 34H

GH13 ZP 011126461 Reinekea blandensis M

ED297

GH13 AKF11772 Sandaracinus amylolyticus DSM

53668

GH13 AKF07527 Sandaracinus amylolyticus DSM

53668

GH13 YP 0016184291 Sorangium cellulosum

So ce56

GH13 ZP 019113391 Plesiocystis pacifica SIR-1

GH13 ZP 036315591 Pedosphaera parvula Ellin514

GH13 YP 0016604791 Microcystis aeruginosa NIES-843

GH13 YP 0043416831 Archaeoglobus veneficus SNP6

GH13 ZP 073351521 Desulfovibrio fructosovorans JJ

GH

13 AKF03605 Sandaracinus amylolyticus D

SM 53668

GH

13 YP 0027853821 Deinococcus deserti VC

D115

GH

13 NP 2951951 D

einococcus radiodurans R1

GH

13 YP 004171

1212

M

SD

sisn

epoc

ira

m su

ccoc

onie

D 1

661

1

GH

13 YP 0041715811 Deinococcus m

aricopensis DSM

21211

GH

13 YP 0037038171 Truepera radiovictrix DSM

17093

GH

13 YP 6052101 Deinococcus geotherm

alis DSM

11300

GH

13 YP 0027555491 A

cidobacterium capsulatum

ATCC

51196

GH

13 Y

P9

XTC

A5P

M alocirdnut allecilunarG 1860612400

GH

13 YP 6335241 M

yxococcus xanthus DK

1622G

H13 Y

PB

S su

cihp

ortid

ica

suhp

ortny

S 1

9412

64

GH

13 A

AW32

490

1 iiklahcsttog acnarborean

A

GH

13 Y

P 00

2316

532

1 An

oxyb

acillu

s fla

vith

erm

us W

K1

GH

13 Y

P 00

3252

634

1 G

eoba

cillu

s sp

Y41

2MC

61G

H13

ZP

0117

0936

1 B

acillu

s sp

NR

RL

B-14

911

GH

13 Z

P 07

7074

981

Bac

illus

sp m

3-13

GH

13 Z

P1-

GS ps sullica

B 116916810

GH

13 Y

P 00

1813

251

1 Ex

iguo

bact

eriu

m s

ibiri

cum

255

-15

GH

13 Y

P 00

2886

871

1 Ex

iguo

bact

eriu

m s

p A

T1b

GH

13 Y

P 00

1568

454

1 Pe

troto

ga m

obilis

SJ9

5

GH

13 Y

P 00

2940

188

1 Ko

smot

oga

olea

ria T

BF 1

95

1G

H13

ZP

0125

8420

1 V

ibrio

alg

inol

ytic

us 1

2G01

GH

13 1

0911

18 T

herm

oact

inom

yces

vul

garis

GH

13 Z

P 06

9705

751

Kte

dono

bact

er ra

cem

ifer D

SM 4

4963

GH

13 Y

P 00

1545

735

1 H

erpe

tosi

phon

aur

antia

cus

DSM

785

GH

13 E

FY95

778

1 M

etar

hizi

um ro

berts

ii AR

SEF

23

GH

13 N

P 58

8205

1 S

chiz

osac

char

omyc

es p

ombe

972

h-

GH

13 N

P 59

4401

1 S

chiz

osac

char

omyc

es p

ombe

972

h-

GH

13 X

P 00

1908

940

1 Po

dosp

ora

anse

rina

S m

at+

GH

13 4

1016

824

Schw

anni

omyc

es o

ccid

enta

lis

GH

13 B

AF98

616

1 H

ypho

pich

ia b

urto

nii

GH13 AKF07406 Sandaracinus amylolyticus DSM 53668

GH13 YP 0043692881 Desulfobacca acetoxidans DSM 11109

GH13 YP 0023716111 Cyanothece sp PCC 8801GH13 YP 3219451 Anabaena variabilis ATCC 29413

GH13 YP 4738811 Synechococcus sp JA-3-3AbGH13 YP 8464671 Syntrophobacter fumaroxidans MPOBGH13 ZP 063702061 Desulfovibrio sp FW1012B

GH13 YP 0033193471 Sphaerobacter thermophilus DSM 20745GH13 YP 0037057361 Truepera radiovictrix DSM 17093GH13 YP 4120971 Nitrosospira multiformis ATCC 25196GH13 YP 6431071 Rubrobacter xylanophilus DSM 9941

GH13 BAD389831 Rhodobacter sphaeroides f sp denitrificans

GH13 YP 3175601 Nitrobacter winogradskyi Nb-255

GH13 YP 8215181 Candidatus Solibacter usitatus Ellin6076

GH13 YP 0034506361 Azospirillum sp B510

GH13 ZP 070169471 Desulfonatronospira thiodismutans ASO3-1

GH13 YP 0041842351 Terriglobus saanensis SP1PR4

GH13 ZP 070315011 Granulicella mallensis MP5ACTX8

GH13 YP 0029438961 Variovorax paradoxus S110

GH13 YP 5918381 Candidatus Koribacter versatilis Ellin345

GH13 YP 7445681 Granulibacter bethesdensis CGDNIH1

GH13 YP 0039098001 Burkholderia sp CCGE1003

GH13 ZP 024903661 Burkholderia pseudomallei NCTC 13177

GH13 YP 0016309761 Bordetella petrii DSM 12804

GH13 YP 0022527351 Ralstonia solanacearum MolK2

GH13 YP 0037759061 Herbaspirillum seropedicae SmR1

GH13 YP 5455251 Methylobacillus flagellatus KT

GH13 YP 0013485041 Pseudomonas aeruginosa PA7

GH13 YP 0016008321 Gluconacetobacter diazotrophicus PA1 5

GH13 AEB584851 Pseudomonas mendocina NK-01

GH13 YP 0027996471 Azotobacter vinelandii DJ

GH

13 N

P 63

5804

1 X

anth

omon

as c

ampe

stris

pv

cam

pest

ris s

tr A

TCC

339

13

GH

13 Y

P 00

3188

581

1 Ac

etob

acte

r pas

teur

ianu

s IF

O 3

283-

01

GH

13 Y

P 74

4570

1 G

ranu

libac

ter b

ethe

sden

sis

CG

DN

IH1

GH

13 Y

P 00

3052

329

1 M

ethy

lovo

rus

gluc

oset

roph

us S

IP3-

4

GH

13 Z

P 06

8981

631

Ros

eom

onas

cer

vica

lis A

TCC

499

57

GH

13 Y

P 00

2943

895

1 Va

riovo

rax

para

doxu

s S1

10

GH

13 Y

P 31

4931

1 T

hiob

acillu

s de

nitri

fican

s AT

CC

252

59

GH

13 Y

P 42

5597

1 R

hodo

spiri

llum

rubr

um A

TCC

111

70

GH

13 Y

P 93

3302

1 A

zoar

cus

sp B

H72

GH

13 N

P 43

7816

1 S

inor

hizo

bium

mel

iloti

1021

GH

13 Z

P 01

1553

281

Oce

anic

ola

gran

ulos

us H

TCC

2516

GH

13 Y

P 00

1167

979

1 R

hodo

bact

er s

phae

roid

es A

TCC

170

25

GH

13 Y

P 00

1440

575

1 C

rono

bact

er s

akaz

akii A

TCC

BAA

-894

GH

13 Z

P 01

4401

991

Ful

vim

arin

a pe

lagi

HTC

C25

06

GH

13 Y

P 00

4234

793

1 Ac

idov

orax

ave

nae

subs

p a

vena

e AT

CC

198

60

GH

13 Y

P 00

1818

894

1 O

pitu

tus

terra

e PB

90-1

GH

13 Y

P 00

3154

808

1 Br

achy

bact

eriu

m fa

eciu

m D

SM 4

810

GH

13 ZP 010940301 Blastopirellula marina D

SM 3645

GH

13 ZP 027320551 Gem

mata obscuriglobus U

QM

2246

GH

13 YP 4273821 Rhodospirillum

rubrum ATC

C 11170

GH

13 YP 0021361131 Anaeromyxobacter sp K

GH

13 YP 0039532711 Stigmatella aurantiaca

GH

13 YP 0021384951 Geobacter bem

idjiensis BemG

H13 Y

P23

-C

RF ii

notl

ad r

etca

boe

G 1

3868

3520

0 G

H13 Y

P65

ec o

S mu

solu

llec

mu

igna

roS

191

8716

100

GH

13 ZPB2

101

WF

ps o

irbi

vofl

use

D 1

5398

6360

GH

13 Y

P 82

1516

1 C

andi

datu

s S

olib

acte

r usi

tatu

s E

llin6

076

GH

13 Y

P24

NFC ilte

muibozihR 1077074

GH

13

86635 M

SD sucityloly

ma sunicaradnaS 43690F

KA

GH

13 Y

P 00

1820

541

1 O

pitu

tus

terra

e P

B90

-1G

H13

ZP

0313

0066

1 C

htho

niob

acte

r fla

vus

Ellin

428

GH

13 YP 0035707081 Salinibacter ruber M8

GH

13 YP 0037057351 Truepera radiovictrix DSM

17093

GH

13 YP 0032689441 Haliangium

ochraceum D

SM 14365

GH

13 YP 0016130311 Sorangium cellulosum

So ce56

GH

13 YP 6288101 Myxococcus xanthus D

K 1622

GH

13 62738967 Deinococcus radiodurans

GH

13 AKF04017 Sandaracinus amylolyticus D

SM 53668

GH

13 YP 0027878991 Deinococcus deserti VC

D115

GH13 YP 5893201 Candidatus Koribacter versatilis Ellin345

GH13 ZP 063702081 Desulfovibrio sp FW

1012B

GH13 YP 0041842341 Terriglobus saanensis SP1PR4

GH13 YP 8731321 Acidotherm

us cellulolyticus 11B

GH13 ZP 070169501 Desulfonatronospira thiodism

utans ASO3-1

GH13 ADX845721 Sulfolobus islandicus REY15A

GH13 ZP 027318151 G

emm

ata obscuriglobus UQM

2246

GH13 YP 0016347811 Chloroflexus aurantiacus J-10-fl

GH

13 NP 9534051 G

eobacter sulfurreducens PCA

GH

13 YP 0025366761 Geobacter daltonii FR

C-32

GH

13 YP 0012121361 Pelotomaculum

thermopropionicum

SI

GH

13 YP 0041781661 Isosphaera pallida ATCC

43644

GH

13 ZP 036310341 Pedosphaera parvula Ellin514

GH13 ZP 031287911 Chthoniobacter flavus Ellin428

GH13 NP 6913271 Oceanobacillus ih

eyensis HTE831

GH13 YP 0025601851 Macro

coccus c

aseolyticus J

CSC5402

GH13 YP 0030110001 Paenibacill

us sp J

DR-2

GH13 CAJ384141 Anaerobranca

gottsch

alkii

GH13 YP 00

1988

1311

Lacto

bacill

us ca

sei B

L23

GH13 YP 00

2459

6561

Des

ulfito

bacte

rium ha

fnien

se D

CB-2

GH13 YP 00

3947

0781

Paenib

acillu

s poly

myxa S

C2

GH13 Y

P 0013

1197

11 C

lostrid

ium be

ijerin

ckii N

CIMB 80

52

GH13 Y

P 0039

6683

31 Ily

obac

ter po

lytrop

us D

SM 2926

GH13 Z

P 0539

1277

1 Clos

tridium

carb

oxidi

vora

ns P

7

GH13 Y

P 0013

9686

01 C

lostrid

ium kl

uyve

ri DSM 55

5

GH13 ZP 02

2128

451

Clostrid

ium ba

rtlettii

DSM 16

795

GH13 ZP 04

4331

841

Bacillu

s coa

gulan

s 36D

1

GH13 C

BL37

8151

Ana

eros

tipes

hadr

us

GH13 Z

P 04

4519

311

Abiotro

phia

defec

tiva A

TCC 49

176

GH13

YP

0031

4407

51

Slac

kia h

eliot

rinire

duce

ns D

SM 2

0476

GH13

ZP 0

2025

300

1 Eu

bacte

rium

vent

riosu

m A

TCC

2756

0

GH1

3 YP

004

3093

801

Clo

strid

ium

lent

ocel

lum

DSM

542

7

GH13

YP 5

1565

71

Chlam

ydop

hila

felis

GH13

ZP 0

6299

217

1 Pa

rach

lamyd

ia ac

anth

amoe

bae

str H

allrsquos

cocc

us

GH13

CBL1

5412

1 R

umino

cocc

us b

rom

ii L2-

63

GH13 Y

P 00

1692

347

1 Fi

nego

ldia

mag

na AT

CC 293

28

GH13 Z

P 07

5262

211

Pep

tostr

epto

cocc

us st

omat

is DSM

176

78

GH1

3 YP

003

4751

021

Clo

strid

iale

s ge

nom

osp

BVA

B3 s

tr U

PII9

-5

GH1

3 YP

004

1716

241

Dei

noco

ccus

mar

icope

nsis

DSM

212

11

GH1

3 YP

022

845

1 Pi

crop

hilu

s to

rridu

s DS

M 9

790

GH1

3 YP

001

8207

181

Opi

tutu

s te

rrae

PB90

-1

GH1

3 YP

003

1672

861

Can

dida

tus A

ccum

ulib

acte

r pho

spha

tis c

lade

IIA

str

UW-1

GH1

3 AK

F038

60 S

anda

racin

us a

mylo

lyticu

s DS

M 5

3668

GH1

3 AK

F069

52 S

anda

racin

us a

mylo

lyticu

s DS

M 5

3668

GH

13 Y

P 00

2762

222

1 G

emm

atim

onas

aur

antia

ca T

-27

GH1

3 ZP

082

0671

01

Gor

doni

a ne

ofel

ifaec

is NR

RL B

-593

95

GH

13 Y

P 00

3964

374

1 Ke

togu

loni

cige

nium

vul

gare

Y25

GH13 ZP 045772811 Oxalobacter fo

rmigenes H

OxBLS

GH13 YP 0033856211 Spirosoma linguale DSM 74

GH13 AEA650561 Burkholderia gladioli B

SR3

GH13 YP 0030725361 Teredinibacter turnerae T7901

GH13 YP 4255961 Rhodospirillum ru

brum ATCC 11170

GH13 YP 0010427851 Rhodobacter sphaeroides ATCC 17029

GH13 ZP 075890671 Sinorhizobium meliloti BL225C

GH13 YP 0030504181 Methylovorus glucosetrophus SIP3-4

GH13 YP 4224271 Magnetospirillum magneticum AMB-1

GH13 YP 0042554001 Deinococcus proteolyticus MRP

GH13 ZP 029300551 Verrucomicrobium spinosum DSM 4136

GH13 ZP 080190951 Lautropia mirabilis ATCC 51599

GH13 YP 5335351 Rhodopseudomonas palustris BisB18

GH13 YP 8654291 Magnetococcus marinus MC-1

GH13 YP 0028938741 Tolumonas auensis DSM 9187

GH13 YP 6612131 Pseudoalteromonas atlantica T6c

GH13 YP 0040654571 Pseudoalteromonas sp SM9913

GH13 YP 8302311 Arthrobacter sp FB24

GH13 YP 0042347941 Acidovorax avenae subsp avenae ATCC 19860

GH13 YP 0016390191 Methylobacterium extorquens PA1

GH13 ZP 068060481 Brevibacterium mcbrellneri ATCC 49030

GH13 YP 0031655431 Candidatus Accumulibacter phosphatis clade IIA str UW-1

GH13 YP 9813411 Polaromonas naphthalenivorans CJ2

GH13 ZP 029305921 Verrucomicrobium spinosum DSM 4136

GH13 YP 8283661 Candidatus Solibacter usitatus Ellin6076

GH13 YP 0015687661 Petrotoga mobilis SJ95

GH13 YP 2560581 Sulfolobus acidocaldarius DSM 639

GH13 YP 0023732201 Cyanothece sp PCC 8801

GH13 AKF07413 Sandaracinus amylolyticus DSM 53668

GH13 AKF03796 Sandaracinus amylolyticus DSM 53668

GH13 EFS357291 Propionibacterium acnes HL013PA1

GH13 YP 0033827501 Kribbella flavida DSM 17836

GH13 YP 9230111 Nocardioides sp JS614

GH13 AEA239681 Pseudonocardia dioxanivorans CB1190

GH13 YP 0031037201 Actinosynnema mirum DSM 43827

GH57 YP 0018695221 Nostoc punctiforme PCC 73102

GH57 YP 0041847961 Terriglobus saanensis SP1PR4

GH57 YP 0033170621 Thermanaerovibrio acidaminovorans DSM 6589

GH57 YP 0025231331 Thermomicrobium roseum DSM 5159

GH57 CBL286501 Fretibacterium fastidiosum

GH57 YP 0041781671 Isosphaera pallida ATCC 43644

GH57 YP 0027549471 Acidobacterium capsulatum ATCC 51196

GH57 ZP 070292791 Granulicella mallensis MP5ACTX8

GH57 YP 0018749261 Elusimicrobium minutum Pei191

GH57 ADL259941 Fibrobacter succinogenes subsp succinogenes S85

GH57 YP 4773871 Synechococcus sp JA-2-3B

GH57 YP 8454221 Syntrophobacter fumaroxidans MPOB

GH57 NP 9534062 Geobacter sulfurreducens PCA

GH57 YP 5020321 Methanospirillum hungatei JF-1

GH57 YP 0019958521 Chloroherpeton thalassium ATCC 35110

GH57 YP 5937791 Candidatus Koribacter versatilis Ellin345

GH57 YP 0010471761 Methanoculleus marisnigri JR1

GH57 YP 0014309471 Roseiflexus castenholzii DSM 13941

GH57 YP 0029516361 Desulfovibrio magneticus RS-1

GH57 YP 0038064671 Desulfarculus baarsii DSM 2075

GH57 YP 0013789531 Anaeromyxobacter sp Fw109-5

GH57 ZP 063702071 Desulfovibrio sp FW1012B

GH57 YP 0035531061 Aminobacterium colombiense DSM 12261

GH57 ZP 064391271 Anaerobaculum hydrogeniformans ATCC BAA-1850

GH57 AKF06951 Sandaracinus amylolyticus DSM 53668

GH57 ZP 063915611 Dethiosulfovibrio peptidovorans DSM 11002

GH57 YP 0024634161 Chloroflexus aggregans DSM 9485

GH57 YP 0043692891 Desulfobacca acetoxidans DSM 11109

GH57 YP 8464661 Syntrophobacter fumaroxidans M

POB

GH57 CAJ738171 Candidatus Kuenenia stuttgartiensis

GH57 YP 0031969181 Desulfohalobium

retbaense DSM 5692

FIG 3mdashPhylogeny of Sandaracinus amylolyticus GH13 and GH57 a-amylases Sandaracinus amylolyticus a-amylases (GH13 and GH57) are colored in

pink along with their domain architecture displayed as Domain Color Shape [GH13 Red rectangle GH57 Black rectangle GH77 dark pink rectangle

CBM48 Dark blue circle CBM20 green circle CBM41 cyan circle] Myxobacterial homologs are colored fluorescent green Bootstrap values correspond-

ing to the tree nodes are provided

Complete Genome of a Starch-Degrading Myxobacteria GBE

Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2525

of the phyla Betaproteobacteria suggestive of a possible hor-

izontal transfer

AKF04017 and AKF03605 have close homologs in the ex-

tremophile Deinococcus desserti VCD115 and shares clade

with certain myxobacterial amylases belonging to

Myxococcus xanthus S cellulosum and Haliangium ocraceum

which further share clades with Deinococcus amylases

AKF04017 and AKF03605 share the highest sequence identity

with D desserti VCD115 plasmid protein (YP_002787899

533) and chromosomal protein (YP_002785382 456)

respectively that might point to its plasmid-borne horizontal

transfer across the clade (fig 3) The sequence conservation of

CSRs and the high percentage identity of these amylases to

Deinococcus support the inference for the putative horizontal

transfers among these members AKF09634 does not share its

sister branch with any member but the clade contains mem-

bers from diverse phyla that is Rhizobium from alphaproteo-

bacteria Opitutus from Chlamydae Chthonibacter from

Verrucomicrobia suggesting the multiple gain and loss of

this amylase among prokaryotes Similarly AKF07413 shares

the clade with members of Cyanobacteria AKF07406 shares

the clade with members of Deltaproteobacteria AKF03861

with members of Alphaproteobacteria and AKF03642 with

members of Bacteroidetes Taken together we can suggest

that many of the S amylolyticus amylases have been acquired

via horizontal gene transfer events from other bacterial species

belonging to the phylum Deinococcus-Thermus

Acidobacteria Cyanobacteria and Alphaproteobacteria

To further support these horizontal transfer events we

checked the sequence conservation between S amylolyticus

amylases and closely related members in the phylogenetic tree

and aligned using PROMALS-3D with Taka-Amylase A of A

oryzae (PDBid 2TAA) as a structural template We also per-

formed synteny studies analyzing proteins present in the vicin-

ity of all identified S amylolyticus GH13 and GH57 amylases

and their closely related homologs as revealed by phylogenetic

studies We could not trace any conserved syntenic regions in

these genomes whereas in the case of AKF03862 and

AKF09634 we identified glycogen debranching enzymes in

Stigmatella aurantiaca DW43-1 [56 identity and 98

query coverage of AKF03860 with WP_0133760911] and

Opitutus terrae PB90-1 [57 identity and 97 query cover-

age of AKF09635 YP_0018205421] which further support

their putative horizontal transfer in S amylolyticus

Similarly the phylogenetic inference was drawn from two

representative domains of g-amylases using the same meth-

odology as followed for a-amylases (fig 4) We found that

one of the g-amylases AKF10340 lies closer to M xanthus

amylase whereas the other AKF10487 shares its sister

branch with Nitrosococcus watsonii a Gammaproteobacteria

This suggests that while some of the a- and g-amylases

are present in many myxobacterial species a majority of the

a- and g-amylases are present only in S amylolyticus and not

in other myxobacterial genomes possibly due to the result of

horizontal transfer events from diverse bacterial phylum such

as Deinococcus-Thermus Cyanobacteria Alphaproteobac-

teria Gammaproteobacteria and Acidobacteria

Agar Degrading Enzymes

Some bacteria are known to degrade agar and use it as a

carbon source and possess agarase enzymes that help in hy-

drolyzing agar in oligosaccharides (Chi et al 2012) Depending

on the site of action they can be of two types a-agarase

(agarose 3-glycanohydrolase 321158 GH96) and

b-agarase (agarose 4-glycanohydrolase 32181 GH16

GH50 GH86 GH118) (table 1) Both families of agarases

GH15 EFV841771 Achrom

obacter xylosoxidans C54

GH

15 ZP 011267341 Nitrococcus m

obilis Nb-231

GH15 YP 4129921 Nitrosospira m

ultiformis ATCC 25196

GH15 YP 5716301 Nitrobacter ham

burgensis X14 plasmid

GH15 ZP 012260811 Aurantim

onas manganoxydans SI85-9A1

GH15 YP 0016032801 Gluconacetobacter diazotrophicus PA1 5

GH15 YP 7451371 Granulibacter bethesdensis CGDNIH1

GH15 AEA616211 Burkholderia gladioli BSR3

GH15 YP 0018344311 Beijerinckia indica subsp indica ATCC 9039

GH15 YP 0027560251 Acidobacterium capsulatum

ATCC 51196

GH15 CBJ387091 Ralstonia solanacearum CMR15

GH15 YP 3189151 Nitrobacter winogradskyi Nb-255

GH15 YP 0012344011 Acidiphilium cryptum JF-5

GH15 YP 0040115781 Rhodomicrobium vannielii ATCC 17100

GH15 YP 0016419391 Methylobacterium extorquens PA1GH15 YP 5252641 Rhodoferax ferrireducens T118

GH15 YP 0041811661 Terriglobus saanensis SP1PR4

GH15 ZP 031333281 Chthoniobacter flavus Ellin428

GH15 CCA537261 Streptomyces venezuelae ATCC 10712

GH15 YP 0015065621 Frankia sp EAN1pec

GH15 YP 0024892701 Arthrobacter chlorophenolicus A6

GH15 YP 0018202091 Opitutus terrae PB90-1

GH15 YP 8729011 Acidothermus cellulolyticus 11B

GH15 YP 0013795591 Anaeromyxobacter sp Fw109-5

GH15 YP 0024929091 Anaeromyxobacter dehalogenans 2CP-1

GH15 YP 6355121 Myxococcus xanthus DK 1622

GH15 YP 0011047051 Saccharopolyspora erythraea NRRL 2338

GH15 YP 0011092191 Saccharopolyspora erythraea NRRL 2338

GH15 AKF10340 Sandaracinus amylolyticus DSM 53668

GH15 YP 6342991 Myxococcus xanthus DK 1622

GH15 YP 0031091351 Acidimicrobium ferrooxidans DSM 10331

GH15 YP 0035703361 Salinibacter ruber M8

GH15 YP 0025241401 Thermomicrobium roseum DSM 5159 plasmid

GH15 YP 0033214441 Sphaerobacter thermophilus DSM 20745

GH15 NP 7336051 Streptomyces coelicolor A3-2

GH15 YP 0035282991 Nitrosococcus halophilus Nc 4

GH15 YP 0043676121 Marinithermus hydrothermalis DSM 14884

GH15 YP 0043687811 Marinithermus hydrothermalis DSM 14884

GH15 YP 0034042871 Haloterrigena turkmenica DSM 5511

GH15 ZP 080461001 Haladaptatus paucihalophilus DX253

GH1

5 YP

023

376

1 Pi

crop

hilu

s to

rrid

us D

SM 9

790

GH1

5 ZP

055

7131

11

Ferr

opla

sma

acid

arm

anus

fer1

GH1

5 BA

B604

801

The

rmop

lasm

a vo

lcan

ium

GSS

1

GH

15 Y

P 00

3323

761

1 Th

erm

obac

ulum

terr

enum

ATC

C B

AA

-798

GH

15 Y

P 00

3899

615

1 C

yano

thec

e sp

PC

C 7

822

plas

mid

GH

15 Y

P 00

3705

927

1 Tr

uepe

ra ra

diov

ictr

ix D

SM 1

7093

GH

15 Y

P 00

2478

443

1 A

rthr

obac

ter c

hlor

ophe

nolic

us A

6 pl

asm

idG

H15

AK

F104

87 S

anda

raci

nus

amyl

olyt

icus

DSM

536

68G

H15

YP

0037

6113

81

Nitr

osoc

occu

s w

atso

nii C

-113

GH1

5 ZP

069

7134

91

Kted

onob

acte

r rac

emife

r DSM

449

63

GH15

ZP

0697

6049

1 K

tedo

noba

cter

race

mife

r DSM

449

63

GH15

YP

0033

9335

81

Cone

xiba

cter

woe

sei D

SM 1

4684

GH15

YP

0042

3629

11 A

cido

vora

x av

enae

sub

sp a

vena

e ATC

C 19

860

GH15 Z

P 03

6273

271

Pedo

spha

era p

arvu

la El

lin51

4

GH15 Y

P 00

1618

7001

Sor

angi

um ce

llulo

sum

So

ce56

GH15 ZP

0145

9561

1 Stig

matella

aura

ntiac

a DW

4-3-1

GH15 ZP

0503

8177

1 Syn

echo

cocc

us sp

PCC 73

35

GH15 YP 64

4038

1 Rubrobac

ter xy

lanophilu

s DSM 99

41

GH15 ZP 064746651 Frankia symbiont of D

atisca glomerata

GH15 ZP 069744041 Ktedonobacter racemife

r DSM 44963

GH15 ZP 069653261 Ktedonobacter racemifer D

SM 44963

GH15 ZP 069661381 Ktedonobacter racemifer D

SM 44963

FIG 4mdashPhylogeny of Sandaracinus amylolyticus GH15 g-amylases

Sandaracinus amylolyticus g-amylases are colored dark pink and myxobac-

terial homologs are colored fluorescent green Bootstrap values corre-

sponding to the tree nodes are provided

Sharma et al GBE

2526 Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016

were absent in S amylolyticus genome which could be related

to its agar nondegrading property (Mohr et al 2012) as sup-

ported by colony morphology where agar was not penetrated

or liquefied by the bacteria (supplementary fig S5

Supplementary Material online)

Cellulose Degrading Enzymes

We identified only GH5 and GH74 in S amylolyticus proteome

in two and three proteins respectively GH5 domain was pre-

sent as a single unit in AKF04963 and AKF09194 whereas

GH74 was identified in AKF04859 AKF06963 and

AKF09775 associated with a GH74 domain CAZY domains

for cellobiohydrolaseexoglucanases (represented by GH6

GH7 GH9 and GH48) were not present in S amylolyticus

Cellobiohydrolaseexoglucanases plays a significant role in cel-

lulose degradation as they cut two to four sugar subunits from

the exposed ends formed by the endoglucanases which are

used as initial substrates for b-glucosidase (Mba Medie et al

2012) The absence of cellobiohydrolase or exoglucanases

could be a possible reason for the lack of cellulose degradation

in S amylolyticus Out of b-glucosidase representative CAZY

families GH1 GH3 GH5 GH9 GH30 and GH116 only GH1

GH3 and GH5 families were found in three two and two

proteins in S amylolyticus respectively AKF03843 AKF0

6028 and AKF10711 have a GH1 domain whereas

AKF09078 and AKF10875 have only the GH3 domain

Chitin Degrading Enzymes

Chitinases (EC32114) are involved in degradation of chitin

a component of the cell wall of fungi and exoskeleton of

arthropods GH18 GH19 GH23 and GH48 families have

been reported in the CAZY database as chitinases that hydro-

lyse b-14 linkages between N-acetylglucosamine units GH18

is a chitinase family with a characteristic ldquo[LIVM

FY][DN]G[LIVMF][DN][LIVMF][DN]Erdquo motif and is expressed

in archaea prokaryotes and eukaryotes (Funkhouser and

Aronson 2007) GH18 chitinases were not found in S amylo-

lyticus genome while members of GH19 (AKF03006 and

AKF10423) and GH23 (AKF04575 AKF05533 AKF06346

AKF08835 and AKF11377) could be identified GH19

family chitinases are mostly reported to be present in plants

with only a few members in prokaryotes (Kasprzewska 2003

Funkhouser and Aronson 2007) Among the two identified

GH19 homologs we found that AKF10423 is approximately

65 identical (~68 query coverage) with cyanobacteria

such as Nodosilinea nodulosa and Limnoraphis robusta

along with another myxobacterium Chondromyces crocatus

Besides these homologs we also found that AKF10423 shows

maximum similarity with eukaryotic GH19 chitinases counter-

parts as present in Acacia mangium Festuca arundinacea

Triticum aestivum and Leucaena leucocephala (55ndash59 iden-

tity with 70 query coverage) We identified that another

chitinase AKF06346 has GH23 domain along with three

copies of CBM50 which is generally present in the enzymes

cleaving either chitin or peptidoglycan

Conclusion

The complete 1033 Mb circular genome of S amylolyticus

type strain DSM 53668T was determined CAZy analysis re-

vealed the presence of 72 GT domains 72 CE domains 58 GH

domains 19 AA domains 4 PL domains and 26 CBM do-

mains Sixteen S amylolyticus proteins were identified of

which 15 are a-amylases of class GH13 and 1 GH57 Two

g-amylases of GH15 family were also identified Phylogeny

analysis revealed that ten GH13 a-amylases and one GH15

g-amylase have been aquired via horizontal gene transfer

from other bacterial phyla such as Deinococcus-Thermus

Cyanobacteria Alphaproteobacteria Gammaproteobacteria

and Acidobacteria

Table 1

Cellulose Starch Chitin and Agar Degrading Enzymes and Their Representative CAZY Domains Distribution

Substrate Enzyme Category Enzyme Name EC Number CAZY Families (protein repertoire in brackets)

Starch Amylase a-Amylase 3211 GH13 (15) GH57 (1) GH119 GH126

Amylase b-Amylase 3212 GH14

Amylase g-Amylase 3213 GH15 (2) GH97

Chitin Chitinase Chitinase 32114 GH18 GH19 (2) GH23 (5) GH48

Agar a-Agarase Agarose 3-glycanohydrolase 321158 GH96

b-Agarase Agarose 4-glycanohydrolase 32181 GH16 GH50 GH86 GH118

Cellulose Cellulase b-Glucosidase 32121 GH1 (3) GH3 (2) GH5 (2) GH9 GH30 GH116

Cellulase Cellobiohydrolase or Exoglucanase 321176 GH48 GH6 GH7 GH9

Cellulase Endoglucanase 3214 GH5 (2) GH6 GH7 GH8 GH9 GH12 GH44

GH45 GH48 GH51 GH74 (3) GH124

NOTEmdashEC number enzyme category and CAZY families for proteins involved in starch chitin agar and cellulose degradation are shown The number of Sandaracinusamylolyticus proteins identified having CAZY domains are represented in bold fonts in respective families

Complete Genome of a Starch-Degrading Myxobacteria GBE

Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2527

Supplementary Material

Supplementary figures S1ndashS5 and tables S1 and S2 are avail-

able at Genome Biology and Evolution online (httpwww

gbeoxfordjournalsorg)

Acknowledgments

This work is supported by a project ldquoExpansion and modern-

ization of Microbial Type Culture Collection and Gene Bank

(MTCC) jointly supported by Council of Scientific and

Industrial Research (CSIR) Grant No BSC0402 and

Department of Biotechnology (DBT) Govt of India Grant No

BTPR7368INF221772012rdquo The authors thank MTCC for

providing help in procuring the cultures from DSMZ They

acknowledge Dr Ramya TNC for providing workbench for cul-

turing myxobacteria GS and IK acknowledge the Council

of Scientific and Industrial Research (CSIR) and University

Grant Commission (UGC) for the research fellowship respec-

tively They also thank the Genome Quebec Innovation

Centre McGill University Canada and C-CAMP next-genera-

tion genomics facility Bangalore India for help in obtaining

PacBio and Illumina NGS data respectively

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Image Generator (BRIG) simple prokaryote genome comparisons

BMC Genomics 12402

Altschul SF Gish W Miller W Myers EW Lipman DJ 1990 Basic local

alignment search tool J Mol Biol 215403ndash410

Auch AF Von Jan M Klenk HP Goker M 2010 Digital DNA-DNA hybrid-

ization for microbial species delineation by means of genome-to-

genome sequence comparison Stand Genomic Sci 2117ndash134

Aziz RK et al 2008 The RAST Server rapid annotations using subsystems

technology BMC Genomics 975

Blesak K Janecek S 2012 Sequence fingerprints of enzyme specificities

from the glycoside hydrolase family GH57 Extremophiles 16497ndash

506

Chi WJ Chang YK Hong SK 2012 Agar degradation by microorganisms

and agar-degrading enzymes Appl Microbiol Biotechnol 94917ndash

930

Chin CS et al 2013 Nonhybrid finished microbial genome assemblies

from long-read SMRT sequencing data Nat Methods 10563ndash569

Eddy SR 2011 Accelerated profile HMM searches PLoS Comput Biol

7e1002195

Edgar RC 2004 MUSCLE multiple sequence alignment with high accu-

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Funkhouser JD Aronson NN Jr 2007 Chitinase family GH18 evolutionary

insights from the genomic history of a diverse protein family BMC Evol

Biol 796

Garcia R Muller R 2014 The family Polyangiaceae In Rosenberg E

Delong E Lory S Stackebrandt E Thompson F editors The prokary-

otes Berlin Heidelberg Springer p 247ndash279

Grissa I Vergnaud G Pourcel C 2007 CRISPRFinder a web tool to identify

clustered regularly interspaced short palindromic repeats Nucleic

Acids Res 35W52ndashW57

Hall TA 1999 BioEdit a user-friendly biological sequence alignment editor

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4195ndash98

Han K et al 2013 Extraordinary expansion of a Sorangium cellulosum

genome from an alkaline milieu Sci Rep 32101

Janecek S Kuchtova A 2012 In silico identification of catalytic residues

and domain fold of the family GH119 sharing the catalytic machinery

with the alpha-amylase family GH57 FEBS Lett 5863360ndash3366

Janecek S Svensson B Macgregor EA 2014 alpha-Amylase an enzyme

specificity found in various families of glycoside hydrolases Cell Mol

Life Sci 711149ndash1170

Kasprzewska A 2003 Plant chitinasesndashregulation and function Cell Mol

Biol Lett 8809ndash824

Lagesen K et al 2007 RNAmmer consistent and rapid annotation of

ribosomal RNA genes Nucleic Acids Res 353100ndash3108

Lombard V Golaconda Ramulu H Drula E Coutinho PM Henrissat B

2014 The carbohydrate-active enzymes database (CAZy) in 2013

Nucleic Acids Res 42D490ndashD495

Lowe TM Eddy SR 1997 tRNAscan-SE a program for improved detection

of transfer RNA genes in genomic sequence Nucleic Acids Res

25955ndash964

Matsuura Y Kusunoki M Harada W Kakudo M 1984 Structure

and possible catalytic residues of Taka-amylase A J Biochem

95697ndash702

Mba Medie F Davies GJ Drancourt M Henrissat B 2012 Genome anal-

yses highlight the different biological roles of cellulases Nat Rev

Microbiol 10227ndash234

Mohr KI Garcia RO Gerth K Irschik H Muller R 2012

Sandaracinus amylolyticus gen nov sp nov a starch-degrading

soil myxobacterium and description of Sandaracinaceae fam

nov Int J Syst Evol Microbiol 621191ndash1198

Noguchi J et al 2011 Crystal structure of the branching enzyme I

(BEI) from Oryza sativa L with implications for catalysis and sub-

strate binding Glycobiology 211108ndash1116

Patel RK Jain M 2012 NGS QC Toolkit a toolkit for quality control of next

generation sequencing data PLoS One 7e30619

Pei J Kim BH Grishin NV PROMALS3D a tool for multiple protein se-

quence and structure alignments Nucleic Acids Res 36 2295ndash2300

Reichenbach H Dworkin M 1992 The myxobacteria In Balows A

Truper HG Dworkin M Harder W Schleifer KH editors The pro-

karyotes a handbook on the biology of bacteria ecophysiology

isolation identification applications New York Springer New

York p 3416ndash3487

Reichenbach H 2005 ldquoOrder VIII Myxococcalesrdquo Bergeyrsquos Manual of

Systematic Bacteriology In Brenner DJ Krieg NR Staley JT Garrity

GM editors Vol 2 2nd ed part C New York Springer-Verlag New

York Inc pp 1074ndash1079

Richter M Rossello-Mora R 2009 Shifting the genomic gold standard for

the prokaryotic species definition Proc Natl Acad Sci U S A 106

19126ndash19131

Schneiker S et al 2007 Complete genome sequence of the myxobacter-

ium Sorangium cellulosum Nat Biotechnol 25 1281ndash1289

Shimkets LJ 1990 Social and developmental biology of the myxobacteria

Microbiol Rev 54 473ndash501

Siguier P Perochon J Lestrade L Mahillon J Chandler M 2006 ISfinder

the reference centre for bacterial insertion sequences Nucleic Acids

Res 34 D32ndashD36

Steinmetz H et al 2012 Indiacens A and B prenyl indoles from the

myxobacterium Sandaracinus amylolyticus J Nat Prod 75 1803ndash

1805

Tamura K Stecher G Peterson D Filipski A Kumar S 2013 MEGA6

molecular evolutionary genetics analysis version 60 Mol Biol Evol

30 2725ndash2729

Van Der Maarel MJ Van Der Veen B Uitdehaag JC Leemhuis H Dijkhuizen

L 2002 Properties and applications of starch-converting enzymes of

the alpha-amylase family J Biotechnol 94 137ndash155

Sharma et al GBE

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Weissman KJ Muller R 2010 Myxobacterial secondary metabolites

bioactivities and modes-of-action Nat Prod Rep 27 1276ndash1295

Whitworth DE 2008 Myxobacteria multicellularity and differentiation

Washington DC ASM Press American Society for Microbiology

Yin Y et al 2012 dbCAN a web resource for automated carbohydrate-

active enzyme annotation Nucleic Acids Res 40 W445ndashW451

Zhao JY et al 2008 Discovery of the autonomously replicating plasmid

pMF1 from Myxococcus fulvus and development of a gene cloning

system in Myxococcus xanthus Appl Environ Microbiol 74 1980ndash

1987

Associate editor Howard Ochman

Complete Genome of a Starch-Degrading Myxobacteria GBE

Genome Biol Evol 8(8)2520ndash2529 doi101093gbeevw151 Advance Access publication June 29 2016 2529