Scholarly article on topic 'Genome analyses of the carboxydotrophic sulfate-reducers Desulfotomaculum nigrificans and Desulfotomaculum carboxydivorans and reclassification of Desulfotomaculum caboxydivorans as a later synonym of Desulfotomaculum nigrificans'

Genome analyses of the carboxydotrophic sulfate-reducers Desulfotomaculum nigrificans and Desulfotomaculum carboxydivorans and reclassification of Desulfotomaculum caboxydivorans as a later synonym of Desulfotomaculum nigrificans Academic research paper on "Biological sciences"

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Academic research paper on topic "Genome analyses of the carboxydotrophic sulfate-reducers Desulfotomaculum nigrificans and Desulfotomaculum carboxydivorans and reclassification of Desulfotomaculum caboxydivorans as a later synonym of Desulfotomaculum nigrificans"

Standards in Genomic Sciences (2014) 9:655-675

DOI:10.4056/sigs.4718645

Genome analyses of the carboxydotrophic sulfate-reducers Desulfotomaculum nigrificans and Desulfotomaculum carboxydivorans and reclassification of Desulfotomaculum caboxydivorans as a later synonym of Desulfotomaculum nigrificans

Michael Visser1, Sofiya N. Parshina2, Joana I. Alves3, Diana Z. Sousa13, Ines A. C. Pereira4, Gerard Muyzer5, Jan Kuever6, Alexander V. Lebedinsky2, Jasper J. Koehorst7, Petra Worm1, Caroline M. Plugge1, Peter J. Schaap7, Lynne A. Goodwin89, Alla Lapidus1011, Nikos C. Kyrpides8, Janine C. Detter9, Tanja Woyke8, Patrick Chain89, Karen W. Davenport89, Stefan Spring12, Manfred Rohde13, Hans Peter Klenk12, Alfons J.M. Stams13

1Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands

2Wingradsky Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia

3Centre of Biological Engineering, University of Minho, Braga, Portugal

4Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, Oeiras, Portugal

5Department of Aquatic Microbiology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands

6Department of Microbiology, Bremen Institute for Materials Testing, Bremen, Germany

7Laboratory of Systems and Synthetic Biology, Wageningen University, Wageningen, The Netherlands

8DOE Joint Genome Institute, Walnut Creek, California, USA

9Los Alamos National Laboratory, Bioscience Division, Los Alamos, New Mexico, USA

10Theodosius Dobzhansky Center for Genome Bionformatics, St. Petersburg State University, St. Petersburg, Russia

11 Algorithmic Biology Lab, St. Petersburg Academic University, St. Petersburg, Russia 12Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany

13HZI - Helmholtz Centre for Infection Research, Braunschweig, Germany

Correspondence: Michael Visser (michaelwendy8@gmail.com)

Keywords: Thermophilic spore-forming anaerobes, sulfate reduction, carboxydotrophic,

Peptococcaceae, Clostridiales.

Desulfotomaculum nigrificans and D. carboxydivorans are moderately thermophilic members of the polyphyletic spore-forming genus Desulfotomaculum in the family Peptococcaceae. They are phylogenetically very closely related and belong to 'subgroup a' of the Desulfotomaculum cluster 1. D. nigrificans and D. caboxydivorans have a similar growth substrate spectrum; they can grow with glucose and fructose as electron donors in the presence of sulfate. Additionally, both species are able to ferment fructose, although fermentation of glucose is only reported for D. carboxydivorans. D. nigrificans is able to grow with 20% carbon monoxide (CO) coupled to sulfate reduction, while D. carboxydivorans can grow at 100% CO with and without sulfate. Hydrogen is produced during growth with CO by D. carboxydivorans. Here we present a summary of the features of D. nigrificans and D. carboxydivorans together with the description of the complete genome sequencing and annotation of both strains. Moreover, we compared the genomes of both strains to reveal their differences. This comparison led us to propose a reclassification of D. carboxydivorans as a later heterotypic synonym of D. nigrificans.

Introduction

In 1965, the genus Desulfotomaculum was created for sulfate-reducing bacteria that form heat-resistant spores [1]. One of the first species that was included in this new genus was D. nigrificans Delft 74, which was originally described as "Clostridium nigrificans" by Werkman and Weaver (1927] [2]. Later, Starkey (1938] renamed it to "Sporovibrio desulfuricans" [3] before it was finally renamed as D. nigrificans [1]. D. nigrificans is a moderate thermophile that typically grows with fructose and glucose coupled to sulfate reduction [1,4]; without sulfate, only growth with fructose was observed. Utilizing sugars is rare among Desulfotomaculum species. Additionally, D. nigrificans was described to be able to grow with a number of other substrates including lactate, eth-anol, alanine, formate, and carbon monoxide (20%] coupled to sulfate reduction [5,6].

Another moderately thermophilic

Desulfotomaculum species that can grow with glucose and CO is D. carboxydivorans CO-1-SRB [6]. D. carboxydivorans was isolated from sludge in an anaerobic bioreactor treating paper mill wastewater [6] and was described to be the first sulfate-reducing bacterium able to grow at 100% CO. D. carboxydivorans converted CO in the presence and absence of sulfate and produced hydrogen during CO conversion. D. carboxydivorans can also grow with glucose. In contrast to D. nigrificans, D. carboxydivorans degrades glucose both with and without sulfate.

Phylogenetically, D. carboxydivorans is most closely related to D. nigrificans. However, D. nigrificans is not able to produce hydrogen from CO. Therefore, by comparing the genomes of these strains, the physiological differences might be explained. Here we present a summary of the features of D. nigrificans and D. carboxydivorans, together with the description of the complete genome sequencing and annotation of both strains. Moreover, we compared the genomes of both strains to reveal differences between these phylogenetically very closely related strains. This comparison led us to propose to that D. carboxydivorans is a later heterotypic synonym of D. nigrificans.

Classification and features

Comparison of the 16S rRNA gene sequences of D. carboxydivorans CO-1-SRB DSM 14880 and D. nigrificans DSM 574 revealed that the two bacteria

are highly related (99% sequence similarity]. Both strains are part of the Desulfotomaculum cluster 1 subgroup a, together with D. aeronauticum, D. putei, D. hydrothermale, "D. reducens" and D. ruminis (Figure 1].

D. nigrificans and D. carboxydivorans are Grampositive, sulfate-reducing, rod shaped bacteria with rounded ends (0.3-0.5 [j.m thick and 3-6 [j.m long [1]; 0.5-1.5 [j.m thick and 5-15 [j.m long [6], respectively] (Figure 2 and Figure 3]. They have a similar temperature range for growth and can both grow optimally at 55°C. Additional similarities can be found in the substrates used for growth. Both D. nigrificans and D. carboxydivorans can grow with fructose, glucose and alanine. These substrates are incompletely oxidized to acetate, coupled to sulfate reduction. Other suitable electron acceptors in addition to sulfate are thiosul-fate and sulfite. Neither nitrate nor elemental sulfur are used as electron acceptors.

In the absence of an electron acceptor, D. nigrificans is able to grow by fermentation of fructose and pyruvate [7]. Additionally, D. nigrificans has been reported to grow with lactate and etha-nol in syntrophic interaction with Methanobacterium thermoautotrophicum [5]. Syntrophic growth of D. carboxydivorans has never been tested. D. carboxydivorans is able to grow in the absence of an electron acceptor with CO (100%], pyruvate, lactate, glucose and fructose [6]. The cellular fatty acid patterns of the two strains were analyzed by Parshina et al. [6] and Krishnamurthi et al. [8]. Both fatty acid patterns are similar and the dominating fatty acids were identified as 16:0, iso 15:0, iso 17:0, anteiso 15:0, 18:0 and iso 16:0. Collins and Widdel [9] analyzed the respiratory lipoquinone content of D. nigrificans DSM 574 and found MK7 as the predominant isoprenoid quinone. A summary of the classification and general features of D. nigrificans and D. carboxydivorans is presented in Table 1 and 2, respectively.

Genome sequencing and annotation Genome project history

D. nigrificans and D. carboxydivorans were selected for sequencing in the DOE Joint Genome Institute Community Sequencing Program 2009, proposal 300132_795700 'Exploring the genetic and physiological diversity of Desulfotomaculum species'. They are important for their position in sub-

group a of the Desulfotomaculum cluster 1. Sequencing the complete genome of the two strains was proposed as it would allow the study of the genetic and physiological diversity within subgroup a. Furthermore, a comparison of the two genomes should reveal the genes involved in CO metabolism and the H2 production in D. carboxydivorans. The genome projects of D. nigrificans and D. carboxydivorans are listed in the

Genome OnLine Database (GOLD) [23] as project Gi03933 and Gc01783, respectively. The two complete genome sequences were deposited in Genbank. Sequencing, finishing and annotation of the two genomes were performed by the DOE Joint Genome Institute (JGI). A summary of the project information of D. nigrificans and D. carboxydivorans is shown in Table 3.

— Desulfotomaculum aeronauticum (X9 84071 Desulfotomaculum putel (AF053929)

— Desulfotomaculum hydrothermale (EF081293) -"Desulfotomaculum reducens" (C P0006121

-Desulfotomaculum ruminis (AB29414Q)

r- DesiJtfotomatidumnlgrlftcans CO-1-SRB (CP002736) — Desulfotomaculum nigrificans {X62176}

-Desulfotomaculum atkatiphitum (AFÜ97024)

-Desulfotomaculum hatophitum (U88891)

-Desulfotomaculum acetoxidans (CP0Q1720)

Desulfotomaculum sapomandens (AFI 68365) - Desulfotomaculum thermosapovorans (Z26315)

-Sporotomaculum hydroxy be nzoicum [Y14845)

-Sporotomaculum syntrophicum (A BO 76610)

-Desulfotomaculum geothermicum (Y11567}

-Desulfotomaculum gibsoniae (AJ 2944311

-Desulfotomaculum alcoholivorax (AY5487781

-Desulfotomaculum arcticum (DQ148942)

— Pelotomaculum proplonlcicum (AB 154390) Pelotomaculum 5CfflnW/(X91170)

-Cryptanaerobacter phenolicus (AY327251)

— Pelotomaculum Isophthalicicum (AB232785)

-Pelotomaculum terephthaticicum [AB 0913231

-Pelotomaculum thermoproplonicum (AB0357231

-Desulfurispora thermophila (AY5487761

— Desulfotomaculum kuznetsovii (Y11S69) Desulfotomaculum solfataricum (AY084078)

-Desulfotomaculum luciae (AF0692931

-Desulfotomaculum thermosubterraneum (DQ208688)

Desulfotomaculum salinum (AY9H8122) -Desulfotomaculum australicum (M96S65)

Desulfotomaculum thermoclstemum [U 3345 5)

Desulfotomaculum thermobenzoicum subsp. thermobenzoicum (AJ 294429) Desulfotomaculum thermobenzoicum subsp, thermosyntrophicumi AY007190) — Desulfotomaculum thermoacetoxldans (Y11573)

Figure 1. Neighbor joining tree based on 1 6S rRNA sequences showing the phylogenetic affiliation of Desulfotomaculum and related species divided in the subgroups of Desulfotomaculum cluster 1. DSM 574 and DSM 14880 are in bold type. The sequences of different Thermotogales were used as outgroup, but were pruned from the tree. Closed circles represent bootstrap values between 75 and 100%. The scale bar represents 10% sequence divergence.

MIGS ID Property Term Evidence codea

Current classification Domain Bacteria Phylum Firmicutes Class Clostridia Order Clostridiales Family Peptococcaceae Genus Desulfotomaculum Species Desulfotomaculum nigrificans Type strain Delft 74 TAS [11] TAS [12-14] TAS [15,16] TAS [17,18] TAS [17,19] TAS [17,20,21] TAS [17,20] TAS [12-14]

Gram stain negative, with a Gram-positive cell wall structure

Cell shape rods, rounded ends, sometimes paired TAS [1]

Motility Slight tumbling, peritrichous flagella TAS [1]

Sporulation oval, terminal or subterminal, slightly swelling the cell TAS [1]

Temperature range 30-70 °C TAS [1]

Optimum temperature 55 °C TAS [1]

Carbon source glucose and other carbohydrates TAS [1,4,5]

Energy source heterotrophic TAS [1,4,5]

Electron acceptor sulfate, thiosulfate and sulfite. TAS [4]

MIGS-6 Habitat soils, compost heaps, thermal spring water, spoiled foods. TAS [1]

MIGS-6.3 Salinity not reported

MIGS-22 Oxygen obligate anaerobic TAS [1]

MIGS-15 Biotic relationship free living TAS [1]

MIGS-14 Pathogenicity none TAS [1]

MIGS-4 Geographic location Delft, The Netherlands

MIGS-5 Sample collection time

MIGS-4.1 Latitude 52.011

MIGS-4.2 Longitude 4.360

MIGS-4.3 Depth not reported

Evidence codes - TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). Evidence codes are from the Gene Ontology project [22].

Table 2. Classification and general features of D. carboxydivorans DSM 14880 according to the MIGS recommendations [1 0]

MIGS ID Property

Evidence codea

MIGS-6

MIGS-6.3

MIGS-22

MIGS-15

MIGS-14

MIGS-4

MIGS-5

MIGS-4.1

MIGS-4.2

MIGS-4.3

Current classification

Gram stain

Cell shape

Motility

Sporulation

Temperature range

Optimum temperature

Carbon source

Energy source

Electron acceptor

Habitat

Salinity

Oxygen

Biotic relationship

Pathogenicity

Geographic location

Sample collection time

Latitude

Longitude

Depth_

Domain Bacteria TAS

Phylum Firmicutes TAS

Class Clostridia TAS

Order Clostridiales TAS

Family Peptococcaceae TAS

Genus Desulfotomaculum TAS

Species Desulfotomaculum carboxydivorans TAS

Type strain CO-1-SRB TAS negative, with a Gram-positive cell wall structure TAS

rods, rounded ends, sometimes paired. TAS

twisting and tumbling motion TAS

oval, terminal or subterminal TAS

30-68°C TAS

55°C TAS

100% CO, with and without sulfate TAS

hydrogenogenic and heterotrophic growth TAS

sulfate, thiosulfate and sulfite. TAS Paper mill waste water sludge

0-1 7 g NaCl l-1 TAS

obligate anaerobe TAS

free living TAS none

Eerbeek, the Netherlands TAS

1999 TAS

52.104217 TAS

6.060133 TAS

not reported_

12-14] 15,16]

17.18]

17.19] 17,20,21]

17.20] 12-14] 6]

6] 6] 6] 6] 6] 6] 6] 6]

6] 6] 6]

6] 6] 6] 6]

Evidence codes - TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). Evidence codes are from the Gene Ontology project [22].

1 \ÂIX\

Figure 3. Scanning electron microscopic photograph of DSM 14880

Growth conditions and DNA isolation

D. nigrificans and D. carboxydivorans were grown anaerobically at 55oC in bicarbonate buffered medium with lactate and sulfate as substrates [6]. DNA of cell pellets was isolated using the standard DOE-JGI CTAB method recommended by the DOE Joint Genome Institute (JGI, Walnut Creek, CA, USA]. Cells were resuspended in TE (10 mM tris; 1 mM EDTA, pH 8.0]. Subsequently, cells were lysed using lysozyme and proteinase K, and DNA was extracted and purified using CTAB and phe-

nol:chloroform:isoamylalcohol extractions. After precipitation in 2-propanol and washing in 70% ethanol, the DNA was resuspended in TE containing RNase. Following a quality and quantity check using agarose gel electrophoresis in the presence of ethidium bromide, and spectrophotometric measurement using a NanoDrop ND-1000 spectrophotometer (NanoDrop® Technologies, Wilmington, DE, USA].

Table 3. Genome sequencing project information of DSM 574 and DSM 14880.

MIGS ID

Property

Term (for DSM 574)

Term (for DSM 14880)

MIGS-31

Finishing quality

Permanent draft

Finished

MIGS-28

Libraries used

Three genomic libraries: Four genomic libraries: one 454

454 standard library, 454 PE pyrosequence standard library, two

libraries (7kb insert size), 454 PE libraries (4kb and 11 kb insert

library size), one Illumina library

one Illumina

MIGS-29 Sequencing platforms

Illumina GAii, 454 GS FLX Titanium

Illumina GAii, 454 GS FLX Titanium

MIGS-31.2

coverage

462.8 x Illumina; 35.2 x pyrosequence

116.8 x Illumina; 50.6 x pyrosequence

MIGS-30

Assemblers

Newbler version 2.3-PreRelease-June 30,2009, VELVET version 1.0.13, phrap version SPS - 4.24

Newbler version 2.3-PreRelease-June 30, 2009, VELVET version 1.0.13, phrap version SPS - 4.24

MIGS-32

MIGS-13

Gene calling method INSDC ID

Prodigal 1.4, GenePRIMP AEVP00000000

Genome Database release December 10, 2010

Genbank Date of Release February 17, 2011

GOLD ID

NCBI project ID

Gi03933

Source material identifier DSM 574T

Prodigal 1.4, GenePRIMP

CP002 736.1

August 13, 2012

May 23, 2011

Gc01783

DSM 14880T

Project relevance

Obtain insight into the phy-logenetic and physiological diversity of

Desulfotomacum species.

Obtain insight into the phylogenetic and physiological diversity of Desulfotomacum species, and hydrogenogenic CO conversion.

Genome sequencing and assembly

The genome of D nigrificans strain Delft 74 (DSM 574] was sequenced using a combination of Illumina and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website [24]. Pyrosequencing reads were assembled using the Newbler assembler (Roche]. The initial Newbler assembly consisting of 75 contigs in two scaffolds was converted into a phrap [25] assembly by making fake reads from the consensus, to collect the read pairs in the 454 paired end library. Illumina GAii sequencing data (3,053.3 Mb] was assembled with Velvet [26] and the consensus sequences were shredded into 1.5 kb overlapped fake reads and assembled together with the 454 data. The 454 draft assembly was based on 127.9 Mb 454 draft data and all of the 454 paired end data. Newbler parameters are -consed -a 50 -l 350 -g -m -ml 21. The Phred/Phrap/Consed software package [25] was used for sequence assembly and quality assessment in the subsequent finishing process. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC]. Whenever possible mis-assemblies were corrected with gap Resolution [24], Dupfinisher [27], or sequencing cloned bridging PCR fragments with subcloning. Some gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F. Chang, unpublished]. Some mis-assembly is still possible in the current assembly that consists in seven contigs and one scaffold. A total of 268 additional reactions and one shatter library were necessary to close gaps and to raise the quality of the final contigs. Illumina reads were also used to correct potential base errors and increase consensus quality using a software Polisher developed at JGI [28]. The error rate of the final genome sequence is less than 1 in 100,000. Together, the combination of the Illumina and 454 sequencing platforms provided 498.0x coverage of the genome. The final assembly contained 332,256 pyrosequence and 37,872,777 Illumina reads. The same protocol applied to the D. carboxydivorans strain CO-1-SRB (DSM 14880] genome allowed to produce finished assembly without gaps. Illumina GAii sequencing data (334.0Mb] was assembled with Velvet 0.7.63 and the 454 draft assembly was based on 138.8 MB of sequence. A total of 290 additional reactions were necessary to close some gaps and to raise the

quality of the final contigs. Illumina reads were also used to correct potential base errors and increase consensus quality using a software Polisher developed at JGI [28]. The error rate of the final genome sequence is less than 1 in 100,000. Together, the combination of the Illumina and 454 sequencing platforms provided 167.4x coverage of the genome. The final assembly contained 543,495 pyrosequence and 9,254,176 Illumina reads

Genome annotation

Genes were identified using Prodigal [29] as part of the DOE-JGI genome annotation pipeline [30], followed by a round of manual curation using the JGI GenePRIMP pipeline [31]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI] nonredundant database, UniProt, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes - Expert Review (IMG-ER] platform [32].

Genome properties

The genome of D. nigrificans and D. carboxydivorans consist of one chromosome of 3,052,787 and 2,892,255 nucleotides with a GC content of 46.28 and 46.63%, respectively (Table 4]. Of the 3,112 genes in the genome of D. nigrificans, 98 are RNA genes of which 6 16S rRNA genes. A total of 2,340 genes of the 3,014 protein coding genes are assigned to COG functional categories. The distribution of these genes into COG functional categories is presented in Table 5. The distribution of the 2,174 COG assigned genes of D. carboxydivorans into COG functional categories is also presented in Table 5. Of the 2,844 predicted genes in the D. carboxydivorans genome, 2,747 are protein coding genes and 97 RNA genes, of which 8 are 16S rRNA genes. Both strains have sets of multiple 16S rRNA genes. Within the sets and among the sets most of the genes are 99.5-99.9% identical. Each strain has one differently deviating 16S rRNA gene, the difference probably originating from differential gene loss. In addition, 3.09% of the total genes of D. carboxydivorans are identified as pseudo genes. More genome statistics of D. nigrificans and D. carboxydivorans are displayed in Table 4.

Table 4. Genome statistics of DSM 574 (A) and DSM 14880 (B)

Attribute A. Genome (total) B. Genome (total)

Value % of total Value % of total

Genome size (bp) 3,052,787 100 2,892,2 55 100.00

DNA coding region (bp) 2,595,629 85.02 2457154 84.96

DNA G+C content (bp) 1,412,511 46.2 8 1,348,537 46.63

Total genes 3,112 100 2,844 100

RNA genes 98 3.15 97 3.41

Protein-coding genes 3,014 96.85 2,747 96.59

Genes in paralog clusters 1,542 49.55 1,363 47.93

Genes assigned to COGs 2,340 75.19 2,174 76.44

Pseudo genes 1 37 4.40 88 3.09

Genes with signal peptides 582 18.70 504 17.72

Genes with transmembrane helices 721 23.17 647 22.75

Figure 4. Graphical map of the DSM 574 (upper) and DSM 14880 (lower) chromosome. In both maps one genome was compared to the other. When genes were not similar or present in the other genome it resulted in gaps. The indicated variable regions with their function can also be found in Table 6 and the supplementary data S1.

Table 5. Number of DSM 574 and DSM 14880 genes associated with the g eneral COG functional categories.

DSM 574 DSM14880

Code Description Value %agea Value %agea

J Translation 153 5.97 152 3.39

A RNA processing and modification 1 0.04 0 0.00

K Transcription 153 5.97 1 39 5.85

L Replication, recombination and repair 210 8.20 172 7.23

B Chromatin structure and dynamics 1 0.04 1 0.04

D Cell cycle control, mitosis and meiosis 45 1.76 45 1.89

Y Nuclear structure 0 0.00 0 0.00

V Defense mechanisms 22 0.86 22 0.93

T Signal transduction mechanisms 171 6.71 148 6.22

M Cell wall/membrane biogenesis 1 32 5.15 126 5.3

N Cell motility 70 2.73 68 2.86

Z Cytoskeleton 0 0.00 0 0.00

W Extracellular structures 0 0.00 0 0.00

U Intracellular trafficking and secretion 65 2.54 64 2.69

O Posttranslational modification, protein turnover, chaperones 83 3.24 85 3.57

C Energy production and conversion 217 8.47 211 8.87

G Carbohydrate transport and metabolism 125 4.88 98 4.12

E Amino acid transport and metabolism 224 8.74 216 9.08

F Nucleotide transport and metabolism 62 2.42 60 2.52

H Coenzyme transport and metabolism 134 5.23 133 5.59

I Lipid transport and metabolism 40 1.56 36 1.51

P Inorganic ion transport and metabolism 104 4.06 101 4.25

Q Secondary metabolites biosynthesis, transport and catabolism 29 1.13 27 1.14

R General function prediction only 261 10.19 250 10.51

S Function unknown 241 9.41 224 9.42

- Not in COGs 772 24.81 670 23.56

a) The total is based on the total number of protein coding genes in the annotated genome.

Table 6. Description of genes present in the variable regions depicted in Figure 4._

Variable region Functions_

Transposases, recombinases, transport proteins, isomerases, histidine kinase and threonine de-1A hydrogenase

1B 1C 1 D

Transposases, recombinases, resolvase and alcohol dehydrogenase Helicases, DNA-methylation, endonuclease and recombinase

TRAP transporter, Threonine dehydrogenase, 2 keto-4-petnenoate hydratase, sugar kinase, aldolase, glycerol dehydrogenase and mannonate dehydratase

1 E 1 F

Pilus assembly, proteases and hypothetical proteins dominate this variable region

Protease, DNA methylase, RNA polymerase, recombinase, cytochrome c biogenesis, Fe2 transport system and many hypothetical proteins

Transposase, secretory protein secB, nucleotide sugar dehydrogenase, glycosyltransferase, sugar epimerase, O-antigen ligase and copper amine oxidase

Pyruvate ferredoxin oxidareductase, transport proteins, sugar phosphate permease, threonine dehydrogenase, transporsase, DNA methylase and endonuclease

Growth inhibitor protein, terminase, phage portal protein, secretory protein, recombinase and many hypothetical proteins

Endonuclease, DNA methylase, transposase, ATP binding protein, ATPase, threonine kinase, 2A pyridoxamine 5'phosphate oxidase, ferric reductase, many hypothetical proteins and the

CODH-ECH complex

2B CRISPR-Cas

2C DNA-helicases, -methyltransferase, and -replication protein, restriction protein and many hypo-

thetical proteins

2D Urea metabolism

2E Mainly transport proteins and agmatinase

Alpha ribazole phosphatase, metal dependent phosphohydrolase, phenylacetate-CoA ligase, 2F methyltransferase, amine oxidase, aldehyde dehydrogenase, transposase, phage tail component

and many hypothetical proteins

2G Pilus associated proteins

2H Recombinase, integrase, AAA ATPase, restriction modification system, deoxyribonuclease 2I_Many transferase proteins_

Insights into the genomes Incomplete oxidation of organic compounds

D. nigrificans and D. carboxydivorans oxidize organic substrates such as lactate, pyruvate, ethanol and sugars incompletely to acetate. Both genomes have gene copies that are predicted to encode L-lactate dehydrogenases (DesniDRAFT_1264, 2906; Desca_0533) and D-lactate dehydrogenase (DesniDRAFT_0054, 1145, 1691; Desca_0863, 2222), which are involved in the oxidation of lactate to pyruvate. For incomplete oxidation of pyruvate to acetate via acetyl-CoA D. nigrificans and D. carboxydivorans have genes encoding a putative pyruvate dehydrogenase (DesniDRAFT_1250, 2504, 1245 and Desca_0770, 0146, 0775, respectively) and subsequently an acetyl-CoA synthetase (DesniDRAFT_2242 and Desca_0484, respectively). Although the two strains cannot grow with succinate, fumarate and malate as electron donors, genes to metabolize these compounds are present in both genomes. D. nigrificans and D. carboxydivorans have genes putatively coding for a fumarate reductase (DesniDRAFT_0617-15 and Desca_1387-89), fumarate hydratase

(DesniDRAFT_0612-13 and Desca_1391-92), malate dehydrogenase (DesniDRAFT_0618 and Desca_1386), and a pyruvate carboxylase (DesniDRAFT_1477-78 and Desca_2116-17) that might be involved in the oxidation of succinate, fumarate and malate to pyruvate. For growth on ethanol, both genomes contain alcohol dehydro-genases (DesniDRAFT_0051, 0320, 0326, 0367, 1219, 2126, 2174, 2779; Desca_0375, 0418, 1671, 1913, 1943, 2553, 2558) and acetaldehyde dehydrogenases (DesniDRAFT_0038; Desca_1928).

For sulfate reducers to oxidize acetate to CO2, either the complete tricarboxylic acid (TCA) cycle or acetyl-CoA pathway has to be present [33]. Since D. nigrificans and D. carboxydivorans cannot grow with acetate, it was expected neither strain would possess a complete TCA cycle; which was verified by a lack of the putative genes that code for ATP-dependent citrate synthase, aconitase, and isocitrate dehydrogenase. All genes coding for the acetyl-CoA pathway are present in both genomes, except for the genes encoding the acetyl-CoA syn-thase subunit and the FeS-protein large and small subunit. Probably the gene coding for the acetyl-CoA synthetase is also involved in the acetyl-CoA production from acetate and coenzyme A.

Sugar metabolism

D. nigrificans and D. carboxydivorans are able to utilize glucose and fructose as electron donors in the presence of sulfate. Additionally, both species are able to ferment fructose, although fermentation of glucose is only reported for D. carboxydivorans [5,6]. The capability of utilizing sugars for growth is unusual among Desulfotomaculum species. The other Desulfotomaculum species that belong to cluster I, sub group a, D. ruminis, D. aeronauticum, D. putei and D. hydrothermale (with the exception of "D. reducens"), are not able to grow with glucose or fructose [34-36]. Glucose metabolism in D. nigrificans was studied before [4]. Akagi and Jackson showed that the majority of the glucose was degraded by the Embden-Meyerhof-Parnas pathway and in several instances the glucose followed the Entner-Doudoroff pathway [4]. The Embden-Meyerhof-Parnas pathway and the pentose phosphate pathway are predicted to be complete in the genome of D. nigrificans and D. carboxydivorans. However, genes coding for the 6-phosphogluconate dehydratase and the 2-keto-3-deoxy-6-phosphogluconate aldolase, the two characteristic enzymes of the Entner-Doudoroff pathway, were not found in the genome of D. nigrificans and D. carboxydivorans. A phosphotransferase system (PTS) for glucose-specific transport was not found in either genome. Such a system is present in the genome of the glucose-utilizer D. reducens (Dred_0332). Genes coding for the fructose-specific PTS are present in an operon structure in D. nigrificans (DesniDRAFT_2286 and 2291) and D. carboxydivorans (Desca_2698 and 2703). This system is likely involved in fructose uptake and its subsequent phosphorylation to fructose-1-phosphate. The fructose-1-phosphate thus formed can be further phosphorylated by 1-phosphofructokinase to fructose-1,6-bisphosphate (DesniDRAFT_2290 and Desca_2702).

Unlike D. nigrificans and D. carboxydivorans, D. ruminis and D. kuznetsovii are not able to grow with glucose or fructose. However, they have the genes that code for all the enzymes involved in the Embden-Meyerhof-Parnas pathway present in their genome. What is missing in their genome is the PTS for fructose-specific transport. This suggests that the absence of this PTS system prevents the use of fructose for growth.

Growth on one-carbon substrates

D. nigrificans and D. carboxydivorans can grow with formate plus sulfate in the presence of yeast extract and acetate as a carbon source. Since the genomes lack a complete acetyl-CoA pathway, D. nigrificans and D. carboxydivorans are not able to produce acetyl-CoA from formate and need an additional carbon source. The two genomes have similar genes that putatively code for three formate dehydrogenases (FDHs). The first FDH consists of an alpha subunit (DesniDRAFT_0989, Desca_1018), which is located next to a hydrogenase (DesniDRAFT_0990, Desca_1017) and a flavoprotein (DesniDRAFT_0988 and Desca_1019). The flavoprotein has one predicted transmembrane helix. Therefore, these genes might code for one intracellular membrane associated FDH. The second FDH gene cluster (DesniDRAFT_1389-1392, Desca_2053-2055) pu-tatively codes for a confurcating cytoplasmic FDH. The third is predicted to code for an extracellular FDH (DesniDRAFT_1396-1397, Desca_2059-2060) associated with the membrane by a proposed 10 transmembrane helixes containing protein (DesniDRAFT_1395, Desca_2058). BLAST results and orthologous BLAST analysis [37] indicate that this transmembrane helix protein is orthologous to cytochrome b. Therefore, electron transport from this FDH might go through cytochrome b. D. nigrificans and D. carboxydivorans are able to grow with CO in the presence of yeast extract. However, D. nigrificans grows with up to 20% of CO coupled to sulfate reduction, while D. carboxydivorans can grow with 100% CO with and without sulfate. These physiological differences should also be visible in the genome for the genes involved with carbon monoxide dehydrogenase (CODH). Figure 5 shows the organization of the CODH catalytic subunit (cooS) and neighboring genes in D. nigrificans and D. carboxydivorans. D. nigrificans has two cooS genes in the genome (DesniDRAFT_0854 and 1323) while D. carboxydivorans has three (Desca_0349, 1148, 1990). The organization of the cooS and neighboring genes in D. nigrificans is similar to that of two of the cooS and neighboring genes in D. carboxydivorans. However, one cooS gene cluster in the D. carboxydivorans genome cannot be found in the genome of D. nigrificans. The genes in this cluster are similar to genes described to be involved in the H2 production from CO oxidation

[38-41]. Carboxydothermus hydrogenoformans was the first bacterium described to have multiple cooS genes, one of which is united in a cluster with hydrogenase genes [40]. The hydrogenase module of this gene cluster represents a membrane-bound energy-converting hydrogenase (ECH) capable of energizing the membrane by proton translocation. Among sequenced Desulfotomaculum species, only D. carboxydivorans, D. acetoxidans, and D. ruminis possess putative genes coding for ECHs. However, in the latter two genomes, ECH encoding genes do not cluster with cooS genes. Earlier analysis showed that clustering of cooS genes and ECH genes is a characteristic feature of hydrogenogenic carboxydotrophs [42]. The presence of the putative ECH-cooS gene cluster in D. carboxydivorans explains its ability to grow hydrogenogenically on CO.

In D. nigrificans there are no CODH involved genes in close proximity of the cooS genes, apart from one cooC gene (DesniDRAFT_0855). Apparently, this is sufficient for D. nigrificans to grow with 20% of CO coupled to sulfate reduction. However, D. ruminis, another Desulfotomaculum species in cluster 1a (Figure 1) of which the genome was recently described [43], also has the cooS gene (Desru_0859) downstream of a transcriptional regulator (Desru_0858) and upstream of the cooC gene (Desru_0860) but that bacterium is not able to grow on CO and sulfate. The reason for this is not yet clear.

A cluster of nitrogenase genes (Dtox_1023 to 1030) has been described in the genome of Desulfotomaculum acetoxidans [44]. In the genomes of D. nigrificans and D. carboxydivorans very similar gene clusters occur (DesniDRAFT_0869-0858 and Desca_1134-1144). Notably, in both cases there are cooS genes in the vicinity (DesniDRAFT_0854 and Desca_1148). They are located on another DNA strand and are convergently directed. Since the low-potential carbon monoxide seems to be a good electron donor for nitrogen fixation, this proximity might be more than mere coincidence. This would suggest that small amounts of CO could be oxidized by D. nigrificans in the absence of sulfate. D. ruminis also has a similar gene cluster (Desru_3454-3445). However, in contrast to the genomes of D. nigrificans and D. carboxydivorans no cooS gene is nearby in the genome of D. ruminis.

Figure 5. Organization of cooS and neighboring genes in DSM 574 (A) and DSM 14880 (B). Abbreviations: HP, hypothetical protein; TR, transcriptional regulator.

Methyltransferase genes as present in D. kuznetsovii that might point to possible growth with methanol or methylated amines were not found in the genomes of D. nigrificans and D. carboxydivorans. These two strains accordingly, do not grow with methanol. Growth on methylated amines were never tested, but the genome suggests there is no growth possible with these compounds.

Hydrogen metabolism

D. nigrificans and D. carboxydivorans have a similar hydrogenase composition that is dominated by [FeFe] hydrogenases, as observed in other Desulfotomaculum spp. Each of the two bacteria has 9 [FeFe] hydrogenases, divided in the following groups: Three copies of trimeric bifurcating

hydrogenases (DesniDRAFT_0775-0777,

DesniDRAFT_0770-0772 and DesniDRAFT_ 13311333; Desca_1224-1226, Desca_1230-1232 and Desca_1996-1998); two copies of a monomeric hydrogenase (DesniDRAFT_0646 and DesniDRAFT_0308; Desca_1356 and Desca_1680); one HsfB-type hydrogenase encoding a PASsensing domain that is likely involved in sensing and regulation (DesniDRAFT_0986 and Desca_1021); one hydrogenase that is part of a 5-gene operon also encoding one membrane protein and two flavin-dependent oxidoreductases (DesniDRAFT_1073-1077 and Desca_0931-0935); and finally two copies of a membrane-associated hydrogenase (DesniDRAFT_1068-1070 and DesniDRAFT_2001-2003; Desca_0940-0938 and Desca_2453-2455). The catalytic subunit

(DesniDRAFT_1068, 2001 and Desca_0940, 2453) of this hydrogenase contains a tat signal motif, which suggests that the hydrogenase complex is positioned extracellular. Moreover, the membrane associated subunit is a 10 transmembrane helix containing protein that is orthologous to cyto-chrome b. This is similar to the extracellular FDH.

The high number of hydrogenases in the genomes of the two bacteria indicate a high metabolic flexibility. This is important for changing growth strategies, from, for example, sulfate respiration to syntrophic growth. A syntrophic co-culture of D. nigrificans and Methanobacterium

thermoautotrophicum on lactate and ethanol was described [5]. Syntrophic consortia are able to grow from very small free energy changes due to their ability to overcome thermodynamically difficult reactions. Reverse electron transfer is an essential part of this. The genes coding for the bifurcating hydrogenases and the confurcating formate dehydrogenase in the D. nigrificans genome are therefore likely candidates to be involved in syntrophic growth on lactate and ethanol.

A membrane-associated ECH is present only in D. carboxydivorans, as mentioned above, and no other [NiFe] hydrogenases are present. Other membrane associated complexes found in the genome of D. nigrificans and D. carboxydivorans are complex I (DesniDRAFT_0902-0892 and Desca_1110-1120) and a H+-pumping membrane-bound pyrophosphatase (DesniDRAFT_2060 and Desca_2506).

Electron acceptor metabolism

The genes for the assimilatory sulfate reduction are organized in an identical way in D. nigrificans and D. carboxydivorans. ATP-sulfurylase (DesniDRAFT_ 1837, Desca_2237) is followed by adenosine-5'-phosphosulfate (APS) reductase (DesniDRAFT_1836-1835, Desca_2378-2377), and the QmoAB complex (DesniDRAFT_1834-1833, Desca_ 2376-2375). A qmoC gene is absent but seems to be substituted by heterodisulfide reductases (Hdr) CB (DesniDRAFT _1838-1839, Desca_ 2381-2380). This organization is also found in D. ruminis and D. reducens. The position of the HdrCB is switched to the other side in D. acetoxidans, D. gibsoniae, D. alcoholicoviorans, Desulfurispora thermophila, and Desulfarculus baarsii (which owns a Gram-positive aprBA [45]). In contrast to these organisms, D. kuznetsovii, Ammonifex degensii, Desulfovirgula thermocuniculi,

and Gram-negative sulfate-reducing bacteria which posses a Gram-positive aprBA [45] like Desulfomonile tiedjei and Syntrophobacter fumaroxidans have a complete qmoABC complex (for D. kuznetsovii: Desku_ 1075, Desku_1076, Desku_1078).

The genes for the dissimilatory sulfite reductase found and their organization are identical to all other six Desulfotomaculum genomes published so far and most other Gram-positive sulfate-reducing bacteria. The dsrAB genes (DesniDRAFT_2256-2255, Desca_2666-2665) are linked to a dsrD gene (DesniDRAFT_2254, Desca_2664). Both organisms also contain a truncated DsrMK complex [46](DesniDRAFT_2267-2268, Desca_2678-2679) which is linked to a dsrC gene (DesniDRAFT_2266, Desca_2677) as it was found in D. ruminis [43]. This truncated DsrMK is generally found in Grampositive sulfate-reducing bacteria and not restricted to members of the genus Desulfotomaculum.

D. nigrificans and D. carboxydivorans lack nitrate reduction genes for reduction of nitrate to N2. Nitrate reductase, nitric-oxide forming nitrite reductase, nitric-oxide reductase and nitrous-oxide reductase are all absent in both genomes. However, a nitrite/sulphite reductase (DesniDRAFT_ 1001, 2506; Desca_0162, 1181) and an ammonia forming nitrite reductase (DesniDRAFT_0204; Desca_2313) are present in the genome of D. nigrificans and D. carboxydivorans. No taurine degradation pathway was detected in the genome of either strain, but it was described for the closely related D. ruminis [43].

Fumarate reductases

Using fumarate as an electron acceptor for growth of D. nigrificans and D. carboxydivorans has not been tested yet. However, a fumarate reductase is present in the genomes of the two bacteria. The three genes encode for a FAD containing catalytic subunit (DesniDRAFT_0617; Desca_1387), an iron sulfur containing subunit (DesniDRAFT_0616; Desca_1388), and a membrane associated cytochrome b (DesniDRAFT_0615 and Desca_1389). This cytochrome b protein might perform an electron interaction with the cytochrome b of the extracellular FDH (Figure 6, panel B). This interaction could occur as described in Wolinella succinogenes, where fumarate can be used as an electron acceptor for growth on formate [47].

Figure 6. Schematic representation of putative formate dehydrogenases in the genome of DSM 574 and DSM 14880 (A). Including the hypothesized electron interaction of the putative extracellular membrane bound formate dehydrogenase with the putative fumarate reductase (B). The electron acceptor fumarate is reduced to succinate by using formate as an electron donor. Gene locus tag numbers and -, |S, and Y-subunits are depicted. Moreover, predicted iron-sulfur clusters and other metal-binding sites are indicated.

Comparative genomics

Distinct genes in Desulfotomaculum carboxydivorans and D. nigrificans

To reveal genomic differences between these two very closely related species, a bidirectional BLAST of the protein coding genes was performed. BLAST analyses were performed using standard settings and best hits were filtered for 70% sequence coverage and 40% identity (supplementary data S1). A total of 2,529 homologous genes were found (Figure 7). The distinct genes were screened for operon structure and function, revealing genes involved in CRISP R, urea metabolism and hydrogenogenic CO metabolism in D. carboxydivorans.

CRISPR genes in D. carboxydivorans were found to have low sequence coverage and or identity with genes in the D. nigrificans genome (Figure 3). These genes involved two CRISPR-Cas systems, which we classified as a I-C subtype (Desca_0534-0540) and a III-A subtype (Desca_0572-0576). D. nigrificans has one CRISPR-Cas system subtype, I-A (DesniD RAFT_2444-2452), which is also present in D. carboxydivorans (Desca_0726-0734). The presence of multiple CRISPR-Cas systems and the

occurrence of the different subtypes in one strain has been described previously [49,50] and shows that the co-occurrence of subtype I-A with I-C and III-A is a common feature. However, it also shows that D. carboxydivorans is part of the 2% of bacteria that have a III-A subtype without a III-B subtype.

The genome of D. carboxydivorans also contains genes coding for a urease (Desca_0743-0749) and urea transport (Desca_0738-0742) (Figure 3). Urease catalyzes the reaction of urea to CO2 and ammonia. Urea is very common in the environment and is a nitrogen source for many bacteria [51]. The genome of D. nigrificans lacks the genes coding for an urease, which indicates that D. nigrificans is relatively more restricted regarding its nitrogen source. Other interesting genes that are present in the D. carboxydivorans genome and not in the D. nigrificans genome are genes involved in the carbon monoxide dehydrogenase (CODH) and hydrogenase as described above.

DSM 574(3014)

Figure 7. Venn diagram showing a comparison of the protein coding genes of DSM 574 and DSM 14880. The number of overlapping protein coding genes is given inside the areas of the circles and the total number of derived protein sequences used for each strain is shown in parentheses. The figure was created using the program Venn diagram plotter available from the Pacific Northwest National Laboratory Software Distribution Center [48].

Taxonomic conclusions

The overall similarity of the genome sequences of the type strains of D. nigrificans and D. carboxydivorans was estimated by using the Genome-To-Genome Distance Calculator (GGDC) as described previously [52]. This program calculates DNA-DNA similarity values by comparing the genomes to obtain high-scoring segment pairs (HSPs) and inferring distances from a set of three formulas (1, HSP length/total length; 2, identi-ties/HSP length; 3, identities/total length). According to the GGDC the average estimated DNADNA similarity value between the two type strains is 86.5 ± 5.5% and thus clearly above 70%, which is the widely accepted threshold value for assigning strains to the same species [53]. The high similarity of the genome sequences of both type strains was further supported by the average nu-cleotide identity of shared genes (ANI), which proved to be above 99%. This ANI value is much higher than the 95 to 96% value shown to correspond to the 70% DNA-DNA hybridization level [54]. Moreover, the two strains have almost identical 16S rRNA gene sequences (>99%) and a high number of shared genes (Figure 7). It should be mentioned that the previously reported and deposited rRNA gene sequence of D. nigrificans DSM 574 contained a lot ambiguities and some missing nucleotides, which are counted as mismatches by BLAST. Therefore, we reanalyzed the rRNA gene sequences of D. nigrificans deposited in the NCIMB culture collections and confirmed the identity of the rRNA gene sequence found in the genome of DSM 574. We propose that the species should be united under one name. According to the rules of priority as given by the Bacteriological Code [55] the name D. nigrificans should be used for the unified taxon, with D. carboxydivorans as a later het-erotypic synonym.

Acknowledgements

We would like to gratefully acknowledge the help of Christine Munk and Megan Lu for finishing the genome sequence (both at JGI). The work conducted by the U.S. Department of Energy Joint Genome Institute is supported by the Office of Science of the U.S. Department

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