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 Visser1Email author,
- Sofiya N. Parshina2,
- Joana I. Alves3,
- Diana Z. Sousa1, 3,
- Inês A. C. Pereira4,
- Gerard Muyzer5,
- Jan Kuever6,
- Alexander V. Lebedinsky2,
- Jasper J. Koehorst7,
- Petra Worm1,
- Caroline M. Plugge1,
- Peter J. Schaap7,
- Lynne A. Goodwin8, 9,
- Alla Lapidus10, 11,
- Nikos C. Kyrpides8,
- Janine C. Detter9,
- Tanja Woyke8,
- Patrick Chain8, 9,
- Karen W. Davenport8, 9,
- Stefan Spring12,
- Manfred Rohde13,
- Hans Peter Klenk12 and
- Alfons J. M. Stams1, 3
© The Author(s) 2014
Published: 15 June 2014
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. carboxydivorans 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.
KeywordsThermophilic spore-forming anaerobes sulfate reduction carboxydotrophic Peptococcaceae Clostridiales
In 1965, the genus Desulfotomaculum was created for sulfate-reducing bacteria that form heat-resistant spores . 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) . Later, Starkey (1938) renamed it to “Sporovibrio desulfuricans”  before it was finally renamed as D. nigrificans . 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, ethanol, 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 . D. carboxydivorans was isolated from sludge in an anaerobic bioreactor treating paper mill wastewater  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
Classification and general features of D. nigrificans DSM 574 according to the MIGS recommendations 
Species Desulfotomaculum nigrificans
Type strain Delft 74
negative, with a Gram-positive cell wall structure
rods, rounded ends, sometimes paired
Slight tumbling, peritrichous flagella
oval, terminal or subterminal, slightly swelling the cell
glucose and other carbohydrates
sulfate, thiosulfate and sulfite.
soils, compost heaps, thermal spring water, spoiled foods.
Delft, The Netherlands
Sample collection time
Classification and general features of D. carboxydivorans DSM 14880 according to the MIGS recommendations 
Species Desulfotomaculum carboxydivorans
Type strain CO-1-SRB
negative, with a Gram-positive cell wall structure
rods, rounded ends, sometimes paired.
twisting and tumbling motion
oval, terminal or subterminal
100% CO, with and without sulfate
hydrogenogenic and heterotrophic growth
sulfate, thiosulfate and sulfite.
Paper mill waste water sludge
0–17 g NaCl l−1
Eerbeek, the Netherlands
Sample collection time
Genome sequencing and annotation
Genome project history
Genome sequencing project information of DSM 574 and DSM 14880.
Term (for DSM 574)
Term (for DSM 14880)
Three genomic libraries: 454 standard library, 454 PE libraries (7kb insert size), one Illumina library
Four genomic libraries: one 454 pyrosequence standard library, two 454 PE libraries (4kb and 11 kb insert size), one Illumina library
Illumina GAii, 454 GS FLX Titanium
Illumina GAii, 454 GS FLX Titanium
462.8 × Illumina; 35.2 × pyrosequence
116.8 × Illumina; 50.6 × pyrosequence
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
Gene calling method
Prodigal 1.4, GenePRIMP
Prodigal 1.4, GenePRIMP
Genome Database release
December 10, 2010
August 13, 2012
Genbank Date of Release
February 17, 2011
May 23, 2011
NCBI project ID
Source material identifier
Obtain insight into the phylogenetic and physiological diversity of Desulfotomacum species.
Obtain insight into the phylogenetic and physiological diversity of Desulfotomacum species, and hydrogenogenic CO conversion.
Growth conditions and DNA isolation
D. nigrificans and D. carboxydivorans were grown anaerobically at 55°C in bicarbonate buffered medium with lactate and sulfate as substrates . 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 phenol: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).
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 . 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  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  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  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 gapResolution , Dupfinisher , 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 . 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 498.0× 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 . 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 167.4× coverage of the genome. The final assembly contained 543,495 pyrosequence and 9,254,176 Illumina reads
Genes were identified using Prodigal  as part of the DOE-JGI genome annotation pipeline , followed by a round of manual curation using the JGI GenePRIMP pipeline . 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 .
Genome statistics of DSM 574 (A) and DSM 14880 (B)
A. Genome (total)
B. Genome (total)
% of total
% of total
Genome size (bp)
DNA coding region (bp)
DNA G+C content (bp)
Genes in paralog clusters
Genes assigned to COGs
Genes with signal peptides
Genes with transmembrane helices
Number of DSM 574 and DSM 14880 genes associated with the general COG functional categories.
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, mitosis and meiosis
Signal transduction mechanisms
Cell wall/membrane biogenesis
Intracellular trafficking and secretion
Posttranslational modification, protein turnover, chaperones
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
General function prediction only
Not in COGs
Description of genes present in the variable regions depicted in Figure 4.
Transposases, recombinases, transport proteins, isomerases, histidine kinase and threonine dehydrogenase
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
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, pyridoxamine 5′phosphate oxidase, ferric reductase, many hypothetical proteins and the CODH-ECH complex
DNA-helicases, -methyltransferase, and -replication protein, restriction protein and many hypothetical proteins
Mainly transport proteins and agmatinase
Alpha ribazole phosphatase, metal dependent phosphohydrolase, phenylacetate-CoA ligase, methyltransferase, amine oxidase, aldehyde dehydrogenase, transposase, phage tail component and many hypothetical proteins
Pilus associated proteins
Recombinase, integrase, AAA ATPase, restriction modification system, deoxyribonuclease
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 dehydrogenases (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 . 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 synthase 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.
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 . 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 . 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) putatively 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  indicate that this transmembrane helix protein is orthologous to cytochrome b. Therefore, electron transport from this FDH might go through cytochrome b.
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 , 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 . 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.
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.
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_1331-1333; 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 PAS-sensing 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 cytochrome 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 . 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 ). In contrast to these organisms, D. kuznetsovii, Ammonifex degensii, Desulfovirgula thermocuniculi, and Gram-negative sulfate-reducing bacteria which posses a Gram-positive aprBA  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 (DesniDRAFT_2267-2268, Desca_2678-2679) which is linked to a dsrC gene (DesniDRAFT_2266, Desca_2677) as it was found in D. ruminis . This truncated DsrMK is generally found in Gram-positive 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 .
Distinct genes in Desulfotomaculum carboxydivorans and D. nigrificans
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 (DesniDRAFT_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 . 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.
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 . 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, identities/HSP length; 3, identities/total length). According to the GGDC the average estimated DNA-DNA 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 . The high similarity of the genome sequences of both type strains was further supported by the average nucleotide 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 . 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  the name D. nigrificans should be used for the unified taxon, with D. carboxydivorans as a later heterotypic synonym.
Emended description of Desulfotomaculum nigrificans (Werkman and Weaver 1927) Campbell and Postgate 1965
The cells are Gram-positive, rod-shaped with rounded ends, 0.3–1.5 × 2–15 µm, single or sometimes paired. Motility with tumbling or twisting movements conferred by peritrichous flagella. Terminal or subterminal oval endospores that are slightly swelling the cells. Thermophilic and neutrophilic with an temperature optimum of 55°C. NaCl is not required for growth. The following substrates are utilized, coupled to the reduction of sulfate to sulfide: DL-lactate, pyruvate, ethanol, L-alanine, D-fructose, D-glucose. Acetate and methanol are not utilized. Substrates are incompletely oxidized to acetate. In the presence of 0.5 g/l yeast extract, lithoheterotrophic growth is possible, such as growth on H2 and CO2 with sulfate or growth on 20% CO with sulfate for D. nigrificans strain Delft 74 and growth on 100% CO with or without sulfate for strain CO-1-SRB. Suitable electron acceptors with lactate as substrate are sulfate, sulfite and thiosulfate, but not elemental sulfur or nitrate. Fermentation of pyruvate and fructose; strain CO-1-SRB is also able to ferment DL-lactate, glucose and CO. The prevalent respiratory lipoquinone is MK7 with only small amounts of MK6. The dominating cytochromes are of type b. Major cellular fatty acids are 16:0, iso 15:0, iso 17:0, anteiso 15:0, 18:0 and iso 16:0. The DNA G+C content is around 46 mol%. The type strain is Delft 74 (=NCIMB 8395 = DSM 574 = ATCC 19998 = NBRC 13698).
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 of Energy under Contract No. DE-AC02-05CH11231, and was also supported by grants CW-TOP 700.55.343 and ALW 819.02.014 of the Netherlands Science Foundation (NWO) and grant 323009 of the European Research Council.
- Campbell LL, Postgate JR. Classification of the spore-forming sulfate-reducing bacteria. Bacteriol Rev 1965; 29:359–363. PubMedPubMed CentralPubMedGoogle Scholar
- Werkman CH, Weaver HJ. Studies in the bacteriology of sulphur stinker spoilage of canned sweet corn. State Coll. J. Sci. 1927; 2:57–67.Google Scholar
- Starkey RL. A study of spore formation and other morphological characteristics of Vibrio desulfuricans. Arch Mikrobiol 1938; 9:268–404. http://dx.doi.org/10.1007/BF00407364View ArticleGoogle Scholar
- Akagi JM, Jackson G. Degradation of glucose by proliferating cells of Desulfotomaculum nigrificans. Appl Microbiol 1967; 15:1427–1430. PubMedPubMed CentralPubMedGoogle Scholar
- Klemps R, Cypionka H, Widdel F, Norbert P. Growth with hydrogen, and further physiological characteristics of Desulfotomaculum species. Arch Microbiol 1985; 143:203–208. http://dx.doi.org/10.1007/BF00411048View ArticleGoogle Scholar
- Parshina SN, Sipma J, Nakashimada Y, Henstra AM, Smidt H, Lysenko AM, Lens PN, Lettinga G, Stams AJ. Desulfotomaculum carboxydivorans sp. nov., a novel sulfate-reducing bacterium capable of growth at 100% CO. Int J Syst Evol Microbiol 2005; 55:2159–2165. PubMed http://dx.doi.org/10.1099/ijs.0.63780-0View ArticlePubMedGoogle Scholar
- Postgate JR. Sulfate-Free Growth of Clostridium nigrificans. J Bacteriol 1963; 85:1450–1451. PubMedPubMed CentralPubMedGoogle Scholar
- Krishnamurthi S, Spring S, Anil Kumar P, Mayilraj S, Klenk HP, Suresh K. Desulfotomaculum defluvii sp. nov., a sulfate-reducing bacterium isolated from the subsurface environment of a landfill. Int J Syst Evol Microbiol 2012. PubMed
- Collins M, Weddel F. Respiratory quinones of sulphate-reducing and sulphur-reducing bacteria: a systematic investigation. Syst Appl Microbiol 1986; 8:8–18. http://dx.doi.org/10.1016/S0723-2020(86)80141-2View ArticleGoogle Scholar
- Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ, Angiuoli SV, et al. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol 2008; 26:541–547. PubMed http://dx.doi.org/10.1038/nbt1360PubMed CentralView ArticlePubMedGoogle Scholar
- Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA 1990; 87:4576–4579. PubMed http://dx.doi.org/10.1073/pnas.87.12.4576PubMed CentralView ArticlePubMedGoogle Scholar
- Gibbons NE, Murray RGE. Proposals Concerning the Higher Taxa of Bacteria. Int J Syst Bacteriol 1978; 28:1–6. http://dx.doi.org/10.1099/00207713-28-1-1View ArticleGoogle Scholar
- Garrity GM, Holt JG. The Road Map to the Manual. In: Garrity GM, Boone DR, Castenholz RW (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume 1, Springer, New York, 2001, p. 119–169.View ArticleGoogle Scholar
- Murray RGE. The Higher Taxa, or, a Place for Everything…? In: Holt JG (ed), Bergey’s Manual of Systematic Bacteriology, First Edition, Volume 1, The Williams and Wilkins Co., Baltimore, 1984, p. 31–34.Google Scholar
- List Editor. List of new names and new combinations previously effectively, but not validly, published. List no. 132. Int J Syst Evol Microbiol 2010; 60:469–472. http://dx.doi.org/10.1099/ijs.0.022855-0
- Rainey FA. Class II. Clostridia class nov. In: De Vos P, Garrity G, Jones D, Krieg NR, Ludwig W, Rainey FA, Schleifer KH, Whitman WB (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume 3, Springer-Verlag, New York, 2009, p. 736.Google Scholar
- Skerman VBD, McGowan V, Sneath PHA. Approved Lists of Bacterial Names. Int J Syst Bacteriol 1980; 30:225–420. http://dx.doi.org/10.1099/00207713-30-1-225View ArticleGoogle Scholar
- Prévot AR. In: Hauderoy P, Ehringer G, Guillot G, Magrou. J., Prévot AR, Rosset D, Urbain A (eds), Dictionnaire des Bactéries Pathogènes, Second Edition, Masson et Cie, Paris, 1953, p. 1–692.Google Scholar
- Rogosa M. Peptococcaceae, a new family to include the Gram-positive, anaerobic cocci of the genera Peptococcus, Peptostreptococcus and Ruminococcus. Int J Syst Bacteriol 1971; 21:234–237. http://dx.doi.org/10.1099/00207713-21-3-234View ArticleGoogle Scholar
- Campbell LL, Postgate JR. Classification of the spore-forming sulfate-reducing bacteria. Bacteriol Rev 1965; 29:359–363. PubMedPubMed CentralPubMedGoogle Scholar
- Campbell LL. Genus IV. Desulfotomaculum Campbell and Postgate 1965, 361. In: Buchanan RE, Gibbons NE (eds), Bergey’s Manual of Determinative Bacteriology, Eighth Edition, The Williams and Wilkins Co., Baltimore, 1974, p. 572–573.Google Scholar
- Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 2000; 25:25–29. PubMed http://dx.doi.org/10.1038/75556PubMed CentralView ArticlePubMedGoogle Scholar
- Pagani I, Liolios K, Jansson J, Chen IM, Smirnova T, Nosrat B, Markowitz VM, Kyrpides NC. The Genomes OnLine Database (GOLD) v.4: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res 2012; 40:D571–D579. PubMed http://dx.doi.org/10.1093/nar/gkr1100PubMed CentralView ArticlePubMedGoogle Scholar
- JGI website. http://www.jgi.doe.gov/.
- The Phred/Phrap/Consed software package. http://www.phrap.com.
- Zerbino DR, Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 2008; 18:821–829. PubMed http://dx.doi.org/10.1101/gr.074492.107PubMed CentralView ArticlePubMedGoogle Scholar
- Han C, Chain P. Finishing repeat regions automatically with Dupfinisher. In: H.R. A, H. V, editors 2006 June 26–29, 2006. CSREA Press. p 141–6.
- Lapidus A, LaButti K, Foster B, Lowry S, Trong S, Goltsman E. POLISHER: An effective tool for using ultra short reads in microbial genome assembly and finishing. Marco Island, FL: AGBT; 2008.Google Scholar
- Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 2010; 11:119. PubMed http://dx.doi.org/10.1186/1471-2105-11-119PubMed CentralView ArticlePubMedGoogle Scholar
- Mavromatis K, Ivanova NN, Chen IM, Szeto E, Markowitz VM, Kyrpides NC. The DOE-JGI Standard operating procedure for the annotations of microbial genomes. Stand Genomic Sci 2009; 1:63–67. PubMed http://dx.doi.org/10.4056/sigs.632PubMed CentralView ArticlePubMedGoogle Scholar
- Pati A, Ivanova NN, Mikhailova N, Ovchinnikova G, Hooper SD, Lykidis A, Kyrpides NC. GenePRIMP: a gene prediction improvement pipeline for prokaryotic genomes. Nat Methods 2010; 7:455–457. PubMed http://dx.doi.org/10.1038/nmeth.1457View ArticlePubMedGoogle Scholar
- Markowitz VM, Mavromatis K, Ivanova NN, Chen IM, Chu K, Kyrpides NC. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics 2009; 25:2271–2278. PubMed http://dx.doi.org/10.1093/bioinformatics/btp393View ArticlePubMedGoogle Scholar
- Goevert D, Conrad R. Carbon isotope fractionation by sulfate-reducing bacteria using different pathways for the oxidation of acetate. Environ Sci Technol 2008; 42:7813–7817. PubMed http://dx.doi.org/10.1021/es800308zView ArticlePubMedGoogle Scholar
- Hagenauer A, Hippe H, Rainey FA. Desulfotomaculum aeronauticum sp. nov., a Sporeforming, Thiosulfate-Reducing Bacterium from Corroded Aluminium Alloy in an Aircraft. Syst Appl Microbiol 1997; 20:65–71. http://dx.doi.org/10.1016/S0723-2020(97)80049-5View ArticleGoogle Scholar
- Haouari O, Fardeau ML, Cayol JL, Casiot C, Elbaz-Poulichet F, Hamdi M, Joseph M, Ollivier B. Desulfotomaculum hydrothermale sp. nov., a thermophilic sulfate-reducing bacterium isolated from a terrestrial Tunisian hot spring. Int J Syst Evol Microbiol 2008; 58:2529–2535. PubMed http://dx.doi.org/10.1099/ijs.0.65339-0View ArticlePubMedGoogle Scholar
- Liu Y, Karnauchow TM, Jarrell KF, Balkwill DL, Drake GR, Ringelberg D, Clarno R, Boone DR. Description of two new thermophilic Desulfotomaculum spp., Desulfotomaculum putei sp. nov., from a deep terrestrial subsurface, and Desulfotomaculum luciae sp. nov., from a hot spring. Int J Syst Bacteriol 1997; 47:615–621. http://dx.doi.org/10.1099/00207713-47-3-615View ArticleGoogle Scholar
- Zhou Y, Landweber LF. BLASTO: a tool for searching orthologous groups. Nucleic Acids Res 2007;35(Web Server issue):W678–82.PubMed CentralView ArticlePubMedGoogle Scholar
- Hedderich R. Energy-converting [NiFe] hydrogenases from archaea and extremophiles: ancestors of complex I. J Bioenerg Biomembr 2004; 36:65–75. PubMed http://dx.doi.org/10.1023/B:JOBB.0000019599.43969.33View ArticlePubMedGoogle Scholar
- Meuer J, Bartoschek S, Koch J, Kunkel A, Hedderich R. Purification and catalytic properties of Ech hydrogenase from Methanosarcina barkeri. Eur J Biochem 1999; 265:325–335. PubMed http://dx.doi.org/10.1046/j.1432-1327.1999.00738.xView ArticlePubMedGoogle Scholar
- Wu M, Ren Q, Durkin AS, Daugherty SC, Brinkac LM, Dodson RJ, Madupu R, Sullivan SA, Kolonay JF, Haft DH, et al. Life in hot carbon monoxide: the complete genome sequence of Carboxydothermus hydrogenoformans Z-2901. PLoS Genet 2005; 1:e65. PubMed http://dx.doi.org/10.1371/journal.pgen.0010065PubMed CentralView ArticlePubMedGoogle Scholar
- Fox JD, He Y, Shelver D, Roberts GP, Ludden PW. Characterization of the region encoding the CO-induced hydrogenase of Rhodospirillum rubrum. J Bacteriol 1996; 178:6200–6208. PubMedPubMed CentralPubMedGoogle Scholar
- Sokolova TG, Henstra AM, Sipma J, Parshina SN, Stams AJ, Lebedinsky AV. Diversity and ecophysiological features of thermophilic carboxydotrophic anaerobes. FEMS Microbiol Ecol 2009; 68:131–141. PubMed http://dx.doi.org/10.1111/j.1574-6941.2009.00663.xView ArticlePubMedGoogle Scholar
- Spring S, Visser M, Lu M, Copeland A, Lapidus A, Lucas S, Cheng JF, Han C, Tapia R, Goodwin LA, et al. Complete genome sequence of the sulfate-reducing firmicute Desulfotomaculum ruminis type strain (DL(T)). Stand Genomic Sci 2012; 7:294–309. PubMed http://dx.doi.org/10.4056/sigs.3226659View ArticleGoogle Scholar
- Spring S, Lapidus A, Schroder M, Gleim D, Sims D, Meincke L, Glavina Del Rio T, Tice H, Copeland A, Cheng JF, et al. Complete genome sequence of Desulfotomaculum acetoxidans type strain (5575). Stand Genomic Sci 2009; 1:242–253. PubMed http://dx.doi.org/10.4056/sigs.39508PubMed CentralView ArticlePubMedGoogle Scholar
- Meyer B, Kuever J. Phylogeny of the alpha and beta subunits of the dissimilatory adenosine-5′-phosphosulfate (APS) reductase from sulfate-reducing prokaryotes—origin and evolution of the dissimilatory sulfate-reduction pathway. Microbiology 2007; 153:2026–2044. PubMed http://dx.doi.org/10.1099/mic.0.2006/003152-0View ArticlePubMedGoogle Scholar
- Junier P, Junier T, Podell S, Sims DR, Detter JC, Lykidis A, Han CS, Wigginton NS, Gaasterland T, Bernier-Latmani R. The genome of the Grampositive metal- and sulfate-reducing bacterium Desulfotomaculum reducens strain MI-1. Environ Microbiol 2010; 12:2738–2754. PubMedPubMedGoogle Scholar
- Kroger A, Biel S, Simon J, Gross R, Unden G, Lancaster CR. Fumarate respiration of Wolinella succinogenes: enzymology, energetics and coupling mechanism. Biochim Biophys Acta 2002; 1553:23–38. PubMed http://dx.doi.org/10.1016/S0005-2728(01)00234-1View ArticlePubMedGoogle Scholar
- Venn diagram plotter available from the Pacific Northwest National Laboratory Software Distribution Center: http://omics.pnl.gov.
- Staals RHJ, Brouns SJJ. Distribution and Mechanism of the Type I CRISPR-Cas Systems. In: Barrangou R, van der Oost J, editors. CRISPR-Cas Systems: Springer; 2012. p 145–96.
- Makarova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E, Horvath P, Moineau S, Mojica FJ, Wolf YI, Yakunin AF, et al. Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 2011; 9:467–477. PubMed http://dx.doi.org/10.1038/nrmicro2577View ArticlePubMedGoogle Scholar
- Mobley HL, Hausinger RP. Microbial ureases: significance, regulation, and molecular characterization. Microbiol Rev 1989; 53:85–108. PubMedPubMed CentralPubMedGoogle Scholar
- Auch AF, von Jan M, Klenk HP, Goker M. Digital DNA-DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison. Stand Genomic Sci 2010; 2:117–134. PubMed http://dx.doi.org/10.4056/sigs.531120PubMed CentralView ArticlePubMedGoogle Scholar
- Wayne LG, Brenner DJ, Colwell RR, Grimont PAD, Kandler O, Krichevsky MI, Moore LH, Moore WEC, Murray RGE, Stackebrandt E, et al. Report of the Ad-Hoc-Committee on Reconciliation of Approaches to Bacterial Systematics. Int J Syst Bacteriol 1987; 37:463–464. http://dx.doi.org/10.1099/00207713-37-4-463View ArticleGoogle Scholar
- Richter M, Rossello-Mora R. Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci USA 2009; 106:19126–19131. PubMed http://dx.doi.org/10.1073/pnas.0906412106PubMed CentralView ArticlePubMedGoogle Scholar
- In: Lapage SP, Sneath PHA, Lessel EF, Skerman VBD, Seeliger HPR, Clark WA, editors. International Code of Nomenclature of Bacteria: Bacteriological Code, 1990 Revision. Washington (DC) 1992.