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Complete genome sequence of Desulfomicrobium baculatum type strain (XT)
Standards in Genomic Sciences volume 1, pages 29–37 (2009)
Desulfomicrobium baculatum is the type species of the genus Desulfomicrobium, which is the type genus of the family Desulfomicrobiaceae. It is of phylogenetic interest because of the isolated location of the family Desulfomicrobiaceae within the order Desulfovibrionales. D. baculatum strain XT is a Gram-negative, motile, sulfate-reducing bacterium isolated from water-saturated manganese carbonate ore. It is strictly anaerobic and does not require NaCl for growth, although NaCl concentrations up to 6% (w/v) are tolerated. The metabolism is respiratory or fermentative. In the presence of sulfate, pyruvate and lactate are incompletely oxidized to acetate and CO2. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first completed genome sequence of a member of the deltaproteobacterial family Desulfomicrobiaceae, and this 3,942,657 bp long single replicon genome with its 3494 protein-coding and 72 RNA genes is part of the GenomicEncyclopedia ofBacteria andArchaea project.
Strain XT (DSM 4028 = CCUG 34229 = VKM B-1378) is the type strain of the species Desulfomicrobium baculatum, which is the type species of the genus Desulfomicrobium. Strain XT was first described as Desulfovibrio baculatus by Rozanova and Nazina [1,2], and later transferred to the novel genus Desulfomicrobium (currently containing seven species)  (Figure 1) because several phenotypic traits were not consistent with the definition of the genus Desulfovibrio. In 1998 the species epithet was corrected to D. baculatum . Three accompanying strains have been described in addition to strain XT: Strain H.L21 (DSM 2555) was isolated from anoxic intertidal sediment at the Ems-Dollard Estuary, Netherlands (16S rRNA gene accession AJ277895) , strain 5174 (DSM 17142) was isolated from a forest pond near Braunschweig, Germany (16S rRNA gene accession AJ277896) , and strain 9974 (DSM 17143) was isolated as a contaminating chemotrophic bacterium from a culture of a green sulfur bacterium designated ’ Chloropseudomonas ethylica’ N2 . These strains were tentatively affiliated with the species D. baculatum based on some phenotypic traits. Although 16S rRNA gene sequence data are now available for two strains, a definitive affiliation of strains to the species Desulfomicrobium requires supplementary DNA-DNA hybridization experiments due to the observed high similarity values of 16S rRNA gene sequences among distinct species of this genus . Other isolates and clones related to the species were isolated from production waters of a low-temperature biodegraded oil reservoir in Canada , and wastewater from penicillin G production in China (clone B19 EU234202). Screening of environmental genomic samples and surveys reported at the NCBI BLAST server indicated no closely related phylotypes that can be linked to the species. Here we present a summary classification and a set of features for D. baculatum strain XT (Table 1), together with the description of the complete genomic sequencing and annotation.
Classification and features
Cells of D. baculatum strain XT are short rods with rounded ends of 0.6 × 1–2 µm (Figure 2). Cells stain Gram-negative, are motile by a single polar flagellum, and do not form endospores. The metabolism is strictly anaerobic and can be respiratory or fermentative [3,9]. Temperature range for growth is 2–41°C (optimum 28–37°C) and NaCl concentrations of 0–6% (w/v) are tolerated (optimum 1% w/v). Sulfate, sulfite and thiosulfate are used as electron acceptors and are reduced to H2S. Nitrate is not reduced. Simple organic compounds are incompletely oxidized to acetate . Malate, fumarate and pyruvate can be fermented with succinate and acetate as end products. Carbohydrates are not fermented. Vitamins are not required for growth . D. baculatum strain 9974 (DSM 1743) is also able to use ethanol as a substrate  and sulfur as an electron acceptor . The use of ethanol as an electron donor for sulfate respiration depends on supplementing the medium with the trace elements tungstate or molybdate . Sulfate uptake in symport with sodium ions has been shown in strain 9974, unlike in other fresh water sulfate reducers which use protons . Distinctive features of D. baculatum strain XT are: (i) NaCl is not required for growth , (ii) fermentation of fumarate and malate to succinate and acetate is preferred against utilization of these substrates as electron donors for sulfate reduction , (iii) sulfur is not used as an electron acceptor and (IV) molecular nitrogen can be assimilated .
A desulfoviridin-type dissimilatory sulfite reductase, which is a hallmark feature of the genus Desulfovibrio, is absent in strain XT, however a sulfite reductase of the desulforubidin-type was reported for strain 9974 . Cells of D. baculatum strain XT contain c- and b-type cytochromes . The tetraheme cytochrome c3 of strain 9974, which is thought to play a role in sulfur reduction and the coupling of electron transfer to hydrogenases, has been analyzed in some detail using advanced biophysical methods [13–15]. Strain 9974 also contains several distinct [NiFeSe] hydrogenases that are located in different cellular compartments . The crystal structure of the periplasmic [NiFeSe] hydrogenase of this strain has been determined  and it is proposed that the selenium ion in the active center plays a role in the oxygen-tolerant hydrogen production of this enzyme, which distinguishes it from most [NiFe] hydrogenases . An active selenocysteine system for usage of the 21st amino acid has been studied in detail for D. baculatum strain 9974 [19–21]. Pyridoxal-5′-phosphate, the prosthetic group of selenocysteine synthases, is bound to a distinct lysine residue (Lys295) within the active site of the enzyme of this strain .
Figure 1 shows the phylogenetic neighborhood of D. baculatum strain XT in a 16S rRNA based tree. Analysis of the two 16S rRNA gene sequences in the genome of strain XT indicated that the two genes are almost identical (1 bp difference), and that both genes differed by one nucleotide from the previously published 16S rRNA sequence generated from DSM 4028 (AJ277894).
The cellular fatty acid patterns of D. baculatum strain XT and the accompanying strains 5174, 9974 and H.L21  were found to be dominated by anteiso- (ai) and iso-methyl branched unsaturated and saturated fatty acids. The most abundant fatty acid is iso-17:1 cis7 (24.2-28.6%), followed by 18:1 cis11 (6.4-12.2%), iso-15:0 (8.2-11.6%), ai-17:0 (4.5-8.3%), ai-15:0 (5.2-7.7%), 18:0 (3.9-7.1%) and 16:0 (3.6-5.7%). Less abundant fatty acids are iso-15:1 (3.1–4.0%), 16:1 cis7 (2.2–5.0%), ai-17:1 (2.4–4.1%), 18:1 cis9 (2.6–4.3%), iso-16:1 (0.5–2.2%), and 17:0 (0.2-0.3%). Branched chain, hydroxylated fatty acids are also present, 3-OH iso-15:0 (1.4–2.4%), 3-OH ai-15:0 (0.7–1.2%), and 3-OH iso-17:0 (1.2–2.2%), which may be derived from a lipopolysaccharide. The polar lipid composition of D. baculatum strain XT has not been investigated. The respiratory quinone composition of D. baculatum strain XT has also not been investigated, but the presence of MK-6 has been reported in D. macestii and D. norvegicum [7,27].
Genome sequencing and annotation
Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position, and is part of the Genomic Encyclopedia of Bacteria and Archaea project. The genome project is deposited in the Genomes OnLine Database  and the complete genome sequence (CP001629) is deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.
Growth conditions and DNA isolation
D. baculatum strain XT (DSM 4028) was grown in DSMZ medium 63 at 30°C. DNA was isolated from 1–1.5 g of cell paste using Qiagen Genomic 500 DNA Kit (Qiagen, Hilden, Germany) with a modified protocol for cell lysis, adding 100 µl lysozyme; 500 µl achromopeptidase, lysostaphin, mutanolysin, each, to standard lysis solution, but reducing proteinase K to 160µl, only. Incubation over night at 35°C.
Genome sequencing and assembly
The genome was sequenced using a combination of Sanger and 454 sequencing platforms. All general aspects of library construction and sequencing performed at the JGI can be found at the JGI website. 454 Pyrosequencing reads were assembled using the Newbler assembler version 1.1.02.15 (Roche). Large Newbler contigs were broken into 4,375 overlapping fragments of 1,000 bp and entered into assembly as pseudo-reads. The sequences were assigned quality scores based on Newbler consensus q-scores with modifications to account for overlap redundancy and to adjust inflated q-scores. A hybrid 454/Sanger assembly was made using the parallel phrap assembler (High Performance Software, LLC). Possible mis-assemblies were corrected with Dupfinisher or transposon bombing of bridging clones . Gaps between contigs were closed by editing in Consed, custom primer walk or PCR amplification. 731 Sanger finishing reads were produced to close gaps, to resolve repetitive regions, and to raise the quality of the finished sequence. The error rate of the completed genome sequence is less than 1 in 100,000. Together all sequence types provided 37.2 x coverage of the genome.
Genes were identified using Prodigal  as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using JGI’s GenePRIMP pipeline . The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and functional annotation were performed within the Integrated Microbial Genomes (IMG-ER) platform. 
The genome is 3,942,657 bp long and comprises one circular chromosome with a 58.7% GC content (Table 3 and Figure 3). Of the 3,565 genes predicted, 3,494 were protein coding genes, and 71 RNAs; 58 pseudogenes were also identified. 74.9% of the genes were assigned a putative function while the remaining ones were annotated as hypothetical proteins. The properties and the statistics of the genome are summarized in Table 3. The distribution of genes into COGs functional categories is presented in Table 4.
Rozanova EP, Nazina TN. A mesophilic, sulfate-reducing, rod-shaped, nonsporeforming bacterium. Mikrobiologiya 1976; 45:825–830
List Editor. Validation of the publication of new names and new combinations previously effectively published outside the ISJB. Int J Syst Bacteriol 1984; 34:355-357
Rozanova EP, Nazina TN, Galushko AS. Isolation of a new genus of sulfate-reducing bacteria and description of a new species of this genus, Desulfomicrobium apsheronum gen. nov., sp. nov. Mikrobiologiya 1988; 57:634–641
Euzeby JP. Taxonomic note: necessary correction of specific and subspecific epithets according to Rules 12c and 13b of the International Code of Nomenclature of Bacteria (1990 Revision). Int J Syst Bacteriol 1998; 48:1073–1075 doi: 10.1099/00207713-48-3-1073
Laanbroek HJ, Pfennig N. Oxidation of short-chain fatty acids by sulfate-reducing bacteria in freshwater and in marine sediments. Arch Microbiol 1981; 128:330–335 PMID:7212933 doi:10.1007/BF00422540
Biebl H, Pfennig, N. Growth of sulfate-reducing bacteria with sulfur as electron acceptor. Arch Microbiol 1977; 112:115–117 PMID:843165 doi:10.1007/BF00446664
Grabowski A, Nercessian O, Fayolle F, Blanchet D, Jeanthon C. Microbial diversity in production waters of a low-temperature biodegraded oil reservoir. FEMS Microbiol Ecol 2005; 54:427–443 PMID:16332340 doi:10.1016/j.femsec.2005.05.007
Hippe H, Vainshtein M, Gogotova Gl, Stackebrandt E. Reclassification of Desulfobacterium macestii as Desulfomicrobium macestii comb. nov. Int J Syst Evol Microbiol 2003; 53:1127–1130 PMID:12892138 doi:10.1099/ijs.0.02574-0
Sharak-Genthner BR, Friedman SD, Devereux R. Reclassification of Desulfovibrio desulfuricans Norway 4 as Desulfomicrobium norvegicum comb. nov. and confirmation of Desulfomicrobium escambiense (corrig., formerly “escambium”) as a new species in the genus Desulfomicrobium. Int J Syst Bacteriol 1997; 47:889–892
Hensgens CMH, Nienhuis-Kuiper ME, Hansen TA. Effect of tungstate on the growth of Desulfovibrio gigas NCIMB 9332 and other sulfate-reducing bacteria with ethanol as a substrate. Arch Microbiol 1994; 162:143–147 doi:10.1007/BF00264388
Cypionka H, Kreke B. Energetics of sulfate transport in Desulfomicrobium baculatum. Arch Microbiol 1995; 163:307–309 doi:10.1007/BF00393385
Lee JP, Yi CS, LeGall J, Peck HD, Jr. Isolation of a new pigment, desulforubidin, from Desulfovibrio desulfuricans (Norway strain) and its role in sulfite reduction. J Bacteriol 1973; 115:453–455 PMID:4717523
Fauque G, Herve D, Le Gall J. Structure-function relationship in hemoproteins: the role of cytochrome c3 in the reduction of colloidal sulfur by sulfate-reducing bacteria. Arch Microbiol 1979; 121:261–264 PMID:229785 doi:10.1007/BF00425065
Coutinho IB, Turner DL, Legall J, Xavier AV. NMR studies and redox titration of the tetraheme cytochrome c3 from Desulfomicrobium baculatum. Identification of the low-potential heme. Eur J Biochem 1995; 230:1007–1013 PMID:7601130 doi:10.1111/j.1432-1033.1995.tb20649.x
Correia IJ, Paquete CM, Coelho A, Almeida CC, Catarino T, Louro RO, Frazao C, Saraiva LM, Carrondo MA, Turner DL, et al. Proton-assisted two-electron transfer in natural variants of tetraheme cytochromes from Desulfomicrobium sp. J Biol Chem 2004; 279:52227–52237 PMID:15456779 doi:10.1074/jbc.M408763200
Teixeira M, Fauque G, Moura I, Lespinat PA, Berlier Y, Prickril B, Peck HD, Jr., Xavier AV, Le Gall J, Moura JJ. Nickel-[iron-sulfur]-selenium-containing hydrogenases from Desulfovibrio baculatus (DSM 1743). Redox centers and catalytic properties. Eur J Biochem 1987; 167:47–58 PMID:3040402 doi:10.1111/j.1432-1033.1987.tb13302.x
Garcin E, Vernede X, Hatchikian EC, Volbeda A, Frey M, Fontecilla-Camps JC. The crystal structure of a reduced [NiFeSe] hydrogenase provides an image of the activated catalytic center. Structure 1999; 7:557–566 PMID:10378275 doi:10.1016/S0969-2126(99)80072-0
Parkin A, Goldet G, Cavazza C, Fontecilla-Camps JC, Armstrong FA. The difference a Se makes? Oxygen-tolerant hydrogen production by the [Ni-FeSe]-hydrogenase from Desulfomicrobium baculatum. J Am Chem Soc 2008; 130:13410–13416 PMID:18781742 doi:10.1021/ja803657d
Tormay P, Wilting R, Heider J, Bock A. Genes coding for the selenocysteine-inserting tRNA species from Desulfomicrobium baculatum and Clostridium thermoaceticum: structural and evolutionary implications. J Bacteriol 1994; 176:1268–1274 PMID:8113164
Tormay P, Wilting R, Lottspeich F, Mehta PK, Christen P, Bock A. Bacterial selenocysteine synthase — structural and functional properties. Eur J Biochem 1998; 254:655–661 PMID:9688279 doi:10.1046/j.1432-1327.1998.2540655.x
Kromayer M, Wilting R, Tormay P, Bock A. Domain structure of the prokaryotic selenocysteinespecific elongation factor SelB. J Mol Biol 1996; 262:413–420 PMID:8893853 doi:10.1006/jmbi.1996.0525
Lee C, Grasso C, Sharlow MF. Multiple sequence alignment using partial order graphs. Bioinformatics 2002; 18:452–464 PMID:11934745 doi:10.1093/bioinformatics/18.3.452
Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 2000; 17:540–552 PMID:10742046
Stamatakis A, Hoover P, Rougemont J. A rapid bootstrap algorithm for the RAxML Web servers. Syst Biol 2008; 57:758–771 PMID:18853362 doi:10.1080/10635150802429642
Liolios K, Mavromatis K, Tavernarakis N, Kyrpides NC. The Genomes On Line Database (GOLD) in 2007: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res 2008; 36:D475–479 PMID:17981842 doi:10.1093/nar/gkm884
Vainshtein M, Hippe H, Kroppenstedt RM. Cellular fatty acid composition of Desulfovibrio species and its use in classification of sulfate-reducing bacteria. Syst Appl Microbiol 1992; 15:554–566
Collins MD, Widdel F. Respiratory quinones of sulphate-reducing and sulphur-reducing bacteria: a systematic investigation. Syst Appl Microbiol 1986; 8:8–18
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 PMID:18464787 doi:10.1038/nbt1360
Kuever J, Rainey FA, Widdel F. Family II Desulfomicrobiaceae fam. nov. NR Krieg JS, GM Garrity, editor. New York: Springer; 2005. 944
Anonymous. Biological Agents: Technical rules for biological agents. <www.baua.de>.
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 PMID:10802651doi:10.1038/75556
Sims D, Brettin T, Detter JC, Han C, Lapidus A, Copeland A, Del Rio TG, Nolan M, Chen F, Lucas S, et al. Complete genome of Kytococcus sedentarius type strain (541T). SIGS 2009; 1:12–20 doi:10.4056/sigs.761
Anonymous. Prodigal Prokaryotic Dynamic Programming Genefinding Algorithm. Oak Ridge National Laboratory and University of Tennessee 2009 <http://compbio.ornl.gov/prodigal>.
Pati A. GenePRIMP: A Gene Prediction Improvement Pipeline for microbial genomes; 2009 (In press)
Markowitz V, Mavromatis K, Ivanova N, Chen IM, Chu K, Palaniappan K, Szeto E, Anderson I, Lykidis A, Kyrpides N. Expert Review of Functional Annotations for Microbial Genomes. (Submitted)
We would like to gratefully acknowledge the help of Maren Schroeder (DSMZ) for growing D. baculatum cultures. This work was performed under the auspices of the US Department of Energy Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344, and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396 as well as German Research Foundation (DFG) INST 599/1-1.
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Copeland, A., Spring, S., Göker, M. et al. Complete genome sequence of Desulfomicrobium baculatum type strain (XT). Stand in Genomic Sci 1, 29–37 (2009). https://doi.org/10.4056/sigs.13134
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