Skip to main content

Complete genome sequence of the acetate-degrading sulfate reducer Desulfobacca acetoxidans type strain (ASRB2T)

Abstract

Desulfobacca acetoxidans Elferink et al. 1999 is the type species of the genus Desulfobacca, which belongs to the family Syntrophaceae in the class Deltaproteobacteria. The species was first observed in a study on the competition of sulfate-reducers and acetoclastic methanogens for acetate in sludge. D. acetoxidans is considered to be the most abundant acetate-degrading sulfate reducer in sludge. It is of interest due to its isolated phylogenetic location in the 16S rRNA-based tree of life. This is the second completed genome sequence of a member of the family Syntrophaceae to be published and only the third genome sequence from a member of the order Syntrophobacterales. The 3,282,536 bp long genome with its 2,969 protein-coding and 54 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

Introduction

Strain ASRB2T (= DSM 11109 = ATCC 700848) is the type strain of the species Desulfobacca acetoxidans, which is the type and sole species of its genus Desulfobacca [1]. The type strain was isolated from granular sludge of a laboratory-scale upflow anaerobic sludge bed (UASB) reactor fed with acetate and sulfate [1]. The generic name derives from the Neo-Latin word ‘desulfo’, meaning desulfuricating, and the Latin word ‘bacca’, berry, especially olive, meaning a sulfate-reducing olive-shaped bacterium. The species epithet is derived from the Neo-Latin words ‘acetum’, vinegar, and ‘oxido’, meaning acetate-oxidizing. The strain is important for the understanding of the competition for acetate between sulfate-reducers and acetoclastic methanogens in sludge [1]. Here we present a summary classification and a set of features for D. acetoxidans strain ASRB2T, together with the description of the complete genomic sequencing and annotation.

Classification and features

The single genomic 16S rRNA sequence of D. acetoxidans DSM ASRB2T was compared using NCBI BLAST [2,3] under default settings (e.g., considering only the high-scoring segment pairs (HSPs) from the best 250 hits) with the most recent release of the Greengenes database [4] and the relative frequencies of taxa and keywords (reduced to their stem [5]) were determined, weighted by BLAST scores. The most frequently occurring genera were Desulfobacca (74.9%) and Desulfomonile (25.1%) (4 hits in total). Regarding the two hits to sequences from members of the species, the average identity within HSPs was 98.9%, whereas the average coverage by HSPs was 96.7%. Among all other species, the one yielding the highest score was Desulfomonile limimaris (NR_025079), which corresponded to an identity of 90.4% and an HSP coverage of 49.8%. (Note that the Greengenes database uses the INSDC (= EMBL/NCBI/DDBJ) annotation, which is not an authoritative source for nomenclature or classification.) The highest-scoring environmental sequence was AY340836 (‘sulfate-reducing fluidized-bed reactor clone SR FBR L13’), which showed an identity of 99.8% and an HSP coverage of 93.0%. The most frequently occurring keywords within the labels of environmental samples which yielded hits were ‘sediment’ (5.2%), ‘microbi’ (3.2%), ‘lake’ (1.9%), ‘water’ (1.7%) and ‘depth’ (1.6%) (246 hits in total). The most frequently occurring keywords within the labels of environmental samples which yielded hits of a higher score than the highest scoring species were ‘sediment’ (5.4%), ‘microbi’ (2.5%), ‘lake’ (2.1%), ‘water’ (1.9%) and ‘contamin’ (1.8%) (152 hits in total). These keywords reflect some of the ecological and properties reported for strain ASRB2T in the original description [1].

Figure 1 shows the phylogenetic neighborhood of D. acetoxidans in a 16S rRNA based tree. The sequence of the single 16S rRNA gene in the genome differs by 20 nucleotides from the previously published 16S rRNA sequence (AF002671), which contains eleven ambiguous base calls

Figure 1.
figure 1

Phylogenetic tree highlighting the position of D. acetoxidans relative to the type strains of the other species within the order Syntrophobacterales. The tree was inferred from 1,457 aligned characters [6,7] of the 16S rRNA gene sequence under the maximum likelihood (ML) criterion [8]. Rooting was done initially using the midpoint method [9] and then checked for its agreement with the current classification (Table 1). The branches are scaled in terms of the expected number of substitutions per site. Numbers adjacent to the branches are support values from 1,000 ML bootstrap replicates [10] (left) and from 1,000 maximum parsimony bootstrap replicates [11] (right) if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [12] are labeled with one asterisk, those also listed as ‘Complete and Published’ with two asterisks (see [13] and CP000478 for Syntrophobacter fumaroxidans).

Cells of strain ASRB2T are oval to rod-shaped with a size of 1.3 x 1.9–2.2 εm, appear singly or in pairs (Figure 2) and occasionally contain gas vacuoles in the late-exponential growth phase [1]. The strain is non-motile, non-spore-forming and stains Gram-negative (Table 1) [1]. Strain ASRB2T has a temperature range for growth between 27 and 47°C, with an optimum at 36–40°C [1]. At the optimum growth temperature with acetate as sole carbon and energy source the shortest doubling time recorded was 1.7–2.2 days [1]. Growth rate in brackish medium was significantly (4.8 x) slower, and no growth was observed in marine medium [1]. The pH range for growth is 6.5–8.3, with an optimum of pH 7.1–7.5 [1]. Desulfoviridin was not observed, but the c-type cytochromes were present [1]. Sulfate or other inorganic sulfur components serve as electron acceptors via reduction to H2S [1]. Strain ASRB2T degrades acetate (as the common carbon source and electron donor) completely to CO2 via the acetyl-CoA/CO-dehydrogenase pathway [1]. The key enzyme of this pathway is encoded by the genes Desac_1965–Desac_1969. Several more putative electron donors were tested but not found to be utilized by strain ASRB2T, such as: propionate, butyrate, lactate, H2/CO2, formate, ethanol, propanol, butanol, pyruvate, fumarate, glucose, crotonate, benzoate, phenol, aspartate and glutamate [1].

Figure 2.
figure 2

Scanning electron micrograph of D. acetocidans ASRB2T

Table 1. Classification and general features of D. acetocidans ASRB2T according to the MIGS recommendations [14] and the NamesforLife database [15].

Chemotaxonomy

No data on cell wall structure, quinones, fatty acid pattern or polar lipids are available for this strain.

Genome sequencing and annotation

Genome project history

This organism was selected for sequencing on the basis of its phylogenetic position [26], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [27]. The genome project is deposited in the Genome On Line Database [12] and the complete genome sequence 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.

Table 2. Genome sequencing project information

Growth conditions and DNA isolation

D. acetoxidans ASRB2T, DSM 11109, was grown anaerobically in DSMZ medium 728 (Desulfobacca medium) [28] at 37°C. DNA was isolated from 0.5–1 g of cell paste using Jetflex Genomic DNA Purification Kit (GENOMED 600100) following the standard protocol as recommended by the manufacturer, but with additional 2 hours incubation with 20 εl proteinase K at 58°C for cell lysis. DNA is available through the DNA Bank Network [29].

Genome sequencing and assembly

The genome 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 [30]. Pyrosequencing reads were assembled using the Newbler assembler (Roche). The initial Newbler assembly consisting of 66 contigs in one scaffold was converted into a phrap [31] assembly by making fake reads from the consensus, to collect the read pairs in the 454 paired end library. Illumina GAii sequencing data (1,042 Mb) was assembled with Velvet [32] 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 159.0 Mb 454 draft data and all of the 454 paired end data. Newbler parameters are -consed -a 50 -l 350 -g -m -ml 20. The Phred/Phrap/Consed software package [31] 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). Possible mis-assemblies were corrected with gapResolution [30], Dupfinisher [33], or sequencing cloned bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F. Chang, unpublished). A total of 55 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. Illumina reads were also used to correct potential base errors and increase consensus quality using a software Polisher developed at JGI [34]. The error rate of the completed genome sequence is less than 1 in 100,000. Together, the combination of the Illumina and 454 sequencing platforms provided 350.7 x coverage of the genome. The final assembly contained 346,781 pyrosequence and 28,710,424 Illumina reads.

Genome annotation

Genes were identified using Prodigal [35] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [36]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) non-redundant database, UniProt, TIGRFam, 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 [37].

Genome properties

The genome consists of a 3,282,536 bp long chromosome with a 52.9% G+C content (Table 3 and Figure 3). Of the 3,023 genes predicted, 2,969 were protein-coding genes, and 54 RNAs; 103 pseudogenes were also identified. The majority of the protein-coding genes (68.2%) were assigned a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.

Figure 3.
figure 3

Graphical circular map of the genome. From outside to the center: Genes on forward strand (color by COG categories), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, GC skew.

Table 3. Genome Statistics
Table 4. Number of genes associated with the general COG functional categories

References

  1. Oude Elferink SJ, Akkermans-van Vliet WM, Bogte JJ, Stams AJM. Desulfobacca acetoxidans gen. nov., sp. nov., a novel acetate-degrading sulfate reducer isolated from sulfidogenic granular sludge. Int J Syst Bacteriol 1999; 49:345–350. PubMed doi:10.1099/00207713-49-2-345

    Article  PubMed  Google Scholar 

  2. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Bascic local alignment search tool. J Mol Biol 1990; 215:403–410. PubMed

    Article  CAS  PubMed  Google Scholar 

  3. Korf I, Yandell M, Bedell J. BLAST, O’Reilly, Sebastopol, 2003.

  4. DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T, Dalevi D, Hu P, Andersen GL. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol 2006; 72:5069–5072. PubMed doi:10.1128/AEM.03006-05

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Porter MF. An algorithm for suffix stripping. Program: electronic library and information systems 1980; 14:130–137.

    Article  Google Scholar 

  6. Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 2000; 17:540–552. PubMed

    Article  CAS  PubMed  Google Scholar 

  7. Lee C, Grasso C, Sharlow MF. Multiple sequence alignment using partial order graphs. Bioinformatics 2002; 18:452–464. PubMed doi:10.1093/bioinformatics/18.3.452

    Article  CAS  PubMed  Google Scholar 

  8. Stamatakis A, Hoover P, Rougemont J. A rapid bootstrap algorithm for the RAxML web servers. Syst Biol 2008; 57:758–771. PubMed doi:10.1080/10635150802429642

    Article  PubMed  Google Scholar 

  9. Hess PN, De Moraes Russo CA. An empirical test of the midpoint rooting method. Biol J Linn Soc Lond 2007; 92:669–674. doi:10.1095-8312.2007.00864.x

    Article  Google Scholar 

  10. Pattengale ND, Alipour M, Bininda-Emonds ORP, Moret BME, Stamatakis A. How many bootstrap replicates are necessary? Lect Notes Comput Sci 2009; 5541:184–200. doi:10.1007/978-3-642-02008-7_13

    Article  CAS  Google Scholar 

  11. Swofford DL. PAUP*: Phylogenetic Analysis Using Parsimony (*and Other Methods), Version 4.0 b10. Sinauer Associates, Sunderland, 2002.

    Google Scholar 

  12. Liolios K, Chen IM, Mavromatis K, Tavernarakis N, Hugenholtz P, Markowitz VM, Kyrpides NC. The Genomes On Line Database (GOLD) in 2009: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res 2010; 38:D346–D354. PubMed doi:10.1093/nar/gkp848

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. McInerney MJ, Rohlin L, Mouttaki H, Kim U, Krupp RS, Rios-Hernandez L, Sieber J, Struchtemeyer CG, Bhattacharyya A, Campbell JW, Gunsalus RP. The genome of Syntrophus aciditrophicus: Life at the thermodynamic limit of microbial growth. Proc Natl Acad Sci USA 2007; 104:7600–7605. PubMed doi:10.1073/pnas.0610456104

    Article  PubMed Central  PubMed  Google Scholar 

  14. 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 doi:10.1038/nbt1360

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Garrity G. NamesforLife. BrowserTool takes expertise out of the database and puts it right in the browser. Microbiol Today 2010; 37:9.

    Google Scholar 

  16. 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 doi:10.1073/pnas.87.12.4576

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Garrity GM, Bell JA, Lilburn T. Phylum XIV. Proteobacteria phyl. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume 2, Part B, Springer, New York, 2005, p. 1.

    Chapter  Google Scholar 

  18. 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.

    Chapter  Google Scholar 

  19. Editor L. Validation of publication of new names and new combinations previously effectively published outside the IJSEM. List no. 106. Int J Syst Evol Microbiol 2005; 55:2235–2238. doi:10.1099/ijs.0.64108-0

  20. Kuever J, Rainey FA, Widdel F. Class IV. Deltaproteobacteria class. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume 2, Part C, Springer, New York, 2005, p. 922.

    Chapter  Google Scholar 

  21. Editor L. Validation List No. 107. List of new names and new combinations previously effectively, but not validly, published. Int J Syst Evol Microbiol 2006; 56:1–6. PubMed doi:10.1099/ijs.0.64188-0

  22. Kuever J, Rainey FA, Widdel F. Order VI. Syntrophobacterales ord. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume 2, Part C, Springer, New York, 2005, p. 1021.

    Google Scholar 

  23. Kuever J, Rainey FA, Widdel F. Family II. Syntrophaceae fam. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume 2, Part C, Springer, New York, 2005, p. 1033.

    Chapter  Google Scholar 

  24. Classification of bacteria and archaea in risk groups. http://www.baua.de TRBA 466.

  25. 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. Nat Genet 2000; 25:25–29. PubMed doi:10.1038/75556

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Klenk HP, Göker M. En route to a genome-based classification of Archaea and Bacteria? Syst Appl Microbiol 2010; 33:175–182. PubMed doi:10.1016/j.syapm.2010.03.003

    Article  CAS  PubMed  Google Scholar 

  27. Wu D, Hugenholtz P, Mavromatis K, Pukall R, Dalin E, Ivanova NN, Kunin V, Goodwin L, Wu M, Tindall BJ, et al. A phylogeny-driven genomic encyclopaedia of Bacteria and Archaea. Nature 2009; 462:1056–1060. PubMed doi:10.1038/nature08656

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. List of growth media used at DSMZ: http://www.dsmz.de/microorganisms/media_list.php.

  29. Gemeinholzer B, Dröge G, Zetzsche H, Haszprunar G, Klenk HP, Güntsch A, Berendsohn WG, Wägele JW. The DNA Bank Network: the start from a German initiative. Biopreservation and Biobanking 2011; 9:51–55. doi:10.1089/bio.2010.0029

    Article  PubMed  Google Scholar 

  30. The DOE Joint Genome Institute. http://www.jgi.doe.gov.

  31. Phrap and Phred for Windows. MacOS, Linux, and Unix. http://www.phrap.com.

  32. Zerbino DR, Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 2008; 18:821–829. PubMed doi:10.1101/gr.074492.107

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  33. Han C, Chain P. 2006. Finishing repeat regions automatically with Dupfinisher. In: Proceeding of the 2006 international conference on bioinformatics & computational biology. Arabina HR, Valafar H (eds), CSREA Press. June 26–29, 2006: 141–146.

  34. 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. AGBT, Marco Island, FL, 2008.

    Google Scholar 

  35. 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 doi:10.1186/1471-2105-11-119

    Article  PubMed Central  PubMed  Google Scholar 

  36. 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 doi:10.1038/nmeth.1457

    Article  CAS  PubMed  Google Scholar 

  37. Markowitz VM, Ivanova NN, Chen IMA, Chu K, Kyrpides NC. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics 2009; 25:2271–2278. PubMed doi:10.1093/bioinformatics/btp393

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We would like to gratefully acknowledge the help of Esther Schüler (DSMZ) for growing D. acetoxidans 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, UT-Battelle, and Oak Ridge National Laboratory under contract DE-AC05-00OR22725, as well as German Research Foundation (DFG) INST 599/1-2.

Author information

Authors and Affiliations

Authors

Rights and permissions

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Reprints and permissions

About this article

Cite this article

Göker, M., Teshima, H., Lapidus, A. et al. Complete genome sequence of the acetate-degrading sulfate reducer Desulfobacca acetoxidans type strain (ASRB2T). Stand in Genomic Sci 4, 393–401 (2011). https://doi.org/10.4056/sigs.2064705

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.4056/sigs.2064705

Keywords