Skip to main content

Advertisement

Complete genome sequence of Tolumonas auensis type strain (TA 4T)

Article metrics

Abstract

Tolumonas auensis Fischer-Romero et al. 1996 is currently the only validly named species of the genus Tolumonas in the family Aeromonadaceae. The strain is of interest because of its ability to produce toluene from phenylalanine and other phenyl precursors, as well as phenol from tyrosine. This is of interest because toluene is normally considered to be a tracer of anthropogenic pollution in lakes, but T. auensis represents a biogenic source of toluene. Other than Aeromonas hydrophila subsp. hydrophila, T. auensis strain TA 4T is the only other member in the family Aeromonadaceae with a completely sequenced type-strain genome. The 3,471,292 bp chromosome with a total of 3,288 protein-coding and 116 RNA genes was sequenced as part of the DOE Joint Genome Institute Program JBEI 2008.

Introduction

Strain TA 4T (= DSM 9187) is the type strain of the species Tolumonas auensis [1], which is the type species of the monotypic genus Tolumonas [1,2]. ‘Tolumonas osonensis’, isolated from anoxic fresh sediment, was recently proposed as the second species of the genus [3]. ‘T. osonensis’ does not produce toluene from phenylalanine or other aromatic substrates [3]. The genus name is derived from the Neo-Latin words toluolum, toluene, and monas, unit, meaning toluene-producing unit. The species epithet originated from the Latin auensis, of Lake Au. Strain TA 4T was originally isolated from anoxic sediments of Lake Au (a separate part of Lake Zurich), Switzerland [1]. Four more strains (TA 1–3 and TA5) were also isolated from this source, but these strains were not able to produce toluene [1]. Here we present a summary classification and a set of features for T. auensis TA 4T, together with the description of the complete genomic sequencing and annotation.

Classification and features

A representative genomic 16S rRNA sequence of T. auensis TA 4T was compared using NCBI BLAST [4] 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 [5] and the relative frequencies of taxa and keywords (reduced to their stem [6]) were determined, weighted by BLAST scores. The most frequently occurring genera were Yersinia (72.3%), Escherichia (8.0%), Tolumonas (7.2%), Cronobacter (6.3%) and Enterobacter (3.6%) (219 hits in total). Regarding the ten hits to sequences from members of the species, the average identity within HSPs was 99.3%, whereas the average coverage by HSPs was 98.5%. Among all other species, the one yielding the highest score was Cronobacter sakazakii (NC_009778), which corresponded to an identity of 91.8% and an HSP coverage of 100.0%. (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 GQ479961 (‘changes during treated process sewage wastewater treatment plant clone BXHA2’), which showed an identity of 99.2% and an HSP coverage of 97.9%. The most frequently occurring keywords within the labels of environmental samples which yielded hits were ‘reduc’ (7.7%), ‘sludg’ (5.6%), ‘activ’ (4.8%), ‘treatment, wastewat’ (4.2%) and ‘comamonadacea’ (4.1%) (31 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 ‘reduc’ (7.9%), ‘sludg’ (5.3%), ‘activ’ (5.0%), ‘treatment, wastewat’ (4.3%) and ‘comamonadacea’ (4.3%) (27 hits in total). These keywords fit reasonably well to the ecological properties reported for strain TA 4T in the original description [1].

Figure 1 shows the phylogenetic neighborhood of T. auensis in a 16S rRNA-based tree. The sequences of the eight 16S rRNA gene copies in the genome differ from each other by up to 29 nucleotides, and differ by up to 19 nucleotides from the previously published 16S rRNA sequence (X92889), which contains eight ambiguous base calls.

Figure 1.
figure1

Phylogenetic tree highlighting the position of T. auensis relative to the type strains of the other species within the family Aeromonadaceae. The tree was inferred from 1,462 aligned characters [7,8] of the 16S rRNA gene sequence under the maximum likelihood (ML) criterion [9] and rooted with the neighboring family Succinivibrionaceae. 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 [13].

Cells of T. auensis strain TA 4T are rod-shaped, 0.9–1.2 × 2.5–3.2 µm (Figure 2, Table1) and occur singly and in pairs [1]. TA 4T cells stain Gram-negative, are non-motile, and grow equally well under oxic and anoxic conditions [1]. Strain TA 4T grows at a pH range from 6.0 to ­7.5, and a temperature range of 12–25°C, with an optimum at 22°C [1]. Oxidase was not produced under any of the growth conditions, whereas catalase was produced only under aerobic conditions [1]. Substrate spectrum and biochemistry of the strain were reported in detail by Fischer-Romero et al. [1]. Toluene production was observed under oxic and anoxic conditions, but only in the presence of phenylalanine, phenyllactate, phenylpyruvate, or phenylacetate and one of the carbon sources specified in [1]. Phenol was produced from tyrosine [1].

Figure 2.
figure2

Scanning Electron micrograph of T. auensis TA 4T

Table 1. Classification and general features of T. auensis according to the MIGS recommendations [14] and the NamesforLife database [15].

Chemotaxonomy

Data on the cell wall structure of strain TA 4T are not available. Ubiquinones and menaquinones were present under oxic and anoxic conditions, with Q-8 being the major ubiqinone and MK-8 being the major menaquinone [1]. Under aerobic conditions a second, as yet uncharacterized menaquinone was observed [1]. Phosphatidylglycerol and phosphatidyl-ethanolamine were the major phospholipids under both oxic and anoxic growth conditions [1]. The major cellular fatty acids were C12:0, C14:0, C16:0, C16:1 ω7cis, C18:0, C18:1 ω7cis, as well as C14:0 3-OH. One half of the latter fatty acid was amide-bound, the other half was ester-linked as were all the other fatty acids [1].

Genome sequencing and annotation

Genome project history

This organism was selected for sequencing on the basis of the DOE Joint Genome Institute Program JBEI 2008. The genome project is deposited in the Genomes OnLine 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

Strain history

The history of strain TA 4T begins with C. Fischer who directly deposited the strain in the DSMZ open collection, where cultures of the strain have been maintained in lyophilized form frozen in liquid nitrogen since 1994.

Growth conditions and DNA isolation

The culture of strain TA 4T, DSM 9187, used to prepare genomic DNA (gDNA) for sequencing was only three transfers removed from the original deposit. A lyophilized sample was cultivated under anoxic conditions at 20°C using DSMZ medium 500 (with 2 g/L glucose as the primary carbon source) [24]. Genomic DNA was isolated using the MasterPure Gram Positive DNA Purification Kit (EpiCentre MGP04100) according to the manufacturer’s instructions. The purity, quality, and size of the bulk gDNA were assessed according to DOE-JGI guidelines. The gDNA ranged in size from 20–125 kb, with most falling in the 75–100 kb range, as determined by pulsed-field gel electrophoresis.

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 can be found at the JGI website [25]. Pyrosequencing reads were assembled using the Newbler assembler (Roche). Large Newbler contigs were broken into 3,816 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 adjust inflated q-scores. A hybrid 454/Sanger assembly was made using the phrap assembler [26]. Possible mis-assemblies were corrected with Dupfinisher and gaps between contigs were closed by editing in Consed, by custom primer walks from sub-clones or PCR products [27]. A total of 764 Sanger finishing reads and four shatter libraries were needed 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, the combination of the Sanger and 454 sequencing platforms provided 29.3 × coverage of the genome. The final assembly contained 20,349 Sanger reads and 409,035 pyrosequencing reads.

Genome annotation

Genes were identified using Prodigal [28] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [29]. 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. These data sources were combined to assert a product description for each predicted protein. Non-coding genes and miscellaneous features were predicted using tRNAscan-SE [30], RNAMMer [31], Rfam [32], TMHMM [33], and signalP [34].

Genome properties

The genome consists of a 3,471,292-bp long chromosome with a 49.0% G+C content (Table 3 and Figure 3). Of the 3,288 genes predicted, 3,172 were protein-coding genes, and 116 RNAs; 42 pseudogenes were also identified. The majority of the protein-coding genes (76.5%) 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.
figure3

Graphical circular map of the chromosome. 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. 1.

    Fischer-Romero C, Tindall BJ, Jüttner F. Tolumonas auensis gen. nov., sp. nov., a toluene-producing bacterium from anoxic sediments of a freshwater lake. Int J Syst Bacteriol 1996; 46:183–188. PubMed doi:10.1099/00207713-46-1-183

  2. 2.

    Euzéby JP. List of Bacterial Names with Standing in Nomenclature: a folder available on the Internet. Int J Syst Bacteriol 1997; 47:590–592. PubMed doi:10.1099/00207713-47-2-590

  3. 3.

    Caldwell ME, Allen TD, Lawson PA, Tanner RS. Tolumonas osonensis sp. nov., isolated from anoxic freshwater sediment. Int J Syst Bacteriol Dec 2010 Epub ahead of print PMID: 21148672.

  4. 4.

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

  5. 5.

    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

  6. 6.

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

  7. 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

  8. 8.

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

  9. 9.

    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

  10. 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

  11. 11.

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

  12. 12.

    Liolios K, Chen IM, Mavromatis K, Tavernarakis N, 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

  13. 13.

    Seshadri R, Joseph SW, Chopra AK, Sha J, Shaw J, Graf J, Haft D, Wu M, Ren Q, Rosovitz MJ, et al. Genome sequence of Aeromonas hydrophila ATCC 7966T: jack of all trades. J Bacteriol 2006; 188:8272–8282. PubMed doi:10.1128/JB.00621-06

  14. 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

  15. 15.

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

  16. 16.

    Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms. Proposal for the domains Archaea and Bacteria. Proc Natl Acad Sci USA 1990; 87:4576–4579. PubMed doi:10.1073/pnas.87.12.4576

  17. 17.

    Garrity GM, Bell JA, Lilburn T. Phylum XIV. Proteobacteria phyl. nov. In: Brenner DJ, Krieg NR, Staley JT, Garrity GM (eds), Bergey’s Manual of Systematic Bacteriology, second edition, vol. 2 (The Proteobacteria), part B (The Gammaproteobacteria), Springer, New York, 2005, p. 1.

  18. 18.

    Garrity GM, Bell JA, Lilburn T. Class III. Gammaproteobacteria class. 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.

  19. 19.

    Validation List 106. Int J Syst Evol Microbiol 2005; 55:2235–2238. doi:10.1099/ijs.0.64108-0

  20. 20.

    Martin-Carnahan A, Joseph SW. Order XII. Aeromonadales ord. 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. 556.

  21. 21.

    Colwell RR, Macdonell MT, De Ley J. Proposal to recognize the family Aeromonadaceae fam. nov. Int J Syst Bacteriol 1986; 36:473–477. doi:10.1099/00207713-36-3-473

  22. 22.

    BAuA. Classification of bacteria and archaea in risk groups. TRBA 2005; 466:348.

  23. 23.

    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 doi:10.1038/75556

  24. 24.

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

  25. 25.

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

  26. 26.

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

  27. 27.

    Sims D, Brettin T, Detter JC, Han C, Lapidus A, Copeland A, Glavina Del Rio T, Nolan M, Chen F, Lucas S, et al. Complete genome sequence of Kytococcus sedentarius type strain (541T). Stand Genomic Sci 2009; 1:12–20. PubMed doi:10.4056/sigs.761

  28. 28.

    Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal Prokaryotic Dynamic Programming Genefinding Algorithm. BMC Bioinformatics 2010; 11:119. PubMed doi:10.1186/1471-2105-11-119

  29. 29.

    Pati A, Ivanova N, Mikhailova N, Ovchinikova G, Hooper SD, Lykidis A, Kyrpides NC. GenePRIMP: A Gene Prediction Improvement Pipeline for microbial genomes. Nat Methods 2010; 7:455–457. PubMed doi:10.1038/nmeth.1457

  30. 30.

    Lowe TM, Eddy SR. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 1997; 25:955–964. PubMed doi:10.1093/nar/25.5.955

  31. 31.

    Lagesen K, Hallin PF, Rødland E, Stærfeldt HH, Rognes T, Ussery DW. RNammer: consistent annotation of rRNA genes in genomic sequences. Nucleic Acids Res 2007; 35:3100–3108. PubMed doi:10.1093/nar/gkm160

  32. 32.

    Griffiths-Jones S, Bateman A, Marshall M, Khanna A, Eddy SR. Rfam: an RNA family database. Nucleic Acids Res 2003; 31:439–441. PubMed doi:10.1093/nar/gkg006

  33. 33.

    Krogh A, Larsson B, von Heijne G, Sonnhammer ELL. Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes. J Mol Biol 2001; 305:567–580. PubMed doi:10.1006/jmbi.2000.4315

  34. 34.

    Bendtsen JD, Nielsen H, von Heijne G, Brunak S. Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 2004; 340:783–795. PubMed doi:10.1016/j.jmb.2004.05.028

Download references

Acknowledgements

The work conducted by the U.S. Department of Energy Joint Genome Institute was supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231, and work conducted by the Joint BioEnergy Institute (H.R.B.) was supported by the Office of Science, Office of Biological and Environmental Research, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.

Author information

Correspondence to Harry R. Beller.

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

Keywords

  • facultatively anaerobic
  • chemoorganotrophic
  • Gram-negative
  • non-motile
  • toluene producer
  • Aeromonadaceae
  • Gammaproteobacteria
  • JBEI 2008