Draft genome sequence of Mesotoga strain PhosAC3, a mesophilic member of the bacterial order Thermotogales, isolated from a digestor treating phosphogypsum in Tunisia
- Wajdi Ben Hania1, 2Email author,
- Khaled Fadhlaoui1, 2Email author,
- Céline Brochier-Armanet3Email author,
- Cécile Persillon4Email author,
- Anne Postec1Email author,
- Moktar Hamdi2Email author,
- Alain Dolla5Email author,
- Bernard Ollivier1Email author,
- Marie-Laure Fardeau1Email author,
- Jean Le Mer1Email author and
- Gaël Erauso1Email author
© Ben Hania et al.; licensee BioMed Central. 2015
Received: 25 May 2014
Accepted: 30 November 2014
Published: 1 May 2015
Mesotoga strain PhosAc3 was the first mesophilic cultivated member of the order Thermotogales. This genus currently contain two described species, M. prima and M. infera. Strain PhosAc3, isolated from a Tunisian digestor treating phosphogypsum, is phylogenetically closely related to M. prima strain MesG1.Ag.4.2T. Strain PhosAc3 has a genome of 3.1 Mb with a G+C content of 45.2%. It contains 3,051 protein-coding genes of which 74.6% have their best reciprocal BLAST hit in the genome of the type species, strain MesG1.Ag.4.2T. For this reason we propose to assign strain PhosAc3 as a novel ecotype of the Mesotoga prima species. However, in contrast with the M. prima type strain, (i) it does not ferment sugars but uses them only in the presence of elemental sulfur as terminal electron acceptor, (ii) it produces only acetate and CO2 from sugars, whereas strain MesG1.Ag.4.2T produces acetate, butyrate, isobutyrate, isovalerate, 2-methyl-butyrate and (iii) sulfides are also end products of the elemental sulfur reduction in theses growth conditions.
KeywordsAnaerobic Mesophilic Thermotogales Mesotoga
Members of the order Thermotogales typically possess a sheath-like structure called a “toga” and are mostly known as thermophilic or hyperthermophilic bacteria. Most species within this order have been isolated from heated sub-seafloors, marine hydrothermal vents, terrestrial hot springs and oil field reservoirs. Interestingly, SSU rRNA genes of Thermotogales were also detected in samples from polluted environments such as sediments of harbors and sludge from waste water treatment plants . Accordingly they were also found in mesothermic enrichment cultures, notably those capable of (i) reductively dechlorinating 2, 3, 4, 5-tetrachlorobiphenyl, (ii) oxidizing hydrocarbons . We reported in 2011 the first cultivation and a preliminary description of a mesophilic bacterium pertaining to the Thermotogales (strain PhosAc3) which was tentatively named “Mesotoga sulfurireducens” . This mesophilic isolate was shown to belong to a large group of uncultivated bacteria that is distantly related to the thermophilic genus Kosmotoga. Soon after, M. prima strain MesG1.Ag.4.2T isolated from sediments from Baltimore Harbor  and M. infera strain VNs100T isolated from a water sample collected in the area of an underground gas storage  were fully characterized and described as new species. Strain PhosAc3 was isolated from a digestor treating phosphogypsum inoculated with a mixture of marine sediments and sludge originating from a dump and a wastewater treatment plant in Tunisia. It grows at temperatures between 30°C and 50°C (optimum 40°C) and uses fructose and lactate as energy sources. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain PhosAc3 is closely related to M. prima strain MesG1.Ag.4.2T.
Here we report on further taxonomic and physiological studies on strain PhosAc3 and describe the draft genome sequence and its annotation. We show that while they belong to the same species, PhosAc3 and MesG1.Ag.4.2T strains exhibit significant phenotypic and metabolic differences and that their genomes differ by about 25% in gene content.
Classification and features
The fatty acid analysis was performed by the DSMZ on a PhosAc3 culture stopped at the end of exponential phase. Fatty acids were extracted using the method of Miller , analyzed by gas chromatography (gas chromatograph, model 6890 N, Agilent Technologies) and the resulting profile was determined using the Microbial Identification System (MIDI, Sherlock Version 6.1; database, TSBA40). The fatty acid pattern of strain Phos Ac3 was similar to that of M. infera (Additional file 1: Table S2). In contrast to these bacteria, C14 was not detected in M. prima type species (MesG1.Ag.4.2T) thus suggesting that strain PhosAc3 should be considered as novel ecotype of M. prima species.
Genome sequencing information
Genome project history
454 paired-end 8- kb non-cloned libraries
454 GS FLX Titanium
Gene calling method
AmiGene 2.0 and IMG/ER
CARH01000001 to CARH01000099
Genbank date of release
April 10, 2013
Evolution (thermophily/mesophily), Bioremediation
Source material identifier
Classification and general features of Mesotoga strain PhosAc3
Evidence code a
Species Mesotoga prima
Gram - negative
Rod to coccoid with spheroids
30°C to 50°C
pH range; Optimum
Acetate as carbon source, Sugars (pentoses and hexoses) and small organic acids (e.g. lactate, pyruvate) as energy sources
Mesothermic anaerobic reactor
0 to 3% (optimum 0.2%)
Tunis - Tunisia
Sample collection time
Genome statistics for M. prima strain PhosAc3
Genome sizea (bp)
DNA coding region (bp)
DNA G+C content (bp)
Genes in internal clusters
Genes with function prediction
Genes assigned to COGs
Genes assigned Pfam domains
Genes with signal peptides
Genes with transmembrane helices
Number of genes associated with general COG functional categories
Translation, ribosomal structure and biogenesis
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, cell division, chromosome partitioning
Signal transduction mechanisms
Cell wall/membrane/envelope biogenesis
Intracellular trafficking, secretion, and vesicular
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
Translation, ribosomal structure and biogenesis
Not in COG
Growth conditions and DNA isolation
Genomic DNA was isolated from an exponentially growing culture of strain PhosAc3 using the protocol of Marteinsson et al. .
Genome sequencing and assembly
De novo whole-genome shotgun sequencing was performed by combining a single and a long paired end (8 kbp) non-cloned libraries sequencing using the Roche Titanium pyrosequencing GS FLX+ technology (MWG Eurofins). This produced 350,813 reads with an average length of 439 bp for a total number of sequenced bases of 154,143,916 representing a sequencing depth of 49×. Using Celera Assembler software (v.6.1) both data sets could be assembled into four scaffolds including 14 large contigs (>1,000 bp) and 127 small contigs.
Gene predictions annotation and comparative genomic analyses were performed using the MicroScope annotation platform . The predicted CDSs were translated and used to search the National Center for Biotechnology Information non-redundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COGs, and InterPro. 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, RNAMMer, Rfam, TMHMM, and signalP. Additional gene prediction analysis and functional annotation were performed within the Integrated Microbial Genomes Expert Review platform . CRISPR were searched using CRISPRFinder . Table 3 presents the project information and its association with MIGS version 2.0 compliance .
The overall genome size estimated for M. prima strain PhosAc3 is 3,113,612 bp, significantly larger than that of the M. prima type strain MesG1.Ag.4.2 (2,974,229 bp)  and is composed of a unique circular chromosome (no plasmid was found in contrast to MesG1.Ag.4.2T). The average genome G+C content of strain PhosAc3 of 45.19% is close to that of MesG1.Ag.4.2T (45.45%). It contains two ribosomal operons, 47 tRNAs and 3,051 predicted protein-coding genes (Table 3; Figure 3).
Insights from the genome sequence
Like Mesotoga prima (strain MesG1.Ag.4.2T), Mesotoga strain PhosAc3 possesses a significantly larger genome (3.11 and 2.97 Mb respectively) than their thermophilic counterparts within the Thermotogales whose genome size ranged from 1.86 to 2.30 Mb. Of the 3051 protein encoding genes (CDS) of strain PhosAc3, 2392 (78.4%) have their best homologs (satisfying the bi-directional best hit criterion) in the complete genome of M. prima and are clustered in 273 syntons (cluster of at least two contiguous genes) in the two strains (SM Figure 1). For comparison sake, the two Mesotoga strains, MesG1.Ag.4.2T and PhosAc3, share respectively 1468 and 1542 CDS with the closely related species Kosmotoga olearia strain TBF 19.5.1 (SM Figure 1). It seems that the supplementary genes found in Mesotoga strain PhosAc3 (not present in K. olearia) have been acquired by LGT mostly from mesophilic members of the Firmicutes (peculiarly within the Clostridiales order) to the Mesotoga (data not shown) with whom they share the same microbial habitat . As previously observed for Mesotogaprima strain MesG1.Ag.4.2T, the largest fractions of the genes presumably acquired by LGT are involved in amino acids transport and metabolism (COG category E), secondary metabolite biosynthesis (COG category Q) and signal transduction mechanisms (COG category T) (Table 4; data not shown). Of the 659 genes of Mesotoga strain PhosAc3 with no detectable homologs in M. prima strain MesG1.Ag.4.2T, the majority (65%) are annotated as unknown function proteins, 58 (8,8%) correspond to transposons and the rest to poorly characterized functions (COG categories S and R).
Based on taxonomic and genomic criteria, Mesotoga strain PhosAc3 should be considered a novel strain of the M. prima species. Besides numerous similarities, both strains exhibit clear differences in their phenotypic features and even to their gene content. They may therefore represent distinct ecotypes as previously defined [20, 21]. Strain PhosAc3, like M. infera, is capable of significant growth on simple substrates (sugars and organic acids) only in the presence of elemental sulfur as terminal electron acceptor suggesting that these substrates are oxidized rather than fermented. This sharply contrasts with the reported fermentative metabolism of sugars of M. prima type species (strain MesG1.Ag.4.2T). Other differences between strain PhosAc3 and M. prima strain MesG1.Ag.4.2T include the end products of sugar metabolism, the optimum NaCl concentration for growth and the range of electron acceptors used (Table 1). The availability of the genome sequences of two Mesotoga strains offers a good opportunity to look in further details the genomic determinants that may be responsible of the metabolic differences observed between the two strains. Moreover, the comparison with other Thermotogales genomes should bring relevant information regarding the bacterial adaptation to novel ecological niches (from hot to mesothermic biotopes) and the importance of lateral gene transfer in such evolutionary processes .
Sequencing cost was supported by Protéus SA. We thank Zoe Rouy from the MicroScope team for providing access to this powerful annotation platform. C.B.-A. is member of the Institut Universitaire de France. C. B-A is funded by the ANR-10-BINF-01-01 (Ancestrome) grants.
- Nesbo CL, Dlutek M, Zhaxybayeva O, Doolittle WF: Evidence for existence of “mesotogas”, members of the order Thermotogales adapted to low-temperature environments. Appl Environ Microbiol 2006, 72: 5061–5068. 10.1128/AEM.00342-06PubMed CentralView ArticlePubMedGoogle Scholar
- Chouari R, Le Paslier D, Daegelen P, Ginestet P, Weissenbach J, Sghir A: Novel predominant archaeal and bacterial groups revealed by molecular analysis of an anaerobic sludge digester. Environ Microbiol 2005, 7: 1104–1115. 10.1111/j.1462-2920.2005.00795.xView ArticlePubMedGoogle Scholar
- Ben Hania W, Ghodbane R, Postec A, Brochier-Armanet C, Hamdi M, Fardeau M-L, et al.: Cultivation of the first mesophilic representative (“ Mesotoga ”) within the order Thermotogales . Syst Appl Microbiol 2011, 34: 581–585. 10.1016/j.syapm.2011.04.001View ArticlePubMedGoogle Scholar
- Nesbo CL, Bradnan DM, Adebusuyi A, Dlutek M, Petrus AK, Foght J, et al.: Mesotoga prima gen. nov., sp nov., the first described mesophilic species of the Thermotogales . Extremophiles 2012, 16: 387–393. 10.1007/s00792-012-0437-0View ArticlePubMedGoogle Scholar
- Ben Hania W, Postec A, Aullo T, Ranchou-Peyruse A, Erauso G, Brochier-Armanet C, et al.: Mesotoga infera sp. nov., a mesophilic member of the order Thermotogales , isolated from an underground gas storage aquifer. Int J Syst Evol Microbiol 2013, 63: 3003–3008. 10.1099/ijs.0.047993-0View 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 U S A 1990, 87: 4576–4579. 10.1073/pnas.87.12.4576PubMed CentralView ArticlePubMedGoogle Scholar
- Reysenbach A: Phylum BII. Thermotogae phy. nov. In Bergey’s Manual® of Systematic Bacteriology. Volume Volume One : The Archaea and the Deeply Branching and Phototrophic Bacteria. Edited by: Boone DR, Castenholz RW, Garrity GM. New York: Springer; 2001:369–387.View ArticleGoogle Scholar
- Reysenbach A: Class I. Thermotogae class nov. In Bergey’s Manual® of Systematic Bacteriology. Volume Volume One : The Archaea and the Deeply Branching and Phototrophic Bacteria. Edited by: Boone DR, Castenholz RW, Garrity GM. New York: Springer; 2001:369–370.View ArticleGoogle Scholar
- Reysenbach A: Order I. Thermotogales ord. nov. In Bergey’s Manual® of Systematic Bacteriology. Volume Volume One : The Archaea and the Deeply Branching and Phototrophic Bacteria. Edited by: Boone DR, Castenholz RW, Garrity GM. New York: Springer; 2001:369–370.View ArticleGoogle Scholar
- Reysenbach A: Thermotogaceae fam. nov. In Bergey’s Manual® of Systematic Bacteriology. Volume Volume One : The Archaea and the Deeply Branching and Phototrophic Bacteria. Edited by: Boone DR, Castenholz RW, Garrity GM. New York: Springer; 2001:369–370.View ArticleGoogle Scholar
- Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al.: Gene ontology: tool for the unification of biology. The gene ontology consortium. Nat Genet 2000, 25: 25–29. 10.1038/75556PubMed CentralView ArticlePubMedGoogle Scholar
- Miller LT: Single derivatization method for routine analysis of bacterial whole-cell fatty acid methyl esters, including hydroxy acids. J Clin Microbiol 1982, 16: 584–586.PubMed CentralPubMedGoogle Scholar
- Zhaxybayeva O, Swithers KS, Lapierre P, Fournier GP, Bickhart DM, DeBoy RT, et al.: On the chimeric nature, thermophilic origin, and phylogenetic placement of the Thermotogales . Proc Natl Acad Sci U S A 2009, 106: 5865–5870. 10.1073/pnas.0901260106PubMed CentralView ArticlePubMedGoogle Scholar
- Marteinsson V, Watrin L, Prieur D, Caprais J-C, Raguenès G, Erauso G: Sulfur-metabolizing hyperthermophilic anaerobic Archaea isolated from hydrothermal vents in the Southwestern Pacific Ocean. Int J Syst Bacteriol 1995, 45: 623–632. 10.1099/00207713-45-4-623View ArticleGoogle Scholar
- Vallenet D, Engelen S, Mornico D, Cruveiller S, Fleury L, Lajus A, et al.: MicroScope: a platform for microbial genome annotation and comparative genomics. Nucleic Acids Res Database (Oxford). 2009;2009:bap021. Epub 2009 Nov 25. Database (Oxford). 2009;2009:bap021. Epub 2009 Nov 25.
- Markowitz VM, Chen I-MA, Palaniappan K, Chu K, Szeto E, Grechkin Y, et al.: IMG: the integrated microbial genomes database and comparative analysis system. Nucleic Acids Res 2012, 40: D115-D122. 10.1093/nar/gkr1044PubMed CentralView ArticlePubMedGoogle Scholar
- Grissa I, Vergnaud G, Pourcel C: CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res 2007, 35: W52-W57. 10.1093/nar/gkm360PubMed CentralView ArticlePubMedGoogle Scholar
- Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, et al.: The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol 2008, 26: 541–547. 10.1038/nbt1360PubMed CentralView ArticlePubMedGoogle Scholar
- Zhaxybayeva O, Swithers KS, Foght J, Green AG, Bruce D, Detter C, et al.: Genome sequence of the mesophilic Thermotogales Bacterium Mesotoga prima MesG1.Ag.4.2 reveals the largest Thermotogales genome to date. Genome Biol Evol 2012, 4: 700–708. 10.1093/gbe/evs059View ArticlePubMedGoogle Scholar
- Fenchel T: Microbiology. Biogeography for bacteria. Science 2003, 301: 925–926. 10.1126/science.1089242View ArticlePubMedGoogle Scholar
- Nesbo CL, Dlutek M, Doolittle WF: Recombination in Thermotoga : implications for species concepts and biogeography. Genetics 2006, 172: 759–769.PubMed CentralView ArticlePubMedGoogle Scholar
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