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Clostridium sulfidigenes sp. nov., a mesophilic, proteolytic, thiosulfate- and sulfur-reducing bacterium isolated from pond sediment

Ahmed Sallam, Alexander Steinbüchel

  • Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany
  • Correspondence
    Alexander Steinbüchel
    steinbu{at}uni-muenster.de

    • International Journal of Systematic and Evolutionary Microbiology 2009; 59(7):1661–1665 · https://doi.org/10.1099/ijs.0.004986-0

    View at publisher PubMed

    Abstract

    A novel strictly anaerobic, endospore-forming, rod-shaped, Gram-positive bacterium, designated strain SGB2T, was isolated from a mixed culture from a pond sediment during screening for sulfate-reducing bacteria capable of degrading cyanophycin (CGP). In this study, the taxonomic characterization of this mesophilic, proteolytic Clostridium isolate and the role which it, and its phylogenetic relatives, may play in peptide degradation and in the sulfur cycle are reported. Strain SGB2T was a commensal strain, utilizing CGP degradation products produced by other micro-organisms. Cells were motile until sporulation, forming oval, terminal spores that swell the cells. It showed optimum growth at 34 °C, pH 6.6 and in the absence of NaCl. Strain SGB2T utilized proteinaceous compounds such as peptone, Casamino acids, gelatin and trypticase soy, in addition to several amino acids and pyruvate. Utilization of many of these compounds was enhanced in the presence of thiosulfate. The isolate was unable to use any of the carbohydrates or alcohols investigated or CGP as carbon and energy sources. Thiosulfate and elemental sulfur were used as terminal electron acceptors. Phylogenetic analysis revealed that strain SGB2T belongs to the low-G+C-containing Clostridiales group. It exhibited 99 % 16S rRNA gene sequence similarity to its closest relatives Clostridium thiosulfatireducens Lup21T and Clostridium subterminale DSM 6970T. DNA–DNA hybridization values with these two strains were 39.4 and 42.1 %, respectively. Based on phenotypic, genotypic and phylogenetic characteristics, we conclude that the isolate represents a novel species of the genus Clostridium, Clostridium sulfidigenes sp. nov. The type strain is SGB2T (=DSM 18982T =ATCC BAA-1538T).

    • The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain SGB2T is EF199998.

    While several Clostridium species are well known for their role in the nitrogen cycle as N2-fixing bacteria (Purushothaman et al., 1981), sulfide toxicity in several habitats such as rice-field soils has been attributed exclusively to sulfate-reducing bacteria that also reduce thiosulfate and sulfur. Thus, the establishment of the taxon Desulfotomaculum resulted in the reclassification of Clostridium nigrificans and a few other bacteria as members of this new genus, with the sulfate reducer Desulfotomaculum nigrificans (previously C. nigrificans) as the type species (Campbell & Postgate, 1965). This led to the distinct definition of clostridia as being strictly anaerobic, Gram-positive, endospore formers that are unable to carry out dissimilatory sulfate reduction (Cato et al., 1986; Collins et al., 1994). However, some exceptional cases have also been reported, such as asporogenous mutants that may appear and proliferate, different degrees of aerotolerance known for several species and mesophilic clostridia that show a transient ability to reduce sulfate (Campbell & Postgate, 1965; Hippe et al., 1992).

    It was found recently that thiosulfate-reducing clostridia unable to reduce sulfate may also contribute to the formation of sulfide from inorganic sulfur compounds (Escoffier et al., 1998, 2001), e.g. the saccharolytic, thermophilic bacteria Clostridium thermohydrosulfuricum, Clostridium thermosaccharolyticum (Hollaus & Sleytr, 1972) and Clostridium thermosulfurigenes (Schink & Zeikus, 1983), which were subsequently reclassified and now belong to the genera Thermoanaerobacter and Thermoanaerobacterium (Lee et al., 1993). Only two mesophilic, proteolytic bacteria, Clostridium peptidivorans (Mechichi et al., 2000) and Clostridium thiosulfatireducens (Hernández-Eugenio et al., 2002), are able to reduce thiosulfate and are currently members of the genus Clostridium. In this study, an isolate belonging to the genus Clostridium, strain SGB2T, which is a strictly anaerobic, spore-forming bacterium isolated from an anaerobic consortium (pond sediment samples, Schloßgraben, Münster, Germany) as a member of a cyanophycin (CGP)-degrading consortium, is reported. The polymer CGP consists of equimolar amounts of aspartic acid and arginine (Simon & Weathers, 1976) and functions as a temporary nitrogen, energy and possibly carbon reserve in cyanobacteria. CGP is degraded by intracellular cyanophycinases or by extracellular cyanophycinases of bacteria unable to synthesize CGP (Obst et al., 2005). Strain SGB2T itself was unable to use CGP as a carbon and energy source, but occurred as a commensal micro-organism in the consortium and utilized CGP degradation products (Asp–Arg dipeptides) (Sallam & Steinbüchel, 2008).

    The enrichment of axenic cultures, DNA extraction and sequencing of the 16S rRNA gene of strain SGB2T were reported recently by Sallam & Steinbüchel (2008). Accordingly, strain SGB2T was assigned to the genus Clostridium, with Clostridium subterminale DSM 6970T and C. thiosulfatireducens Lup21T as its closest phylogenetic relatives (99 % 16S rRNA gene sequence similarity for both). However, analysis of the G+C content and DNA–DNA hybridizations (both performed at the DSMZ) showed that strain SGB2T had a slightly higher G+C content (32.3 mol%) than these two strains (28 mol% and 31.4 mol%, respectively) and DNA–DNA hybridization values were only 42.1 and 39.4 %, respectively, which indicated that strain SGB2T is clearly not a strain of C. subterminale or C. thiosulfatireducens.

    The next closest phylogenetic relative to strain SGB2T was ‘Clostridium tunisiense’ DSM 15206 (96 % 16S rRNA gene sequence similarity), whereas C. peptidivorans TMC4T showed much lower similarity to strain SGB2T (93 %). Using only a trimmed part (1434 bp) of the 16S rRNA gene sequence of strain SGB2T and the available sequences for related strains in the NCBI database, a phylogenetic tree was generated (Fig. 1⇓). This tree shows the relationship between strain SGB2T, C. subterminale DSM 6970T, the sulfur-reducer ‘C. tunisiense’ DSM 15206 and related species reported to reduce thiosulfate. DNA G+C content analysis and the phylogenetic tree showed that strain SGB2T is a member of the low-G+C-containing Gram-positive bacteria and represents a member of cluster I of the order Clostridiales (Collins et al., 1994; Stackebrandt & Rainey, 1997).

    Figure image not available in archive
    Fig. 1.

    Neighbour-joining tree based on 16S rRNA gene sequences showing the estimated phylogenetic relationships of Clostridium sulfidigenes sp. nov. SGB2T and closely related species. Accession numbers are given in parentheses. Bootstrap values are shown as percentages of 100 replicates. Bar, 2 % sequence divergence.

    Growth studies were performed in duplicate under anaerobic conditions in Hungate tubes containing basal medium (Sallam & Steinbüchel, 2008) with 0.5 % (w/v) proteose peptone (Difco) and were inoculated from similar precultures. Cells of strain SGB2T multiplied only in an oxygen-free anaerobic atmosphere and at temperatures between 18 and 48 °C, with optimum growth at 34 °C, which is lower than its taxonomic relatives. Strain SGB2T could grow at pH 5.5–9.0, with optimum growth at pH 6.6. Optimum growth occurred in the absence of NaCl; however, retarded growth occurred up to an NaCl concentration of 20 g l−1. Additionally, strain SGB2T differed from its closest relatives in several other criteria including reduction of electron acceptors and the range of utilizable amino acids (Table 1⇓).

    Table 1.

    Comparison between the characteristics of strain SGB2T and its closest phylogenetic relatives

    Strains: 1, strain SGB2T (data from this study); 2, C. subterminale DSM 6970T (unless indicated, data from Elsden & Hilton, 1979; Cato et al., 1986; Hippe et al., 1992); 3, C. thiosulfatireducens DSM 13105T (unless indicated, data from Hernández-Eugenio et al., 2002); 4, C. peptidivorans DSM 12505T (unless indicated, data from Mechichi et al., 2000); 5, ‘C. tunisiense’ DSM 15206 (data from Thabet et al., 2004). Studies on the utilization of electron acceptors and substrates were performed in liquid basal medium containing 0.5 % (w/v) Casamino acids for electron acceptors, or 1 g yeast extract l−1, 20 mM sodium thiosulfate and no cysteine for substrate utilization. The following concentrations were used in electron acceptor tests: 20 mM sodium sulfate, 20 mM sodium thiosulfate and 2 % (w/v) elemental sulfur. Substrate concentrations were: 10 mM for amino acids, 5 g l−1 for peptides and 20 mM for carbohydrates and organic acids. All experiments were performed in Hungate tubes and incubated at 34 °C for at least 15 days. Additionally, l-proline, l-isoleucine, l-serine, peptone, Casamino acids, gelatin and CGP dipeptides were utilized by all strains. The following substrates were tested and were not utilized by any of the strains: lactose, glucose, galactose, mannose, arabinose, xylose, cellobiose, ribose, maltose, sucrose, fructose, lactate, acetate, succinate, ethanol, 1-propanol and CGP. nd, No data available.

    C. thiosulfatireducens DSM 13105T and C. peptidivorans DSM 12505T were used as reference strains to investigate the use of inorganic electron acceptors by strain SGB2T. This was determined photometrically by the increase in growth (turbidity) and H2S production as described by Sallam & Steinbüchel (2008). Table 1⇑ shows the characteristics of strain SGB2T and other related strains regarding reduction of inorganic sulfur compounds. Notably, sulfate was not reduced by axenic cultures of strain SGB2T except, non-reproducibly, in a few experimental repetitions (two of 18 independent repetitions) whereas, in co-cultivations with other isolates from the same mixed consortium, sulfate reduction by strain SGB2T was stable over several repetitions (Sallam & Steinbüchel, 2008). In contrast, growth of strain SGB2T in the presence of thiosulfate consistently started after 1 day of incubation, yielding a maximum OD578 of 0.7 after 10 days and producing 2.6 mM H2S.

    Growth and H2S production by strain SGB2T on the utilized substrates is shown in Fig. 2⇓. The presence of thiosulfate improved substrate utilization as revealed by growth to higher OD, in particular on proteose peptone (Difco) (22.8 %), Casamino acids (10 %), trypticase soy (17.2 %), l-arginine (21.5 %), l-ornithine (14.5 %) and l-citrulline (9.2 %). Relatively large amounts of H2S were produced in the presence of thiosulfate, especially on proteose peptone (4.8 mM), Casamino acids (3.8 mM), trypticase soy (5.5 mM), gelatin (8.0 mM), l-arginine (4.0 mM), l-valine (3.4 mM) and l-cysteine (3.7 mM) (Fig. 2⇓). In the absence of thiosulfate, only negligible H2S production was observed.

    Figure image not available in archive
    Fig. 2.

    Growth and H2S production of strain SGB2T in the presence of various substrates. Cultivation experiments were done in basal medium containing 1 g yeast extract l−1 and 20 mM sodium thiosulfate, in addition to the indicated substrate at concentrations of 10 mM for amino acids, 5 g l−1 for peptides and 20 mM for pyruvate, incubated at 34 °C for 6 days. Shaded bars show growth (change in OD578 compared with control tubes); open bars show H2S production (mM). Data shown represent means and standard deviations from duplicate cultivation tubes.

    Fermentation products of the utilized substrates were measured by HPLC (LaChrom Elite HPLC; VWR-Hitachi) as described by Sallam & Steinbüchel (2008). The main fermentation end-products from Casamino acids, trypticase soy and gelatin were acetate, propionate, valerate and butyrate. Peptone was converted mainly to acetate, succinate, propionate and valerate, whereas yeast extract was fermented to acetate and ethanol. The main fermentation end product detected from citrulline, ornithine, alanine, threonine, serine and cysteine was acetate. Utilization of arginine produced acetate and ethanol, whereas utilization of methionine and phenylalanine produced mainly propionate. Valine was converted to butyrate, leucine to valerate, and pyruvate to acetate and ethanol.

    Based on the phenotypic, genotypic and phylogenetic characteristics mentioned above and in Table 1⇑, the novel isolate was designated the type strain of a novel species of the genus Clostridium; Clostridium sulfidigenes sp. nov. This study provides another example, in addition to previous observations, that strain SGB2T and other proteolytic clostridium strains probably play a significant role in the sulfur cycle and in the degradation of peptides, proteins and amino acids in different environments. They are greatly affected by the presence of different forms of inorganic and organic sulfur compounds. In addition, the increasing number of reports on Clostridium species with such special traits extends the known diversity of this genus.

    Description of Clostridium sulfidigenes sp. nov.

    Clostridium sulfidigenes (sul.fi.di′ge.nes. N.L. n. sulfidum sulfide; N.L. neut. suff. -genes producing from Gr. v. gennaio to produce; N.L. neut. part. adj. sulfidigenes sulfide-producing).

    Cells are rods, about 3–6 μm long and 0.5 μm in diameter, occurring as single cells or in pairs, and are motile until sporulation. Subterminal to terminal spores are formed. Gram staining is positive. Colonies appearing after 48 h of incubation on peptone at 34 °C and after only 24 h on yeast extract are round with regular margins, translucent on peptone and yellowish brown on yeast extract. Optimum growth temperature is 34 °C, with growth at 18–48 °C. Optimum pH for growth is 6.6 and growth occurs at pH 5.5–9.0. Optimum growth occurs in the absence of NaCl; however, concentrations up to 20 g NaCl l−1 are tolerated. The following substrates are used as carbon and energy sources: peptone, Casamino acids, gelatin, trypticase soy, l-citrulline, l-ornithine, l-arginine, l-alanine, l-leucine, l-isoleucine, l-methionine, l-threonine, l-serine, l-phenylalanine, l-valine, l-cysteine and pyruvate. The following substrates are not used: l-proline, l-aspartate, l-lysine, l-histidine, l-glutamate, glycine, dl-tryptophan, l-tyrosine, lactose, glucose, galactose, mannose, arabinose, xylose, cellobiose, ribose, maltose, sucrose, fructose, lactate, acetate, succinate, ethanol and 1-propanol. The main fermentation end products from Casamino acids, trypticase soy and gelatin are acetate, propionate, valerate and butyrate. Peptone is converted mainly to acetate, succinate, propionate and valerate, whereas yeast extract is converted to acetate and ethanol. The main fermentation end product detected from citrulline, ornithine, alanine, threonine, serine and cysteine is acetate; from arginine utilization, end-products are acetate and ethanol, and from methionine and phenylalanine, the end-product is mainly propionate. Valine is converted to butyrate, leucine to valerate, and pyruvate to acetate and ethanol. Uses thiosulfate and sulfur, but not sulfite, nitrate or nitrite as electron acceptors. Thiosulfate improves the utilization of proteose peptone (Difco), Casamino acids, trypticase soy, l-arginine, l-ornithine and l-citrulline.

    The type strain is SGB2T (=DSM 18982T =ATCC BAA-1538T), isolated from pond sediment in Münster, Germany. Adverse effects on humans and animals by strain SGB2T are not known, but cannot be excluded. Cautious handling and autoclaving of cultures before disposal is recommended. Strain SGB2T has a DNA G+C content of 32.3 mol% (HPLC).

    Acknowledgments

    We thank Dr Peter Schumann and Dr Cathrin Spröer from the DSMZ for custom analysis of G+C content and DNA–DNA hybridization and discussion.

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