SGM Journals
Proteobacteria

Stenotrophomonas terrae sp. nov. and Stenotrophomonas humi sp. nov., two nitrate-reducing bacteria isolated from soil

Kim Heylen, Bram Vanparys, Filip Peirsegaele, Liesbeth Lebbe, Paul De Vos

  • Laboratory of Microbiology, Department of Biochemistry, Physiology and Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium
  • Correspondence
    Kim Heylen
    Kim.Heylen{at}UGent.be

    • International Journal of Systematic and Evolutionary Microbiology 2007; 57(9):2056–2061 · https://doi.org/10.1099/ijs.0.65044-0

    View at publisher PubMed

    Abstract

    Three Gram-negative, rod-shaped, non-spore-forming, nitrate-reducing isolates (R-32746, R-32768T and R-32729T) were obtained from soil. Analysis of repetitive sequence-based PCR showed that the three isolates represented two different strains. 16S rRNA gene sequence analysis and DNA–DNA hybridization placed them within the genus Stenotrophomonas and revealed that they were genotypically different from each other and from all recognized Stenotrophomonas species. Analysis of the fatty acid composition and physiological and biochemical tests allowed differentiation from their closest phylogenetic neighbours. They are therefore considered to represent two novel species, for which the names Stenotrophomonas terrae sp. nov. and Stenotrophomonas humi sp. nov. are proposed, with strains R-32768T (=LMG 23958T=DSM 18941T) and R-32729T (=LMG 23959T=DSM 18929T), respectively, as the type strains.

    • The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains R-32768T and R-32729T are AM403589 and AM403587, respectively.

    • A table giving the cellular fatty acid compositions of strains R-32768T and R-32729T and their closest phylogenetic neighbours is available as supplementary material with the online version of this paper.

    In the large-scale phenotypic study of Stanier et al. (1966), Pseudomonas maltophilia was regarded as an authentic member of the genus Pseudomonas. Subsequent allocation of this species to the genus Xanthomonas as Xanthomonas maltophilia was supported by rRNA hybridization data (Swings et al., 1983), but resulted in a reclassification at the genus level into Stenotrophomonas Palleroni and Bradbury 1993, the genus being differentiated from both Pseudomonas and Xanthomonas based on various taxonomic methods. At the time of writing, the genus Stenotrophomonas comprises six recognized species, Stenotrophomonas maltophilia (Palleroni & Bradbury, 1993), S. nitritireducens (Finkmann et al., 2000), S. acidaminiphila (Assih et al., 2002), S. rhizophila (Wolf et al., 2002), S. koreensis (Yang et al., 2006) and S. dokdonensis (Yoon et al., 2006).

    A cultivation-dependent study on soil used selective isolation conditions to focus on the microbial diversity involved in nitrogen removal. Within the dominant gammaproteobacterial nitrate reducers, seven isolates were retrieved on the defined isolation medium G3M12 – with ethanol as carbon source and nitrate as nitrogen source (Heylen et al., 2006) – that could be assigned to Stenotrophomonas based on partial 16S rRNA gene sequences. Three of these isolates, R-32746, R-32768T and R-32729T, could possibly represent novel species based on partial 16S rRNA gene sequence similarities and were analysed further in a polyphasic study. With all of the techniques employed the strains were identified as not representing S. maltophilia (recommended comparison). The type strains of all recognized Stenotrophomonas species, except that for the phylogenetically most distant S. dokdonensis (Fig. 1⇓), were re-examined for phenotyping, chemotaxonomy and biochemical analysis to guarantee comparable data. In addition, S. maltophilia LMG 22072, which was the proposed type strain of Stenotrophomonas africana, was included [Stenotrophomonas africana was found to be a later heterotypic synonym of Stenotrophomonas maltophilia (Coenye et al., 2004a)].

    Figure image not available in archive
    Fig. 1.

    Phylogenetic dendrogram of 16S rRNA gene sequences showing the position of strains R-32729T and R-32768T among the type strains of Stenotrophomonas species. Neighbour-joining clustering was performed with BioNumerics version 4.6. Bootstrap values (expressed as percentages of 1000 replicates) are shown at branch points. EMBL accession numbers are shown in parentheses. ‘Stenotrophomonas daejeonensis’ is also included, but this name has not been validly published and only the 16S rRNA gene sequence is available.

    In order to avoid studying duplicate isolates of the same strain, genotyping by random amplified polymorphic DNA PCR analysis (Coenye et al., 2002) and repetitive sequence-based PCR analysis with REP and BOX primers (Heyrman et al., 2005) were carried out. The three fingerprint methods generated identical patterns for isolates R-32746 and R-32768T, but showed clear genetic differences for isolate R-32729T (data not shown). As R-32746 and R-32768T are most probably members of a single strain, only R-32768T was subject to further study. The DNA G+C contents of strains R-32768T and R-32729T, based on single HPLC determinations (Mesbah et al., 1989), were 65 and 64 mol%, respectively. Nearly complete 16S rRNA gene sequences of strains R-32768T and R-32729T were determined as described by Vanparys et al. (2005). Phylogenetic analysis was performed by using BioNumerics software version 4.6 after multiple alignment with clustal_x (Thompson et al., 1997). Cluster analysis via the neighbour-joining algorithm with or without corrections for evolutionary distances as described by Jukes & Cantor (1969) and Kimura (1980) were in agreement with the maximum-parsimony and maximum-likelihood methods. Strains R-32729T and R-32768T clustered together with S. nitritireducens LMG 22074T and S. acidaminiphila LMG 22073T, supported by high bootstrap values (Fig. 1⇑). Therefore, DNA–DNA hybridization experiments were performed within this cluster, by using a modification of the microplate method of Ezaki et al. (1989) as described by Willems et al. (2001). A hybridization temperature of 45 °C (calculated with correction for the presence of 50 % formamide) was used. Strains R-32768T and R-32729T showed a mean DNA–DNA relatedness of 44.2±2.8 % (n=2 for all determinations) with each other. Strain R-32768T showed a mean DNA–DNA relatedness of 41.3±7 and 35.8±4.7 % with S. nitritireducens LMG 22074T and S. acidaminiphila LMG 22073T, respectively. Strain R-32729T showed a mean DNA–DNA relatedness of 37.2±6.6 and 38.1±5.1 % with S. nitritireducens LMG 22074T and S. acidaminiphila LMG 22073T, respectively. These results confirmed that strains R-32729T and R-32768T represent two novel genotypic species.

    Cell morphology, motility and possible sporulation were investigated using phase-contrast microscopy at a magnification of 1000× with cells grown on tryptone soy agar (TSA; Oxoid) for 48 h at 28 °C. Cells were Gram stained and examined for catalase and oxidase activity. Utilization of carbon sources and enzyme production were tested with the API 20NE (48 h, 28 °C), API ZYM (4 h, 28 °C) and API 50 CH (inoculated with AUX medium, 48 h, 28 °C) systems (bioMérieux), according to the manufacturer's instruction. Strains R-32729T and R-32768T were identified as not representing S. maltophilia with these conventional phenotypic taxonomic tests. The temperature range (at 4, 15, 28, 37 and 52 °C), pH range (4.5–10.5 at 28 °C) and salinity range (0.5–5 % w/v, at 28 °C) for growth were recorded after incubation for 72 h on TSA. The ability to reduce nitrate was tested, as described by Smibert & Krieg (1994), after growth for 2 weeks on tryptone soy broth (TSB; Oxoid) supplemented with 10 mM potassium nitrate at 37 °C and on liquid G3M11 medium containing 18 mM potassium nitrate and 22.5 mM ethanol (Heylen et al., 2006) at 20 °C – the solid variant of the latter was used as isolation medium for the Stenotrophomonas strains. For all strains, these results were in agreement with the nitrate reduction test in the API 20NE system. Lipolytic activity was tested via hydrolysis of Tween 80, as described by Sierra (1957). The phenotypic and biochemical characteristics of all strains tested are given in Table 1⇓.

    Table 1.

    Physiological characteristics of strains R-32768T and R-32729T and their closest phylogenetic neighbours in the genus Stenotrophomonas

    Strains: 1, R-32768T (Stenotrophomonas terrae sp. nov.); 2, R-32729T (Stenotrophomonas humi sp. nov.); 3, S. nitritireducens LMG 22074T; 4, S. acidaminiphila LMG 22073T; 5, S. koreensis LMG 23369T; 6, S. maltophilia LMG 958T; 7, S. maltophilia LMG 22072; 8, S. rhizophila CCUG 47042T. The type strain of S. dokdonensis is not included. Data are from this study unless indicated. +, Positive; w, weakly positive; −, negative; v, variable. All strains characterized in this study are positive for catalase, alkaline phosphatase, esterase, esterase lipase, trypsin, acid phosphatase and glucose fermentation. All are negative for lipase, cystine arylamidase, chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, α-mannosidase, α-fucosidase, indole production, arginine dihydrolase, urease, β-galactosidase, assimilation of caprate, adipate, phenyl acetate, glycerol, erythritol, d-arabinose, l-arabinose, d-ribose, l-xylose, d-adonitol, methyl β-d-xylopyranoside, d-galactose, l-sorbose, l-rhamnose, dulcitol, inositol, d-mannitol, d-sorbitol, methyl α-d-mannopyranoside, methyl α-d-glucopyranoside, inulin, d-melezitose, d-raffinose, starch, xylitol, d-lyxose, d-tagatose, d-fucose, l-fucose, d-arabitol, l-arabitol, potassium gluconate, potassium 2-ketogluconate and potassium 5-ketogluconate.

    Cells of all strains were incubated under identical conditions for 24 h at 28 °C on TSA. One loopful of cells was harvested, fatty acid methyl esters were prepared and these were then extracted according to the standardized protocol of the Microbial Identification System (MIS; Microbial ID Inc.). The MIDI with the TSBA50 database was used for identification. All test strains contained the fatty acids iso-C11 : 0, iso-C11 : 0 3-OH and iso-C13 : 0 3-OH characteristic for the genus Stenotrophomonas (Assih et al., 2002; Yang et al., 1993), and iso-C15 : 0 as the dominant fatty acid. Strains R-32768T and R-32729T had highly similar fatty acid profiles, only differing in the amount of specific components, and mostly containing iso-branched compounds. Characteristic fatty acids for strains R-32768T and R-32729T were iso-C14 : 0 (14.2 and 15.7 %, respectively), iso-C15 : 1 (4.6 and 2 %), iso-C16 : 0 (8 and 12.7 %) and iso-C17 : 1ω9c (7.2 and 4.6 %). The complete fatty acid profiles of all the test strains are available as Supplementary Table S1 in IJSEM Online. Numerical analysis of the fatty acid profiles (Fig. 2⇓) revealed that strains R-32768T and R-32729T formed a distinct cluster, supported with a high co-phenetic correlation, and grouping closely with their phylogenetically nearest neighbours S. nitritireducens LMG 22074T and S. acidaminophila LMG 22073T. The MIDI fatty acid identification system showed no relevant matches for strains R-32768T and R-32729T. However, the position of these strains within the group Stenotrophomonas–Xanthomonas was further confirmed by comparing the fatty acid profiles qualitatively and quantitatively with an in-house database containing over 60 000 fatty acid profiles. The failure of the MIDI identification system to allocate these strains within the genus Stenotrophomonas was also observed for several highly similar profiles of members of the genus Xanthomonas (K. Heylen and P. De Vos, unpublished data) and reported previously for S. acidaminiphila by Assih et al. (2002).

    Figure image not available in archive
    Fig. 2.

    Numerical comparison of the fatty acid profiles obtained. UPGMA clustering with Pearson's correlation similarity coefficients was performed by using BioNumerics version 4.6. The co-phenetic correlation tool, which distinguishes reliable from unreliable subclusters, was used for cluster significance analysis.

    Coenye et al. (2004b) and Hauben et al. (1999) suggested that, prior to their description, the relationship of novel species within the genus Stenotrophomonas with the heterogeneous species S. maltophilia should be investigated. gyrB-restriction fragment length polymorphism (RFLP) analysis of S. maltophilia strains has proven to be in accordance with DNA–DNA hybridization results (Coenye et al.., 2004b). Therefore, the gyrB-RFLP profiles of strains R-32768T and R-32729T were analysed as described by Coenye et al. (2004b), together with an additional set of Stenotrophomonas strains representing gyrB-RFLP clusters A, B, C, E, F, G and I. Strains R-32768T and R-32729T did not render a gyrB amplicon, even after several repeats. The same observation was made for two Stenotrophomonas spp. strains (T. Coenye, personal communication). It was concluded that this approach was not suitable for the novel strains, which, therefore, must differ from the strains that did render gyrB amplicons.

    To substantiate further that strains R-32729T and R-32768T do not represent S. maltophilia, SDS-PAGE analysis of whole-cell proteins was performed on all strains included in the gyrB-RFLP trials. Aerobically grown cells were harvested after incubation at 28 °C for 24 h on phosphate-buffered nutrient agar (pH 6.8). An SDS-PAGE banding pattern for all strains was generated according to the standardized protocol of Pot et al. (1994). Pearson's correlation similarity coefficients were clustered with UPGMA and analysed with the co-phenetic correlation method in BioNumerics version 4.6 (Fig. 3⇓). The grouping of the whole-cell protein profiles was supported by high co-phenetic correlation values but did not correlate with the gyrB-RFLP grouping, except for gyrB-RFLP cluster G. Strains R-32729T and R-32768T did not group together. Their phylogenetically closest neighbours, S. nitritireducens LMG 22074T and S. acidaminophila LMG 22073T, together with strain R-12772, grouped separately from all other Stenotrophomonas strains.

    Figure image not available in archive
    Fig. 3.

    Grouping of normalized digitized SDS-PAGE patterns. UPGMA clustering with Pearson's correlation similarity coefficients was performed by using BioNumerics version 4.6. The co-phenetic correlation tool, which distinguishes reliable from unreliable subclusters, was used for cluster significance analysis. The identification of non-type strains and the assignment to gyrB-RFLP clusters were taken from Coenye et al. (2004b).

    Based on the data presented from the polyphasic study, strains R-32768T and R-32729T are considered to represent two novel species of the genus Stenotrophomonas, for which the names Stenotrophomonas terrae sp. nov. and Stenotrophomonas humi sp. nov. are proposed.

    Description of Stenotrophomonas terrae sp. nov.

    Stenotrophomonas terrae (ter′rae. L. gen. n. terrae of/from soil).

    After 24 h incubation at 28 °C on TSA, colonies are irregular in shape and light yellow. Cells are motile, non-spore-forming, Gram-negative rods. Catalase- and oxidase-positive. Growth is observed at 15–37 °C (but not at 4 or 52 °C), at pH 5–10.5 (but not at pH 4.5) and at salinity of 0.5–5 %. Anaerobic growth is possible through nitrate reduction. Enzyme activities and carbon utilization results are given in Table 1⇑. Can be differentiated from the type strains of its closest phylogenetic neighbours, S. humi, S. nitritireducens and S. acidaminiphila, by SDS-PAGE analysis and the presence of leucine arylamidase and protease, the assimilation of glucose and the absence of N-acetyl-β-glucosaminidase.

    The type strain is R-32768T (=LMG 23958T=DSM 18941T), which has a DNA G+C content of 65 mol%, and was isolated from soil from a university test field in Ghent, Belgium.

    Description of Stenotrophomonas humi sp. nov.

    Stenotrophomonas humi (hu′mi. L. gen. n. humi of/from soil).

    After 24 h incubation at 28 °C on TSA, colonies are round, smooth and beige. Cells are motile, non-spore-forming, Gram-negative rods. Catalase- and oxidase-positive. Growth is observed at 15–37 °C (but not at 4 or 52 °C), at pH 5–10.5 (but not at pH 4.5) and at salinity of 0.5–4 % (but not at 5 %). Anaerobic growth is possible through nitrate reduction. Enzyme activities and carbon utilization results are given in Table 1⇑. Can be differentiated from the type strains of its closest phylogenetic neighbours, S. terrae, S. nitritireducens and S. acidaminiphila, by SDS-PAGE analysis and the assimilation of malate.

    The type strain is R-32729T (=LMG 23959T=DSM 18929T), which has a DNA G+C content of 64 mol%, and was isolated from soil from a university test field in Ghent, Belgium.

    Acknowledgments

    This work was supported by project G.O.A. 1205073 (2003–2008) of the ‘Ministerie van de Vlaamse Gemeenschap, Bestuur Wetenschappelijk Onderzoek’ (Belgium) and FWO project G20156.02.

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