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1 Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain
2 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 100101 Beijing, People's Republic of China
3 Department of Biotechnology, University of the Western Cape, Bellville 7535, Cape Town, South Africa
4 Genencor International BV, Archimedesweg 30, 2333 CN Leiden, The Netherlands
5 Department of Infection, Immunity and Inflammation, University of Leicester, Leicester LE1 9HN, UK
Correspondence
M. C. Márquez
cmarquez{at}us.es
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). Aerobic, Gram-positive, spore-forming rods constitute an important category within the moderate halophiles. They were originally placed in the genus Bacillus (Claus & Berkeley, 1986) but, on the basis of 16S rRNA gene sequence and chemotaxonomic analyses, they have been reclassified as members of novel genera or transferred to other genera. Halophilic micro-organisms are also often alkaliphilic or alkali-tolerant. In this study, we describe a novel alkali-tolerant, moderately halophilic, Gram-positive, spore-forming bacterial strain, CH9dT, isolated from sediment from Lake Chagannor, an alkaline, saline lake in Inner Mongolia, China.
Strain CH9dT was isolated in September 2003 during the course of a broad microbiological study of saline and/or alkaline lakes in China. Lake Chagannor is located at 4 ° 21' N 11 ° 08' E. At the time of sampling the temperature of the water was 17.1 °C, the pH was 9.5 and the conductivity was very low (<5.66 mS cm–1), probably because of recent heavy rains that had occurred before sampling. The sediment (0.1 g) was suspended in 10 % (w/v) marine salts containing the following (g l–1): NaCl, 8.1; MgCl2, 0.7; MgSO4, 0.96; CaCl2, 0.036; KCl, 0.2; NaHCO3, 0.006; and NaBr, 0.0026 (Rodriguez-Valera et al., 1980). This suspension was vortexed for 1 min, allowed to settle, serially diluted in 10 % (w/v) marine salts and then spread-plated in duplicate on alkaline, saline medium; the plates were then incubated aerobically at 30 °C. The alkaline, saline isolation medium contained the following (g l–1): glucose, 10.0; peptone (Difco), 5.0; yeast extract (Difco), 5.0; KH2PO4, 1.0; MgSO4 . 7H2O, 0.2; NaCl, 80; and Na2CO3, 20. The salts NaCl and Na2CO3 were autoclaved separately and added to the medium prior to the incubation (Duckworth et al., 1996). This medium was solidified by the addition of 2.0 % (w/v) agar. Strain CH9dT was isolated and a pure culture was obtained after several transfers on the same medium.
Genomic DNA from strain CH9dT was obtained using the method described by Marmur (1961). The 16S rRNA gene sequence of the strain was amplified by using a PCR with forward primer 16F27 and reverse primer 16R1488 (Mellado et al., 1995). Direct sequence determination of the PCR-amplified DNA was carried out by using an automated DNA sequencer (model ABI 3130XL; Applied Biosystems). The 16S rRNA gene sequence analysis was performed with the ARB software package (Ludwig et al., 2004). The 16S rRNA gene sequence was aligned with those of phylogenetically closely related organisms (as determined by BLAST analysis) (Maidak et al., 1996) and the alignment was confirmed and checked against both primary and secondary structures of the 16S rRNA molecule by using the alignment tool of the ARB software package. Phylogenetic trees were constructed using three different methods, namely the maximum-likelihood (Felsenstein, 1981), maximum-parsimony (Fitch, 1971) and neighbour-joining (Saitou & Nei, 1987) algorithms integrated in the ARB software for phylogenetic inference. The 16S rRNA gene sequences used for the phylogenetic comparisons were obtained from the GenBank database: their strain designations and accession numbers are shown in Fig. 1.
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). The topologies of the phylogenetic trees constructed using the maximum-likelihood and neighbour-joining algorithms were similar to that of the tree constructed using maximum-parsimony analysis (data not shown). With respect to the 16S rRNA gene sequence similarity values, the closest neighbours of strain CH9dT were Thalassobacillus devorans DSM 16966T (91 % similarity), Bacillus agaradhaerens DSM 8721T (90.5 % similarity) and the Halobacillus species H. trueperi DSM 10404T, H. litoralis DSM 10405T, H. yeomjeoni DSM 17110T, H. karajensis DSM 14948T and H. dabanensis JCM 12772T, which showed 90 % similarity. However, strain CH9dT shared only 88.7 and 88.1 % 16S rRNA gene sequence similarity with respect to M. halophilus and M. halotolerans. These rather low levels of sequence similarity suggest that strain CH9dT belongs to a novel taxonomic group.
To characterize strain CH9dT phenotypically, standard phenotypic tests were performed. The Gram-stain reaction was determined using the method described by Dussault (1955). To determine the cellular morphology and motility, an exponential culture grown in liquid medium was examined by phase-contrast light microscopy. The morphology, pigmentation and size of colonies were observed after 4 days incubation on the alkaline, saline medium at various salt concentrations. Growth at different concentrations of salts was determined in the liquid isolation medium with 0, 0.5, 1, 3, 5, 7, 10, 15, 20, 25 or 30 % (w/v) total salts. The pH range for growth was determined in the liquid isolation medium at pH values ranging from 5.0 to 11.0 by using the appropriate biological buffers (Na2HPO4/NaH2PO4, for pH <8.0; Na2CO3/NaHCO3 for pH 8.0–10.0; Na2HPO4/NaOH, for pH 11.0), as described previously (Gomori, 1955). The pH was readjusted after sterilization, and growth was scored as optical density at 600 nm. The temperature range for growth was determined over the range 15–55 °C. Catalase was assayed by adding 3 % (v/v) H2O2 to culture plates. The oxidase reaction was performed on filter paper moistened with a 1 % (w/v) aqueous solution of N,N,N',N'-tetramethyl-p-phenylenediamine. Other tests (shown in Table 1 or included in the genus or species description) were carried out using methods described previously (Ventosa et al., 1982; Quesada et al., 1984; García et al., 1987). Unless otherwise indicated, the tests were carried out in the alkaline, saline medium and incubated at 37 °C in sealed containers.
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). Conversely, species of the genus Marinococcus comprise non-spore-forming cocci and are characterized by their orange-pigmented colonies (Hao et al., 1984; Li et al., 2005). Strain CH9dT was moderately halophilic and alkali-tolerant, growing in media containing 3–20 % (w/v) salts and optimally in media containing 10 % (w/v) salts. No growth was observed in the absence of NaCl. It grew at pH 5.8–10.0 and showed optimal growth at pH 8.5, whereas optimal growth for T. devorans and B. agaradhaerens occurs at pH 7.0 and at pH 10 or above, respectively (García et al., 2005; Nielsen et al., 1995). B. agaradhaerens does not grow at pH 7 and is able to grow in media containing 0–16 % NaCl: it is therefore considered to be a strictly alkaliphilic and halotolerant species (Nielsen et al., 1995). Strain CH9dT also differs from T. devorans and B. agaradhaerens in its inability to hydrolyse gelatin. In contrast to species of the genus Halobacillus, strain CH9dT is negative for oxidase and is able to reduce nitrate to nitrite (Spring et al., 1996). Several other features that distinguish strain CH9dT from its closest phylogenetic relatives are shown in Table 1.
The G+C content of the genomic DNA of strain CH9dT was determined from the midpoint value (Tm) of the thermal denaturation profile (Marmur & Doty, 1962) by using the equation of Owen & Hill (1979). The G+C content of the total DNA of strain CH9dT was 44.7 mol%.
Preparation of cell-wall extracts from strain CH9dT and analysis of the peptidoglycan structure was carried out using the method described by Schleifer (1985), with the modification that TLC on cellulose sheets was performed instead of paper chromatography. The peptidoglycan type of the isolate was A1
, based on meso-diaminopimelic acid, which matches the type found in the vast majority of endospore-forming, Gram-positive bacilli. However, this characteristic clearly distinguishes strain CH9dT from species in the genus Halobacillus, which have been described as having a cell-wall type based on Orn–D-Asp, a key marker that differentiates the genus Halobacillus from other aerobic or facultatively anaerobic, endospore-forming, rod-shaped genera (Spring et al., 1996; Shida et al., 1997; Wainø et al., 1999; Yoon et al., 2001).
The whole-cell fatty acid composition of strain CH9dT was determined by the Identification, Characterization and Molecular Typing Service of the BCCM/LMG Bacteria Collection (University of Ghent, Belgium) by using gas chromatography. Cell biomass was obtained from a culture grown in the alkaline, saline medium at 37 °C for 24 h. The major fatty acids of strain CH9dT were anteiso-C15 : 0 (50 %), anteiso-C17 : 0 (16 %), iso-C17 : 0 (8 %) and iso-C15 : 0 (6.9 %); the complete profile of the cellular fatty acids is given in detail in the species description. Table 1 shows the distinctive major fatty acids of strain CH9dT and phylogenetically closely related taxa.
Respiratory lipoquinone and polar lipid analyses of strain CH9dT were carried out by the Identification Service of the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). The isoprenoid quinones of this strain were found to be MK-7 (88 %) and MK-6 (12 %). This lipoquinone composition is similar to that of the phylogenetically related taxa, which have MK-7 as the major isoprenoid quinone. With respect to the polar lipid composition, strain CH9dT contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and two phospholipids of unknown structure.
Overall, the data from the phenotypic, chemotaxonomic, genotypic and phylogenetic analyses suggest that strain CH9dT cannot be assigned to any known taxon. The differences in some phenotypic features, as well as the genomic DNA, G+C content, cell-wall type and fatty acid composition, clearly differentiate this strain from the phylogenetically related taxa (Table 1) and indicate that strain CH9dT represents a novel genus and species, for which the name Salsuginibacillus kocurii gen. nov., sp. nov. is proposed.
Description of Salsuginibacillus gen. nov.
Salsuginibacillus (Sal.su.gin'i.ba.cil.lus. L. n. salsugo -inis salted water; L. n. bacillus rod; N.L. masc. n. Salsuginibacillus a rod living in salted water).
Cells are Gram-positive, spore-forming and rod-shaped and occur singly or in pairs. Motile. Catalase-positive and oxidase-negative. Moderately halophilic; do not grow in media without salt. Alkali-tolerant. Nitrate is reduced to nitrite. Ellipsoidal endospores are present in a central or subterminal position. Cell-wall peptidoglycan type is A1 with meso-diaminopimelic acid. Major cellular fatty acids are anteiso-C15 : 0, anteiso-C17 : 0, iso-C17 : 0 and iso-C15 : 0. Predominant menaquinone is MK-7. The type species is Salsuginibacillus kocurii.
Description of Salsuginibacillus kocurii sp. nov.
Salsuginibacillus kocurii (ko.cu'ri.i. N.L. gen. n. kocurii of Kocur, named for the Czech microbiologist M. Kocur, a pioneer in the study of halophilic micro-organisms).
Displays the following properties in addition to those given in the genus description. Cells are 0.7–0.8x2.2–4.3 µm in size. Colonies are 1 mm in diameter, non-pigmented, circular and entire on alkaline, saline medium after 2 days cultivation at 37 °C. Grows over a wide range (3–20 %, w/v) of salt concentrations; optimal growth occurs at 10 % (w/v) salts. Grows at 20–50 °C (optimally at 37 °C) and pH 5.8–10.0 (optimally at pH 8.5). Strictly aerobic. Aesculin, casein, DNA, gelatin, starch and Tween 80 are not hydrolysed. H2S is not produced. Indole, phenylalanine deaminase and phosphatase tests are negative. The following compounds are utilized as sole carbon and energy sources: acetate, aesculin, butyrate, D-cellobiose, citrate, formate, fumarate, ethanol, glucose, hippurate, D-mannitol, pyruvate, D-ribose and sucrose. The following compounds are not utilized as sole carbon and energy sources: amygdalin, D-arabinose, benzoate, D-fructose, L-fucose, D-galactose, inulin, D-lactose, maltose, D-mannose, D-melibiose, propionate, L-raffinose, salicin, trehalose, D-xylose, butanol, dulcitol, glycerol, myo-inositol, propanol, D-sorbitol and xylitol. Susceptible to bacitracin (10 U), cephalothin (30 µg), streptomycin (30 µg), tetracycline (30 µg) and vancomycin (30 µg). Resistant to kanamycin (30 µg), nalidixic acid (30 µg) and penicillin (10 U). Menaquinones are MK-7 (88 %) and MK-6 (12 %). The polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and two unknown phospholipids. The fatty acids comprise anteiso-C15 : 0 (49.7 %), anteiso-C17 : 0 (16.4 %), iso-C17 : 0 (8.0 %), iso-C15 : 0 (6.9 %), iso-C16 : 0 (5.7 %), iso-C14 : 0 (2.0 %), iso-C17 : 1
5c (1.8 %), anteiso-C17:1 (1.6 %) and small amounts of C14 : 0 (0.3 %) and iso-C18 : 0 (0.23 %). The DNA G+C content of the type strain is 44.7 mol% (Tm).
The type strain, CH9dT (=CCM 7365T=CECT 7154T=CGMCC 1.6287T=DSM 18087T), was isolated from sediment from Lake Chagannor in Inner Mongolia, China.
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ACKNOWLEDGEMENTS |
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