Indibacter alkaliphilus gen. nov., sp. nov., an alkaliphilic bacterium isolated from a haloalkaline lake

Soda lakes are naturally occurring alkaline habitats with pH values reaching 12 due to the presence of sodium carbonate (Na₂CO₃) in the water (Joshi et al., 2008). Several novel haloalkaliphilic bacterial species (close to 55 species) representing 41 genera have been described to date. These genera belong to the phyla ‘Proteobacteria’ (19 genera, 26 species), ‘Firmicutes’ (15 genera, 21 species), ‘Actinobacteria’ (five genera, five species), ‘Bacteroidetes’ (one genus, one species) and ‘Spirochaetes’ (one genus, two species). The majority of the alkaliphiles have been isolated from soda lakes (Milford et al., 2000; Borsodi et al., 2003; Doronina et al., 2003; Sorokin et al., 2003; Zavarzina et al., 2006; Carrasco et al., 2007; Delgado et al., 2006; Zhilina et al., 2004; Hoover et al., 2003). Six novel archaeal species representing five genera and belonging to the phylum ‘Euryarchaeota’ have also been isolated from soda lakes (Kanal et al., 1995; Feng et al., 2005).

Lonar Lake is a haloalkaline water body in India. Although several isolates representing 16 genera and belonging to the phyla ‘Firmicutes’ (seven genera), ‘Proteobacteria’ (five genera) and ‘Actinobacteria’ (four genera) have been isolated from Lonar Lake (Joshi et al., 2008), no novel species have been reported so far. In the present study, a novel bacterium isolated from Lonar Lake and assigned to a new genus in the family ‘Cyclobacteriaceae’ is described.

Strain LW1^T was isolated from a water sample (pH 10) collected from Lonar Lake, located in the town of Lonar, Buldhana district, Maharashtra, a state in India (1 ° 58′ N 7 ° 30′ E) on 25 August 2008. For isolation of bacteria, a water sample (100 μl) was plated on Zobell marine agar medium (Zobell, 1941) adjusted to pH 10.0 with Na₂CO₃ solution (20 %, w/v) and incubated at room temperature for 15 days. The viable bacterial cell counts obtained were approximately 4.0–6.0×10⁵ c.f.u. Based on colony morphology, a reddish-orange coloured colony was selected and characterized in the present study.

Cell morphology was studied using light microscopy. Motility was assessed by light microscopy and on TSA medium containing (g l⁻¹): pancreatic digest of casein (17); papaic digest of soybean meal (3); NaCl (5); K₂HPO₄ (2.5); glucose (2.5) and agar (0.4). Growth at different temperatures, salt tolerance, biochemical characteristics, carbon assimilation, H₂S production and antibiotic sensitivity were determined by previously described methods (Lányí, 1987; Smibert & Krieg, 1994). Biochemical characteristics were also double-checked with a Hi25 Enterobacteriaceae identification kit and HiCarbohydrate kit parts A, B and C (both from HiMedia) according to the manufacturer's protocol. Growth of strain LW1^T was checked at different pH values on TSA medium buffered with citric acid/NaOH (for pH 5 and 6), phosphate (for pH 7 and 8), glycine/NaOH (for pH 9 and 10) or Tris (for pH 11 and 12) buffers.

Fatty acid methyl esters were prepared and analysed by the Sherlock Microbial Identification System (MIDI) according to the protocol described by Agilent Technologies. For this purpose, strain LW1^T was grown on TSA medium at 3 °C for 2 days. Polar lipids were extracted and analysed according to the method described by Komagata & Suzuki (1987). Menaquinones and polar lipids were determined in freeze-dried cells. Menaquinones were extracted as described by Collins et al. (1977) and analysed by HPLC (Groth et al., 1997). DNA was isolated according to the procedure of Marmur (1961) and the G+C content was determined from melting point (T_m) curves (Sly et al., 1986) obtained by using a Lambda 2 UV-Vis spectrophotometer (Perkin Elmer) equipped with the Templab 2.0 software package (Perkin Elmer). Escherichia coli was used as a standard in determining DNA G+C content. For 16S rRNA gene sequencing, DNA was prepared using the Mo Bio microbial DNA isolation kit and sequenced as described previously (Lane, 1991). The resultant almost complete 16S rRNA gene sequence contained 1502 nt and a blast sequence similarity search (Altschul et al., 1990) and EzTaxon (Chun et al., 2007) were used to identify the nearest taxa. All the 16S rRNA gene sequences belonging to the family ‘Cyclobacteriaceae’ were downloaded from the database () and aligned using the clustal_x program (Thompson et al., 1997); the alignment was corrected manually. Phylogenetic trees were constructed using two tree-making algorithms, maximum-likelihood (ML) using the program phyml (Guindon & Gascuel, 2003) and neighbour-joining (NJ) (Saitou & Nei, 1987) using the phylip package, version 3.5 (Felsenstein, 1993), and the resultant tree topologies were evaluated by bootstrap analysis based on 1000 resamplings using the seqboot and consense programs in the phylip package. Pairwise evolutionary distances were computed using dnadist program with the Kimura two-parameter model as developed by Kimura (1980).

Cells of strain LW1^T were rods, 0.5–0.7 μm wide and 2.0–3.0 μm long, and multiplied by binary fission. Cells stained Gram-negative and were non-motile. Colonies were circular, 2–3 mm in diameter, smooth, reddish-orange in colour, opaque, convex and entire on half-strength marine agar. The isolate was positive for catalase and oxidase and negative for gelatinase and urease. Strain LW1^T was able to reduce nitrate to nitrite and hydrolyse aesculin and starch. It was negative for indole production, cellulose degradation, DNase and caseinase activity, and did not grow on medium containing only 0.5 % yeast extract. Growth was observed at 15–40 °C; optimum growth was exhibited at 30–37 °C. Growth was observed in 0–8 % (w/v) NaCl, with optimum growth in 0.5–6.0 % (w/v) NaCl. Growth occurred at pH 7.5–12.0, with optimum growth at pH 10.0. The absorption spectrum of an ethanol extract of strain LW1^T showed a broad peak with a maximum around 479 nm and a shoulder at 505 nm, peaks that are typical for carotenoids. Alkalinization did not show a bathochromatic shift of the peaks. Therefore, strain LW1^T contains carotenoids, but no flexirubins, as often observed for marine Cytophaga–Flavobacterium–Bacteroides bacteria (Brettar et al., 2004b).

The cellular fatty acid composition of strain LW1^T showed a spectrum of 29 fatty acids with a pronounced dominance of branched-chain C_15 : 0 and C_17 : 0 fatty acids (Table 1⇓). The branched-chain fatty acids were predominant and constituted 76.86 % of the total fatty acids, with the iso-fatty acids forming the major fraction (67.59 %). When compared with Belliella baltica and Aquiflexum balticum, the number of fatty acids detected in strain LW1^T was greater and the composition differed considerably (Table 1⇓). The menaquinones present in LW1^T were MK-4 (26 %) and MK-7 (74 %) and the phospholipids were phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine and one unidentified lipid. Other characteristics are listed in the species description. The DNA G+C content of strain LW1^T was 42.7±1 mol%.

The phylogenetic relationship of the novel strain was ascertained based on the 16S rRNA gene sequence similarity of LW1^T with strains of other reported species using a blast sequence similarity search (NCBI-blast/EzTaxon). The results indicated that, at the 16S rRNA gene sequence level, strain LW1^T was similar to various cultivable bacterial strains of the genus Belliella that do not have validly published names and uncultured bacterial clone sequences with similarities of 90–93 %. The phylogenetic neighbours of strain LW1^T with validly published names included the type strains of B. baltica (Brettar et al., 2004a), Aquiflexum balticum (Brettar et al., 2004b), Cyclobacterium marinum (Raj & Maloy, 1990), Echinicola vietnamensis (Nedashkovskaya et al., 2007a), Algoriphagus ornithinivorans (Nedashkovskaya et al., 2007b), Echinicola pacifica (Nedashkovskaya et al., 2006), Cyclobacterium lianum (Ying et al., 2006), Cyclobacterium amurskyense (Nedashkovskaya et al., 2005), Algoriphagus winogradskyi (Nedashkovskaya et al., 2004), Algoriphagus hitonicola (Copa-Patiño et al., 2008), Algoriphagus terrigena (Yoon et al., 2006) and Algoriphagus ratkowskyi (Bowman et al., 2003), with similarities of 88.6–92.3 %. blast results indicated that strain LW1^T probably belongs to a new genus. Phylogenetic analyses based on ML and NJ trees further indicated that strain LW1^T formed a deep-rooted lineage that was distinct from the clades represented by the genera Algoriphagus, Aquiflexum, Belliella, Cyclobacterium and Echinicola (Fig. 1⇓).

Strain LW1^T could also be differentiated phenotypically from the closely related species B. baltica and Aquiflexum balticum (Brettar et al., 2004a, b) (Table 2⇓). For instance, strain LW1^T differed from both with respect to colony colour, cell size, optimal salt concentration for growth, salt tolerance, growth at pH 12 and DNA G+C content (Table 2⇓). In addition, specific differences were observed between strain LW1^T and B. baltica and Aquiflexum balticum with respect to acid production and utilization of various carbon sources (Table 2⇓). Thus, the cumulative differences that strain LW1^T exhibits with the above two closely related species unambiguously support the creation of a novel species within a new genus, Indibacter alkaliphilus gen. nov., sp. nov., to accommodate this isolate.