Litoribacter ruber gen. nov., sp. nov., an alkaliphilic, halotolerant bacterium isolated from a soda lake sediment

Bacteria belonging to the phylum Bacteroidetes (Garrity & Holt, 2001) are considered to be of special importance in aquatic environments. In marine and freshwater environments, bacteria of the phylum Bacteroidetes can be abundant. This phylum includes Gram-negative, heterotrophic bacteria with the capacity to degrade complex organic compounds such as chitin, cellulose (Cottrell & Kirchman, 2000) and agar (Nedashkovskaya et al., 2003), and they play an important role in carbon cycling in the environment. In volume 4 of Bergey's Taxonomic Outline (Ludwig et al., 2008), the phylum Bacteroidetes includes four classes, ‘Bacteroidia’, Flavobacteria, ‘Sphingobacteria’ and ‘Cytophagia’. The class ‘Cytophagia’ includes three families, of which the Cytophagaceae (previously ‘Flexibacteraceae’) is the largest. In this paper, the characterization and classification of a strain that represents a novel genus-level group of the family Cytophagaceae is reported.

During the investigation of cultured microbial diversity of alkaline environments in Yunnan, south-west China, strain YIM CH208^T was isolated from a sediment sample collected from the shore of a soda lake in Yongsheng. The strain was isolated by the standard dilution-plating technique on marine agar 2216 (MA; Difco) at 28 °C. Strain YIM CH208^T was subsequently maintained on MA slants and stored as 20 % (v/v) glycerol suspensions at −80 °C.

Morphological, physiological and biochemical characteristics of strain YIM CH208^T were investigated using routine cultivation on MA. Gram staining was carried out using the standard Gram reaction combined with the KOH lysis test method (Gregersen, 1978). Cell morphology was examined by light microscopy (Olympus) after staining. Gliding motility was determined using phase-contrast light microscopy (Olympus) after growth on quarter-strength MA solidified with 1 % agar according to Bowman (2000). The pigment composition was examined using sonicated cell suspensions, as well as acetone/methanol (7 : 2, v/v) extracts. Absorption spectra were determined between 300 and 800 nm with a UV spectrophotometer. Growth was also tested on trypticase soy agar (TSA; BBL) and nutrient agar. Growth at 4–50 °C and pH 5.0–11.0 was determined in trypticase soy broth (TSB). To test NaCl tolerance, TSA containing 0–15 % (w/v) NaCl was used. Growth under anaerobic conditions was determined after incubation in an anaerobic jar (GasPak Anaerobic Systems; BBL) on TSA. Degradation of aesculin, cellulose, gelatin, starch and Tweens 20, 40 and 80 was determined according to the protocols described by Cowan & Steel (1965). Catalase activity was determined by assessing bubble production in 3 % (v/v) H₂O₂, and oxidase activity was determined using a 1 % (w/v) solution of tetramethyl-p-phenylenediamine (Kovács, 1956). Carbon utilization was tested using artificial seawater medium (Cho & Giovannoni, 2006) as the basal medium containing each carbon source at a final concentration of 0.5 % (w/v or v/v). Acid production from carbon sources was assessed by using API 50CH and enzyme activity tests were performed using the API ZYM system (bioMérieux) according to the manufacturer's instructions. Other biochemical tests were carried out with the API 20NE kit (bioMérieux). Antibiotic sensitivities were determined using the disc-diffusion method (Bauer et al., 1966).

Isoprenoid quinones were extracted according to the method of Collins et al. (1977) and analysed by HPLC as described by Tamaoka et al. (1983). Biomass for quantitative fatty acid analysis of the isolate was obtained by scraping cells from TSA plates that had been incubated for 5 days at pH 8.5 and at 28 °C. Analysis of the whole-cell fatty acid pattern followed the method of Sasser (1990) using the Microbial Identification System (MIDI).

Genomic DNA extraction, PCR amplification of the 16S rRNA gene and sequencing of the purified PCR product were carried out as described previously (Cui et al., 2001). The sequence was compared to those available in GenBank/EMBL/DDBJ using blast (Altschul et al., 1990). Alignments and similarities were obtained by the clustal_x method. Phylogenetic analyses were carried out using mega3 (Kumar et al., 2004). Distances (corrected by Kimura's two-parameter model; Kimura, 1980) were calculated and clustering was performed with the neighbour-joining method (Saitou & Nei, 1987). A maximum-likelihood (Felsenstein, 1981) tree was generated using the treeing algorithm contained in the phylip package (Felsenstein, 1993). Bootstrap analysis was used to evaluate the tree topology by means of 1000 resamplings (Felsenstein, 1985).

Phylogenetic analysis of the almost-complete 16S rRNA gene sequence (1450 bp) revealed that the isolate occupied a distinct lineage with Echinicola species within the family Cytophagaceae (Fig. 1⇓ and Supplementary Fig. S1, available in IJSEM Online). The topology of the phylogenetic tree constructed using the neighbour-joining algorithm was similar to that of the tree constructed using maximum-likelihood analysis. Sequence analysis revealed that strain YIM CH208^T was most closely related to Echinicola pacifica KMM 6172^T (91.7 % 16S rRNA gene sequence similarity), Rhodonellum psychrophilum GCM71^T (91.6 %), Algoriphagus hitonicola 7-UAH^T (91.6 %), Echinicola vietnamensis LMG 23754^T (91.3 %) and Belliella baltica BA134^T (91.2 %) and exhibited less than 91 % sequence similarity to other members of the phylum Bacteroidetes. Because of these low similarities, DNA–DNA hybridization experiments were not performed. The rather low levels of sequence similarity also suggest that the isolate belongs to a novel genus-level taxonomic group.

Cells were Gram-negative, catalase- and oxidase-positive rods, 0.3–0.5 μm wide and 1.2–1.5 μm long. Gliding motility could not be observed by phase-contrast microscopy. Growth occurred on TSA and nutrient agar. Colonies were smooth, circular, convex, opaque and bright red on MA and TSA. Growth did not occur under anaerobic conditions. Growth occurred in media containing 0–9 % (w/v) NaCl and optimal growth was observed in media containing 0.5–2.5 % (w/v) NaCl. Strain YIM CH208^T grew at pH 7.5–10.5 and showed optimal growth at pH 8.5. Strain YIM CH208^T was alkaliphilic and halotolerant, but the isolate differs from two recently described alkaliphilic members of the family ‘Cyclobacteriaceae’, Nitritalea halalkaliphila and Indibacter alkaliphilus (Anil Kumar et al., 2010a, b), in pH and salinity ranges for growth, nitrate reduction, hydrolysis of gelatin and starch and DNA G+C content (Table 1⇓). The temperature range for growth of strain YIM CH208^T was 20–37 °C and the optimal temperature was 28 °C, whereas the temperature ranges for growth of Echinicola and Rhodonellum species are 6–44 °C and 0–22 °C, respectively (Table 1⇓). In addition, strain YIM CH208^T differs from Echinicola and Rhodonellum species in the capacity for nitrate reduction and salinity ranges (Table 1⇓). Strain YIM CH208^T shows differences from Belliella species with respect to salinity and temperature ranges and DNA G+C content (Table 1⇓). Other features that distinguish the isolate from its phylogenetic relatives are shown in Table 1⇓.

The genomic DNA G+C content was determined by HPLC according to Mesbah et al. (1989) after DNA extraction according to the method of Cui et al. (2001). Genomic DNA from Escherichia coli DH5α was used as a standard. The DNA G+C content of the isolate was 45.1 mol%.

The major cellular fatty acids of strain YIM CH208^T were iso-C_17 : 1ω9c (19.9 %), C_15 : 0 3-OH (12.1 %), iso-C_17 : 0 3-OH (11.3 %), summed feature 3 (iso-C_15 : 0 2-OH and/or C_16 : 1ω7c; 10.7 %) and C_17 : 1ω6c (8.7 %). Strain YIM CH208^T could be differentiated chemotaxonomically from closely related species of the family ‘Cyclobacteriaceae’. For instance, strain YIM CH208^T differs from related species on the basis of the relatively large amount of C_15 : 0 3-OH and small amount of iso-C_15 : 0 (Table 2⇓). Nitritalea halalkaliphila (Anil Kumar et al., 2010b) differs clearly from strain YIM CH208^T in its fatty acid composition and respiratory quinone (Table 2⇓; Anil Kumar et al., 2010a). The isoprenoid quinones of the strain were MK-7 (97 %) and MK-5 (3 %). The absorbance spectrum of strain YIM CH208^T showed a broad peak with maximum absorbance at 473 nm with two shoulders at 450 and 504 nm (Supplementary Fig. S2), which suggests the presence of carotenoids. Bacteriochlorophyll a was not synthesized. The close relative Rhodonellum psychrophilum showed a similar carotenoid absorption spectrum, with absorption peaks at 460, 489 and 517 nm (Schmidt et al., 2006).

The data from our phenotypic, chemotaxonomic, genotypic and phylogenetic analyses suggest that the isolate cannot be assigned to any known taxon. Differences in some phenotypic features, as well as fatty acid compositions and 16S rRNA gene sequences, differentiate the isolate clearly from phylogenetically related taxa (Table 1⇑) and indicate that the isolate represents a novel genus and species, for which the name Litoribacter ruber gen. nov., sp. nov. is proposed.