Pontibacter niistensis sp. nov., isolated from forest soil

The genus Pontibacter belongs to the phylum Bacteroidetes and was described by Nedashkovskaya et al. (2005). At the time of writing, the genus Pontibacter comprises five species with validly published names: Pontibacter actiniarum, the type species (Nedashkovskaya et al., 2005), Pontibacter akesuensis, Pontibacter korlensis and Pontibacter xinjiangensis (Zhou et al., 2007; Zhang et al. 2008; Wang et al., 2010) and Pontibacter roseus, resulting from the recent reclassification of Effluviibacter roseus (Suresh et al., 2006; Wang et al., 2010). The type strains of recognized species of the genus are Gram-negative, aerobic, heterotrophic, pink-pigmented bacteria and have been isolated from a marine actinian, desert soil, other soils and muddy water.

Strain NII-0905^T was isolated from a soil sample collected in a dense forest of the Western Ghats in Kerala Province, southern India. A pure culture was obtained by the dilution-plating method on LB agar after 5 days of incubation at 30 °C and was maintained on LB agar slants at 4 °C and as glycerol suspensions (20 %, v/v) at −80 °C. Biomass for molecular systematic and chemotaxonomic studies was obtained after 3 days of incubation at 30 °C in shake flasks (about 150 r.p.m.) containing LB broth adjusted to pH 7.0 using NaOH.

Cell morphology of the isolate was observed after 24 h on LB agar by light microscopy (BH2; Olympus) and transmission electron microscopy (H-7650; Hitachi). Acid production from carbohydrates was tested using the media and methods described by Gordon et al. (1974). Utilization of sole carbon and nitrogen sources was investigated using the API CH50 system (bioMérieux), according to the manufacturer's instructions. Growth at 4, 10, 20, 30, 35, 37, 40 and 45 °C, with 0, 1, 4, 7 and 10 % NaCl and at pH 3.0, 6.0, 7.0, 8.0, 10.0, 11.0 and 12.0 was tested on LB agar.

Gram-staining and other phenotypic characteristics were determined according to the method described by Gerhardt et al. (1994). Menaquinones were isolated as described by Minnikin et al. (1984) and separated using HPLC (Kroppenstedt, 1982). Biomass for quantitative fatty acid analysis was prepared by harvesting cells from shake flasks of LB broth after incubation for 3 days at 30 °C. Fatty acids were extracted, methylated and analysed using the standard Microbial Identification system (MIDI) as described by Sasser (1990).

For the determination of G+C content, genomic DNA was prepared according to the method of Marmur (1961) and analysed using the thermal denaturation method (Mandel & Marmur, 1968). PCR amplification and sequencing of the 16S rRNA gene were performed as described by Li et al. (2007). Multiple alignments with sequences from the most closely related actinobacteria and calculations of sequence similarity were carried out using clustal x (Thompson et al., 1997). A phylogenetic tree (Fig. 1⇓) was constructed by means of the neighbour-joining method of Saitou & Nei (1987), from K_nuc values (Kimura, 1980) using mega version 3.1 (Kumar et al., 2004). Alignment positions with insertions or deletions were excluded from the calculations. The topology of the phylogenetic tree was evaluated by using the bootstrap resampling method of Felsenstein (1985) with 1000 replicates.

Cells of strain NII-0905^T were collected for morphological observations after being cultured on NA for 24 h. Cells were prepared for scanning electron microscopy as described by Nedashkovskaya et al. (2005). Cells of strain NII-0905^T were 0.5–0.6 mm wide and 1.0–2.8 mm long. Colonies of strain NII-0905^T were circular, convex, shiny and smooth. Other physiological and biochemical characteristics are given in Table 1⇓ and the species description. The predominant menaquinone was MK-7. The major fatty acids of strain NII-0905^T were anteiso-C_17 : 0 (28.38 %), anteiso-C_15 : 0 (26.30 %), iso-C_16 : 0 (16.73 %), iso-C_15 : 0 (14.01 %) and iso-C_17 : 0 (6.18 %). Minor amounts (>5 %) of C_16 : 0, summed feature 4 (iso-C_17 : 1 I and/or anteiso-C_17 : 1 B), iso-C_14 : 0, C_15 : 0 and iso-C_11 : 0 3-OH were also detected. The G+C content of the DNA was 51.47 mol%.

Almost-complete 16S rRNA gene sequences (1439 bp) from all strains were compared by sequence similarity calculations. The closest relatives of strain NII-0905^T were P. korlensis X14-1^T (96.3 % 16S rRNA gene sequence similarity), P. actiniarum KMM 6156^T (94.3 %), P. roseus SRC-1^T (94.2 %), P. akesuensis AKS-1^T (93.9 %) and P. xinjiangensis 311-10^T (93.9 %). Given the low levels of 16S rRNA gene sequence similarity, DNA–DNA hybridization studies between strain NII-0905^T and type strains of species of the genus Pontibacter were not carried out. In the phylogenetic dendrogram based on 16S rRNA gene sequences, strain NII-0905^T formed a separate lineage within the Pontibacter group (Fig. 1⇑). It was clear from the phylogenetic analysis that the isolate belonged to the genus Pontibacter and represented a distinct phyletic line that could be equated to a separate genomic species (Stackebrandt & Goebel, 1994).

Strain NII-0905^T shared chemotaxonomic features with members of the genus Pontibacter: the major menaquinone was MK-7, the predominant cellular fatty acids were iso- and anteiso-branched components and the DNA G+C content was 51.47 mol%. However, strain NII-0905^T showed considerable phenotypic and genotypic differences from the type strains of the recognized species of the genus Pontibacter (Table 1⇑). Therefore, strain NII-0905^T is considered to represent a novel species of the genus Pontibacter, for which we propose the name Pontibacter niistensis sp. nov.