Hansschlegelia zhihuaiae sp. nov., isolated from a polluted farmland soil

The family Methylocystaceae was proposed by Bowman (2005, 2006) to harbour the group II methanotrophs, and the type genus is Methylocystis (Bowman et al., 1993). At the time of writing, the family contains six established genera: Albibacter, Methylocystis, Methylopila, Methylosinus, Pleomorphomonas and Terasakiella. Most members of the family have been isolated from soil, groundwater or other complex environmental samples (Bowman et al., 1993; Doronina et al., 1998, 2001; Satomi et al., 2002; Dedysh et al., 2007). The genus Hansschlegelia, which was described by Ivanova et al. (2007), is also a member of family Methylocystaceae; the name has recently been validly published (Ivanova et al., 2010). Many methanotrophic bacteria represent critical links in the global carbon cycle, act as N₂ fixers, degrade a wide array of organic contaminants and display many enzymic functions (Gulledge et al., 2001). 16S rRNA gene sequence analysis has shown that members of the family Methylocystaceae are able to utilize methanol as a carbon source and exhibit the serine or ribulose-bisphosphate pathway for formaldehyde assimilation (Gallego et al., 2005).

In the course of an investigation of the bacterial community in a farmland soil that was seriously contaminated by sulfonylurea herbicides in Jiangsu province, China, strain S 113^T was isolated and subjected to a polyphasic taxonomic analysis. We propose here that it represents a novel species in the genus Hansschlegelia.

Strain S 113^T was isolated using the medium and methods described by Huang et al. (2007) from a sample of polluted farmland soil. The pure culture was preserved in a 25 % (v/v) glycerol solution in distilled water at −80 °C.

The morphological characteristics of the strain were observed by transmission electron microscopy (model H-7650; Hitachi) after incubation for 3 days at 30 °C on YTC agar (0.3 % yeast, 0.5 % tryptone and 0.001 % CaCl₂). Electron microscopy preparations were performed as described by Yoon et al. (2003). Growth was tested in liquid mineral medium K in which methanol or methylamine was added at a concentration of 0.5 % (v/v) and formaldehyde was added at 0.05 % (v/v), as described by Doronina et al. (1998). The method of Yamaguchi & Yokoe (2000) was followed to determine acid production from carbohydrates. Utilization of sole carbon sources was performed as described by Zhou et al. (2007). Growth was tested at 5, 10, 25, 30, 35, 37 and 40 °C in YTC broth and pH tolerance was tested using modified YTC broth adjusted to pH 3.0, 5.0, 6.0, 6.5, 7.0, 7.5, 8.0 and 9.0. Antibiotic susceptibility was examined by placing antibiotic discs on YTC plates as described by Zhou et al. (2007). Gram-staining and other physiological characteristics were assessed according to the methods of Doronina et al. (1998) and Gerhardt et al. (1994). API 20NE strips (bioMérieux) were used to investigate additional biochemical features.

Biomass for molecular systematic and chemotaxonomic tests was obtained using YTC broth or methanol medium K by incubation at 30 °C for 10 days in shaking flasks (about 180 r.p.m.). Respiratory quinones were extracted from lyophilized cells and the sample was purified and analysed by HPLC using the procedures reported by Hu et al. (2001). Cellular fatty acid composition was determined as described by Sasser (1990) using the Microbial Identification System (MIDI, Inc.). Polar lipid analyses were carried out as described by Minnikin et al. (1984). The predominant respiratory quinone of strain S 113^T was ubiquinone 10 (Q-10). The major fatty acids were C_18 : 1ω7c (77 %) and C_16 : 0 (14.3 %); the detailed fatty acid profile is displayed in Supplementary Table S1, available in IJSEM Online. The dominant phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine and phosphatidylcholine.

Genomic DNA for base composition analysis was prepared following the procedure of Marmur (1961). DNA G+C content was determined by reversed-phase HPLC according to Tamaoka & Komagata (1984); the DNA G+C content of S 113^T was 65.7 mol%. DNA–DNA relatedness was determined by the initial renaturation rate method in 2× SSC (De Ley, 1970). Genomic DNA–DNA relatedness between S 113^T and Hansschlegelia plantiphila NCIMB 14035^T was 44.9 %, which is below the value of 70 % recommended for species definition (Wayne et al., 1987).

PCR amplification of the 16S rRNA gene was performed as described by Xu et al. (2003). The 16S rRNA gene sequence was aligned manually with reference sequences retrieved from the GenBank database following blast searches. Phylogenetic trees were reconstructed using the software package mega version 3.1 (Kumar et al., 2004) after multiple alignment of the data by clustal_x (Thompson et al., 1997). Distances (distance options according to Kimura’s two-parameter model; Kimura, 1980, 1983) and clustering were based on the neighbour-joining and maximum-parsimony methods (Saitou & Nei, 1987). Bootstrap analysis was applied to evaluate the topology of the neighbour-joining tree by performing 1000 resamplings (Felsenstein, 1985).

The almost-complete 16S rRNA gene sequence (1444 bp) of strain S 113^T was determined. The neighbour-joining phylogenetic tree (Fig. 1) reconstructed from 16S rRNA gene sequences showed clearly that strain S 113^T belongs to the genus Hansschlegelia; the strain showed 96.8 % 16S rRNA gene sequence similarity value to its closest neighbour, H. plantiphila S₁^T.

Phylogenetic analysis, genomic DNA–DNA hybridization and chemotaxonomy support the inclusion of the new isolate as a member of genus Hansschlegelia. Strain S 113^T can be differentiated easily from the only species currently classified in this genus, H. plantiphila, by some important phenotypic properties such as motility, catalase activity, H₂S production and NaCl tolerance (Table 1). The type strains of the two species also displayed significant differences in fatty acid and polar lipid compositions (Table 1 and Supplementary Table S1). On the basis of the phylogenetic and chemotaxonomic evidence together with the phenotypic characteristics presented in this study, the newly isolated strain S 113^T is assigned to a novel species in the genus Hansschlegelia, for which the name Hansschlegelia zhihuaiae sp. nov. is proposed.