Methylotenera mobilis gen. nov., sp. nov., an obligately methylamine-utilizing bacterium within the family Methylophilaceae

Methylotrophs are microbes capable of utilizing single C₁ compounds as sole sources of energy and carbon. Methylotrophic ability is especially widespread within the Proteobacteria, encompassing the alpha, beta and gamma subdivisions (Anthony, 1982; Hanson & Hanson, 1996). Methylotrophs of the Alphaproteobacteria include four genera of obligate and restricted facultative methanotrophs (Methylosinus, Methylocystis, Methylocapsa and Methylocella) and a broad group (over ten genera) of facultatively methylotrophic bacteria (Methylobacterium, Methylopila, Hyphomicrobium, etc.). Methylotrophs of the Gammaproteobacteria are represented by nine genera of obligately methanotrophic bacteria (Methylomonas, Methylococcus, Methylothermus, Methylomicrobium, Methylosphaera, Methylobacter, Methylocaldum, Methylohalobius and Methylosarcina) and three genera of non-methanotrophic methylotrophic bacteria (Methylophaga, Marinobacterium and Pseudomonas). Betaproteobacterial methylotrophs are represented by three genera (Methylobacillus, Methylophilus and Methylovorus) within the family Methylophilaceae and so far by a single genus (Methylibium) within the Burkholderiales (Nakatsu et al., 2006). 16S rRNA gene sequences with close similarity to those of methylotrophs of the family Methylophilaceae have been detected in a variety of ecosystems; a few representative strains have been cultured but not formally characterized (Connon & Giovannoni, 2002; Lueders et al., 2004; De Marco et al., 2004; Fuchs et al., 2005; Gich et al., 2005). In this study we describe a novel betaproteobacterial obligate methylotroph isolated from lake sediment. Based on 16S rRNA gene sequence analysis, this strain is shown to be more closely related to uncultured or unidentified Betaproteobacteria than to known betaproteobacterial methylotrophs. We propose that this new isolate represents a novel species of a new genus within the family Methylophilaceae.

Strain JLW8^T was isolated from sediment recovered from Lake Washington, Washington State, USA, after enrichment in a basal salts medium (Harder et al., 1973) diluted five-fold and supplemented with 0.1 % methylamine as described previously (Miller et al., 2005). The purity of the culture was monitored by microscopy, by 16S rRNA gene sequence amplification and analysis, and by testing the ability to grow on tryptone–glucose–yeast extract (TGY; Murray, 1992), Luria–Bertani (LB; Sambrook et al., 1989) or Nutrient (Difco) media. Characterization of the ultrastructural, phenotypic and genotypic properties of strain JLW8^T was performed as described previously (Miller et al., 2005). Transmission election microscopy was performed as described previously (Kalyuzhnaya et al., 2005). Negatively stained preparations and thin sections were viewed by using a 1200 Ex II transmission electron microscope (JEOL) at an operating voltage of 80 kV. The strain was routinely grown in the basal salts medium supplemented with 0.1 or 0.2 % methylamine. Cells were stored in the same basal salts medium supplemented with 10 % DMSO, at −80 °C. 16S rRNA gene sequences were aligned using the clustalw program (Higgins et al., 1996). Phylogenetic analysis was carried out using the phylip package (Felsenstein, 2003). The distance method was employed, and 100 bootstrap analyses were performed.

Colonies of strain JLW8^T were cream to light brown and 1–2 mm in diameter when grown at 30 °C for 4–7 days. No pigmentation was observed when cells were grown in liquid culture. Cells did not form aggregates in liquid culture. Microscopy revealed that cells were rod-shaped, 0.6–1.2×0.3–0.4 μm, occurred singly, were motile by means of a single polar flagellum (Fig. 1⇓) and had a Gram-negative cell wall structure. Some cells possessed cell wall extrusions, or ‘prostheca’-like structures. Cells reproduced by binary fission and did not form resting bodies. Strain JLW8^T utilized only methylamine as a growth substrate, but not methanol, formate, dimethylamine, trimethylamine, organic acids, sugars, amino acids, C₂–C₆ alcohols or methane. No growth occurred on TGY, LB or Nutrient media. The optimal concentration of methylamine required for growth was tested using a Bioscreen C (MBR) plate reader at the following concentrations (w/v): 0, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1 and 2 %. Growth occurred at 0.01–0.5 % methylamine with an optimum at 0.05–0.1 %. The specific growth rate in liquid basal medium supplemented with 0.1 % methylamine was 0.134 h⁻¹. Strain JLW8^T grew in the temperature range 10–34 °C, and a pH range 5–8.5, with optimal growth occurring at 30 °C and a pH of 7.5. Elimination of ammonium from the basal mineral medium did not affect growth, indicating that methylamine could serve as a nitrogen source. However, replacing ammonium salts with nitrate salts (0.1 %) resulted in growth inhibition. The test for nitrate reduction was negative. Because strain JLW8^T was restricted to growth using methylamine as a single source of carbon, we were not able to verify its ability to grow without a nitrogen source. However, a PCR amplification test using primers specific for the nifH gene (Zehr & McReynolds, 1989) was negative, suggesting that the strain was unable to utilize N₂. No growth was observed at concentrations of NaCl above 0.1 %, SDS concentrations above 0.001 % or H₂O₂ concentrations above 0.00003 %. Tests for oxidase and catalase were positive. Urease activity was not detected. Cells were not resistant to desiccation, heating to 70 °C for 5 min or to 60 °C for 10 min, and growth was observed after heating to 60 °C for 5 min.

Sensitivity to antibiotics was examined in liquid cultures. The following antibiotics were tested (μg ml⁻¹): ampicillin (30), chloramphenicol (10), gramicidin (10), kanamycin (30), nalidixic acid (30), penicillin (30), tetracycline (10), rifampicin (30), rifomycin (10) and streptomycin (10). The effect of antibiotics on cell growth was assessed after 1 week. No growth was observed in the presence of gramicidin, kanamycin or tetracycline, whereas chloramphenicol, ampicillin, nalidixic acid and streptomycin inhibited growth.

Activities of key enzymes for methylotrophy were measured in cell-free extracts of strain JLW8^T, as described by Kalyuzhnaya et al. (2005). Tests for methylamine dehydrogenase were positive [23±5 nmol min⁻¹ (mg protein)⁻¹; n=3], suggesting its role in methylamine oxidation. Tests for methanol dehydrogenase activity were negative. Accordingly, no PCR product with primers specific for the mxaF gene (McDonald & Murrell, 1997) was obtained. Combined activities of the key enzymes of the ribulose monophosphate (RuMP) cycle, hexulose phosphate synthase and 6-phospho-3-hexuloisomerase, were detected [13±3 nmol min⁻¹ (mg protein)⁻¹; n=3], indicating that the RuMP cycle was operational, whereas no activities of key enzymes of the serine cycle, hydroxypyruvate reductase and serine-glyoxylate aminotransferase, were detected. No activities of formaldehyde dehydrogenase (measured with or without glutathione) or formate dehydrogenase (measured with NAD) were detected.

Cellular phospholipid fatty acid analysis was performed by Microbial Insights (). The fatty acid profile was dominated by C_16 : 1ω7c (66 %) and C_16 : 0 (32 %), which is typical of the known obligate or restricted facultative methylotrophs within the Betaproteobacteria. However, strain JLW8^T did not contain the C_16 : 1ω7t isomer found so far in all representatives of the Methylophilaceae. The major phospholipid, as detected by TLC (Findlay & Evans, 1987), was phosphatidylethanolamine (>80 % of the total phospholipid fraction).

The G+C content of the genomic DNA was 54.3±0.3 mol% (n=3). Analysis of a nearly complete sequence of the 16S rRNA gene indicated that strain JLW8^T was only distantly related to known methylotrophic bacteria of the family Methylophilaceae, sharing 94.3–95.6 % similarity with representatives of the genus Methylophilus and 93.1–94.8 % similarity with representatives of the genera Methylovorus and Methylobacillus. However, it was more closely related (97–98 % similarity) to environmental 16S rRNA gene sequences and to the sequence of the uncharacterized strain HTCC349 isolated from a trichloroethene- and dichloroethene-contaminated aquifer (Connon et al., 2005). Phylogenetic analysis revealed that the 16S rRNA gene sequence of strain JLW8^T grouped with environmental sequences and the sequence of strain HTCC349, and that these were separated from the sequences of species representing the genera Methylobacillus, Methylovorus and Methylophilus (Fig. 2⇓).

All representatives of the Methylophilaceae described thus far have been reported to be obligate or restricted facultative methylotrophs capable of utilizing methanol as a sole source of carbon and energy and possessing methanol dehydrogenase activity (Jenkins & Jones, 1987; Doronina et al., 2004, 2005a). Strain JLW8^T is the first example of a representative of the Methylophilaceae lacking the ability to grow on methanol and restricted to methylamine as a single C₁ substrate. Based on this feature as well as other distinctive features such as the lack of C_16 : 1ω7t fatty acid (Table 1⇓) and low level of 16S rRNA gene sequence similarity with members of the genera Methylophilus, Methylobacillus and Methylovorus, strain JLW8^T is proposed as the type strain of a novel species in a new genus within the family Methylophilaceae, Methylotenera mobilis gen. nov., sp. nov.

16S rRNA gene sequences closely related to that of strain JLW8^T have been amplified from a variety of environments, such as wastewater treatment plants, contaminated aquifers, mining waste and sedimentary rocks (based on descriptions of the sequence sources in the NCBI database), pointing to the widespread distribution and potential ecologically important function of this group of organisms.