Bacillus methylotrophicus sp. nov., a methanol-utilizing, plant-growth-promoting bacterium isolated from rice rhizosphere soil

Methylotrophy has been recognized as a property of specialized bacteria that are capable of growth on C1-compounds and members of numerous genera and species have been found to possess this ability (Whittenbury et al., 1970; Arfman et al., 1992; Kalyuzhnaya et al., 2006; Lidstrom, 2006). It has been found that the occurrence of methylotrophy in prokaryotes does not correlate with traditional bacterial classification (Brautaset et al., 2004), as facultative methylotrophy has been shown to be a property of diverse typically heterotrophic genera (Boden et al., 2008). One of the earliest methylotrophic organisms to be isolated was the Gram-positive bacterium, ‘Bacillus methylicus’ (later renamed as ‘Bacterium methylicum’, but no longer available in culture). This aerobic, non-spore-forming, facultative methylotroph produced red pigment when grown on formate or methanol and also grew on formaldehyde (Loew, 1892; Migula, 1900; Bergey et al., 1939). Numerous specialized methylotrophs have since been described, including a great diversity of methanotrophs, some of which are obligate methane users, many that also use methanol and a few that are capable of growth on multicarbon compounds (Whittenbury et al., 1970; Dedysh et al., 2005). At the time of writing, the genus Bacillus, in the phylum Firmicutes, consisted of 225 species with validly published names () with Bacillus subtilis as the type species (Cohn, 1872; Skerman et al., 1980). The genus includes the methanol-utilizing bacterium Bacillus methanolicus, which was isolated from soil samples (Arfman et al., 1992). Methylotrophic strains of the genus Bacillus display a strong resistance to high methanol concentrations and the molar growth yields on methanol at the optimum growth temperatures in methanol-limited chemostats are among the highest reported for any methylotrophic bacteria (Anthony, 1982; Dijkhuizen et al., 1988). Currently Gram-negative bacteria remain the best-studied plant-interacting microbes, but representatives of high and low G+C Gram-positive bacteria also have excellent biocontrol, plant-growth-promoting and bioremediation activities. In addition, actinorhizal symbioses largely contribute to global biological nitrogen fixation and many Gram-positive bacteria promote other types of symbioses in tripartite interactions (Francis et al., 2010). In this paper, a novel species of the genus Bacillus was isolated from the rice rhizoplane and was found to have the ability to produce 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase).

Strain CBMB205^T was isolated from the rhizoplane of rice (Oryza sativa L. cv O-dae) from samples collected by the Chungbuk Provincial Agricultural Research and Extension Services (Cheongwon, Republic of Korea). The strain was isolated on selective ammonium mineral salts (AMS) medium (Whittenbury et al., 1970) supplemented with filter-sterilized cycloheximide (10 μg ml⁻¹) and methanol 0.5 % (v/v) at 28 °C for 5 days. The cells were maintained on AMS medium with 0.5 % (v/v) methanol or on nutrient agar (NA, Difco) with 1 % methanol (v/v). Morphological properties were studied according to general protocols (Gerhardt et al., 1994). The nutritional features were determined using Biolog GP2 microplates (Madhaiyan et al., 2007a; Poonguzhali et al., 2006) and carbon-source utilization tests (excluding Biolog) were performed using standard protocols (Green & Bousfield, 1982). Gram staining was performed by using a Gram staining kit (Difco) and bacterial suspensions were examined by phase-contrast microscopy for cell morphology and motility. The pH range for growth was determined on R2A agar. The growth temperature range (4, 10, 20, 25, 30, 37, 45 °C) and NaCl tolerance [0, 1, 2, 3, 4, 5, 10 % (w/v)] were tested on R2A medium. All tests were incubated at 28 °C, unless otherwise indicated. Growth on nutrient agar, Luria–Bertani (LB), R2A, trypticase soy agar (Difco), King's B medium (KB), 2 % MH (see DSMZ list of media 620, ), and Colby and Zathman medium was also evaluated at 30 °C. Catalase activity was determined by assessing bubble production in 3 % (v/v) H₂O₂ and oxidase activity was determined using 1 % (w/v) tetramethyl-ρ-phenylenediamine using Difco BBL catalase and oxidase reagent droppers according to the manufacturer's instructions (Difco). Tests for the hydrolysis of casein, glycerol tributyrate, gelatin, aesculin and pectin were performed and evaluated after 5 days (Atlas, 1993; Kouker & Jaeger, 1987; Ten et al., 2004). Other physiological and biochemical characteristics were tested using the API ZYM, API 20NE and API 32GN galleries (bioMérieux) following the manufacturer's instructions. Preparation and examination of cells under a scanning electron microscope (SEM) was carried out according to Bozzola & Russell (1998). Cells were visualized using a Hitachi S-2500C SEM with a GEMINI column equipped with a field-emission electron source. Cells of strain CBMB205^T were Gram-positive rods, aerobic, motile, endospore-forming, frequently branched and occurred singly or in pairs on solid AMS medium. Colonies were creamy white and convex with regular margins. Photomicrographs of cells of strain CBMB205^T grown on the solid surface of AMS medium supplemented with 0.5 % methanol are shown in Supplementary Fig. S1 (available with IJSEM Online). The novel strain grew on methanol and on diethanolamine and showed weak growth on trimethylamine and formaldehyde when they were supplemented as a carbon source in AMS medium (0.5 % v/v). The results of nutrient utilization tests and the results from the API 20NE, API 32GN galleries for strain CBMB205^T are compared with those of closely related species of the genus Bacillus in Table 1⇓.

The 16S rRNA gene was amplified by PCR from extracted DNA using the universal primers 27F and 1492R (DeLong, 1992). The full gene sequence was determined by the fluorescent dye terminator method using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit (version 3.1). The products were run on a ABI3730XL capillary DNA sequencer (ABI Prism 310 Genetic Analyzer). Sequence comparisons with close relatives available from GenBank were performed using blast () to determine an approximate phylogenetic affiliation and sequences were aligned using clustal w (Thompson et al., 1994). Phylogenetic trees were constructed with the neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony (Fitch, 1971) methods, using the mega 4.1 program (Tamura et al., 2007) and bootstrap values based on 1000 replications (Felsenstein, 1985). The results revealed that strain CBMB205^T was phylogenetically affiliated to the genus Bacillus, forming a phyletic lineage within the genus with a bootstrap value of 37 %. Strain CBMB205^T was closely related to Bacillus vallismortis, Bacillus licheniformis, Bacillus subtilis, Bacillus mojavensis, Bacillus atrophaeus and Bacillus amyloliquefaciens showing pairwise sequence similarities ranging from 98.2 to 99.2 % (Fig. 1⇓).

Determination of ACC deaminase activity by plate and quantitative assays and tests for indole-3-acetic acid (IAA) production, sodium thiosulfate oxidation and siderophore production were carried out as described previously (Poonguzhali et al., 2006; Madhaiyan et al., 2006). A gnotobiotic growth pouch assay was performed to measure the degree of root elongation induced by ACC deaminase in tomato, canola and red pepper as these species are good representatives of ethylene sensitive plants (Glick et al., 1994; Li et al., 2000; Penrose & Glick, 2003; Ghosh et al., 2003; Madhaiyan et al., 2006, 2007b). Strain CBMB205^T possessed ACC deaminase activity, showed siderophore production and sulfur oxidation activity when tested by plate assays and produced negligible amounts of IAA (data not shown). The ACC deaminase activity in cell-free extracts of strain CBMB205^T was 8.25 nmol α-ketobutyrate per mg protein h⁻¹. The promotion of root elongation by strain CBMB205^T was checked with ethylene sensitive plants instead of with rice (the host crop), to verify the role of ACC deaminase in lowering the ethylene level. Inoculation of strain CBMB205^T increased the root lengths of tomato, red pepper and canola plants by 38.3, 4.2 and 22.4 % over controls, respectively. Inoculation also increased the dry biomass of the plants (data not shown). The presence of the acdS gene (558 bp), encoding ACC deaminase, was confirmed in the novel strain by detection with PCR amplification using the non-degenerate primers F1936 and F1939 (Blaha et al., 2006). The presence of ACC deaminase in members of the genus Methylobacterium and various other bacterial genera has been reported previously (Madhaiyan et al., 2006, Penrose & Glick, 2001; Belimov et al., 2001). The novel strain did not show acetylene reduction activity, which was performed in order to indicate the presence of the nitrogenase enzyme. However, the presence of the nifH gene in the novel strain was confirmed by PCR amplification carried out with specific primers (Ueda et al., 1995) 19F (5′-GCIWTYTAYGGIAARGGIGG-3′) and 407R (5′-AAICCRCCRCAIACIACRTC-3′).

For the analysis of cellular fatty acids, the novel strain was cultured in trypticase soy agar at 28 °C for 48 h. The fatty acids were extracted, derivatized to methyl esters and analysed by GC (6890; Hewlett Packard) using the Microbial Identification System (MIDI; Microbial ID) software package, according to standard protocols (Sasser, 1990). Polar lipids and menaquinones were extracted and analysed according to Minnikin et al. (1984) and Kroppenstedt (1982). Preparation of cell walls from strain CBMB205^T and the analysis of peptidoglycan were carried out using methods described by Schleifer (1985), with the modification that TLC was performed on cellulose sheets rather than by paper chromatography. The fatty acid profile of the strain CBMB205^T consisted mainly of C_15 : 0 anteiso, C_15 : 0 iso, C_16 : 0 iso and C_17 : 0 anteiso (summarized in Table 2⇓).

DNA–DNA hybridization experiments were performed to determine the relatedness of strain CBMB205^T to its closest relatives. The filter hybridization method was used (Seldin & Dubnau, 1985). The hybridization temperature was 65 °C and DNA–DNA relatedness was quantified by using a densitometer (Bio-Rad Laboratories). Probe labelling was conducted by using the non-radioactive DIG-High prime system (Roche Diagnostics GmbH) and hybridized DNA was visualized using the DIG Luminescent Detection kit according to the manufacturer's instructions (Roche Diagnostics GmbH). The G+C content of the genomic DNA of the novel strain was determined by HPLC analysis using a reverse-phase column (Supelcosil LC-18 S, Supelco) of individual nucleosides as previously described (Mesbah et al., 1989). Strain CBMB205^T showed a low level of DNA–DNA relatedness that ranged from 19.0 to 6.0 % with its closest relatives B. amyloliquefaciens (36.0 %), B. vallismortis (32 %), B. subtilis subsp. subtilis (31 %), B. subtilis subsp. spizizenii (30 %), B. atrophaeus (28 %), B. licheniformis (25 %) and B. mojavensis (19 %). These results indicated that strain CBMB205^T did not belong to the above species if the recommended threshold value of 70 % DNA–DNA similarity for the delineation of species is considered (Wayne et al., 1987). The DNA G+C content of strain CBMB205^T was 45.0 mol%, which fell within the range previously described for the genus Bacillus.

The 16S rRNA gene sequence similarity data, DNA–DNA hybridization values and other phenotypic characteristics allowed strain CBMB205^T to be recognized as separate from other members of the genus Bacillus. On the basis of these results, strain CBMB205^T is considered to represent a novel species of the genus Bacillus, for which the name Bacillus methylotrophicus sp. nov. is proposed.