Methylobacterium oryzae sp. nov., an aerobic, pink-pigmented, facultatively methylotrophic, 1-aminocyclopropane-1-carboxylate deaminase-producing bacterium isolated from rice

The genus Methylobacterium includes a group of strictly aerobic, Gram-negative, pink-pigmented, facultatively methylotrophic (PPFM) bacteria characterized by their ability to utilize single-carbon compounds like methanol and formaldehyde via the serine pathway, as well as a wide range of multicarbon growth substrates (Green, 1992). Methylobacterium is classified in the α2 subgroup of the Proteobacteria and presently consists of 22 species with validly published names (Heumann, 1962; Ito & Iizuka, 1971; Kouno & Ozaki, 1975; Patt et al., 1976; Rock et al., 1976; Austin & Goodfellow, 1979; Green & Bousfield, 1983; Urakami & Komagata, 1984; Bousfield & Green, 1985; Green et al., 1988; Urakami et al., 1993; Wood et al., 1998; Doronina et al., 2000, 2002; McDonald et al., 2001; Van Aken et al., 2004; Jourand et al., 2004; Anesti et al., 2004; Gallego et al., 2005a, b, c, 2006). Methylobacterium organophilum, the type species, remained the only PPFM bacterium reported with the ability to grow on methane until the description of Methylobacterium populi, a novel methane-utilizing species isolated from poplar trees. Members of the genus Methylobacterium are ubiquitous in nature, detected in soil, freshwater and lake sediments, as well as on other solid surfaces (Corpe & Rheem, 1989; Lidstrom & Chistoserdova, 2002), and are known particularly for their close association with plants (Holland & Polacco, 1994; Lidstrom & Chistoserdova, 2002; Sy et al., 2005). Associations of members of Methylobacterium with plants range from epiphytic to endophytic and symbiotic relations (Sy et al., 2001; Koenig et al., 2002; Pirttilä et al., 2000; Idris et al., 2006). Possible mechanisms of plant-growth promotion by Methylobacterium include production of phytohormones, such as indole-3-acetic acid (IAA), cytokinins or vitamins (Basile et al., 1985; Koenig et al., 2002; Trotsenko et al., 2001). In addition, members of Methylobacterium are associated with nitrogen metabolism of plants by means of bacterial urease (Holland & Polacco, 1992). Methylobacterium strains are able to establish efficient nitrogen-fixing symbioses by nodulating legume roots (Sy et al., 2001) and have been identified as endosymbionts within the buds of Scots pine (Pirttilä et al., 2000). Furthermore, we have reported the enhancement of plant growth in canola by 1-aminocyclopropane-1-carboxylate (ACC) deaminase-producing Methylobacterium strains (Madhaiyan et al., 2006). The data presented in this paper include the formal taxonomic description of a novel species of the genus Methylobacterium for two strains isolated from rice tissues with the ability to produce ACC deaminase.

Strains CBMB20^T and CBMB110 were respectively isolated from surface-disinfected stem and leaf tissues of rice (Oryza sativa L. ‘Nam-Pyeoung’). The strains were recovered on 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. The strains were maintained routinely on nutrient agar (NA; Difco) medium, supplemented with 1 % (v/v) methanol, or on selective AMS medium. Morphological properties were studied according to general protocols (Gerhardt et al., 1994). Scanning electron microscope (SEM) observations were performed on fixed material that was prepared for routine examinations as described by Bozzola & Russell (1998). Samples were critical-point-dried, mounted on stubs, sputter-coated with gold/palladium and visualized by using a Hitachi S-2500C SEM with GEMINI column equipped with a field-emission electron source.

Chromosomal DNA was extracted and purified from cells cultured on AMS medium as described by Sambrook et al. (1989). 16S rRNA genes were amplified using universal primers fD1 and rP2 (Weisberg et al., 1991) and 16S rRNA gene sequencing was performed by the Big-Dye primer method using an automated DNA sequencer (ABI Prism 310 Genetic Analyzer) as described previously (Brosius et al., 1978; Shima et al., 1994). Phylogenetic analyses were performed using mega version 3.1 (Kumar et al., 2004), after multiple alignment of the data by clustal w (Thompson et al., 1994). Distances were obtained using options according to the Kimura two-parameter model (Kimura, 1980) and clustering was performed using the neighbour-joining (Saitou & Nei, 1987) method. Bootstrap values from 1000 replications were used to obtain the confidence level of the branches (Felsenstein, 1985).

Nutritional features were determined using the Biolog Microstation (MicroLog-3, 4.01B). The analysis was carried out in Biolog GN2 microtitre plates according to the manufacturer's instructions; the reactions were observed after incubating the plates at 28 °C for 7 days. Carbon-source utilization tests (excluding Biolog) were performed by using a standard protocol described by Green & Bousfield (1982). Other physiological and biochemical characteristics were tested using the API ZYM and API 20NE galleries (bioMérieux) following the manufacturer's instructions. Cellular fatty acids were analysed in organisms grown on NA with 1 % methanol (v/v) for 48 h. The cellular fatty acids were derivatized to methyl esters (Sasser, 1990) and analysed by gas chromatography (Hewlett Packard 6890) using the Microbial Identification System (MIDI; Microbial ID) software package.

The G+C content of genomic DNA was determined by HPLC analysis of individual nucleosides resulting from DNA hydrolysis and dephosphorylation (Mesbah et al., 1989) using a reversed-phase column (Supelcosil LC-18-S; Supelco). DNA–DNA hybridization was carried out following the filter hybridization method as described by Seldin & Dubnau (1985). Probe labelling was conducted by using the non-radioactive DIG-High Prime system (Roche Diagnostics) and hybridized DNA was visualized using the DIG luminescent detection kit (Roche Diagnostics). Hybridization temperatures were 60 and 65 °C and DNA–DNA relatedness was quantified by using a densitometer (Bio-Rad Laboratories).

Strains CBMB20^T and CBMB110 were strictly aerobic, Gram-negative, non-spore-forming and formed pink- to red-pigmented colonies. Cells were rod-shaped, frequently branched and occurred singly or in rosettes on solid AMS and NA media. SEM photomicrographs of strain CBMB20^T are available as Supplementary Figs S1 and S2 in IJSEM Online. The presence of an mxaF gene encoding methanol dehydrogenase required for methanol utilization was examined by PCR using the non-degenerate primers f1003 and r1561 (McDonald & Murrell, 1997); a product of the expected size (560 bp) was detected, similar to the mxaF genes of other Methylobacterium species. The 16S rRNA gene sequence of strain CBMB20^T obtained was a continuous stretch of 1416 bp. Sequence similarity calculations and phylogenetic analysis identified the strains as members of the genus Methylobacterium as a separate group and indicated the type strains of Methylobacterium fujisawaense (99.1 % similarity), Methylobacterium radiotolerans (98.0 %) and Methylobacterium mesophilicum (97.4 %) as the closest relatives (Fig. 1⇓). The 16S rRNA gene sequence similarity matrix is available in Supplementary Table S1.

Phenotypic differences between Methylobacterium species are limited and often rely on utilization of carbon and energy sources (Green & Bousfield, 1982; Green, 1992). Like other members of the genus, strain CBMB20^T grew on C₁ substrates, such as methanol and formate, but not methylamine, trimethylamine or formaldehyde. Strain CBMB20^T differed from its closest relatives in utilization of several carbon sources (summarized in Table 1⇓). Carbon-source utilization, enzyme activities and other biochemical reactions that differentiate the strains from other related species are available as Supplementary Tables S2–S4. The fatty acid profile of strains CBMB20^T and CBMB110 consisted mainly of cis-vaccenic acid (C_18 : 1ω7c) and octadecanoic acid (stearic acid, C_18 : 0) (Table 2⇓).

Strains CBMB20^T and CBMB110 utilize ACC as a nitrogen source, and the presence of ACC deaminase activity in cell-free extracts and the lowering of ethylene levels and promotion of root elongation in canola seedlings under gnotobiotic conditions have been reported elsewhere (Madhaiyan et al., 2006). This particular ability has not been tested among other members of the genus. The type strains of the closely related Mtb. fujisawaense, Mtb. radiotolerans and Mtb. organophilum were tested by preliminary plate assays on AMS medium spread with 30 μmol ACC as a nitrogen source, and the results revealed that all three strains are positive for ACC deaminase activity. The presence of ACC deaminase in bacteria plays an important role in plant growth promotion; the bacterial cells act as a sink for ACC, the immediate biosynthetic precursor of ethylene, thereby lowering plant ethylene levels and decreasing the negative effects of various environmental stresses (Stearns et al., 2005). The presence of ACC deaminase in various bacterial genera has been reported previously (Penrose & Glick, 2001; Belimov et al., 2001).

The DNA G+C contents of strains CBMB20^T and CBMB110 are respectively 70.6 and 69.2 mol%. DNA–DNA reassociation performed to confirm the species status of the novel isolates in relation to their closest phylogenetic neighbours showed 88.63 % relatedness, confirming the close relationship between the two strains. However, the type strains of closely related Methylobacterium species showed 42.09–63.09 % relatedness with CBMB20^T, indicating that strain CBMB20^T can be separated from other members of the genus Methylobacterium (Table 3⇓). These results indicate that strain CBMB20^T does not belong to any of these species when the recommendation of a threshold value of 70 % DNA–DNA relatedness for species definition is considered (Wayne et al., 1987).

The 16S rRNA gene sequence similarity data, DNA–DNA hybridization values and phenotypic characteristics allowed strains CBMB20^T and CBMB110 to be separated from other members of the genus Methylobacterium. Strain CBMB20^T is proposed as the type strain of a novel Methylobacterium species, for which the name Methylobacterium oryzae sp. nov. is proposed.