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1 Laboratoire des Symbioses Tropicales et Méditerranéennes, UMR 1063, IRD/CIRAD/INRA/Agro-M/UMII, TA 10/J, Campus International de Baillarguet, 34398 Montpellier cedex 5, France
2 Laboratory of Microbiology, University of Gent, Ledeganckstraat 35, 9000 Gent, Belgium
Correspondence
Philippe Jourand
jourand{at}mpl.ird.fr
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and partial nifH gene sequences of strain ORS 2060T are AF220763 and AJ512205, respectively.
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TOPABSTRACTMAIN TEXTNote added in proofREFERENCES |
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). At the time of writing, the genus Methylobacterium consists of 14 PPFM species (Doronina et al., 2002), with Methylobacterium organophilum as the type species (Patt et al., 1976). Methylobacterium strains have been found on many plant tissues but no symbiotic association with plants has been reported (Holland, 1997). Recently and surprisingly, 16S rRNA gene-based phylogenetic analysis classified non-pigmented bacteria isolated from legume root nodules of three Crotalaria species (subfamily Papilionoideae, tribe Crotalarieae), i.e. Crotalaria glaucoides, Crotalaria perrottetii and Crotalaria podocarpa, in the genus Methylobacterium (Sy et al., 2001a, b). More recently, other nitrogen-fixing isolates from root nodules of the legume Lotononis bainesii (Papilionoideae, Crotalarieae) were characterized as pigmented methylotrophic bacteria (Jaftha et al., 2002). Here we review previous reports on Methylobacterium strains that nodulate Crotalaria species and add additional phenotypic and genotypic characterization data, such as morphology, physiology, enzymic and carbon sources assimilation tests, antibiotic resistance and nifH gene sequence, and conclude with the proposal of Methylobacterium nodulans sp. nov.
A group of 72 bacterial strains was isolated from root nodules samples from three Crotalaria species (C. glaucoides, C. perrottetii and C. podocarpa) sampled in five different geographical areas of Senegal (West Africa) (Samba et al., 1999). SDS-PAGE protein pattern analysis clearly indicated that these strains constituted a homogeneous group that was separate from other known legume-nodule-forming bacteria (Samba et al., 1999; Sy et al., 2001a). Eleven strains, representative of the different SDS-PAGE subclusters and for plant and geographical origins, were chosen and studied further. The 16S rRNA PCR-RFLP profile analysis on these strains confirmed the homogeneity of this group and the 16S rRNA gene sequence analysis of two of them, ORS 2060T and ORS 1924 presenting 100 % identity, showed their close phylogenetic relationship with members of the genus Methylobacterium (Sy et al., 2001a, b). The methylotrophic metabolism of the same 11 strains was confirmed by growth on MMS medium (Green, 1992) with C1 compounds as sole carbon source: methanol, formate and formaldehyde but not methylamine (Dreyfus et al., 1999; Sy et al., 2001b). In addition, an mxaF gene, encoding the methanol dehydrogenase required for methanol utilization, was detected by PCR in the 11 strains and the mxaF gene sequence of ORS 2060T showed 88 % identity to that of M. organophilum (Sy et al., 2001b). Taken together, these data confirmed that the bacterial strains isolated from root nodules of C. glaucoides, C. perrottetii and C. podocarpa constituted a homogeneous bacterial group that belonged to the genus Methylobacterium.
Cross-inoculation and nitrogen fixation tests on legume plants revealed both nodulation specificity and nitrogen-fixing efficiency within the genus Crotalaria (Sy et al., 2001a). Representative strains for the main Methylobacterium species, i.e. Methylobacterium extorquens, Methylobacterium organophilum, Methylobacterium radiotolerans, Methylobacterium rhodinum, Methylobacterium mesophilicum, Methylobacterium rhodesianum and Methylobacterium zatmanii, and two Methylobacterium spp. were tested for plant nodulation but none of them was able to induce any legume root nodule (Sy et al., 2001b). In addition, the nodA gene, present in all legume-nodule-forming bacteria and encoding a key enzyme in Nod factor biosynthesis that induces legume nodulation (Martinez Romero, 1994; van Rhijn & Vanderleyden, 1995), was detected by PCR in the strains of the novel Methylobacterium species isolated from C. glaucoides, C. perrottetii and C. podocarpa. In contrast, nodA was not detected in any of the main representative strains of the genus Methylobacterium mentioned above (Sy et al., 2001b). A comparative analysis of the NodA protein deduced from the nodA gene sequence showed a range of 53·1 % similarity with the NodA protein sequence from Azorhizobium caulinodans to 74·1 % similarity with that of Bradyrhizobium elkanii (Sy et al., 2001b).
As a consequence of the above-mentioned results, Sy et al. (2001b) concluded that the group of strains made up of facultatively methylotrophic, root-nodule-forming and nitrogen-fixing bacteria may be regarded as a novel Methylobacterium species. In this report, we formally propose the name Methylobacterium nodulans sp. nov. to include these strains, with ORS 2060T as the type strain.
Since the report of Sy et al. (2001b), novel Methylobacterium species have been described (Doronina et al., 2002). Fig. 1 shows a 16S rRNA gene-based phylogenetic tree that includes M. nodulans ORS 2060T, representative strains of Methylobacterium sp. isolated from L. bainesii (Jaftha et al., 2002), 13 of the 14 validly published Methylobacterium species (the sequence of Methylobacterium aminovorans is not available) and their nearest phylogenetic neighbours. All Methylobacterium species and strains form a separate branch, consisting of three sub-branches. One sub-branch consists of M. nodulans ORS 2060T and Methylobacterium sp. (isolated from L. bainesii) strains xct10, xct14 and xct17. M. nodulans ORS 2060T shows sequence identity values of 95·8–97·6 % with Methylobacterium sp. strains isolated from L. bainesii (Jaftha et al., 2002), and less than 95·2 % sequence identity with the other Methylobacterium species. These similarity values confirm that M. nodulans constitutes a separate species in the genus Methylobacterium and that it is distinct from Methylobacterium sp. strains isolated from L. bainesii.
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, and sequenced (GenBank/EMBL/DDBJ accession no. AJ512205). Fig. 2 depicts a nifH-based phylogenetic tree showing the relationships of the nifH sequences of Crotalaria-nodulating strains with those of related nitrogen-fixing bacteria. A maximum-likelihood method was applied for the reconstruction of the phylogenetic tree based on inferred amino acid sequences, excluding third codon positions from the alignment (due to the saturation of this position). The phylogeny shows a close relationship among M. nodulans ORS 2060T, Methylobacterium sp. isolated from L. bainesii and the nifH gene sequence from Gluconacetobacter diazotrophicus, a sugarcane endophyte of the 
-Proteobacteria. Jaftha et al. (2002) suggested the closest relationship between their Methylobacterium sp. nifH sequence was with Azospirillum brasilense. However, they did not include the G. diazotrophicus sequence in their analysis and applied a phenetic method, including all nucleotide positions in their phylogenetic reconstruction, which is thus much more sensitive to homoplasy and false reconstruction than our maximum-likelihood approach that excluded saturated positions. To the best of our knowledge, nitrogen fixation and the presence of the nifH gene have never been reported in any other species belonging to the genus Methylobacterium.
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; Sy et al., 2001b): ORS 2026, ORS 2045 and ORS 2076 isolated from C. glaucoides; ORS 1917, ORS 1991 and ORS 2060T isolated from C. podocarpa; and ORS 1924, ORS 1928, ORS 1937, ORS 2030 and ORS 2092 isolated from C. perrottetii. The following substrates were readily used as sole source of carbon in MMS medium at 30 °C (Green, 1992): succinate, citrate, pyruvate, glutamate and ethanol. In addition, bacterial enzymic activities were determined using the API 20NE galleries according to the manufacturer's protocol (bioMérieux); tests for nitrate reductase and urease were positive; tests for 
-galactosidase, -glucosidase, protease, indole production and glucose fermentation were negative. API Biotype 100 galleries (bioMérieux) were also used to check assimilation and growth on 100 carbon sources as according to Kersters et al. (1984). M. nodulans strains were able to grow on the following substrates as sole carbon sources: (+)-D-galactose, (–)-D-ribose, (+)-L-arabinose, (+)-D-xylose, glycerol, D-lyxose, D-saccharate, mucate, (+)-L- and (–)-D-tartrate, (+)-D- and (–)-L-malate, cis- and trans-aconitate, tricarballylate, glucuronate, 2- and 5-keto-D-gluconate, D-gluconate, phenylacetate, p-hydroxybenzoate, quinate, benzoate, betaine, -DL-amino-n-butyrate, DL-lactate, fumarate, glutarate, DL-glycerate, -DL-hydroxybutyrate, L-aspartate, L-proline, L-alanine, L-serine and 2-oxoglutarate. Strains were unable to use -(+)-D-glucose, -(+)-D-fructose, (+)-D-trehalose, (+)-D-mannose, (+)-L-sorbose, -(+)-D-melibiose, sucrose, -lactose, (+)-D-raffinose, maltotriose, maltose, lactulose, (+)-D-cellobiose, -gentobiose, aesculin, -L-rhamnose, -(–)-L-fucose, (+)-D-arabitol, xylitol, dulcitol, myo-inositol, D-mannitol, D-sorbitol, tryptophan, N-acetylglucosamine, coumarate, trigonelline, putrescine, histamine, L-histidine, ethanolamine, tryptamine, D-glucosamine, D-alanine, malonate, propionate and L-tyrosine. The intrinsic antibiotic resistance patterns of the strains show fairly high resistance to ampicillin, carbenicillin and nalidixic acid but sensitivity to kanamycin, gentamicin and tetracycline. Table 1 summarizes features useful for distinguishing M. nodulans sp. nov. from the 14 recognized species of the genus Methylobacterium (Green, 1992; Doronina et al., 2002).
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Short asporogenous Gram-negative rods (0·8–1·0x1·0–1·5 µm) that occur singly or occasionally in pairs; some are motile with one or more polar flagella. Colonies on MMS medium+agar (Green, 1992) with methanol as sole carbon source are shiny, smooth, raised, entire and 0·5–1 mm in diameter after 3 days at 30 °C and are not pigmented. Optimal growth occurs at pH 6·8–7·5 and at 30–37 °C. Strictly aerobic, catalase-positive and weakly oxidase-positive; urease-positive and able to reduce nitrate into nitrite. Table 1
shows other phenotypic traits of the species. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA, mxaF, partial nodA and partial nifH gene sequences of strain ORS 2060T are AF220763, AF220764, AF266748 and AJ512205, respectively. Can form nitrogen-fixing root nodules in symbiosis with Crotalaria glaucoides, Crotalaria perrottetii and Crotalaria podocarpa.
The type strain is ORS 2060T (=CNCM I 2342T=LMG 21967T), isolated from C. podocarpa from the Bel-Air area, Dakar, Senegal. Most of the molecular and physiological studies were conducted on this strain.
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ACKNOWLEDGEMENTS |
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3+I model). The reconstruction is based on the first and second codon positions only. Bootstrap values are indicated in bold and were estimated from 100 replicates. GenBank/EMBL/DDBJ accession numbers are in parentheses.