There is a need for easy, practical, reliable and robust techniques for the identification and classification of bacterial isolates to the species level as alternatives to 16S rRNA gene sequence analysis and DNA-DNA hybridization. Here, we demonstrate that multilocus sequence analysis (MLSA) of housekeeping genes is a valuable alternative technique. An MLSA study of 10 housekeeping genes (atpD, dnaK, gap, glnA, gltA, gyrB, pnp, recA, rpoB and thrC) was performed on 34 representatives of the genus Ensifer. Genetic analysis and comparison with 16S and 23S rRNA gene sequences demonstrated clear species boundaries and a higher discrimination potential for all housekeeping genes. Comparison of housekeeping gene sequence data with DNA-DNA reassociation data revealed good correlation at the intraspecies level, but indicated that housekeeping gene sequencing is superior to DNA-DNA hybridization for the assessment of genetic relatedness between Ensifer species. Our MLSA data, confirmed by DNA-DNA hybridizations, support the suggestion that Ensifer xinjiangensis is a later heterotypic synonym of Ensifer fredii.

Multilocus sequence analysis (MLSA) was performed on representatives of Ensifer (including species previously assigned to the genus Sinorhizobium) and related taxa. Neighbour-joining (NJ), maximum-parsimony (MP) and maximum-likelihood (ML) phylogenies of dnaK, gltA, glnA, recA, thrC and 16S rRNA genes were compared. The data confirm that the potential for discrimination of Ensifer species is greater using MLSA of housekeeping genes than 16S rRNA genes. In incongruence-length difference tests, the 16S rRNA gene was found to be significantly incongruent with the other genes, indicating that this gene should not be used as a single indicator of relatedness in this group. Significant congruence was detected for dnaK, glnA and thrC. Analyses of concatenated sequences of dnaK, glnA and thrC genes yielded very similar NJ, MP and ML trees, with high bootstrap support. In addition, analysis of a concatenation of all six genes essentially produced the same result, levelling out potentially conflicting phylogenetic signals. This new evidence supports the proposal to unite Ensifer and Sinorhizobium in a single genus. Support for an alternative solution preserving the two genera is less strong. In view of the opinions expressed by the Judicial Commission, the name of the genus should be Ensifer, as proposed by Young [Young, J. M. (2003). Int J Syst Evol Microbiol 53, 2107-2110]. Data obtained previously and these new data indicate that Ensifer adhaerens and 'Sinorhizobium morelense' are not heterotypic synonyms, but represent separate species. However, transfer to the genus Ensifer is not possible at present because the species name is the subject of a pending Request for an Opinion, which would affect whether a novel species in the genus Ensifer or a new combination based on a basonym would be created.

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During a study of polysaccharide-hydrolysing bacteria present in different plant sources, two strains were isolated from pulverized decaying wood of Populus alba and classified in the genus Rhizobium on basis of their almost complete 16S rRNA gene sequences. Their closest phylogenetic relatives were Rhizobium galegae USDA 4128T and Rhizobium huautlense S02 T , with 98.2 and 98.1 % 16S rRNA gene sequence similarity, respectively. recA and atpD sequence analysis showed that these species have less than 88 and 92 % similarity, respectively, to the novel strains. In contrast to their closest phylogenetic relatives, the two strains showed strong cellulase activity on plates containing CM-cellulose as a carbon source. They were also distinguishable from these species on the basis of other phenotypic characteristics. The strains were able to induce ineffective nodules on Medicago sativa and the sequence of their nodD gene was phylogenetically close to that of Ensifer meliloti 1021 (99.6 % similarity). DNA-DNA hybridization values ranged from 10 to 22 % with respect to R. galegae USDA 4128T and 14 to 25 % with respect to R. huautlense S02 T , showing that the strains from this study belong to a novel species, for which the name Rhizobium cellulosilyticum sp. nov. is proposed. The type strain is ALA10B2 T (=LMG 23642The species of the genus Rhizobium are traditionally considered as legume endosymbionts and, although Rhizobium daejeonense was isolated from a cyanide-treatment bioreactor (Quan et al., 2005), most of them have been isolated from nodules. Decaying wood is a good source of polysaccharide-hydrolysing micro-organisms, and we have previously isolated several species, mainly actinomycetes, able to hydrolyse xylan and cellulose from similar sources (Rivas et al., 2003(Rivas et al., , 2004a. In this work, from pulverized decaying wood of Populus alba, we isolated two Gram-negative strains, ALA10B2 T and ALA38.2, that actively hydrolyse CM-cellulose in vitro. The 16S rRNA gene sequences of these strains allowed their classification in the genus Rhizobium, near to Rhizobium galegae and Rhizobium huautlense, two species that do not produce cellulases under the conditions used in this study. A polyphasic study of these strains, including phenotypic and molecular taxonomic approaches, showed that they belong to a novel species of the genus Rhizobium.For isolation of strains ALA10B2 T and ALA38.2, a sample of pulverized decaying wood was collected aseptically from a cavity in the trunk of a healthy Populus alba tree in Salamanca, Spain. From this sample, 1 g was ground, placed in 9 ml sterile water and stirred for 60 min. Aqueous portions (100 ml of the mixture) were spread on CEA medium containing 0.7 % CM-cellulose as the only carbon source, 0.3 % yeast extract and 2.5 % agar. Colonies were picked from these plates and inoculated on the same medium. Cellulase activity was detected after 5 days incubation at 28 uC after staining with a 1 % aqueous Congo red solution. Two bacterial strains, ALA10B2T and ALA38.2, showing ...

Nine isolates from Argyrolobium uniflorum, Lotus creticus, Medicago sativa (Tunisia) and Lotus arabicus (Senegal) were analysed by multilocus sequence analysis (MLSA) of five housekeeping genes (recA, atpD, glnA, gltA and thrC), the 16S rRNA gene and the nodulation gene nodA. Analysis of the individual and concatenated gene sequences demonstrated that the nine new strains constituted three stable, well-supported (bootstrap and gene sequence similarity values) monophyletic clusters, A, B and C, all belonging to the branch of the genus Ensifer, regardless of the phylogenetic reconstruction method used (maximum likelihood, maximum-parsimony, neighbour-joining). The three groups were further characterized by API 100 auxanographic tests, host specificity and nodA gene sequence analysis. On the basis of these data, clusters A and C are suggested as representing two novel species within the genus Ensifer, for which the names Ensifer numidicus sp. nov. (type strain ORS 1407T=LMG 24690T=CIP 109850T) and Ensifer garamanticus sp. nov. (type strain ORS 1400T=LMG 24692T=CIP 109916T) are proposed. The cluster B strains were assigned to Ensifer adhaerens genomovar A.

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