The 16S rRNA gene sequences of 34 named and unnamed clostridial strains were determined by PCR direct sequencing and were compared with more than 80 previously determined clostridial sequences and the previously published sequences of representative species of other low- G+C-content gram-positive genera, thereby providing an almost complete picture of the genealogical interrelationships of the clostridia. The results of our phylogenetic analysis corroborate and extend previous findings in showing that the genus Clostridium is extremely heterogeneous, with many species phylogenetically intermixed with other sporeforming and non-sporeforming genera. The genus Clostridium is clearly in need of major revision, and the rRNA structures defined in this and previous studies may provide a sound basis for future taxonomic restructuring. The problems and different possibilities for restructuring are discussed in light of the phenotypic and phylogenetic data, and a possible hierarchical structure for the clostridia and their close relatives is presented. On the basis of phenotypic criteria and the results of phylogenetic analyses the following five new genera and 11 new combinations are proposed: Caloramator gen. nov., with Caloramator fervidus comb. nov.; Filifactor gen. nov., with Filifactor villosus comb. nov.; Moorella gen. nov., with Moorella thermoacetica comb. nov. and Moorella thermoautotrophica comb. nov.; Oxobacter gen. nov., with Oxobacter pfennigii comb. nov.; Oxalophagus gen. nov., with Oxalophagus oxalicus comb. nov.; Eubacterium barkeri comb. nov.; Paenibacillus durum comb. nov.; Thermoanaerobacter kivui comb. nov.; Thermoanaerobacter thermocopriae comb. nov.; and Thermoanerobacterium thermosaccharolyticum comb. nov.

Rhizobia described so far belong to three distinct phylogenetic branches within the α-2 subclass of Proteobacteria . Here we report the discovery of a fourth rhizobial branch involving bacteria of the Methylobacterium genus. Rhizobia isolated from Crotalaria legumes were assigned to a new species, “ Methylobacterium nodulans ,” within the Methylobacterium genus on the basis of 16S ribosomal DNA analyses. We demonstrated that these rhizobia facultatively grow on methanol, which is a characteristic of Methylobacterium spp. but a unique feature among rhizobia. Genes encoding two key enzymes of methylotrophy and nodulation, the mxaF gene, encoding the α subunit of the methanol dehydrogenase, and the nodA gene, encoding an acyltransferase involved in Nod factor biosynthesis, were sequenced for the type strain, ORS2060. Plant tests and nodA amplification assays showed that “ M. nodulans ” is the only nodulating Methylobacterium sp. identified so far. Phylogenetic sequence analysis showed that “ M. nodulans ” NodA is closely related to Bradyrhizobium NodA, suggesting that this gene was acquired by horizontal gene transfer.

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.

The taxonomic position of nitrogen-fixing strains that were isolated from rhizosphere macerates of rice cultivated in the Binh Thanh region of Vietnam was determined by using polyphasic taxonomy. We determined the phylogenetic relationships of these organisms by performing DNA-rRNA hybridization experiments with a labeled rRNA probe from the type strain of Burkholderia cepacia, and we found that they belong to a single rRNA complex. Other members of this rRNA complex were also studied, and the N2-fixing strains were found to be closely related to B. cepacia. In addition, all members of the rRNA complex containing B. cepacia were studied by performing auxanographic and DNA-DNA hybridization experiments. Phenotypically and genotypically, the N2-fixing isolates constitute a single cluster together with two strains of clinical origin. These organisms constitute a new Burkholderia species, for which the name Burkholderia vietnamiensis is proposed; the type strain of this species is TVV75 (= LMG 10929). All members of this species can fix nitrogen. On the basis of our polyphasic taxonomy results and previously published data we concluded that the genus Burkholderia should be restricted to the following species: B. cepacia (the type species), Burkholderia mallei, Burkholderia pseudomallei, B. vietnamiensis, Burkholderia gladioli, Burkholderia caryophylli, Burkholderia plantarii, Burkholderia glumae, Burkholderia vandii, Burkholderia cocovenenans comb. nov., and Burkholderia andropogonis comb. nov. On the basis of genotypic and phenotypic results [Alcaligenes] eutrophus, [Burkholderia] solanacearum, and [Burkholderia] pickettii belong to two other clusters whose internal structures must be studied further.

Rhizobia are the common bacterial symbionts that form nitrogen-fixing root nodules in legumes. However, recently other bacteria have been shown to nodulate and fix nitrogen symbiotically with these plants. Neptunia natans is an aquatic legume indigenous to tropical and subtropical regions and in African soils is nodulated by Allorhizobium undicola. This legume develops an unusual root-nodule symbiosis on floating stems in aquatic environments through a unique infection process. Here, we analyzed the low-molecular-weight RNA and 16S ribosomal DNA (rDNA) sequence of the same fast-growing isolates from India that were previously used to define the developmental morphology of the unique infection process in this symbiosis with N. natans and found that they are phylogenetically located in the genus Devosia , not Allorhizobium or Rhizobium . The 16S rDNA sequences of these two Neptunia -nodulating Devosia strains differ from the only species currently described in that genus, Devosia riboflavina. From the same isolated colonies, we also located their nodD and nifH genes involved in nodulation and nitrogen fixation on a plasmid of approximately 170 kb. Sequence analysis showed that their nodD and nifH genes are most closely related to nodD and nifH of Rhizobium tropici , suggesting that this newly described Neptunia -nodulating Devosia species may have acquired these symbiotic genes by horizontal transfer.

A total of 80 bacterial strains isolated from different Sesbania and Acacia species growing in various sites in Senegal (West Africa) were compared with 35 reference strains of Rhizobium, Bradyrhizobium, Azorhizobium, and Agrobacterium species and with 33 representative strains of the different groups of Brazilian isolates described on the basis of the results of a numerical analysis of the whole-cell protein patterns obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fifty-two strains could be placed in three protein electrophoretic clusters, two of which were different from the clusters containing various reference or representative strains, while 30 other strains could not be placed in any group. The strains belonging to the three clusters were studied by determining their nodulation host ranges and their morphological, physiological, and auxanographic characteristics. Representative strains of the three clusters were also genotypically characterized by determining their DNA base compositions, by performing DNA-DNA and DNA-rRNA hybridization experiments, and by determining their 16S rRNA gene sequences. Our results showed that two of the clusters identified on the basis of SDS-PAGE data are genotypically and phenotypically distinct groups that belong on the Rhizobium meliloti-Rhizobium fredii rRNA branch. The third cluster is localized on the Rhizobium loti rRNA branch in the vicinity of Rhizobium huakuii and contains strains isolated in Africa, in Brazil, and in New Zealand from different leguminous species. On the basis of the results of the present study, we propose to emend the genus Sinorhizobium and to reclassify R. meliloti as Sinorhizobium meliloti comb. nov. In addition, two new species, Sinorhizobium saheli and Sinorhizobium teranga, are proposed for isolates from Senegal.