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-spore-forming 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. ~ ~~Great advances, have been made over the past decade in unravelling the phylogenetic complexities within the grampositive endospore-forming bacteria. For example, 16s rRNA oligonucleotide cataloging and, more recently, almost complete rRNA (or gene) sequencing have shown that the aerobic endospore-forming bacilli are phylogenetically very heterogeneous, consisting of at least six highly divergent lines (1,11,51). As a result of these studies, taxonomic reorganization of the genus Bacillus was initiated with the introduction of the genus Alicyclobacillus for some acidophilic species (51) and the genus Paenibacillus (2) for Bacillus polymyxa and its close relatives (rRNA group 3 [l]). Although the remainder of the genus is still in need of taxonomic revision, the phylogenetic groups established by rRNA analysis are already forming the foundation for a new molecular data-based taxonomy for this group of organisms. Knowledge of the natural interrelationships within the anaerobic genus Clostridium is more fragmented than knowledge of the interrelationships among the aerobic bacilli. The earliest, and until recently the most comprehensive, phylogenetic study of the genus Clostridium was published by Johnson and Francis (22), who demonstrated that there is considerable diversity within the genus by u...

Rhizobia described so far belong to three distinct phylogenetic branches within the ␣-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 ␣ 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.Symbioses between leguminous plants and soil bacteria commonly referred to as rhizobia are of considerable environmental and agricultural importance since they are responsible for most of the atmospheric nitrogen fixed on land. Rhizobia are able to elicit on most of the 18,000 species of the Leguminosae family the formation of specialized organs, called nodules, in which they reduce atmospheric nitrogen to ammonia to the benefit of the plant. Nodule formation is controlled by extracellular bacterial signal molecules, called Nod factors, which are recognized by the host plant (21, 34). The rhizobial species described so far are very diverse and do not form an evolutionary homogenous clade. They belong to three distinct branches within the ␣-2 subclass of Proteobacteria and are phylogenetically intertwined with non-symbiotic bacteria (40) (Fig. 1). A first large branch groups the genera Rhizobium, Sinorhizobium, Mesorhizobium, and Allorhizobium with Agrobacterium, a pathogenic bacterium of plants. A second branch contains the genus Bradyrhizobium together with photosynthetic free-living Rhodopseudomonas, whereas the third branch includes the genus Azorhizobium as well as the chemiautotroph Xanthobacter. Each rhizobial species has a defined host range, varying from very narrow, as in the case of Azorhizobium caulinodans (6), to very broad, as in the case of Sinorhizobium sp. strain NGR234 (30). Symbionts of legumes exhibiting ecological and agronomic potential should be characterized prior to their use in sustainable agriculture and environment management.Crotalaria spp. are herbs and shrubs of the subfamily Papilionoideae; it is the largest plant genus in Africa. More than 500 species commonly occur in diverse climatological situations, from semidesert to rain forests and high mountains (1, 29). Some Crotalaria spp. are of great agronomic interest since they are used as green manure to improve soil fertility o...

F-54501 Vandewre-les-Nancy, and URA CNRS 1977, Ecologie Microbienne, USBSE,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 N,-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 N,-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 glumue, Burkholderia vandii, BurkhoMeria cocovenenans comb. nov., and Burkholderia andropogonis comb. nov. On the basis of genotypic and phenotypic results [Alcaligenes] eutrophus, [BurkhoZderia] solanacearum, and[Burkholderia] pickettii belong to two other clusters whose internal structures must be studied further.On the basis of the results of extensive phenotypic studies and DNA-rRNA and DNA-DNA hybridization experiments performed by Palleroni, Stanier, and their collaborators, the genus Pseudomonas was divided into five groups (24, 43, 46, 56). Additional phylogenetic data have shown that these groups are only very remotely related and that each of them contains species belonging to other genera (21-23, 70, 71). In addition to the former pseudomonads that have been shown to belong to the genus Xanthomonas and to a smaller group related to Pseudomonas diminuta and Pseudomonas vesicularis (21-23,43) classified in the recently described genus Brevundirnonas (52), three large groups of pseudomonads can be considered. Pseudomonas rRNA group I (43) is part of rRNA superfamily I1 (18) or the gamma subclass of the Proteobacteria (S), where it constitutes a separate rRNA complex (18,21,22,68); this group represents the authentic pseudomonads which are grouped with Pseudomonas aeruginosa, the type species (43). Although the internal relationships within this group have not been determined yet, it is evident that the genus Pseudomonas must be limited to this group and that all other Pseudomonas species have been generically misnamed as determined by phylogenetic da...

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