Showing papers in "International Journal of Systematic and Evolutionary Microbiology in 2001"

Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and 'HGE agent' as subjective synonyms of Ehrlichia phagocytophila.

Abstract: The genera Anaplasma, Ehrlichia, Cowdria, Neorickettsia and Wolbachia encompass a group of obligate intracellular bacteria that reside in vacuoles of eukaryotic cells and were previously placed in taxa based upon morphological, ecological, epidemiological and clinical characteristics. Recent genetic analyses of 16S rRNA genes, groESL and surface protein genes have indicated that the existing taxa designations are flawed. All 16S rRNA gene and groESL sequences deposited in GenBank prior to 2000 and selected sequences deposited thereafter were aligned and phylogenetic trees and bootstrap values were calculated using the neighbour-joining method and compared with trees generated with maximum-probability, maximum-likelihood, majority-rule consensus and parsimony methods. Supported by bootstrap probabilities of at least 54%, 16S rRNA gene comparisons consistently clustered to yield four distinct clades characterized roughly as Anaplasma (including the Ehrlichia phagocytophila group, Ehrlichia platys and Ehrlichia bovis) with a minimum of 96.1% similarity, Ehrlichia (including Cowdria ruminantium) with a minimum of 97.7% similarity, Wolbachia with a minimum of 95.6% similarity and Neorickettsia (including Ehrlichia sennetsu and Ehrlichia risticii) with a minimum of 94.9% similarity. Maximum similarity between clades ranged from 87.1 to 94.9%. Insufficient differences existed among E. phagocytophila, Ehrlichia equi and the human granulocytic ehrlichiosis (HGE) agent to support separate species designations, and this group was at least 98.2% similar to any Anaplasma species. These 16S rRNA gene analyses are strongly supported by similar groESL clades, as well as biological and antigenic characteristics. It is proposed that all members of the tribes Ehrlichieae and Wolbachieae be transferred to the family Anaplasmataceae and that the tribe structure of the family Rickettsiaceae be eliminated. The genus Anaplasma should be emended to include Anaplasma (Ehrlichia) phagocytophila comb. nov. (which also encompasses the former E. equi and the HGE agent), Anaplasma (Ehrlichia) bovis comb. nov. and Anaplasma (Ehrlichia) platys comb. nov., the genus Ehrlichia should be emended to include Ehrlichia (Cowdria) ruminantium comb. nov. and the genus Neorickettsia should be emended to include Neorickettsia (Ehrlichia) risticii comb. nov. and Neorickettsia (Ehrlichia) sennetsu comb. nov.

Taxonomic study of aerobic thermophilic bacilli: descriptions of Geobacillus subterraneus gen. nov., sp. nov. and Geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustophilus, Bacillus thermodenitrificans to Geobacillus as the new combinations G. stearothermophilus, G. th.

Abstract: Five hydrocarbon-oxidizing strains were isolated from formation waters of oilfields in Russia, Kazakhstan and China. These strains were moderately thermophilic, neutrophilic, motile, spore-forming rods, aerobic or facultatively anaerobic. The G+C content of their DNA ranged from 49.7 to 52.3 mol%. The major isoprenoid quinone was menaquinone-7; cellular fatty acid profiles consisted of significant amounts of iso-15:0, iso-16:0 and iso-17:0 fatty acids (61.7-86.8% of the total). Based on data from 16S rDNA analysis and DNA-DNA hybridization, the subsurface isolates could be divided into two groups, one of which consisted of strains UT and X and the other of which consisted of strains K, Sam and 34T. The new strains exhibited a close phylogenetic relationship to thermophilic bacilli of 'Group 5' of Ash et al. [Ash, C., Farrow, J. A. E., Wallbanks, S. & Collins, M. D. (1991). Lett Appl Microbiol 13, 202-206] and a set of corresponding signature positions of 16S rRNA. Comparative analysis of the 16S rDNA sequences and fatty acid compositions of the novel isolates and established species of thermophilic bacilli indicated that the subsurface strains represent two new species within a new genus, for which the names Geobacillus subterraneus gen. nov., sp. nov., and Geobacillus uzenensis sp. nov. are proposed. It is also proposed that Bacillus stearothermophilus, Bacillus thermoleovorans, Bacillus thermocatenulatus, Bacillus kaustophilus, Bacillus thermoglucosidasius and Bacillus thermodenitrificans be transferred to this new genus, with Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) as the type species.

Proposal of the genus Sphingomonas sensu stricto and three new genera, Sphingobium, Novosphingobium and Sphingopyxis, on the basis of phylogenetic and chemotaxonomic analyses.

Abstract: Phylogenetic analyses of 16S rRNA gene sequences by distance matrix and parsimony methods indicated that the currently known species of the genus Sphingomonas can be divided into four clusters. Some chemotaxonomic and phenotypic differences were noted among these clusters. Three new genera, Sphingobium, Novosphingobium and Sphingopyxis, are proposed in addition to the genus Sphingomonas sensu stricto. The genus Sphingobium is proposed to accommodate Sphingomonas chlorophenolica, Sphingomonas herbicidovorans and Sphingomonas yanoikuyae. The genus Novosphingobium is proposed for Sphingomonas aromaticivorans, Sphingomonas capsulata, Sphingomonas rosa, Sphingomonas stygia, Sphingomonas subarctica and Sphingomonas subterranea. Sphingomonas macrogoltabidus and Sphingomonas terrae are reclassified in the genus Sphingopyxis. The type species of Sphingobium, Novosphingobium and Sphingopyxis are Sphingobium yanoikuyae, Novosphingobium capsulatum and Sphingopyxis macrogoltabida, respectively.

A revision of Rhizobium Frank 1889, with an emended description of the genus, and the inclusion of all species of Agrobacterium Conn 1942 and Allorhizobium undicola de Lajudie et al. 1998 as new combinations: Rhizobium radiobacter, R. rhizogenes, R. rubi, R. undicola and R. vitis.

Abstract: Rhizobium, Agrobacterium and Allorhizobium are genera within the bacterial family Rhizobiaceae, together with Sinorhizobium. The species of Agrobacterium, Agrobacterium tumefaciens (syn. Agrobacterium radiobacter), Agrobacterium rhizogenes, Agrobacterium rubi and Agrobacterium vitis, together with Allorhizobium undicola, form a monophyletic group with all Rhizobium species, based on comparative 16S rDNA analyses. Agrobacterium is an artificial genus comprising plant-pathogenic species. The monophyletic nature of Agrobacterium, Allorhizobium and Rhizobium and their common phenotypic generic circumscription support their amalgamation into a single genus, Rhizobium. Agrobacterium tumefaciens was conserved as the type species of Agrobacterium, but the epithet radiobacter would take precedence as Rhizobium radiobacter in the revised genus. The proposed new combinations are Rhizobium radiobacter, Rhizobium rhizogenes, Rhizobium rubi, Rhizobium undicola and Rhizobium vitis.

Muricauda ruestringensis gen. nov., sp. nov., a facultatively anaerobic, appendaged bacterium from German North Sea intertidal sediment.

Abstract: A gram-negative, facultatively anaerobic bacterium with appendages was isolated from continuous cultures with a seawater-sediment suspension containing hexadecane as the sole carbon source. Although this organism was isolated from a hexadecane-degrading bacterial community, it was not able to degrade hexadecane. However, this bacterium was able to use different sugars and amino acids for growth, indicating that it probably profits from the lysis or from products like surfactants of other cells in the community. 16S rDNA analysis demonstrated that the isolated strain is phylogenetically related to the family Flavobacteriaceae of the phylum 'Cytophaga-Flavobacterium-Bacteroides'. Evidence based on phenotypic characteristics and 16S rDNA analysis supports the conclusion that this bacterium is distinct from its nearest relative, Zobellia uliginosa (90.72% similarity in 16S rRNA gene sequence), and from the other genera of the Flavobacteriaceae. It is therefore proposed that the isolated marine bacterium represents a novel taxon, designated Muricauda ruestringensis gen. nov., sp. nov. The type strain is strain B1T (= DSM 13258T = LMG 19739T).

Ralstonia taiwanensis sp. nov., isolated from root nodules of Mimosa species and sputum of a cystic fibrosis patient.

Abstract: A polyphasic taxonomic study, including 16S rDNA sequence analysis, DNA-DNA hybridizations, DNA base ratio determinations, amplified 165 rDNA restriction analysis, whole-cell protein analyses and extensive biochemical characterization, was conducted to clarify the relationships of eight isolates from root nodules of Mimosa species and one isolate from sputum of a cystic fibrosis patient. All nine isolates were classified as a novel Ralstonia species, for which the name Ralstonia taiwanensis sp. nov. is proposed. The type strain is LMG 19424T (= CCUG 44338T). R. taiwanensis effectively nodulated the Mimosa species and is the first beta-proteobacterium that is known to be capable of root nodule formation and nitrogen fixation.

Phylogenetic analyses of Klebsiella species delineate Klebsiella and Raoultella gen. nov., with description of Raoultella ornithinolytica comb. nov., Raoultella terrigena comb. nov. and Raoultella planticola comb. nov.

Abstract: The phylogenetic relationships of the type strains of 9 Klebsiella species and 20 species from 11 genera of the family Enterobacteriaceae were investigated by performing a comparative analysis of the sequences of the 16S rRNA and rpoB genes. The sequence data were phylogenetically analysed by the neighbourjoining and parsimony methods. The phylogenetic inference of the sequence comparison confirmed that the genus Klebsiella is heterogeneous and composed of species which form three clusters that also included members of other genera, including Enterobacter aerogenes, Erwinia clusters I and II and Tatumella. Cluster I contained the type strains of Klebsiella pneumoniae subsp. pneumoniae, Klebsiella pneumoniae subsp. rhinoscleromatis and Klebsiella pneumoniae subsp. ozaenae. Cluster II contained Klebsiella ornithinolytica, Klebsiella planticola, Klebsiella trevisanii and Klebsiella terrigena, organisms characterized by growth at 10 degrees C and utilization of L-sorbose as carbon source. Cluster III contained Klebsiella oxytoca. The data from the sequence analyses along with previously reported biochemical and DNA-DNA hybridization data support the division of the genus Klebsiella into two genera and one genogroup. The name Raoultella is proposed as a genus name for species of cluster II and emended definitions of Klebsiella species are proposed.

Phylogenies of atpD and recA support the small subunit rRNA-based classification of rhizobia.

Abstract: The current classification of the rhizobia (root-nodule symbionts) assigns them to six genera. It is strongly influenced by the small subunit (16S, SSU) rRNA molecular phylogeny, but such single-gene phylogenies may not reflect the evolution of the genome as a whole. To test this, parts of the atpD and recA genes have been sequenced for 25 type strains within the alpha-Proteobacteria, representing species in Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium, Azorhizobium, Agrobacterium, Phyllobacterium, Mycoplana and Brevundimonas. The current genera Sinorhizobium and Mesorhizobium are well supported by these genes, each forming a distinct phylogenetic clade with unequivocal bootstrap support. There is good support for a Rhizobium clade that includes Agrobacterium tumefaciens, and the very close relationship between Agrobacterium rhizogenes and Rhizobium tropici is confirmed. There is evidence for recombination within the genera Mesorhizobium and Sinorhizobium, but the congruence of the phylogenies at higher levels indicates that the genera are genetically isolated. rRNA provides a reliable distinction between genera, but genetic relationships within a genus may be disturbed by recombination.

The taxonomy of Streptomyces and related genera.

Abstract: The streptomycetes, producers of more than half of the 10,000 documented bioactive compounds, have offered over 50 years of interest to industry and academia. Despite this, their taxonomy remains somewhat confused and the definition of species is unresolved due to the variety of morphological, cultural, physiological and biochemical characteristics that are observed at both the inter- and the intraspecies level. This review addresses the current status of streptomycete taxonomy, highlighting the value of a polyphasic approach that utilizes genotypic and phenotypic traits for the delimitation of species within the genus.

Phylogenetic analysis and taxonomic study of marine Cytophaga-like bacteria: proposal for Tenacibaculum gen. nov. with Tenacibaculum maritimum comb. nov. and Tenacibaculum ovolyticum comb. nov., and description of Tenacibaculum mesophilum sp. nov. and Tenacibaculum amylolyticum sp. nov.

Abstract: Bacterial strains were isolated from sponge and green algae which were collected on the coast of Japan and Palau. The phylogenetic relationships of these isolates among marine species of the Cytophaga-Flavobacterium-Bacteroides complex were analysed by using their gyrB nucleotide sequences and translated peptide sequences (GyrB) in addition to 16S rDNA sequences. These isolates were closely related to the previously characterized marine Flexibacter species, [Flexibacter] maritimus and [Flexibacter] ovolyticus. These Flexibacter species are distantly related to Flexibacter flexilis, the type species of the genus Flexibacter, and phylogenetically belong to the family Flavobacteriaceae (according to analysis using both 16S rDNA and GyrB sequences). Their phylogenetic, chemotaxonomic and phenotypic characteristics prompted the proposal that these two species should be transferred to the new genus Tenacibaculum, as Tenacibaculum maritimum and Tenacibaculum ovolyticum, respectively. Two additional new species of the genus Tenacibaculum, Tenacibaculum mesophilum gen. nov., sp. nov. (= MBIC 1140T = IFO 16307T) and Tenacibaculum amylolyticum gen. nov., sp. nov. (= MBIC 4355T = IFO 16310T), which were isolated from sponges and macroalgae, are also reported. For taxonomic considerations at the species level, the resolution of gyrB sequences was superior to that of 16S rDNA sequences, and the grouping based on the gyrB phylogram was consistent with DNA-DNA hybridization results.

Proposal to transfer some members of the genera Haemobartonella and Eperythrozoon to the genus Mycoplasma with descriptions of 'Candidatus Mycoplasma haemofelis', 'Candidatus Mycoplasma haemomuris', 'Candidatus Mycoplasma haemosuis' and 'Candidatus Mycoplasma wenyonii'.

Abstract: Cell-wall-less uncultivated parasitic bacteria that attach to the surface of host erythrocytes currently are classified in the order Rickettsiales, family Anaplasmataceae, in the genera Haemobartonella and Eperythrozoon. Recently 16S rRNA gene sequences have been determined for four of these species: Haemobartonella felis and Haemobartonella muris and Eperythrozoon suis and Eperythrozoon wenyonii. Phylogenetic analysis of these sequence data shows that these haemotrophic bacteria are closely related to species in the genus Mycoplasma (class Mollicutes). These haemotrophic bacteria form a new phylogenetic cluster within the so-called pneumoniae group of Mycoplasma and share properties with one another as well as with other members of the pneumoniae group. These studies clearly indicate that the classification of these taxa should be changed to reflect their phylogenetic affiliation and the following is proposed: (i) that Haemobartonella felis and Haemobartonella muris should be transferred to the genus Mycoplasma as 'Candidatus Mycoplasma haemofelis' and 'Candidatus Mycoplasma haemomuris' and (ii) that Eperythrozoon suis and Eperythrozoon wenyonii should be transferred to the genus Mycoplasma as 'Candidatus Mycoplasma haemosuis' and 'Candidatus Mycoplasma wenyonii'. The former Haemobartonella and Eperythrozoon species described here represent a new group of parasitic mycoplasmas that possess a pathogenic capacity previously unrecognized among the mollicutes. These haemotrophic mycoplasmas have been given the trivial name haemoplasmas. These results call into question the affiliation of the remaining officially named species of Haemobartonella and Eperythrozoon which should be considered species of uncertain affiliation pending the resolution of their phylogenetic status.

Construction and bootstrap analysis of DNA fingerprinting-based phylogenetic trees with the freeware program FreeTree : application to trichomonad parasites

Abstract: The Win95/98/NT program FreeTree for computation of distance matrices and construction of phylogenetic or phenetic trees on the basis of random amplified polymorphic DNA (RAPD), RFLP and allozyme data is presented. In contrast to other similar software, the program FreeTree (available at http://www.natur.cuni.cz/~flegr/programs/freetree or http://ijs.sgmjournals.org/content/vol51/issue3/) can also assess the robustness of the tree topology by bootstrap, jackknife or operational taxonomic unit-jackknife analysis. Moreover, the program can be also used for the analysis of data obtained in several independent experiments performed with non-identical subsets of taxa. The function of the program was demonstrated by an analysis of RAPD data from 42 strains of 10 species of trichomonads. On the phylogenetic tree constructed using FreeTree, the high bootstrap values and short terminal branches for the Tritrichomonas foetus/suis 14-strain branch suggested relatively recent and probably clonal radiation of this species. At the same time, the relatively lower bootstrap values and long terminal branches for the Trichomonas vaginalis 20-strain branch suggested more ancient radiation of this species and the possible existence of genetic recombination (sexual reproduction) in this human pathogen. The low bootstrap values and the star-like topology of the whole Trichomonadidae tree confirm that the RAPD method is not suitable for phylogenetic analysis of protozoa at the level of higher taxa. It is proposed that the repeated bootstrap analysis should be an obligatory part of any RAPD study. It makes it possible to assess the reliability of the tree obtained and to adjust the amount of collected data (the number of random primers) to the amount of phylogenetic signals in the RAPD data of the taxon analysed. The FreeTree program makes such analysis possible.

Streptimonospora salina gen. nov., sp. nov., a new member of the family Nocardiopsaceae.

Abstract: Actinomycete strain YIM 90002T (= CCTCC 99003T = CCRC 16284T) was isolated from a soil sample collected from a salt lake in the west of China. The aerial mycelium of this organism is well developed but not fragmented and, at maturity, forms short chains of spores. Spores in short chains are oval- to rod-shaped and have wrinkled surfaces. Substrate mycelium is branched with non-fragmenting hyphae and forms single oval to round spores borne on sporophores or dichotomously branching sporophores. Single spores have wrinkled surfaces. Single spores and spores in short chains are non-motile. Strain YIM 90002T contains meso-diaminopimelic acid, DD-diaminopimelic acid, glycine, lysine and aspartic acid in its cell wall and has glucose, galactose, ribose, xylose, arabinose and mannose as whole-cell sugars (no diagnostic sugars). The phospholipids are phosphatidylglycerol, phosphatidylinositol and phosphatidylethanolamine. The major menaquinones are MK-9(H6), MK-10(H2) and MK-10(H4). Phylogenetic data indicate that this strain belongs to the family Nocardiopsaceae. The morphological and physiological characteristics and chemotaxonomic and phylogenetic data for this strain differ from those of previously described actinomycetes. Therefore, a new genus, Streptimonospora, is proposed for this organism; the type species of the genus is Streptimonospora salina gen. nov., sp. nov., and the type strain of S. salina is strain YIM 90002T.

Dechloromonas agitata gen. nov., sp. nov. and Dechlorosoma suillum gen. nov., sp. nov., two novel environmentally dominant (per)chlorate-reducing bacteria and their phylogenetic position.

Abstract: Previous studies on the ubiquity and diversity of microbial (per)chlorate reduction resulted in the isolation of 20 new strains of dissimilatory (per)chlorate-reducing bacteria. Phylogenetic analysis revealed that all of the isolates were members of the Proteobacteria with representatives in the alpha-, beta- and gamma-subclasses. The majority of the new isolates were located in the beta-subclass and were closely related to each other and to the phototrophic Rhodocyclus species. Here an in-depth analysis of these organisms which form two distinct monophyletic groups within the Rhodocyclus assemblage is presented. Two new genera, Dechloromonas and Dechlorosoma, are proposed for these beta-subclass lineages which represent the predominant (per)chlorate-reducing bacteria in the environment. The type species and strains for these new genera are Dechloromonas agitata strain CKBT and Dechlorosoma suillum strain PST, respectively.

Vibrio shiloi sp. nov., the causative agent of bleaching of the coral Oculina patagonica.

Abstract: The aetiological agent of bleaching of the coral Oculina patagonica was characterized as a new Vibrio species on the basis of 16S rDNA sequence, DNA-DNA hybridization data and phenotypic properties, including the cellular fatty acid profile. Based on its 16S rDNA and DNA-DNA hybridization, the new Vibrio species is closely related to Vibrio mediterranei. The name Vibrio shiloi sp. nov. is proposed for the new coral-bleaching species, the type strain being AK1T (= ATCC BAA-91T = DSM 13774T).

Molecular characterization of planktic cyanobacteria of Anabaena, Aphanizomenon, Microcystis and Planktothrix genera.

Abstract: Toxic and non-toxic cyanobacterial strains from Anabaena, Aphanizomenon, Calothrix, Cylindrospermum, Nostoc, Microcystis, Planktothrix (Oscillatoria agardhii), Oscillatoria and Synechococcus genera were examined by RFLP of PCR-amplified 16S rRNA genes and 16S rRNA gene sequencing With both methods, high 16S rRNA gene similarity was found among planktic, anatoxin-a-producing Anabaena and non-toxic Aphanizomenon, microcystin-producing and non-toxic Microcystis, and microcystin-producing and non-toxic Planktothrix strains of different geographical origins The respective sequence similarities were 999-100%, 942-999% and 993-100% Thus the morphological characteristics (eg Anabaena and Aphanizomenon), the physiological (toxicity) characteristics or the geographical origins did not reflect the level of 16S rRNA gene relatedness of the closely related strains studied In addition, cyanobacterial strains were fingerprinted with repetitive extragenic palindromic (REP)- and enterobacterial repetitive intergenic consensus (ERIC)-PCR All the strains except two identical pairs of Microcystis strains had different band profiles The overall grouping of the trees from the 16S rRNA gene and the REP- and ERIC-PCR analyses was similar Based on the 16S rRNA gene sequence analysis, four major clades were formed (i) The clade containing filamentous heterocystous cyanobacteria was divided into three discrete groups of Anabaena/Aphanizomenon, Anabaena/Cylindrospermum/ Nodularia/Nostoc and Calothrix strains The three other clades contained (ii) filamentous non-heterocystous Planktothrix, (iii) unicellular non-heterocystous Microcystis and (iv) Synechococcus strains

Phylogenetic diversity of Klebsiella pneumoniae and Klebsiella oxytoca clinical isolates revealed by randomly amplified polymorphic DNA, gyrA and parC genes sequencing and automated ribotyping.

Abstract: The infra-specific phylogenetic diversity and genetic structure of both Klebsiella pneumoniae and Klebsiella oxytoca was investigated using a combination of randomly amplified polymorphic DNA (RAPD) analysis, sequencing of gyrA and parC genes, and automated ribotyping. After RAPD analysis with four independent primers of 120 clinical isolates collected from 22 European hospitals in 13 countries, K. pneumoniae isolates fell into three clusters and K. oxytoca isolates fell into two clusters, while Klebsiella planticola isolates formed a sixth cluster. Each cluster was geographically widespread. K. pneumoniae cluster I (KpI) accounted for 80% of the isolates of this species and included reference strains of the three subspecies K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis. Clusters KpII and KpIII were equally represented, as were the two K. oxytoca clusters. Individualization of each cluster was fully confirmed by phylogenetic analysis of gyrA and parC gene sequences. In addition, sequence data supported the evolutionary separation of K. pneumoniae from a phylogenetic group including K. oxytoca, Klebsiella terrigena, K. planticola and Klebsiella ornithinolytica. Automated ribotyping using Mlu I appeared suitable for identification of each Klebsiella cluster. The adonitol fermentation test was found to be useful for cluster identification in K. pneumoniae, since it was negative in all strains of clusters KpIII and in some KpII strains, but always positive in cluster KpI. The usefulness of gyrA and parC sequence data for population genetics and cluster identification in bacteria was demonstrated, even for the phylogenetic positioning of quinolone-resistant isolates.

Burkholderia ambifaria sp. nov., a novel member of the Burkholderia cepacia complex including biocontrol and cystic fibrosis-related isolates

Abstract: A polyphasic taxonomic study, including amplified fragment length polymorphism (AFLP) fingerprinting, DNA-DNA hybridizations, DNA base-ratio determinations, phylogenetic analysis, whole-cell fatty acid analyses and an extensive biochemical characterization, was performed on 19 Burkholderia cepacia-like isolates from the environment and cystic fibrosis (CF) patients. Several of the environmental isolates have attracted considerable interest due to their biocontrol properties. The polyphasic taxonomic data showed that the strains represent a new member of the B. cepacia complex, for which the name Burkholderia ambifaria sp. nov. is proposed. The type strain is strain LMG 19182T. B. ambifaria can be differentiated from the other members of the B. cepacia complex by means of AFLP fingerprinting, whole-cell fatty acid analysis, biochemical tests (including ornithine and lysine decarboxylase activity, acidification of sucrose and beta-haemolysis) and a newly developed recA gene-based PCR assay. 16S rDNA-based RFLP analysis and PCR tests allowed differentiation of B. ambifaria from Burkholderia multivorans, Burkholderia vietnamiensis and B. cepacia genomovar VI, but not from B. cepacia genomovars I and III and Burkholderia stabilis. The finding that this new taxon includes both strains isolated from CF patients and potentially useful biocontrol strains supports the general consensus that the large-scale use of biocontrol strains belonging to the B. cepacia complex would be ill-advised until more is known about their potential pathogenic mechanisms.

Azospirillum doebereinerae sp. nov., a nitrogen-fixing bacterium associated with the C4-grass Miscanthus.

Abstract: A new group of nitrogen-fixing Azospirillum sp. bacteria was isolated from the roots of the C4-gramineous plant Miscanthus. Polyphasic taxonomy was performed, including auxanography using API galleries, physiological tests and 16S rRNA sequence comparison. The ability of the isolates to fix dinitrogen was evaluated by amplification of the nifD gene, immunodetection of the dinitrogenase reductase and acetylene-reduction assay. On the basis of these results, the nitrogen-fixing isolates represent a new species within the genus Azospirillum. Its closest phylogenetic neighbours, as deduced by 16S rDNA-based analysis, are Azospirillum lipoferum, Azospirillum largimobile and Azospirillum brasilense with 96.6, 96.6 and 95.9% sequence similarity, respectively. Two 16S rRNA-targeting oligonucleotide probes were developed which differentiate the new species from the other Azospirillum species by whole-cell fluorescence hybridization. Strains of the new species are curved rods or S-shaped, 1.0-1.5 microm in width and 2,0-3.0 microm in length, Gram-negative and motile with a single polar flagellum. Optimum growth occurs at 30 degrees C and at pH values between 6.0 and 7.0. No growth takes place at 37 degrees C. They have a respiratory type of metabolism, grow well on arabinose, D-fructose, gluconate, glucose, glycerol, malate, mannitol and sorbitol. They differ from A. largimobile and A. lipoferum by their inability to use N-acetylglucosamine and D-ribose, from A. lipoferum by their ability to grow without biotin supplementation and from A. brasilense by their growth with D-mannitol and D-sorbitol as sole carbon sources. Nitrogen fixation occurs in microaerobic nitrogen-limited conditions. For this species, the name Azospirillum doebereinerae sp. nov. is suggested, with strain GSF71T as the type strain (= DSM 13131T; reference strain Ma4 = DSM 13400). Its G+C content is 70.7 mol%.

DNA-DNA hybridization study of Bradyrhizobium strains

Abstract: DNA-DNA hybridizations were performed between Bradyrhizobium strains, isolated mainly from Faidherbia albida and Aeschynomene species, as well as Bradyrhizobium reference strains. Results indicated that the genus Bradyrhizobium consists of at least 11 genospecies, I to XI. The genospecies formed four subgeneric groups that were more closely related to each other (>40% DNA hybridization) than to other genospecies (<40% DNA hybridization): (i) genospecies I (Bradyrhizobium japonicum), III (Bradyrhizobium liaoningense), IV and V; (ii) genospecies VI and VIII; (iii) genospecies VII and IX; and (iv) genospecies II (Bradyrhizobium elkanii), X and XI. Photosynthetic Aeschynomene isolates were found to belong to at least two distinct genospecies in one subgeneric group. DNA-DNA hybridization data are compared with data from amplified fragment length polymorphism analysis and 165-23S rDNA spacer sequence analysis.

Geobacter hydrogenophilus, Geobacter chapellei and Geobacter grbiciae, three new, strictly anaerobic, dissimilatory Fe(III)-reducers.

Abstract: Recent studies on the diversity and ubiquity of Fe(III)-reducing organisms in different environments led to the isolation and identification of four new dissimilatory Fe(III)-reducers (strains H-2T, 172T, TACP-2T and TACP-5). All four isolates are non-motile, Gram-negative, freshwater, mesophilic, strict anaerobes with morphology identical to that of Geobacter metallireducens strain GS-15T. Analysis of the 16S rRNA sequences indicated that the new isolates belong to the genus Geobacter, in the delta-Proteobacteria. Significant differences in phenotypic characteristics, DNA-DNA homology and G+C content indicated that the four isolates represent three new species of the genus. The names Geobacter hydrogenophilus sp. nov. (strain H-2T), Geobacter chapellei sp. nov. (strain 172T) and Geobacter grbiciae sp. nov. (strains TACP-2T and TACP-5) are proposed. Geobacter hydrogenophilus and Geobacter chapellei were isolated from a petroleum-contaminated aquifer and a pristine, deep, subsurface aquifer, respectively. Geobacter grbiciae was isolated from aquatic sediments. All of the isolates can obtain energy for growth by coupling the oxidation of acetate to the reduction of Fe(III). The four isolates also coupled Fe(III) reduction to the oxidation of other simple, volatile fatty acids. In addition, Geobacter hydrogenophilus and Geobacter grbiciae were able to oxidize aromatic compounds such as benzoate, whilst Geobacter grbiciae was also able to use the monoaromatic hydrocarbon toluene.

Classification of metal-resistant bacteria from industrial biotopes as Ralstonia campinensis sp. nov., Ralstonia metallidurans sp. nov. and Ralstonia basilensis Steinle et al. 1998 emend.

Abstract: Thirty-one heavy-metal-resistant bacteria isolated from industrial biotopes were subjected to polyphasic characterization, including 16S rDNA sequence analysis, DNA-DNA hybridizations, biochemical tests, whole-cell protein and fatty-acid analyses. All strains were shown to belong to the Ralstonia branch of the beta-Proteobacteria. Whole-cell protein profiles and DNA-DNA hybridizations revealed two clearly distinct groups, showing low similarity to known Ralstonia species. These two groups, of 8 and 17 isolates, were assigned to two new species, for which the names Ralstonia campinensis sp. nov. and Ralstonia metallidurans sp. nov. are proposed. The type strains are WS2T (= LMG 19282T = CCUG 44526T) and CH34T (= LMG 1195T = DSM 2839T), respectively. Six isolates were allocated to Ralstonia basilensis, which presently contains only the type strain; an emendation of the latter species description is therefore proposed.

Proposal of the genera Anaerococcus gen. nov., Peptoniphilus gen. nov. and Gallicola gen. nov. for members of the genus Peptostreptococcus.

Abstract: Members of genus Peptostreptococcus have previously been found to be distantly related to the type species, Peptostreptococcus anaerobius, on the basis of 16S rDNA sequence similarities. They were divided into three major phylogenetic groups, and their peptidoglycan structure and biochemical traits differed between groups. The reclassification of the species of these three groups into three new genera, Peptoniphilus gen. nov., Anaerococcus gen. nov. and Gallicola gen. nov., is proposed. The genus Peptoniphilus gen. nov. includes the following butyrate-producing, non-saccharolytic species that use peptone and amino acids as major energy sources: Peptoniphilus asaccharolyticus comb. nov. (type species), Peptoniphilus lacrimaris comb. nov., Peptoniphilus harei comb. nov., Peptoniphilus indolicus comb. nov. and Peptoniphilus ivorii comb. nov. The genus Anaerococcus gen. nov. contains the saccharolytic, butyrate-producing species Anaerococcus prevotii comb. nov. (type species), Anaerococcus tetradius comb. nov., Anaerococcus lactolyticus comb. nov., Anaerococcus hydrogenalis comb. nov., Anaerococcus vaginalis comb. nov. and Anaerococcus octavius sp. nov. The genus Gallicola gen. nov. contains a single species, Gallicola barnesae comb. nov.

Thioalkalimicrobium aerophilum gen. nov., sp. nov. and Thioalkalimicrobium sibericum sp. nov., and Thioalkalivibrio versutus gen. nov., sp. nov., Thioalkalivibrio nitratis sp. nov. and Thioalkalivibrio denitrificans sp. nov., novel obligately alkaliphilic and obligately chemolithoautotrophic sulfur-oxidizing bacteria from soda lakes

Abstract: Forty-three strains of obligately chemolithoautotrophic sulfur-oxidizing bacteria were isolated from highly alkaline soda lakes in south-east Siberia (Russia) and in Kenya using a specific enrichment procedure at pH 10. The main difference between the novel isolates and known sulfur bacteria was their potential to grow and oxidize sulfur compounds at pH 10 and higher. The isolates fell into two groups that were substantially different from each other physiologically and genetically. Most of the Siberian isolates belonged to the group with a low DNA G+C content (48.0-51.2 mol%). They were characterized by a high growth rate, a low growth yield, a high cytochrome content, and high rates of oxidation of sulfide and thiosulfate. This group included 18 isolates with a DNA homology of more than 40%, and it is described here as a new genus, Thioalkalimicrobium, with two species Thioalkalimicrobium aerophilum (type species) and Thioalkalimicrobium sibericum. The other isolates, mainly from Kenyan soda lakes, fell into a group with a high DNA G+C content (61.0-65.6 mol%). In general, this group was characterized by a low growth rate, a high molar growth yield and low, but relatively equal, rates of oxidation of thiosulfate, sulfide, elemental sulfur and polythionates. The group included 25 isolates with a DNA homology of more than 30%. It was less compact than Thioalkalimicrobium, containing haloalkalophilic, carotenoid-producing, nitrate-reducing and facultatively anaerobic denitrifying strains. These bacteria are proposed to be assigned to a new genus, Thioalkalivibrio, with three species Thioalkalivibrio versutus (type species), Thioalkalivibrio denitrificans and Thioalkalivibrio nitratis. Phylogenetic analysis revealed that both groups belong to the gamma-Proteobacteria. The Thioalkalimicrobium species were closely affiliated with the neutrophilic chemolithoautotrophic sulfur bacteria of the genus Thiomicrospira, forming a new alkaliphilic lineage in this cluster. In contrast, Thioalkalivibrio was not related to any known chemolithoautotrophic taxa, but was distantly associated with anaerobic purple sulfur bacteria of the genus Ectothiorhodospira.

Emended descriptions of Clostridium acetobutylicum and Clostridium beijerinckii, and descriptions of Clostridium saccharoperbutylacetonicum sp. nov. and Clostridium saccharobutylicum sp. nov.

Abstract: On the basis of 16S rRNA gene sequencing and DNA-DNA reassociation, industrial solvent-producing clostridia have been assigned to four species. In this study, the phenotypic characteristics of Clostridium acetobutylicum, Clostridium beijerinckii, 'Clostridium saccharoperbutylacetonicum', and an unnamed Clostridium sp. represented by the strains NCP 262T and NRRL B643 are compared. In addition, a further 40 strains of solvent-producing clostridia have been classified by biotyping, DNA fingerprinting and 16S rRNA gene sequencing. These included 14 C. beijerinckii strains, two strains currently designated as 'Clostridium kaneboi' and 'Clostridium butanologenum', and 24 production strains used in the commercial acetone-butanol fermentation. All of the C. beijerinckii strains were confirmed to have been classified correctly. The 'C. kaneboi' and 'C. butanologenum' strains require reclassification as C. acetobutylicum and C. beijerinckii, respectively. The commercial production strains were found to belong either to C. beijerinckii or to the unnamed Clostridium sp. For the comparative phenotypic studies of the four species, representative strains were selected from each of the DNA-fingerprint subgroups within each species. These strains were analysed for their ability to utilize different carbohydrates, hydrolyse gelatin or aesculin, and produce indole, and were tested for the presence of catalase and urease. On the basis of these results, several phenotypic traits were found to be useful for differentiating between the four species. The descriptions of C. acetobutylicum and C. beijerinckii have been emended. The names Clostridium saccharoperbutylacetonicum sp. nov. [type strain = N1-4 (HMT) = ATCC 27021T] and Clostridium saccharobutylicum sp. nov. (type strain = DSM 13864T = ATCC BAA-117T) are proposed for the two new species.

Phylogenetic analyses of Synechococcus strains (cyanobacteria) using sequences of 16S rDNA and part of the phycocyanin operon reveal multiple evolutionary lines and reflect phycobilin content.

Abstract: The genus Synechococcus (cyanobacteria), while containing morphologically similar isolates, is polyphyletic and organisms presently classified as such require reclassification into several independent genera. Studies based on analysis of 16S rRNA gene sequences have shown that members of the genus Synechococcus are affiliated to three of seven deeply branching cyanobacterial lineages. In addition, some strains do not appear to be associated with any of these lineages and may represent novel clades. In this report, a cyanobacterial phylogeny based on 16S rDNA sequences, including 14 newly sequenced Synechococcus isolates, is presented. One newly sequenced Synechococcus strain (PCC 7902) did not have any close relatives amongst cyanobacterial isolates currently contained in 16S rDNA sequence databases and was only loosely affiliated to a cyanobacterial lineage in which no other Synechococcus strains were found. Three hot-spring Synechococcus isolates, including two that were newly sequenced in this study (PCC 6716 and PCC 6717), formed an additional cyanobacterial lineage. These results indicated that Synechococcus species are affiliated to five of eight deeply branching cyanobacterial lineages. Part of the phycocyanin (PC) gene sequence (cpc), including the intergenic spacer (IGS) between cpcB and cpcA and the corresponding flanking regions (cpcBA-IGS), was used to investigate relationships between closely related Synechococcus isolates. Previously described PCR primers did not amplify this region from the majority of strains under investigation, so a new set of primers was designed that allowed amplification and sequencing of the cpcBA-IGS and flanking regions from 38 Synechococcus species. Phylogenetic analysis of this region was largely consistent with that obtained from 16S rDNA sequence analysis and revealed a relationship between the primary PC DNA sequence and the phycobilin content of cells.

Phylogeny of Rickettsia spp. inferred by comparing sequences of 'gene D', which encodes an intracytoplasmic protein.

Abstract: 'Gene D' is the PS120-protein-encoding gene, first described in Rickettsia conorii and Rickettsia japonica. Sequence analysis of a 3030 bp fragment of 'gene D' in 24 representatives of the genus Rickettsia was carried out to complete phylogenetic analyses previously inferred by comparison of gene sequences encoding citrate synthase, 17 kDa antigen and rOmpA and rOmpB. The phylogenetic relationships between rickettsiae were inferred from the comparison of both the gene and the derived protein sequences, using the parsimony, neighbour-joining and maximum-likelihood methods. Five distinct groups of rickettsiae were identified. These were: the Rickettsia massiliae group, including R. massiliae, Bar 29, Rickettsia rhipicephali and Rickettsia aeschlimannii; the Rickettsia rickettsii group containing Rickettsia sibirica, 'Rickettsia mongolotimonae', Rickettsia parkeri, strain S, Rickettsia africae, the R. conorii complex, Rickettsia slovaca, Rickettsia honei, R. rickettsii, R. japonica and Rickettsia montanensis; the group currently containing only Rickettsia helvetica; the Rickettsia akari group including Rickettsia australis, R. akari and the ELB agent; Rickettsia prowazekii and Rickettsia typhi clustered in the typhus group. As significant bootstrap values were obtained for most of the nodes, sequence comparison of 'gene D' should be considered as a complementary approach in phylogenetic studies of rickettsiae.

Mycobacterium immunogenum sp. nov., a novel species related to Mycobacterium abscessus and associated with clinical disease, pseudo-outbreaks and contaminated metalworking fluids: an international cooperative study on mycobacterial taxonomy.

Abstract: PCR-restriction enzyme pattern analysis of a 439 bp hsp65 gene segment identified 113 unique isolates among non-pigmented rapidly growing mycobacteria (RGM) from clinical and environmental sources that failed to match currently recognized species patterns. This group represented 40% of isolates recovered from bronchoscope contamination pseudo-outbreaks, 0% of disease-associated nosocomial outbreaks and 4% of routine clinical isolates of the Mycobacterium abscessus/Mycobacterium chelonae group submitted to the Mycobacteria/Nocardia laboratory for identification. It is grouped within the Mycobacterium fortuitum complex, with growth in less than 7 d, absence of pigmentation, positive 3-d arylsulfatase reaction and growth on MacConkey agar without crystal violet. It exhibited overlapping biochemical, antimicrobial susceptibility and HPLC characteristics of M. abscessus and M. chelonae. By 16S rRNA gene sequencing, these isolates comprised a homogeneous group with a unique hypervariable region A sequence and differed by 8 and 10 bp, respectively, from M. abscessus and M. chelonae. Surprisingly, this taxon contained two copies of the ribosomal operon, compared with single copies in the two related species. By DNA-DNA hybridization, this new group exhibited <30% homology with recognized RGM species. The name Mycobacterium immunogenum sp. nov. is proposed for this new taxon.

Implications of alternative classifications and horizontal gene transfer for bacterial taxonomy.

Abstract: Following the publication of the Approved Lists, there has been a tendency to regard all subsequent revisions of classification as providing improved nomenclature, to be accepted without question. This takes no account of the fact that such revisions may be based on one of three alternative concepts, phenetic, phylogenetic or polyphasic classification, sometimes leading to different, valid, but incompatible nomenclature, or that some investigations are based only on subsets of relevant taxa and on limited data, leading to incomplete and sometimes confusing revisions of nomenclature. The polyphasic approach to classification has widespread support, although there appears to be a tendency to allow comparative sequence analyses of 16S rDNA to determine classification contrary to the indications of other data. In some cases, classification is based solely on 16S rDNA data. Examples are considered. Consideration is given to the criteria by which taxa are circumscribed, particularly at the level of genus and species. It is suggested that there is a need for reconciliation of the criteria by which taxa at these levels are circumscribed. Recent studies demonstrating the widespread occurrence of horizontal gene transfer suggest that there is a need for caution in monophyletic interpretations, especially when these are based on the analysis of single sequences.

Burkholderia fungorum sp. nov. and Burkholderia caledonica sp. nov., two new species isolated from the environment, animals and human clinical samples.

Abstract: A polyphasic taxonomic study that included DNA-DNA hybridizations, DNA base ratio determinations, 16S rDNA sequence analyses, whole-cell protein and fatty acid analyses and an extensive biochemical characterization was performed on 16 strains isolated from the environment, animals and human clinical samples. The isolates belonged to the genus Burkholderia, were phylogenetically closely related to Burkholderia graminis, Burkholderia caribensis and Burkholderia phenazinium and had G+C contents between 61.9 and 62.2 mol%. Seven strains isolated from the rhizosphere were assigned to Burkholderia caledonica sp. nov. [type strain LMG 19076T (= CCUG 42236T)]. Nine strains isolated from the environment, animals and human clinical samples were assigned to Burkholderia fungorum sp. nov. [type strain LMG 16225T (= CCUG 31961T)]. Differential tests for B. graminis, B. caribensis, B. phenazinium, B. caledonica and B. fungorum include the following: assimilation of trehalose, citrate, DL-norleucine, adipate and sucrose; nitrate reduction; growth in the presence of 0.5% NaCl; and beta-galactosidase activity.