- Volume 46, Issue 4, 1996

A new aerobic, gram-positive, nonsporulating rod-shaped organism is described. Strain IFO 15245T (T = type strain) has the following characteristics: The menaquinone contains a side chain with 11 isoprenyl units; the guanine-plus-cytosine content of the DNA is 66.2 moI%; 2,4-diaminobutyric acid, glutamic acid, alanine, glycine, and γ -aminobutyric acid are present in the cell wall at a molar ratio of ca. 1:1:2:1:1; and glucose and galactose are also present in the cell wall. A comparison of partial 16S rRNA sequences revealed that IFO 15245T represents a distinct line of descent within the gram-positive bacteria with high guanine-plus-cytosine contents. The taxonomic characteristics of this organism are different from those of previously described aerobic, gram-positive, nonsporulating, rod-shaped bacteria. The name Leucobacter komagatae gen. nov., sp. nov., is proposed for this organism. The type strain is strain IFO 15245.

The taxonomic position of isolates of a new genomic species (designated genomic species 2) obtained from several annual Medicago species and originating from different geographical locations was established through the results of phenotypic tests (including the of auxanographic and biochemical tests and symbiotic properties) and 16S rRNA phylogenetic inferences. A comparison of the complete 16S rRNA sequence of a representative of genomic species 2 (strain A 321T [T = type strain]) with the 16S rRNA sequences of other members of the Rhizobiaceae and closely related taxa showed that genomic species 2 was phylogenetically related to Sinorhizobium meliloti, Sinorhizobium fredii, Sinorhizobium saheli, and Sinorhizobium teranga. The levels of sequence similarity and observed numbers of nucleotide substitutions in Sinorhizobium strains indicated that A 321T and S. meliloti exhibited the highest level of sequence similarity (99.7%), with four nucleotide substitutions and one deletion. The results of a numerical analysis based on data from 63 auxanographic and biochemical tests clearly separated genomic species 2 isolates from S. meliloti. Genomic species 2 isolates nodulated and fixed nitrogen with Medicago polymorpha, whereas S. meliloti isolates were ineffective and formed rudimentary nodules on this host plant. On the basis of phenotypic and 16S sequence analysis data, genomic species 2 isolates cannot be assigned to a previously described species. We propose that these isolates belong to a new species, Sinorhizobium medicae.

A gram-negative bacterium which was capable of oxidizing reduced inorganic sulfur compounds was isolated from agricultural soil and designated BI-42. This new isolate grew on a wide range of organic substrates but was not able to grow autotrophically and lacked ribulose 1,5-bisphosphate carboxylase, a key enzyme of carbon dioxide fixation. These results suggested that strain BI-42 was a chemolithoheterotroph. Ammonia and nitrate were not used as sole nitrogen sources for growth, and strain BI-42 lacked glutamate synthase activity, which resulted in glutamate auxotrophy. The glutamate dehydrogenase activity of this organism was apparently insufficient for ammonia assimilation. On the basis of the results of additional biochemical tests, the G+C content of the DNA, the results of a respiratory ubiquinone analysis, the results of a 16S ribosomal DNA sequence analysis, the fatty acid composition, and the results of a membrane lipid analysis, strain BI-42 was identified as a phylogenetically and physiologically distinct taxon belonging to the alpha subclass of the Proteobacteria. Bosea thiooxidans gen. nov., sp. nov. is the name proposed for this taxon.

Seventy-seven strains representing 10 species in the Paenibacillus polymyxa 16S rRNA group and 3 other species that exhibit phenetic relatedness to members of this group, Bacillus lautus, “Bacillus longisporus,” and Bacillus peoriae, were characterized genotypically and phenotypically by performing an amplified ribosomal DNA restriction analysis, a randomly amplified polymorphic DNA analysis, a fatty acid methyl ester analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins, pyrolysis mass spectrometry, and API and other routine phenotypic tests. These analyses revealed distinct clusters representing Paenibacillus alvei, Paenibacillus amylolyticus, Paenibacillus azotofixans, Paenibacillus durum, Paenibacillus larvae subsp. larvae. Paenibacillus larvae subsp. pulvifaciens, B. lautus, Paenibacillus macerans, Paenibacillus macquariensis, B. peoriae, P. polymyxa, and Paenibacillus validus, which confirmed the distinctness of these species, but appreciable within-species heterogeneity was observed in P. alvei, B. lautus, P. macerans, P. polymyxa, and P. validus. The type strain of Paenibacillus pabuli did not cluster with other strains of this species, and in several analyses a relationship between strains of P. pabuli and “B. longisporus” was observed. As the analyses showed that B. lautus and B. peoriae are closely related to the genus Paenibacillus, these species are reclassified as members of this genus.

rRNA sequencing has shown that Leuconostocs comprise three distinct phylogenetic lineages which have been designated separate genera (viz., the genera Leuconostoc sensu stricto, Oenococcus, and Weissella). In addition, the 16S rRNA line formed by Oenococcus oeni (formerly Leuconostoc oenos) is exceptionally long: This fact, together with variations in the compositions of conserved positions in the 16S rRNA, has led to the hypothesis (D. Yang and C. R. Woese, Syst. Appl. Microbiol. 12:145-149, 1989) that this organism is a fast-evolving bacterium. Previous evidence that the leuconostocs should be divided into three genera and that O. oeni is an example of tachytelic evolution has come solely from rRNA analyses. In this study we sequenced the rpoC gene encoding the β’ subunit of DNA-dependent RNA polymerase of leuconostocs and performed a comparative phylogenetic analysis. The subdivision of the leuconostocs into three distinct lineages was confirmed by the rpoC gene data, but no evidence that indicated that O. oeni is evolving at an extraordinary rate was found. If O. oeni is truly tachytelic, then fast-evolving phenomena would be expected to occur throughout the whole genome, including this independent molecular chronometer.

An anaerobic bacterium, strain PCP-1T (T = type strain), which dechlorinates pentachlorophenol (PCP) to 3-chlorophenol, was isolated from a methanogenic consortium. This organism is a spore-forming rod-shaped bacterium that is nonmotile, asaccharolytic, and Gram stain negative but Gram type positive as determined by electron microscopic observations. Inorganic electron acceptors, such as sulfite, thiosulfate, and nitrate (but not sulfate), stimulate growth in the presence of pyruvate and yeast extract. The optimum pH and optimum temperature for growth are 7.5 and 38°C, respectively. The dechlorination pathway is: PCP → 2,3,4,5-tetra-chlorophenol → 3,4,5-trichlorophenol → 3,5-dichlorophenol → 3-chlorophenol. This bacterium dechlorinates several different chlorophenols at ortho, meta, and para positions; exceptions to this are 2,3-dichlorophenol, 2,5-dichlorophenol, 3,4-dichlorophenol, and the monochlorophenols. The time course of PCP dechlorination suggests that two enzyme systems are involved in dehalogenation in strain PCP-1T. One system is inducible for ortho dechlorination, and the second system is inducible for meta and para dechlorinations. A 16S rRNA analysis revealed that strain PCP-1T exhibits 95% homology with Desulfitobacterium dehalogenans JW/IU-DC1, an anaerobic bacterium which can dehalogenate chlorophenols only in ortho positions. These results suggest that strain PCP-1T is a member of a new species and belongs to the recently proposed genus Desulfitobacterium. Strain PCP-1T differs from D. dehalogenans JW/IU-DC1 by its broader range of chlorophenol dechlorination. Strain PCP-1 is the type strain of the new species, Desulfitobacterium frappieri.

The diversity of two populations of rhizobia isolated from Lathyrus japonicus (30 strains) and Lathyrus pratensis (49 strains) growing in northern regions of Quebec, Canada, was determined on the basis of phenotypic characteristics, multilocus enzyme electrophoresis, DNA-DNA homology, and 16S ribosomal DNA sequencing. According to numerical analysis of phenotypic characteristics, strains were divided into four groups. Strains isolated from L. pratensis fell in groups I to III; the latter included reference strains of Rhizobium leguminosarum. All strains isolated from L. japonicus were included in group IV. All strains had nodulation characteristics similar to those of R. leguminosarum bv. viciae. Strains isolated from L. japonicus originating from an arctic region were usually able to grow at 5°C and were more likely to be tolerant to copper (CuCI2 · H2O, 100 μg/ml) and lead [Pb(CH3COO)2, 500 μg/ml] than strains isolated from L. pratensis from a boreal zone. However, both populations of Lathyrus strains were adapted to the cold in comparison to reference strains from temperate regions. Each population had similar genetic diversity (H = 0.45), determined by multilocus enzyme electrophoresis of the loci encoding eight enzymes, but the diversity obtained by analyzing all strains including the reference strains (H = 0.58) was higher. Representative strains of both populations showed high levels of DNA homology among themselves and with R. leguminosarum. Partial sequences of the 16S ribosomal RNA genes were similar to those reported for R. leguminosarum bv. viciae. We conclude that the strains isolated from L. japonicus and L. pratensis belong to R. leguminosarum bv. viciae but are distinguishable by growth at 5°C, which is a characteristic related to their geographic origin.

More than 40 isolates of a novel, ubiquitous, proteolytic, moderately alkaliphilic, thermophilic obligate anaerobe were obtained from geothermally and anthropogenically heated environments and mesobiotic environments located on three continents. Whole-cell protein sodium dodecyl sulfate gel electrophoresis revealed that most of these organisms are very similar. Eight of the isolates were characterized in detail; this analysis included 16S ribosomal DNA sequence analysis. The cells of those organisms are (depending on the isolate) 0.5 to 0.8 μm in diameter and 1.5 to 13 μm long, exhibit tumbling motility, and have a positive Gram stain reaction. The temperature range for growth is 43° to 75°C (optimum temperature, 66°C), and the pH range for growth is 5.4 to 9.5 (optimum pH, 8.2); the shortest doubling time is around 10 min. Yeast extract is required for growth, and (depending on the strain) glucose, sucrose, fructose, galactose, and ribose are utilized. The fermentation products from glucose in the presence of yeast extract are CO2, H2, acetate, formate, and ethanol. The G+C content is 30 to 31 mol%. On the basis of these properties, which differentiate these strains from all alkalitolerant thermophiles described previously, and the results of a comparison of the 16S ribosomal DNA sequences of these organisms with previously described sequences, we propose that our isolates be placed in a single species of the new genus Thermobrachium; strain JW/YL-NZ35 is the type strain of the the type species, Thermobrachium celere.

Bacterial strains isolated from wide ranges of nematode hosts and geographic sources and strains isolated from human clinical specimens were used to assess the taxonomic structure of the genus Photorhabdus. The following two methods were used: DNA relatedness and phenotypic characterization. Analysis of the DNA relatedness data revealed that all of the strains studied were congeneric and that the genus Photorhabdus is, on the basis of DNA relatedness data, more homogeneous than the other genus of nematode-symbiotic bacteria, the genus Xenorhabdus. In contrast to previous reports, only two DNA relatedness groups were identified in the genus Photorhabdus. These groups corresponded to the symbiotic strains and the clinical strains. There appeared to be some subgroups within the symbiotic strain group on the basis of the interactions of the strains with nematodes, which corresponded to some extent with the DNA relatedness data. However, there were significant ambiguities in the DNA relatedness data, and this group could not be subdivided on the basis of DNA relatedness data or phenotypic data. The distinct functional differences within and between the DNA relatedness groups of symbiotic Photorhabdus strains indicated that there are biologically significant subgroups within the genus Photorhabdus that cannot be defined at this time. Further investigation of the taxonomy of Photorhabdus by using different approaches and a suitably wide range of strains is recommended. However, it is clear that the clinical strains form a recognizable subgroup within the genus even though no formal subtaxon can be defined at this time.

Gram-negative polychlorophenol-degrading bacterial strains KF1T (T = type strain), KF3, and NKF1, which were described previously as Pseudomonas saccharophila strains, were studied by chemotaxonomic, genetic, and physiological methods and by electron microscopy and compared with selected xenobiotic compound-degrading bacteria. These strains contained sphingolipids with d-18:0, d-20:1, and d-21:1 as the main dihydrosphingosines, ubiquinone 10 as the main respiratory quinone, and spermidine as the major polyamine, and the DNA G+C content was 66 mol%. The cellular fatty acids included about 60% octadecenoic acid, 9% 2-hydroxymy-ristic acid, 14% cis-9-hexadecenoic acid, and 10% hexadecanoic acid. These strains exhibited less than 97% 16S ribosomal DNA sequence similarity to all of the other taxa studied. In the DNA-DNA reassociation studies the highest levels of reassociation between these strains and previously described species were less than 40%. Thin sections of cells of strains KF1T, KF3, and NKF1 were examined by electron microscopy, and the results showed that the cells had peculiar concentrically arranged layered membranous blebs that extruded from the outer membrane, especially at the cell division points. On the basis of the results of this study, polychlorophenol-degrading strains KF1T, KF3, and NKF1 are considered members of a new species of the genus Sphingomonas, Sphingomonas subarctica. The polycyclic aromatic hydrocarbon-degrading organism Sphingomonas paucimobilis EPA 505 was closely related to Sphingomonas chlorophenolica as determined by chemotaxonomic, phylogenetic, and physiological criteria. The xenobiotic compound degraders Alcaligenes sp. strain A175 and Pseudomonas sp. strain BN6 were identified as members of species of the genus Sphingomonas.

An acidophilic, disulfide-oxidizing, mesophilic, aerobic bacterium was isolated from wastewater sludge. The new organism is a gram-positive sporulated rod. It can use elemental sulfur and pyrite as sole energy sources and grows on organic substrates such as glutamate and glucose. It also grows on the following organic sulfur substrates: Oxidized and reduced glutathione, cysteine, cystine, and dithio(bis)benzothiazole and clearly shows a preference for disulfide bond-containing substrates. The optimal pH of growth is between 1.5 and 2.5, depending on the substrate used, and the growth temperature range varies from 4 to 40°C, with an optimal value at 35°C. The G+C chromosomal DNA content was measured at 53 ± 1 mol%. Phylogenetic analysis of 16S genes coding for rRNA sequences places the new isolate in the genus Sulfobacillus. In addition, unique phenotypic and physiologic characteristics and DNA homology values assign the isolate to a new species in the genus. Therefore, this new isolate has been named Sulfobacillus disulfidooxidans and has been assigned ATCC number 51911.

The glycolate-oxidizing, sulfate-reducing bacterium strain PerGlyS and the syntrophically glycolate-oxidizing bacterium strain FIGIyR were studied with respect to their phylogenetic relationships on the basis of in vitro amplification and direct sequencing of 16S rRNA-encoding DNA. Strain PerGlyS clustered with representatives of the δ subclass of the class Proteobacteria, close to “Desulforhopalus vacuolatus” but sufficiently distinct to preclude its assignment to this genus. These organisms, together with Desulfobulbus propionicus, represent a phylogenetic subgroup among members of the δ subclass of Proteobacteria. Strain FIGIyR was found to cluster with the gram-positive bacteria with low-G+C DNA, and Desulfitobacterium dehalogenans and Desulfotomaculum orientis are its closest relatives. Other species of the genus Desulfotomaculum are phyloge-netically only moderately closely related to these organisms. These results necessitate the establishment of new genera and species for these two strains. Strain PerGlyS was designated the type strain of Desulfofustis glycolicus gen. nov., sp. nov., and strain FIGIyR was designated the type strain of Syntrophobotulus glycolicus gen. nov., sp. nov.

A novel aerobic hyperthermophilic archaeon was isolated from a coastal solfataric vent at Kodakara-Jima Island, Japan. The new isolate, strain K1, is the first strictly aerobic organism growing at temperatures up to 100°C. It grows optimally at 90 to 95°C, pH 7.0, and a salinity of 3.5%. The cells are spherical shaped and 0.8 to 1.2 μm in diameter. Various proteinaceous complex compounds served as substrates during aerobic growth. Thiosulfate stimulates growth without producing H2S. The core lipids consist solely of C25-isopranyl archaeol(glycerol diether). The G+C content of the genomic DNA is 67 mol%. Phylogenetic analysis based on 16S rRNA sequence indicates that strain K1 is a new member of Crenarchaeota. On the basis of our results, the name Aeropyrum pernix gen. nov., sp. nov. is proposed (type strain: K1; JCM 9820).

Whipple’s disease is a rare chronic illness associated with an unculturable bacterium that is constantly present in affected tissues. This bacterium was previously characterized at the molecular level by PCR and sequencing of the 16S rRNA gene. On the basis of 1,321 nucleotides of the sequence of its gene coding for 16S rRNA (16S rDNA), a phylogenetic relationship to the actinomycetes was established. In this study, we determined an almost complete 16S rDNA sequence (1,495 nucleotides), the 16S-23S ribosomal intergenic spacer sequence, and 200 nucleotides of the 23S rRNA gene. The 16S rDNA sequence was compared with the large number of actinomycete sequences that have been added to the database since the original study. Phylogenetic analysis revealed a branching position as the deepest branch of the cluster comprising the actinomycetes with group B peptidoglycan between this group and the family Cellulomonadaceae. This provides additional information on the phylogenetic position of this bacterium and some clues as to its characteristics. The spacer region between the 16S and 23S rRNA genes is 294 nucleotides long and does not contain tRNA genes. As has been shown in other instances, the increased variability of the ribosomal intergenic spacer compared with the 108 rRNA gene makes it a potential target for use in the differentiation of strains of the bacterium associated with Whipple’s disease.

16S rRNA gene sequences of the type strains of the seven previously described Acetobacterium species were determined. The Acetobacterium species were found to form a tight phylogenetic cluster within the Clostridium subphylum of the gram-positive bacteria. Within this subphylum these organisms belong to cluster XV as defined by Collins et al. (M. D. Collins, P. A. Lawson, A. Willems, J. J. Cordoba, J. Fernandez-Garayzabal, P. Garcia, J. Cai, H. Hippe, and J. A. E. Farrow, Int. J. Syst. Bacteriol. 44:812-826, 1994) together with Eubacterium alactolyticum, Eubacterium barkeri, Eubacterium callanderi, and Eubacterium limosum. Our data indicate that Clostridium cluster XV consists of at least the following three genera: The genus Acetobacterium, the genus Eubacterium sensu stricto (comprising E. limosum, E. barkeri, and E. callanderi), and the genus Pseudoramibacter gen. nov., which is created for E. alactolyticum, which we reclassify as Pseudoramibacter alactolyticus comb. nov.

The genus Nocardiopsis was shown to be phylogenetically coherent and to represent a distinct lineage within the radiation of the order Actinomycetales. The closest relatives of the genus Nocardiopsis are members of the genera Actinomadura, Thermomonospora, Streptosporangium, and Microtetraspora. The intrageneric structure of the genus Nocardiopsis is shown to consist of a highly related species group containing Nocardiopsis dassonvillei, Nocardiopsis alborubida, and Nocardiopsis antarctica and a second group of less highly related species comprising Nocardiopsis alba subsp. alba, Nocardiopsis alba subsp. prasina, and Nocardiopsis listeri. Nocardiopsis lucentensis occupies a position intermediate between the two species groups. The results of a 16S ribosomal DNA sequence analysis are generally consistent with the available chemotaxonomic, phenotypic, and DNA-DNA hybridization data. The phylogenetic position and the morpho- and chemotaxonomic properties of Nocardiopsis species support the creation of a family for the genus Nocardiopsis, Nocardiopsaceae fam. nov.

Almost complete (>96%) 16S rRNA sequences from nine ruminant mycoplasmas have been determined by solid-phase DNA sequencing. Polymorphisms were found in four of the 16S rRNA sequences, which indicated the existence of two different rRNA operons. Seven polymorphisms were found in Mycoplasma agalactiae, three were found in Mycoplasma bovis, one was found in Mycoplasma alkalescens, and one was found in Mycoplasma bovirhinis. The sequence data were used for construction of phylogenetic trees. All but one of the ruminant mycoplasmas sequenced in this work clustered in the hominis group. A close relationship was found between M. agalactiae and M. bovis, with a 99% nucleotide similarity between their 16S rRNA sequences. They were also found to be members of the Mycoplasma lipophilum cluster of the hominis group. Furthermore, the 16S rRNA comparisons showed that Mycoplasma alkalescens and Mycoplasma canadense are closely related (>98.5%), and these species were found to cluster in the Mycoplasma hominis cluster of the hominis group. Interestingly, M. bovirhinis grouped in a new phylogenetic cluster of the hominis group. The new cluster, which was supported by bootstrap percentage values, signature nucleotide analysis, and higher-order structural elements, was named the Mycoplasma synoviae cluster. Mycoplasma bovoculi, Mycoplasma conjunctivae, and Mycoplasma ovipneumoniae clustered in the Mycoplasma neurolyticum cluster of the hominis group. Mycoplasma alvi clustered with Mycoplasma pirum in the M. pneumoniae cluster of the pneumoniae group.

A novel extremely thermophilic bacterium was isolated from a shallow marine hydrothermal vent environment (depth, 22 m) in Tachibana Bay, Nagasaki Prefecture, Japan. The cells of this organism were gramnegative rods. Growth occurred at temperatures between 50 and 85°C (optimum temperature, 80°C; doubling time at optimum temperature, 90 min), at pH 5.5 and 9.0 (optimum pH, 7.0), and in the presence of 1 and 5% NaCl (optimum NaCl concentration, 3%). The new isolate was an aerobic heterotroph which utilized the following compounds as sole energy and carbon sources: Yeast extract, peptone, starch, casein, Casamino Acids, a variety of sugars, some carboxylic acids, and amino acids. As determined by a sequence analysis of the 16S rRNA, the new isolate belongs to the genus Rhodothermus and represents a modern lineage of extreme thermophiles within the domain Bacteria. On the basis of the physiological and molecular properties of the new isolate, we describe a new species, Rhodothermus obamensis. The type strain of R. obamensis is strain OKD7 (= JCM 9785).

The cluster of genes encoding components of the botulinum neurotoxin (BoNT) complex was mapped in proteolytic (group I) Clostridium botulinum strains encoding BoNT types A, B, and F. Two different arrangements of genes were found: Type A strain 62A and type B strain NCTC 7273 have similar organizations of genes encoding BoNT, the nontoxic nonhemagglutinin component (NTNH), hemagglutinin components, and P-21; type F strain Langeland has genes encoding BoNT, NTNH, and P-21, and a previously unidentified open reading frame encoding a protein of 416 amino acids. A group of type A strains typified by infant strain Kyoto-F, which is unlike type A strain 62A, lacks genes for hemagglutinin components and exhibits an organization similar to that of type F. Sequencing and pairwise analysis revealed the presence of possible chimeric sequences in some NTNH genes of proteolytic C. botulinum. Discordance in genealogical trees derived from different regions of the NTNH genes was observed which could be symptomatic of recombination and which may indicate that the NTNH gene represents a hot spot for such events within the cluster of genes encoding the BoNT complex. It is also evident that the phylogenetics of the NTNH gene, which is linked to the gene encoding BoNT, does not mirror the evolutionary history of the BoNT, upon which the C. botulinum species complex is defined and subdivided.