- Volume 46, Issue 4, 1996

Eubacterium exiguum sp. nov. is the name proposed for organisms formerly described as Eubacterium group S strains and similar bacteria isolated from various types of oral lesions. This new species was established on the basis of the results of DNA-DNA hybridization experiments and DNA base composition determinations (G+C contents, 60 to 64 mol%). The results of an API ZYM analysis, Western blotting (immunoblotting) reactions, and phenotypic tests are also given. The type strain of E. exiguum is strain S-7.

An acetone-degrading, nitrate-reducing, coccoid to rod-shaped bacterium, strain L1, was isolated from soil on the site of a natural gas company. Cells of the logarithmic growth phase reacted gram positive, while those of the stationary growth phase were gram negative. Single organisms were 0.4 to 0.5 by 0.9 to 1.5 μm in size, nonmotile, and non-spore forming and had poly-β-hydroxybutyrate inclusions. The doubling time of strain L1 on acetone-CO2-nitrate at the optimal pH of 7 to 8 and the optimal temperature of 30 to 37°C was 12 h. More than 0.2% NaCl or 10 mM thiosulfate inhibited growth. For oxygen or nitrate respiration, acetone and a few organic chemicals were utilized as carbon sources whereas many others could not be used (for details, see Results). Bicarbonate (or CO2) was essential for growth on acetone but not for growth on acetoacetate. The growth yields for acetone-CO2 and acetoacetate were 28.3 and 27.3 g/mol, respectively. With acetone as the carbon source, poly-β-hydroxybutyrate accounted for up to 40% of the cellular dry weight The DNA of strain L1 had a G+C content of 68.5 mol% (as determined by high-performance liquid chromatography of nucleotides) or 70 mol% (as determined by the Tm method). The sequence of the gene coding for the 16S rRNA led to the classification of strain L1 in the paracoccus group of the alpha subclass of the Proteobacteria. The new isolate is named Paracoccus solventivorans sp. nov. DSM 6637.

Three strains of an anaerobic thermophilic organoheterotrophic lipolytic alkalitolerant bacterium, Thermosyntropha lipolytica gen. nov., sp. nov. (type strain JW/VS-265T; DSM 11003), were isolated from alkaline hot springs of Lake Bogoria (Kenya). The cells were nonmotile, non-spore forming, straight or slightly curved rods. At 60°C the pH range for growth determined at 25°C [pH25°C] was 7.15 to 9.5, with an optimum between 8.1 and 8.9 (pH60°C of 7.6 and 8.1). At a pH25°C of 8.5 the temperature range for growth was from 52 to 70°C, with an optimum between 60 and 66°C. The shortest doubling time was around 1 h. In pure culture the bacterium grew in a mineral base medium supplemented with yeast extract, tryptone, Casamino Acids, betaine, and crotonate as carbon sources, producing acetate as a major product and constitutively a lipase. During growth in the presence of olive oil, free long-chain fatty acids were accumulated in the medium but the pure culture could not utilize olive oil, triacylglycerols, short- and long-chain fatty acids, and glycerol for growth. In syntrophic coculture (Methanobacterium strain JW/VS-M29) the lipolytic bacteria grew on triacylglycerols and linear saturated and unsaturated fatty acids with 4 to 18 carbon atoms, but glycerol was not utilized. Fatty acids with even numbers of carbon atoms were degraded to acetate and methane, while from odd-numbered fatty acids 1 mol of propionate per mol of fatty acid was additionally formed. 16S rDNA sequence analysis identified Syntrophospora and Syntrophomonas spp. as closest phylogenetic neighbors.

The taxonomic position of eight strains isolated from mineral water and previously grouped in the authentic pseudomonads on the basis of a phenotypic analysis (cluster Ib of M. Elomari, L. Coroler, D. Izard, and H. Leclerc [J. Appl. Bacteriol. 78:71-81, 1995]) has been further studied by DNA-DNA hybridizations. Using the S1 nuclease method at 60°C and labeled reference DNA from a representative strain, CFML 92-134, we showed that members of cluster Ib constituted a homogeneous group with a relative binding ratio of greater than 80% and changes in melting temperature of less than 1°C. With a total of 67 strains representing known or partially characterized species of the genus Pseudomonas, only 4 to 47% DNA hybridization and changes in melting temperature of between 8 and 20°C were found, the highest hybridization values being measured with members of the saprophytic fluorescent pseudomonads. Since cluster Ib could also be clearly differentiated from members of the latter group and from other phenotypic clusters containing isolates from mineral water, we designated the Ib strains members of a new Pseudomonas species for which the name Pseudomonas veronii sp. nov. has been proposed. Members of this species grew on α-aminobutyrate, sucrose, butyrate, isobutyrate, erythritol, l-tryptophan, and trigonelline as sole sources of carbon and energy. The average G+C content of the DNA of the eight strains of P. veronii was 61.5 ± 0.5 mol%. The type strain is CFML 92-134T (CIP 104663T), with a G+C content of 61 mol%. The clinical significance of P. veronii is unknown.

A syntrophic acetate-oxidizing bacterium, strain BST (T = type strain), was isolated from a previously described mesophilic triculture that was able to syntrophically oxidize acetate and form methane in stoichiometric amounts. Strain BST was isolated with substrates typically utilized by homoacetogenic bacteria. Strain BST was a spore-forming, gram-positive, rod-shaped organism which utilized formate, glucose, ethylene glycol, cysteine, betaine, and pyruvate. Acetate and sometimes formate were the main fermentation products. Small amounts of alanine were also produced from glucose, betaine, and cysteine. Strain BST grew optimally at 37°C and pH 7. The G+C content of the DNA of strain BST was 32 mol%. A 16S rRNA sequence analysis revealed that strain BST was a member of a new species of the genus Clostridium. We propose the name Clostridium ultunense for this organism; strain BS is the type strain of C. ultunense.

A new strictly anaerobic bacterium (strain BAL-1T) has been isolated from a reed bed at Ballarat Goldfields in Australia. The organism grew by reducing arsenate [As(V)] to arsenite [As(III)], using acetate as electron donor and carbon source; acetate alone did not support growth. When BAL-1T was grown with arsenate as the terminal electron acceptor, acetate could be replaced by pyruvate, l- and d-lactate, succinate, malate, and fumarate but not by H2, formate, citrate, glutamate, other amino acids, sugars, or benzoate. With acetate was the electron donor, arsenate could be replaced by nitrate or nitrite but not by sulfate, thiosulfate, or iron oxide. Nitrate was reduced to ammonia via nitrite. The doubling time for growth on acetate (5 mM) plus arsenate (5 mM) or nitrate (5 mM) was 4 h. The G+C content of the DNA is 49 mol%. The 16S rRNA sequence data for the organism support the hypothesis that this organism is phylogenetically unique and at present is the first representative of a new deeply branching lineage of the Bacteria. This organism is described as Chrysiogenes arsenatis gen. nov., sp. nov.

Lactobacillus curvatus and Lactobacillus sake are each genetically homogeneous species, as indicated by the high levels of DNA homology (≥76%) exhibited by strains of these taxa. However, the results of a numerical analysis of total soluble cell protein patterns and biochemical test data revealed that there are two phenotypic subgroups within L. curvatus and two phenotypic subgroups within L. sake. The overall randomly amplified polymorphic DNA (RAPD)-PCR band patterns obtained for the majority of L. curvatus strains corresponded well to the pattern obtained for the type strain of L. curvatus (strain DSM 20019). However, six strains of L. curvatus had different, but similar, RAPD-PCR profiles and grouped in a separate genetic cluster, which was linked to one of the clusters of L. sake strains. On the basis of these results, differences in biochemical and physiological characteristics, and total soluble cell protein profiles, we describe the subspecies L. curvatus subsp. curvatus subsp. nov. and L. curvatus subsp. melibiosus subsp. nov. for L. curvatus Abo-Elnaga and Kandler 1965 (Klein et al. 1996, emended description). Strains of L. sake grouped in two RAPD-PCR clusters, which was consistent with previous reports of phenotypic heterogeneity. Strains of Lactobacillus bavaricus, including type strain LMG 9844, clustered with the type strain of L. sake (strain NCFB 2714), indicating that these organisms belong to the same genetic group. We propose that strains of L. sake Katagiri, Kitahara, and Fukami 1934 (Klein et al. 1996, emended description) should be reclassified as members of L. sake subsp. sake subsp. nov. and L. sake subsp. carnosus subsp. nov. Strains of L. bavaricus are reclassified as members of L. sake subsp. sake, and the name L. bavaricus Stetter and Stetter 1980 is rejected.

Sixteen strains of cultivable mycobacteria were grown in Sauton’s medium, and Mycobacterium leprae was purified from armadillo liver. Cell extracts were prepared from log-phase growths of each of the cultivable mycobacterial strains. Superoxide dismutase (SOD) enzyme was purified from all cultivable mycobacterial strains included in the study, and antibodies against purified SOD enzyme were raised in rabbits. Immunological distances (ImDs) between these anti-SOD antibodies and SOD antigens were determined by a previously described immunoprecipitation method and by a recently developed enzyme-linked immunosorbent assay (ELISA) technique. The reciprocal ImDs among mycobacterial strains were constant, reproducible and consistent by these two methods. An evolutionary tree was constructed on the basis of estimated ImDs. Except for M. duvalii and M. terrae, slowly and rapidly growing mycobacterial species appeared to be separately grouped by this analysis. Rapid growers clustered into a group which is near that of some slow-growing mycobacteria. M. avium falls almost in the middle of the evolutionary tree and the position of M. leprae was found to be between those of M. avium and M. bovis BCG. Measurement of immunological relatedness of SODs provides an alternative system with which to study the taxonomical relatedness among mycobacteria.

The mcrBDCGA operon that encodes methyl coenzyme M reductase (MR) in the hyperthermophile Methanopyrus kandleri was cloned and sequenced. The results of a phylogenetic analysis of the nine MR sequences now available support the position that M. kandleri is a separate methanogen lineage. As in other methanogens, the M. kandleri mcr operon is located immediately upstream of the mtrE gene, the promoter-proximal gene in an operon that encodes the N 5-methyltetrahydromethanopterin:coenzyme M methyltransferase that catalyzes the step preceding the MR-catalyzed reaction in methanogenesis. In contrast to other methanogens and hyperthermophilic members of the Archaea. CG dinucleotides and CG-containing codons occur frequently in M. kandleri DNA. The MR subunit-encoding genes are preceded by sequences consistent with ribosome binding sites, indicating that mRNA-rRNA base pairing can still direct translation initiation in cells growing at temperatures above 100°C.

The sequences of the 16S rRNA genes (rrs genes) of Clostridium chauvoei, the causative agent of blackleg in cattle, and the phenotypically related organism Clostridium septicum were determined. After amplification of 1,507-bp PCR fragments from the corresponding rrs genes, the sequences were determined in a single round of sequencing by using conserved region primers. A sequence similarity analysis of the sequences revealed the close phylogenetic relationship of C. chauvoei and C. septicum in Clostridium cluster I (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), which includes Clostridium carnis, Clostridium perfringens, Clostridium botulinum, and Clostridium tetani. We found that 99.3% of the nucleotides in the genes of C. chauvoei and C. septicum are identical.

Pulsed-field gel electrophoresis was used to characterize the genomes of 15 Chlorobium strains. An analysis of chromosomal macrorestriction patterns allowed us to determine the chromosome sizes, which ranged from 1,435 to 3,342 kb depending on the strain. Moreover, this analysis revealed that, even though the genus Chlorobium is a phylogenetically coherent taxon, there is a great deal of genomic heterogeneity within it. This heterogeneity was corroborated by performing a ribotyping analysis, which showed that the rRNA hybridization patterns of most strains are unique.

As the genus Halorubrum was validated before the genus Halorubrobacterium, we propose that the species Halorubrobacterium distributum and Halorubrobacterium coriense should be reclassified as Halorubrum distributum comb. nov. and Halorubrum coriense comb. nov., respectively.

The phylogenetic positions of the porcine mycoplasmas Mycoplasma hyosynoviae and Mycoplasma hyopharyngis were determined by using PCR-amplified 16S rRNA gene sequences. M. hyosynoviae is a member of the Mycoplasma hominis group, while M. hyopharyngis belongs to the Mycoplasma fermentans group of mollicutes. Neither species is closely related to previously characterized porcine mycoplasmas belonging to the Mycoplasma hyorhinis group.

Ureaplasma gallorale is a urease-containing mycoplasma (a member of the Mollicutes) which is pathogenic for chickens, from which it was originally isolated. We amplified the 16S rRNA gene of this bacterium and then cloned and sequenced the amplicon. A phylogenetic analysis based on an alignment of the 16S rRNA sequences of U. gallorale and several other Ureaplasma species revealed that U. gallorale is more closely related to Ureaplasma urealyticum than to other Ureaplasma species.