- Volume 45, Issue 2, 1995

Neorickettsia helminthoeca (tribe Ehrlichieae, family Rickettsiaceae) is the agent of salmon poisoning disease, which affects members of the family Canidae. This bacterium is unusual in that it is the only known obligately intracellular bacterium that is transmitted via a helminth vector. The nucleotide sequence of the N. helminthoeca 16S rRNA gene was determined and compared with the sequences of intracellular bacteria belonging to the alpha subgroup of the Proteobacteria. The N. helminthoeca sequence was most similar to the sequences of two Ehrlichia species, Ehrlichia risticii and Ehrlichia sennetsu (levels of sequence similarity, >95%). All other members of the tribe Ehrlichieae, including members of the other Ehrlichia species, and the related species Cowdria ruminantium and Anaplasma marginale, were only distantly related phylogenetically (levels of sequence similarity, 84 to 86%). Our results corroborate the results of previous ultrastructural and Western blot (immunoblot) comparisons of N. helminthoeca with other ehrlichial species. The genus Ehrlichia is phylogenetically incoherent and can be separated into three identifiable clusters of species. Each cluster is closely associated with a species classified in another non-Ehrlichia bacterial genus. The close relationships among N. helminthoeca, E. risticii, and E. sennetsu and the striking differences between these organisms and other members of the tribe Ehrlichieae suggest that in the future, these organisms should be treated as members of a new bacterial genus separate from the genus Ehrlichia.

Random amplified polymorphic DNA fingerprinting was used to examine 31 mosquito-pathogenic and 14 nonpathogenic strains of Bacillus sphaericus. We verified that DNA bands that migrated the same distance in an agarose gel were homologous by using PCR-generated probes made from the random amplified polymorphic DNA bands. The band patterns obtained with eight primers were analyzed by using the Jaccard coefficient and unweighted pair group with arithmetic average clustering. Pathogenic strains belonging to DNA homology group IIA were similar to strains belonging to nonpathogenic homology groups at an average level of similarity of 6.3%. Individual serotypes were clearly identified among the pathogenic strains. This suggests that there is overall genetic homogeneity among strains within serotypes. It is also consistent with the uniform toxicity pattern found for each serotype (unlike the toxin diversity found in Bacillus thuringiensis serotypes). These results, together with DNA homology data, support the proposal that a new species should be described for the pathogenic strains.

Strain MLFT (T = type strain), a new thermophilic, spore-forming sulfate-reducing bacterium, was characterized and was found to be phenotypically, genotypically, and phylogenetically related to the genus Desulfotomaculum. This organism was isolated from a butyrate enrichment culture that had been inoculated with a mixed compost containing rice hulls and peanut shells. The optimum temperature for growth was 50°C. The G+C content of the DNA was 51.2 mol%. Strain MLFT incompletely oxidized pyruvate, butyrate, and butanol to acetate and presumably CO2. It used long-chain fatty acids and propanediols. We observed phenotypic and phylogenetic differences between strain MLFT and other thermophilic Desulfotomaculum species that also oxidize long-chain fatty acids. On the basis of our results, we propose that strain MLFT is a member of a new species, Desulfotomaculum thermosapovorans.

Nine streptomycete strains that cause deep-pitted scab of potato were characterized by performing 138 physiological and morphological tests. A numerical analysis revealed that the deep-pitted scab-inducing organisms were related to strains belonging to cluster 1 of Williams et al. (Streptomyces albidoflavus). The levels of similarity between the deep-pitted scab-inducing strains and strains of Streptomyces scabies and Streptomyces acidiscabies were low (52 and 54%, respectively). The fatty acid profiles of the deep-pitted scab-inducing organisms differed considerably from those of S. scabies and S. acidiscabies. The deep-pitted scab-inducing bacteria were characterized by the predominance in their profiles of the 15:0 anteiso, 15:0 iso, 16:0 iso, 16:0, and 17:0 anteiso acids. The results of physiological characterization experiments, a fatty acid analysis, and whole-cell protein electrophoresis showed that the deep-pitted scab-inducing strains characterized in this study formed a relatively homogeneous group. These pathogenic strains could also be differentiated from S. scabies by their high cellulolytic and proteolytic activities.

Strains of a previously undescribed species of purple nonsulfur phototrophic bacteria were isolated from coastal seawater in Japan. These new isolates were gram-negative, motile, budding rods that contained lamellar intracytoplasmic membranes and produced pink to red cultures. Cell extracts of photosynthetic cultures exhibited absorption maxima at 377, 468, 500, 530, 591, 802, and 870 nm, indicating that bacterio-chlorophyll a and carotenoids of the spirilloxanthin series were present. The new isolates were halophilic, facultatively aerobic photoheterotrophs that grew anaerobically in the light or aerobically in the dark. Maximum growth occurred in the presence of 4 to 5% NaCl. Anaerobic growth in the dark with nitrate as a terminal electron acceptor also occurred. Various organic compounds were used as photosynthetic electron donors and carbon sources. Sulfate was used as a sulfur source. Both menaquinone 10 and ubiquinone 10 were produced; these quinones were the major quinones. A phylogenetic analysis based on 16S rRNA gene sequences revealed that strain MB312T(T = type strain), a representative of the new phototrophs, was a member of a lineage that was distinct from members of the genus Rhodopseudomonas; Rhodopseudomonas marina was the closest relative. On the basis of the data described above, we propose the name Rhodobium orientis gen. nov., sp. nov. for the new isolates. We also propose that Rhodopseudomonas marina Imhoff 1983 should be transferred to the genus Rhodobium as Rhodobium marinum comb. nov.

Isolates belonging to the “Streptococcus milleri” species group that appear to exhibit a gliding type of motility, which is expressed as spreading growth on certain types of agar media, are described. These strains resembled a biotype of “S. milleri” that is usually isolated from genitourinary sources and is notable for its ability to ferment a wide array of carbohydrates. This biotype, which is currently included in the species Streptococcus anginosus, has been implicated in cases of neonatal infection. The “S. milleri” isolates which we studied lacked any observable organelles of motility and gave negative results when they were tested in conventional motility test medium stab cultures. Colonies growing on certain agar media, however, spread over the surfaces of plates and increased in area with increasing time of incubation. Chocolate agar supported maximum spreading, while this characteristic was barely discernible on blood agar. Electron microscopy studies revealed that there was more production of extracellular glycocalyx by motile strains than by a nonmotile isolate having a similar biotype. The results of an analysis of 16S rRNA gene sequences suggested that the motile strains are closely related to S. anginosus and represent a distinct rRNA population within the “S. milleri” species complex.

Partial sequences of the 16S rRNA genes of the type strains of nine species of the genus Nocardia were determined following the isolation and cloning of the amplified genes. These sequences were aligned with the sequences of representatives of the genera Corynebacterium, Gordona, Mycobacterium, Rhodococcus, and Tsukamurella, and phylogenetic trees were inferred by using the Fitch-Margoliash and neighbor-joining methods. The genus Nocardia formed a distinct clade that was most closely associated with the genus Rhodococcus. The average level of sequence similarity found among the type strains of the Nocardia species was 97.2 ± 0.7%. Two sublines were recognized within the Nocardia clade; one encompassed Nocardia asteroides and related species, and the other encompassed Nocardia otitidiscaviarum and allied taxa. Separation of the two sublines is based on differences in helix 37-1. The results of isoprenoid quinone analyses provided evidence that nocardiae can be distinguished from all other actinomycete taxa on the basis of their characteristic menaquinone profiles. Nocardiae typically contain hexahydrogenated menaquinones with eight isoprene units in which the two end units are cyclized.

Members of the genera Bacteroides, Prevotella, and Porphyromonas were investigated for their glucose 6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) activities by using spectrophotometric (SP) and alloenzyme elecrophoresis (AE) detection methods. When the SP and AE methods were compared, the AE method failed to detect activity in two of the six strains which exhibited G6PDH and 6PGDH activities as determined by the SP detection method. On the basis of the results of SP detection, Bacteroides levii ATCC 29147T(T = type strain) (G6PDH and 6PGDH negative) is not a member of the genus Bacteroides as currently defined, which reflects recent 16S rRNA placement, nor do Prevotella heparinolytica ATCC 35895T, Prevotella zoogleoformans ATCC 33285T, Porphyromonas canoris 12835T, and Porphyromonas salivosa NCTC 11632T(all G6PDH and 6PGDH positive) conform to their respective genus descriptions. When these organisms were grown in prereduced peptone-yeast extract broth containing 10% (wt/vol) glucose, the amounts of glucose remaining after 5 days were less than the amounts present initially for members of the genus Bacteroides (Bacteroides fragilis ATCC 25285T and B. levii) and members of the genus Prevotella (Prevotella melaninogenica ATCC 25845T, Prevotella buccae ATCC 33574T, Prevotella heparinolytica, and Prevotella zoogleoformans). In addition, the glucose levels were lower after 5 days of incubation in broth media containing Porphyromonas asaccharolytica ATCC 25845Tand Porphyromonas salivosa, but not in media containing the other members of the genus Porphyromonas tested (Porphyromonas canoris, Porphyromonas circumdentaria NCTC 12469T, Porphyromonas endodontalis ATCC 35406T, and Porphyromonas gingivalis ATCC 33277T). The reductions in glucose levels were not directly related to the final pH values. Our results suggest that the descriptions of the genera Bacteroides, Prevotella, and Porphyromonas may require emendation to reflect variability in G6PDH and 6PGDH activities and glucose utilization.

The 16S rRNA gene of a new infectious agent, strain AS145T(T = type strain), which was isolated from a wild mouse in Japan, was amplified by using the PCR. The amplimers were directly sequenced by dideoxynucleotide methods with Taq DNA polymerase. Sequence comparisons with other members of the tribe Ehrlichieae and related species revealed that the infectious agent isolated from the mouse is a new species of the genus Ehrlichia that is most closely related to Ehrlichia chaffeensis (level of sequence similarity, 97.9%), an agent of human ehrlichiosis in the United States. This result was consistent with the results of an immunoblot analysis performed with immune sera against different ehrlichiosis agents. On the basis of these findings and other morphological, biological, and serological characteristics of the organism, we propose that ehrlichiae with these properties belong to a new species, Ehrlichia muris.

We investigated expression of acid phosphatases containing low-molecular-mass (25 to 27-kDa) polypeptides (Lmmp-APs) similar to those described previously for Salmonella enterica serovar typhimurium and Morganella morganii in strains that were representatives of 43 different enterobacterial species by using a zymogram technique developed for detection of Lmmp-AP activities and for analysis of some of the properties of these enzymes. Under conditions that were suitable for detection of the previously described Lmmp-APs, production of similar enzymes appeared to be widespread but not universal among enteric bacteria, and heterogeneous patterns of expression were found among strains belonging to different genera and, in some cases, among strains belonging to different species of the same genus. We found that class A Lmmp-APs (i.e., Lmmp-Aps similar to the Morganella morganii PhoC and Salmonella enterica serovar typhimurium PhoN acid phosphatases) were also expressed in Cedecea spp., Enterobacter aerogenes, Hafitia alvei, Klebsiella spp., Providencia stuartii, Serratia plymuthica, and Yokenella regensburgei strains and that class B Lmmp-APs (i.e., Lmmp-APs similar to the Morganella morganii NapA and Salmonella enterica serovar typhimurium NapII acid phosphatases) were also expressed in strains of Citrobacter spp., Escherichia coli, Escherichia fergusonii, Hafnia alvei, Proteus mirabilis, Providencia spp., Salmonella enterica serovar typhi, ShigeUa dysenteriae, and Shigella flexneri. No Lmmp-AP activity was detected in strains of Enterobacter spp. other than Enterobacter aerogenes, Escherichia hermanii, Kluyvera ascorbata, Leclercia adecarboxylata, Leminorella grimontii, Moellerella wisconsensis, Proteus vulgaris, Proteus penneri, Serratia spp. other than Serratia plymuthica, and Yersinia spp. Because of the heterogeneous patterns of expression of Lmmp-APs, analysis of these enzymes could be useful for evolutionary studies of the enterobacterial genome and for precise phylogenetic positioning of enteric bacteria.

The Mycobacterium chelonae-like organism (MCLO) is a recently described member of the Mycobacterium fortuitum complex which causes posttraumatic skin infections and catheter sepsis. This taxon is a distinct group biochemically and has a unique mycolic acid profile as determined by high-performance liquid chromatography. Its phylogenetic relationships to other mycobacteria, however, have not been studied previously. We sequenced 1,062 bp of the 16S rRNA genes from three MCLO strains obtained from the American Type Culture Collection and compared our results with the sequences of previously described taxa of rapidly growing and slowly growing mycobacteria. Two biochemically typical strains (ATCC 49650T[T = type strain] and ATCC 49651) had identical sequences, while the sequence of a biochemically atypical strain (ATCC 49649) differed by 4 bp from the sequence of the two typical strains. The Hamming distances between these MCLO strains and related rapidly growing mycobacteria are comparable to the Hamming distances among taxa of rapidly growing mycobacteria established as species by DNA-DNA hybridization. We propose the name Mycobacterium mucogenicum sp. nov. for this new taxon because of the highly mucoid nature of most isolates on solid media.

I analyzed various families of ribosomal proteins obtained from selected species belonging to the genus Pseudomonas sensu stricto and allied organisms which were previously classified in the genus Pseudomonas. Partial amino acid sequencing of L30 preparations revealed that the strains which I examined could be divided into three clusters. The first cluster, which was assigned to the genus Pseudomonas sensu stricto, included Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas mendocina, and Pseudomonas fluorescens. The second cluster included Burkholderia pickettii and Burkholderia plantarii. The third cluster, which was a deeply branching cluster in the stem of gram-negative bacteria, included Brevundimonas diminuta and Brevundimonas vesicularis. Despite the different levels of conservation of the N-terminal sequences of ribosomal protein families (the highest level of similarity was 74% for L27 proteins and the lowest level of similarity was 42% for L30 proteins), similar phylogenetic trees were constructed by using data obtained from sequence analyses of various ribosomal protein families, including the S20, S21, L27, L29, L31, L32, and L33 protein families. Thus, I demonstrated the efficacy of ribosomal protein analysis in bacterial taxonomy.

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

We describe a new species on the basis of phenotypic characteristics and the results of an analysis of small-subunit rRNA sequences. Three strains of this organism were isolated from a culture of the toxin-producing dinoflagellate Prorocentrum lima. These bacteria are gram-negative, strictly aerobic, ovoid organisms that are motile by means of one or two subpolar flagella. They grow at temperatures ranging from 10 to 37°C and in the presence of NaCl concentrations ranging from 0.1 to 2 M and have an absolute requirement for sodium ions. They are strictly aerobic with a nonfermentative type of metabolism and are not able to grow anaerobically in presence or absence of nitrate. They do not denitrify. They exhibit oxidase, catalase, gelatinase, esculinase, β-galactosidase, and (to a lesser extent) amylase activities. The three strains which we examined require thiamine and biotin for growth. They grow only when glucose, trehalose, saccharose, fructose, maltose, pyruvate, malate, citrate, esculin, 2-ketoglutarate, 5-ketogluconate, glutamate, or shikimate is present as a sole carbon source. The three strains have identical small-subunit rRNA sequences. A phylogenetic analysis of these sequences revealed that these bacteria belong to the alpha subdivision of the Proteobacteria and that they form a distinct and robust monophyletic group with Roseobacter denitrificans and Roseobacter litoralis. This result and the general phenotypic characteristics of the organisms place them in the genus Roseobacter, although they do not produce bacteriochlorophyll a, in contrast to previously described Roseobacter species. On the basis of the phenotypic and genetic similarities of these strains, we assigned them to a single species, for which the name Roseobacter algicola is proposed. The type strain is R. algicola FF3 (= ATCC 51440).

A gram-negative, anaerobic, nonmotile, non-spore-forming, rod-shaped bacterium that fermented succinate quantitatively to propionate was isolated from a high dilution of rumen ingesta obtained from a dairy cow fed a production diet containing grass silage as the main roughage source. This organism did not grow on any of the following energy sources: 12 carbohydrates, pyruvate, lactate, 7 dicarboxylic acids, aspartate, citrate, and trans-aconitate. Both rumen fluid and yeast extract were necessary for good growth on succinate. The organism was negative for the following characteristics: production of propionate from threonine, protein digestion, sulfide production, nitrate reduction, catalase activity, and urease activity. There was no growth at 22°C and reduced growth at 45°C compared with growth at 39°C. The DNA base composition was 52 mol% G+C. The complete 16S rRNA sequence (EMBL accession number, X81137) was obtained, and the phylogenetic relationships of the organism were determined. The most closely related genera were the genera Acidaminococcus and Phascolarctobacterium. The name proposed for this bacterium is Succiniclasticum ruminis gen. nov., sp. nov.; the type strain is strain SE10 (= DSM 9236). Additional isolation attempts revealed that S. ruminis is a common inhabitant of the rumina of cows that are fed production diets and of cows on pasture.

A strictly anaerobic, moderately halophilic, gram-negative, rod-shaped bacterium was isolated from Great Salt Lake, Utah, sediments and designated GSLST(T = type strain). Strain GSLSTgrew optimally at pH 6.7 to 7.0 but had a very broad pH range for growth (pH 5.8 to 10.0). The optimum temperature for growth was 37°C, and no growth occurred at 15 or 55°C. The optimum salt concentration for growth was 10%. Strain GSLSTrequired yeast extract and Trypticase peptone to ferment carbohydrates, pyruvate, and glycine betaine. Strain GSLSTwas resistant to penicillin, d-cycloserine, tetracycline, and streptomycin. The G+C content of this isolate was 31 mol%. The fermentation products from glucose utilization were acetate, butyrate, lactate, CO2, and H2, and in addition strain GSLSTfermented glycine betaine to acetate and trimethylamine. All of these traits distinguish this organism from all previously described halophilic anaerobes. The 16S rRNA gene sequence of strain GSLSTwas found to be similar to, but also significantly different from, the 16S rRNA sequences of Haloanaerobium salsugo and Haloanaerobium praevalens. Therefore, strain GSLST(= DSM 8275T) is described as a new species, Haloanaerobium alcaliphilum.

A thermophilic, glucose-fermenting, strictly anaerobic, rod-shaped bacterium, strain SEBR 6459T(T = type strain), was isolated from an African oil-producing well. This organism was identified as a member of the genus Thermotoga on the basis of the presence of the typical outer sheath-like structure (toga) and 16S rRNA signature sequences and its ability to grow on carbohydrates (glucose, arabinose, fructose, lactose, maltose, and xylose). Major differences in its 16S rRNA gene sequence, its lower optimum temperature for growth (66°C), its sodium chloride range for growth (0 to 2.8%), its lack of lactate as an end product from glucose fermentation, and its peritrichous flagella indicate that strain SEBR 6459Tis not similar to the three previously described Thermotoga species. Furthermore, this organism does not belong to any of the other genera related to the order Thermotogales that have been described. On the basis of these findings, we propose that this strain should be described as a new species, Thermotoga elfii. The type strain of T. elfii is SEBR 6459 (= DSM 9442).

Ehrlichia risticii is the causative agent of Potomac horse fever. Variations among the major antigens of different local E. risticii strains have been detected previously. To further assess genetic variability in this species or species complex, the sequences of the 16S rRNA genes of several isolates obtained from sick horses diagnosed as having Potomac horse fever were determined. The sequences of six isolates obtained from Ohio and three isolates obtained from Kentucky were amplified by PCR. Three groups of sequences were identified. The sequences of five of the Ohio isolates were identical to the sequence of the type strain of E. risticii, the Illinois strain. The sequence of one Ohio isolate, isolate 081, was unique; this sequence differed in 10 nucleotides from the sequence of the type strain (level of similarity, 99.3%). The sequences of the three Kentucky isolates were identical to each other, but differed by five bases from the sequence of the type strain (level of similarity, 99.6%). The levels of sequence similarity of isolate 081, the Kentucky isolates, and the type strain to the next most closely related Ehrlichia sp., Ehrlichia sennetsu, were 99.3, 99.2, and 99.2%, respectively. On the basis of the distinct antigenic profiles and the levels of 16S rRNA sequence divergence, isolate 081 is as divergent from the type strain of E. risticii as E. sennetsu is. Therefore, we suggest that strain 081 and the Kentucky isolates may represent two new distinct Ehrlichia species.

Several strains of phototrophic purple nonsulfur bacteria were isolated from colored blooms occurring in tidal and seawater pools in Japan. All of these isolates had ovoid to rod-shaped cells that were motile by means of single polar flagella and contained vesicular intracytoplasmic membranes together with bacteriochlorophyll a and carotenoids of the spheroidene series. They produced ubiquinone 10 as the major quinone and contained straight-chain fatty acids, with C18:1predominating. They were mesophilic, halophilic, and photoheterotrophic, utilized sulfide and thiosulfate as electron donors for phototrophic growth, and photoassimilated a wide variety of organic compounds as carbon sources. Our results suggested that all of these isolates are members of the recently described genus Rhodovulum. The isolates were classified into four groups (designated groups I through IV) on the basis of phenotypic and genotypic data. The group I isolates, which were the most abundant purple nonsulfur bacteria recovered from the blooms, grew in the presence of NaCl concentrations ranging from 0.5 to 3.0% (optimum NaCl concentration, 0.8%) and at pH values ranging from 7.5 to 9.0 (optimum pH, 8.0 to 8.5). On the basis of these unique physiological traits, together with genotypic and phylogenetic data, we propose that the group I isolates should be classified as members of a new species, Rhodovulum strictum. The group II isolates were identified definitely as Rhodovulum sulfidophilum, and the group III and IV isolates were phenotypically most similar to R. sulfidophilum and Rhodovulum adriaticum, respectively, but could be differentiated from these specks by DNA-DNA pairing data.

In the past workers have isolated several pseudomonad strains which have been used for studies of anaerobic aromatic metabolism. The best studied of these strains are strains KB 740T (T = type strain) and K172T. The taxonomic positions of these two organisms were determined by classical methods, including experiments to determine substrate spectrum, quinone type, and total fatty acid composition. Our results clearly excluded these strains from the authentic genus Pseudomonas, which belongs to the gamma subclass of the Proteobacteria. Instead, the properties of these organisms indicated that they belong to the beta subclass of the Proteobacteria. The sequences of the 16S ribosomal DNA genes confirmed this conclusion and indicated that strain K 172T represents a new species of the genus Thauera, Thauera aromatica, and that strain KB 740T represents a new species of the genus Azoarcus, Azoarcus evansii.

The taxonomic positions of 10 strains of 3-ketolactose-forming bacteria which were isolated from the roots of plants (Rosa sp., Psychotria nairobiensis, Ardisia crispa, Prunus persica, and apple trees) were investigated. The DNA base compositions of these strains ranged from 64.0 to 65.7 mol%, the isoprenoid quinone of each strain was ubiquinone 10,3-hydroxy fatty acids were lacking in the cellular fatty acids of these organisms, and all of the strains contained a sphingolipid with the long-chain base dihydrosphingosin. These are characteristics of the genus Sphingomonas. On the basis of morphological, physiological, and chemotaxonomic characteristics, together with DNA-DNA hybridization and 16S ribosomal DNA sequence comparison data, we propose the following four new species of the genus Sphingomonas: Sphingomonas rosa (type strain, IFO 15208) for the strains isolated from rose plants and formerly named [Agrobacterium rhizogenes]; Sphingomonas pruni (type strain, IFO 15498) for the strains isolated from Prunus persica; and Sphingomonas asaccharolytica (type strain, IFO 15499) and Sphingomonas mali (type strain, IFO 15500) for the strains isolated from apple trees. Two strains which were isolated from Psychotria nairobiensis and formerly named [Chromobacterium lividum] were identified as Sphingomonas yanoikuyae strains.

The distribution of 3-ketolactose formation among members of the alpha subclass of the Proteobacteria was investigated by the Benedict reagent test and by a more sensitive quantitative method in which high-performance liquid chromatography was used. 3-Ketolactose formation activity was found in strains of Agrobacterium biovar 1 and in strains of eight species of the genus Sphingomonas, including Sphingomonas paucimobilis (the type species), which belong to the alpha-2 and alpha-4 subclasses of the Proteobacteria, respectively. Weak activity was found in Erythrobacter longus IFO 14126T (T = type strain), a member of the alpha-4 subclass of the Proteobacteria. The ketosugar was not produced by members of the other taxa of the alpha subclass of the Proteobacteria tested. The ketosugar was isolated from culture broths of S. paucimobilis IFO 13935T and Sphingomonas yanoikuyae IFO 15102T and was identified as 3-ketolactose [4-O-(β-d-xylo-hexopyranosyl-3-ulose)-d-glucopyranose] by chromatographic analyses and 1H nuclear magnetic resonance spectroscopy.

The nucleotide sequences of the 16S rRNA genes of two mycoplasmas, Mycoplasma agassizii (proposed sp. nov.) and Mycoplasma testudinis, isolated from tortoises were determined and used for taxonomic comparisons. Signature nucleotide sequence motifs and overall sequence similarities to other mollicutes positioned these mycoplasmas in the M. hyorhinis and M. pneumoniae phylogenetic groups, respectively. A third, previously unrecognized tortoise mycoplasma was detected by 16S rRNA gene amplification and sequence analysis and was positioned in the M. fermentans phylogenetic group. The 16S rRNA gene of Acholeplasma laidlawii was similarly detected in a tortoise isolate, showing that diverse mollicutes can share the same family of reptilian host.

Nearly complete sequences of 16S rRNA genes of representative strains of the genus Saccharomonospora were determined following the isolation and cloning of the amplified genes. The sequences were aligned with those of representatives of the family Pseudonocardiaceae, and a phylogenetic tree was inferred by the neighbor-joining method. The genus Saccharomonospora formed a distinct clade within the evolutionary radiation encompassed by the family Pseudonocardiaceae. The average nucleotide similarity value found between the type strains of the four validly described Saccharomonospora species was 97.5% ± 1.0%. The most distant relationship was found between Saccharomonospora azurea and Saccharomonospora viridis K73 (96.3% similarity). In contrast, Saccharomonospora azurea K161 and “Saccharomonospora caesia” K163 had identical 16S rRNA gene sequences. The nucleotide sequence data suggest that the genus Saccharomonospora contains several new centers of variation.

We describe a new genus of mesophilic actinomycetes, for which we propose the name Lentzea. The strains of this genus form abundant aerial hyphae that fragment into rod-shaped elements. Whole-cell hydrolysates contain the meso isomer of diaminopimelic acid and no characteristic sugar (wall chemotype III). The phospholipid pattern type is type PII (phosphatidylethanolamine is the characteristic phospholipid); the major menaquinone is MK-9. The fatty acid profile comprises saturated, unsaturated, and branched-chain fatty acids of the iso and anteiso types in addition to tuberculostearic acid (10Me-C18:0). A 16S ribosomal DNA sequence analysis revealed that the genus Lentzea is phylogenically related to the genera Actinosynnema, Saccharothrix, and Kutzneria. The type species of this genus is Lentzea albidocapillata sp. nov.; the type strain of this species is strain IMMIB D-958 (= DSM 44073).

The total cellular fatty acid contents of 40 recently isolated cyanobacterial symbionts obtained from seven species of Azolla host plants were determined by gas-liquid chromatography-mass spectroscopy. A total of 63 fatty acids belonging to seven distinct chemical classes were identified. Fatty acid compositions varied among the cyanobacteria depending on the hosts species. Parameters that differed significantly (at the 99% level of probability) included the concentrations of the 16:0 and 18:3 fatty acids, the total concentrations of the polyunsaturated acids, the total concentrations of the 16-carbon and 18-carbon fatty acids, the ratios of unsaturated fatty acids to saturated fatty acids, and the total percentages of straight-chain even-carbonnumber fatty acids, unsaturated fatty acids, and branched-chain unsaturated fatty acids. The results of an analysis of variance suggested statistical regression for the total percentages of these fatty acids and chemical classes according to the following linear alignment of cyanobacteria by host: Azolla filiculoides, Azolla microphylla, Azolla caroliniana, Azolla mexicana, Azolla rubra, Azolla nilotica, and Azolla pinnata (including Azolla pinnata subsp. pinnata and Azolla pinnata subsp. imbricata). The seven groups could be divided into two distinct clusters on the basis of the results of a dendrogram analysis of Euclidian distances. The symbionts obtained from A. filiculoides, A. microphylla, A. mexicana, and A. caroliniana constituted one cluster, and the symbionts obtained from A. rubra, A. nilotica, and A. pinnata constituted a second cluster. A minor dichotomy separated the A. filiculoides symbionts from the other members of the first cluster. The clustering of Azolla cyanobacterial symbionts based on the results of our fatty acid analysis correlates remarkably well with the taxonomic grouping of the American Azolla species. This correlation suggests that the cyanobacterial symbionts of Azolla spp. coevolved into distinct genetic groups with their hosts.

The taxonomic positions of motile actinomycetes that were isolated from soil and fallen leaves obtained from around a pond and a lake and from fallen leaves of persimmons were studied. The aerial mycelia of all of the isolates exhibited fragmentation during growth, and motile spores arranged in chains were produced within the mycelia. Sporangia were not observed. These isolates contained menaquinone MK-9(H4), their DNA G+C contents were 69 to 70 mol%, they contained glutamic acid, glycine, alanine, and meso-diaminopimelic acid as cell wall amino acids, and arabinose and galactose were found in their whole-cell hydrolysates. These taxonomic characteristics are the same as those of Actinokineospora riparia. On the basis of morphological, physiological, and chemotaxonomic characteristics and DNA-DNA hybridization data, we propose the following four new species of the genus Actinokineospora for these strains: Actinokineospora inagensis for a single isolate, type strain YU4-1 (= IFO 15663); Actinokineospora globicatena for isolates YU5-1, YU6-1, YU6-2, YU7-1, and YU7-2 (type strain, YU6-1 [= IFO 15664]); Actinokineospora terrae for a single isolate, type strain YU6-3 (= IFO 15668); and Actinokineospora diospyrosa for a single isolate, type strain YU8-1 (= IFO 15665).

Comparative analysis of the genes coding for 16S rRNA of the type strains of Xenorhabdus and Photorhabdus species indicates the close phylogenetic relationship of these two genera. However, distance matrix analyses do not unambiguously separate the symbionts of entomopathogenic nematodes according to their assignment into different genera. When various 16S rRNA gene sequences from a selection of members of the gamma subclass of Proteobacteria and outgroup taxa were used, the intrageneric relationships of Xenorhabdus species and the positions of Photorhabdus luminescens and related species changed significantly.

Restriction fragment length polymorphisms of the 16S rRNA genes of Erysipelothrix strains were studied by cleavage of the chromosomal DNA with restriction endonuclease EcoRI, followed by hybridization to a 420-bp internal fragment of the 16S rRNA gene. Thirty-two Erysipelothrix type and reference strains were classified, together with seven field strains. Reference strains of all serotypes and the type strains of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum were included. Nine ribopatterns were observed. Pattern A was represented by 16 strains and included strains of serotypes 1b, 2 to 8, 11 to 13, 15 to 17, 19, and 23. Pattern B was represented by two strains (serotypes la and 9). Pattern C was represented by five strains (serotypes 5, 6, and 21). Pattern D was represented by one strain of serotype 4. Pattern E was represented by 11 strains of serotypes 2, 7, 10, 20, 22, 24, and 25. Patterns F, G, H, and I were each represented by a single strain of serotypes 26,2,18, and 3, respectively. All the different ribopatterns had some bands in common. Patterns B, C, and D were most similar to pattern A, while patterns F, G, H, and I resembled pattern E. Partial sequencing of the 16S rRNA gene of nine selected strains resulted in three different sequences, i.e., the typical E. rhusiopathiae sequence, the E. tonsillarum sequence, and a third sequence found for two strains. Strains of the same serotype were found to have different ribopatterns as well as different partial 16S rDNA sequences.

The genomic relatedness among three Aeromonas allosaccharophila strains, including the type strain, and other Aeromonas type and reference strains that were assigned to DNA hybridization groups was estimated by DNA-DNA hybridization (competition procedure using a membrane method). All A. allosaccharophila strains were highly related (70 to 100%) to strains 289T (= CECT 4199T) and ATCC 35942. Type strains of other validated Aeromonas species, reference strains of DNA groups 8 and 11, and the Aeromonas sp. strain ATCC 43946 (enteric group 501) were 0 to 41% related to A. allosaccharophila 289T and ATCC 35942. The G+Cs content of A. allosaccharophila strains were in the range 55.9 to 57.3 mol%. The G+C content of the type strain of this species was 56.9 mol%, a value somewhat lower than that reported in the original description.

Using a modified Eckhardt method, we visualized replicons larger than 1,000 kb in Rhizobium tropici strains belonging to both subgroup A and subgroup B. The megaplasmid of R. tropici CFN299 was characterized. This megaplasmid is different from a cointegrate of various plasmids and from the chromosome. Hybridization of Eckhardt blots of 15 R. tropici strains with fragments derived from the megaplasmids of the type strains of subgroups A and B revealed that the megaplasmids are subgroup specific.

Wine strains belonging to the genus Leuconostoc were classified as Leuconostoc oenos by Garvie in 1967, and this name was confirmed on the Approved Lists of Bacterial Names in 1980. L. oenos is distinguished from other Leuconostoc spp. by its growth in acidic media, by its requirement for a growth factor in tomato juice, and by a number of carbohydrate fermentation characteristics. In addition, the results of a total soluble cell protein analysis, an electrophoretic analysis of NAD-dependent d-(–)-lactate dehydrogenase, 6-phosphogluconate dehydrogenase, and alcohol dehydrogenase, and an analysis of cross-reactivity with anti-glucose-6-phosphate dehydrogenase and anti-NAD-dependent d-(–)-lactate dehydrogenase performed with other Leuconostoc spp. clearly indicated that L. oenos should be distinguished from the other Leuconostoc species. Phylogenetic studies, in particular 16S and 23S rRNA sequencing studies, have revealed that L. oenos represents a distinct subline that is separate from other Leuconostoc spp. and lactic acid bacteria. In view of the phenotypic and phylogenetic distinctiveness of L. oenos, we propose that this species should be assigned to a new genus as Oenococcus oeni [corrig.] gen. nov., comb. nov. The type strain of O. oeni is NCDO 1674 (= ATCC 23179).

The taxonomic relationships of six strains of Lactobacillus bavaricus, including type strain 136 (= DSM 20269) and reference strain Tro-8/78, Lactobacillus curvatus, and Lactobacillus sake were assessed by performing DNA-DNA hybridization experiments. We confirmed that L. curvatus and L. sake are genotypically distinct species. However, the previously described differentiation of L. bavaricus from L. sake could not be substantiated. We found that L. sake is specifically related to the type strain of L. bavaricus and all L. bavaricus reference strains except “L. bavaricus” Tro-8/78, which is related to L. curvatus. Clearly, strains identified as L. bavaricus Stetter and Stetter 1980 do not form a separate genotypic group, and the name L. bavaricus is a junior synonym of L. sake Katagiri, Kitahara, and Fukami 1934.

Methanolobus vulcani PL-12/MT (T = type strain) grew on methylamines and methanol but not on dimethyl sulfide, formate, acetate, or H2-CO2. The cells grew rapidly at mesophilic temperatures, at neutral pH values (pH 6 to 7.5), and in medium supplemented with 0.1 to 1.2 M NaCl and 13 mM Mg2+. The cells grew in mineral medium containing biotin and a catabolic substrate, but growth was stimulated by yeast extract and peptones. M. vulcani was physiologically similar to Methanolobus tindarius and had a similar 16S rRNA sequence, although the results of DNA-DNA hybridization experiments indicated that these organisms should be considered separate species.

The nucleotide sequence of the 16S rRNA gene of the type strain of Dialister pneumosintes was determined. Phylogenetic analysis revealed that this species belongs to the Sporomusa branch of the Clostridium subphylum of the gram-positive bacteria and should therefore be excluded from the family Barteroidaceae. Within this branch, which encompasses several other gram-negative taxa, such as Acidaminococcus, Pectinatus, Phascolarcobacterium, Quinella, Selenomonas, and Zymophilus, Dialister showed a specific, albeit distant, affinity with the genera Megasphaera and Veillonella.

We determined the 16S rRNA sequences of the type strains of Streptococcus mitis and Streptococcus gordonii and calculated the phylogenetic distances between those organisms and other members of the genus Streptococcus. The viridans group streptococci were separated into five phylogenetic groups; we named these groups the anginosus group, the mitis group, the salivarius group, the bovis group, and the mutans group. S. mitis and S. gordonii clustered in the mitis group together with Streptococcus pneumoniae, Streptococcus oralis, Streptococcus sanguis, and Streptococcus parasanguis at levels of sequence homology of more than 96%. Within this group, S. mitis, S. oralis, and S. pneumoniae exhibited more than 99% sequence homology with each other, although the DNA-DNA similarity values for their total chromosome DNAs were less than 60%.

A large number of Bacillus strains assigned to different species were tested to determine their susceptibilities to antibiotics. Some clear differences between species were observed. The antibiotic susceptibilities of strains isolated from natural sources seemed to be stable and to reflect adaptation of the strains to specific conditions in certain ecological niches. A method for data processing which can be used for rapid species identification is described.

Clostridium putrificum (Trevisan 1889) Reddish and Rettger 1922; Clostridium botulinum (van Ermengem 1896) Bergey, Harrison, Breed, Hammer, and Huntoon 1923; and Clostridium sporogenes (Mechnikoff 1908) Bergey, Harrison, Breed, Hammer, and Huntoon 1923 are genetically related at the species level. We propose rejection of the name C. putrificum (which has priority) and conservation of the name C. botulinum on the basis of Rules 23a and 56a of the International Code of Nomenclature of Bacteria and conservation of the name C. sporogenes for nontoxigenic strains according to Rules 23a and 56b.