- Volume 41, Issue 3, 1991

The phylogenetic interrelationships of 14 members of the genus Fusobacterium were investigated by performing a comparative analysis of the 16S rRNA sequences of these organisms. The sequence data revealed considerable intrageneric heterogeneity. The four species Fusobacterium nucleatum (including F. nucleatum subsp. nucleatum, F. nucleatum subsp. polymorphum, “F. nucleatum subsp. fusiforme,” and “F. nucleatum subsp. animalis”), Fusobacterium alocis, Fusobacterium periodonticum, and Fusobacterium simiae, which colonize oral cavities, exhibited high levels of sequence homology with each other and formed a distinct group within the genus. Fusobacterium mortiferum, Fusobacterium varium, and Fusobacterium ulcerans also formed a phylogenetically coherent group, as did the two species Fusobacterium gonidiaformans and Fusobacterium necrophorum. Fusobacterium russii and Fusobacterium necrogenes displayed no specific relationship with any of the other fusobacteria. The sequence data are discussed in the context of previous physiological and chemical findings.

Clostridium orbiscindens sp. nov. is an obligate anaerobe that is capable of cleaving the C-3-C-4 bond of the natural anticarcinogen quercetin. The metabolic products, 3,4-dihydroxyphenylacetic acid and presumably phlorglucinol, are not known to possess anticarcinogen properties. This organism was isolated from human feces. On sheep blood agar plates C. orbiscindens forms minute, irregular, convex, gray or white, shiny, smooth, nonhemolytic colonies. It is beta-hemolytic on rabbit blood agar. The motile peritrichous rods are gram variable. Subpolar spores are common. Cultures are resistant to 80°C for 10 min. Capsules are absent. This asaccharolytic organism does not metabolize esculin, urea, meat, gelatin, casein, or nitrate. The G+C content is 56 to 57 mol%. DNA hybridization experiments did not reveal relatedness to phenotypically similar Clostridium strains. Strain 265 (= ATCC 49531) is the type strain.

Mesophilic Methanothrix sp. strains MTAS and MTKO were isolated as pure cultures from the sludge of anaerobic digestors. We examined the relationships between these two isolates and three other Methanothrix strains (strains OpfikonT [T = type strain], GP6T, and FE) by using an immunological method, by performing DNA homology experiments, and by analyzing polar lipids. Our results indicate that all five strains belong to the same species.

Arthrobacter sp. strain NRRL B-3381T (T = type strain) is a nonmycelial, nonsporulating actinomycete that produces the macrolide antibiotic erythromycin. This bacterium differs in many ways from the type species of the genus Arthrobacter (Arthrobacter globiformis), suggesting that a taxonomic revision is appropriate. The G+C content of strain NRRL B-3381T DNA is 71 to 73 mol%, and the peptidoglycan of this organism contains LL-diaminopimelic acid. Evolutionary distance data obtained from 16S rRNA sequences identified NRRL B-3381T as the deepest branching member of the Nocardioides group of actinomycetes. The principal long-chain fatty acids which we identified that distinguished strain NRRL B-3381T from related G+C-rich bacteria were 10-methyloctadecanoic (tuberculosteric), octadecenoic, and hexadecanoic acids. These characteristics, together with phage typing and biochemical characteristics, form the basis for our recommendation that strain NRRL B-3381 should be the type strain of a new taxon, for which we propose the name Aeromicrobium erythreum.

Azotobacter salinestris sp. nov. was isolated from slightly saline soils of Western Canada, where strains of this species accounted for ≤5% of the aerobic nitrogen-fixing isolates. Also, one isolate was obtained from a saline Egyptian soil. These bacteria shared many physiological traits with Azotobacter chroococcum, but were absolutely dependent on Na+ ions for growth and frequently used melibiose as a carbon and energy source. Aerobically grown cells were melanized dark brown to black; the cells were large, gram negative, oval with pointed ends, and motile by means of peritrichous flagella and formed pairs and chains of six to eight cells during active growth. Capsule production was variable. Nitrogen fixation by A. salinestris occurred optimally at 35°C in the presence of molybdate or vanadate ions in a microaerophilic, aeroadaptive manner. The cells were very sensitive to H2O2 and catalase negative. However, a single weak catalase electromorph was observed in cell extracts. This contrasted with a very active catalase represented by multiple electromorphs in A. chroococcum. Iron was absolutely required for growth and aeroadaptation. Other growth-promoting substrates included fructose, galactose, glucose, mannitol, starch, and sucrose. Acid was formed from growth-promoting sugars and also from the non-growth-promoting substrates arabinose, cellobiose, lactose, mannose, rhamnose, and xylose. Incubation in the presence of increased NaCl concentrations promoted acidification of the culture to inhibitory levels, and sufficient acid was released from nitrogen-fixing cells in the presence of 1.0 to 1.5% NaCl to solubilize CaCO3 suspended in solid medium. The cells grew well in marine broth alone, producing an alkaline reaction. An acid reaction was produced both oxidatively and fermentatively in marine broth containing glucose. Nitrate was used in an assimilatory fashion, and there was no evidence of NO2- or N2 formation. The type strain is strain 184 (=ATCC 49674), which was isolated from soils of Alberta, Canada.

Deleya salina sp. nov., which was isolated from hypersaline habitats, is described. Our description is based on 26 strains that exhibit high levels of phenotypic similarity (simple matching coefficient, >74%) and DNA-DNA homology (<71%). The organisms are moderately halophilic gram-negative rods; growth occurs at salt concentrations between 2.5 and 20% (wt/vol) (optimal growth occurs at a salt concentration of 5% [wt/vol]). D. salina is aerobic, nonmotile, and catalase and oxidase positive. The cells accumulate poly-β-hydroxybutyrate, and the G+C contents of the DNAs range from 60.4 to 64.2 mol%. The type strain is strain F8-11 (= ATCC 49509).

On the basis of the results of a numerical analysis in which 112 characters were used, strains ATCC 271T (T = type strain) and ATCC 4004, which were previously classified as Rhodococcus roseus by Tsukamura but were not included on the 1980 Approved Lists of Bacterial Names, were differentiated from all previously described species of the genus Rhodococcus. The mycolic acids of these strains contained 36 to 46 carbon atoms, and the number of double bonds was zero or one. The predominant menaquinone was MK-8(H2), and the moles percent guanine plus cytosine was 67.5. We propose that the species Rhodococcus roseus be revived as a new species of the genus Rhodococcus. The type strain is strain ATCC 271.

We found that Mycobacterium porcinum ATCC 33776T (T = type strain) contains a new kind of mycolic acid with a methoxy group at the ω-1 position. This mycolic acid was identified by comparing it with the previously described methoxymycolic acids. The patterns of mycolic acid methyl esters from 418 strains belonging to 44 species of mycobacteria were studied by using thin-layer chromatography. In addition to M. porcinum ATCC 33776T, representative strains of M. porcinum, Mycobacterium fortuitum, “Mycobacterium peregrinum,” Mycobacterium senegalense, and a recently isolated Mycobacterium sp. contained appreciable amounts of the newly described mycolic acid.

The biological and biochemical properties, DNA base compositions, and levels of DNA-DNA homology of two biovars of Fusobacterium necrophorum were examined. Some differences were found between the two biovars in biological and biochemical properties. The G+C contents of DNAs from biovar A strains VPI 2891T (T = type strain), NCTC 10576, N167, Fn47, and Fn43, were 32, 30, 29, 28, and 31 mol%, respectively. The G+C contents of DNAs from biovar B strains Fn524T, 606, Fn49, Fn45, and 1260 were 30, 31, 27, 31, and 30 mol%, respectively. Labeled DNA from biovar A strain VPI 2891T exhibited 100 to 80% relatedness to DNAs from biovar A strains and 59 to 51% relatedness to DNAs from biovar B strains. Labeled DNA from biovar B strain Fn524T exhibited 100 to 81% relatedness to DNAs from biovar B strains and 71 to 60% relatedness to DNAs from biovar A strains. Therefore, the names Fusobacterium necrophorum subsp. necrophorum subsp. nov., nom. rev. (ex Flügge 1886), and Fusobacterium necrophorum subsp. funduliforme subsp. nov., nom. rev. (ex HallÉ 1898), are proposed for Fusobacterium necrophorum biovars A and B, respectively. The type strain of F. necrophorum subsp. necrophorum is strain VPI 2891 (= JCM 3718 = ATCC 25286), and the type strain of F. necrophorum subsp. funduliforme is strain Fn524 (= JCM 3724).

The taxonomic status of strains belonging to the Streptomyces violaceusniger phenotypic cluster, including eight S. violaceusniger strains, three Streptomyces hygroscopicus strains, and the type strains of Streptomyces sparsogenes and Streptomyces melanosporofaciens, was evaluated by using DNA relatedness data. The S. violaceusniger strains were separated into seven different DNA homology groups, including four single-member clusters, at a level of DNA relatedness of >70%. S. hygroscopicus NRRL 2387T (T = type strain) clustered with the type strain of Streptomyces endus and several S. violaceusniger strains. The other strains of S. hygroscopicus, S. sparsogenes, and S. melanosporofaciens formed single-member homology groups.

We analyzed ribosomal AT-L30 proteins from 11 type strains of wall chemotype IV actinomycete species which lack mycolic acids. The electrophoretic mobilities of the AT-L30 preparations from these strains, as determined by two-dimensional polyacrylamide gel electrophoresis, revealed that the genera Actinopolyspora, Amycolatopsis, Amycolata, Pseudonocardia, Saccharopolyspora, and Saccharomonospora can be separated from each other on the basis of the relative electrophoretic mobilities of their AT-L30 proteins. Overlapping relative electrophoretic mobilities were observed only for the genera Amycolata and Pseudonocardia. The electrophoretic mobility of protein AT-L30 from Actinopolyspora mortivallis was similar to the electrophoretic mobility of Actinopolyspora halophila protein AT-L30 but not to the electrophoretic mobilities of the AT-L30 proteins of the reference genera. Our results confirm the taxonomic status of A. mortivallis as a species of the genus Actinopolyspora and illustrate the efficacy of ribosomal protein analysis in actinomycete taxonomy.

The properties and taxonomic position of bacterial strains isolated from diseased specimens of cultured yellowtail and eels were examined. The isolates were gram-positive, short-chain-forming, catalase-negative, facultatively anaerobic cocci. Growth at 10 and 45°C in 6.5% NaCl (pH 9.6) with 40% bile and in 0.1% methylene blue-milk were both positive. The isolates could be distinguished from other species of the genus Enterococcus by several biochemical characteristics and by Lancefield’s group antigen. Guanine-plus-cytosine content of DNA was 44 mol% as determined by the thermal melting temperature. The value for DNA-DNA hybridization was sufficiently low to warrant distinguishing this species from reported Enterococcus species. The name Enterococcus seriolicida is proposed. The type strain is YT-3 (=ATCC 49156).

We isolated strain HI350 from a gas and oil well in the Gulf of Mexico, characterized it, and found that it is closely related to Methanolobus siciliae T4/MT (T = type strain), which we also characterized. The previously published characterization of the type strain of M. siciliae was limited to the optimum temperature for growth, and our characterization suggested the species description given below. Cells are irregular, nonmotile, coccoid, and 1.5 to 3 μm in diameter. The catabolic substrates used include methanol, trimethylamine, and dimethyl sulfide, but not H2-CO2, formate, or acetate. Growth is fastest in the presence of 0.4 to 0.6 M Na+, in the presence of 60 to 200 mM Mg2+, at pH 6.5 to 6.8, and at 40°C. Growth on trimethylamine is stimulated by yeast extract. An electrophoretic analysis confirmed that strain HI350 is closely related to strain T4/MT and indicated that major changes in the intracellular proteins of M. siciliae HI350 occur when the growth substrate is switched between dimethyl sulfide and trimethylamine.

A new Rhizobium species that nodulates Phaseolus vulgaris L. and Leucaena spp. is proposed on the basis of the results of multilocus enzyme electrophoresis, DNA-DNA hybridization, an analysis of ribosomal DNA organization, a sequence analysis of 16S rDNA, and an analysis of phenotypic characteristics. This taxon, Rhizobium tropici sp. nov., was previously named Rhizobium leguminosarum biovar phaseoli (type II strains) and was recognized by its host range (which includes Leucaena spp.) and nif gene organization. In contrast to R. leguminosarum biovar phaseoli, R. tropici strains tolerate high temperatures and high levels of acidity in culture and are symbiotically more stable. We identified two subgroups within R. tropici and describe them in this paper.

We used DNA-rRNA hybridization, DNA base composition, polyacrylamide gel electrophoresis of whole-cell proteins, DNA-DNA hybridization, numerical analysis of phenotypic features, and immunotyping to study the taxonomy of the genus Comamonas. The relationships of this genus to Aquaspirillum aquaticum and a group of clinical isolates (E. Falsen group 10 [EF 10]) were studied. Our DNA and rRNA hybridization results indicate that the genus Comamonas consists of at least the following five genotypic groups: (i) Comamonas acidovorans, (ii) Comamonas testosteroni, (iii) Comamonas terrigena, (iv) A. aquaticum and a number of EF 10 strains, and (v) other EF 10 strains, several unnamed clinical isolates, and some misnamed strains of Pseudomonas alcaligenes and Pseudomonas pseudoalcaligenes subsp. pseudoalaligenes. The existence of these five groups was confirmed by the results of immunotyping and protein gel electrophoresis. A numerical analysis of morphological, auxanographic, and biochemical data for the same organisms revealed the existence of three large phena. Two of these phena (C. acidovorans and C. testosteroni) correspond to two of the genotypic groups. The third phenon contains strains belonging to the other three genotypic groups, including most EF 10 strains and the type strains of C. terrigena and A. aquaticum. The strains belonging to the third phenon were all incorporated into C. terrigena, and we propose that the use of the name Aquaspirillum aquaticum should be discontinued. Emended descriptions of the genus Comamonas and C. terrigena are presented.

A new family, the Comamonadaceae, is proposed for the organisms belonging to the acidovorans rRNA complex in the beta subclass of the Proteobacteria. This family includes the genera Comamonas, Acidovorax, Hydrogenophaga, Xylophilus, and Variovorax (formerly Alcaligenes paradoxus), as well as a number of phylogenetically misnamed Aquaspirillum and phytopathogenic Pseudomonas species.

The genera Campylobacter, Arcobacter, Helicobacter, Wolinella, and “Flexispira” constitute, within the class Proteobacteria, a separate eubacterial lineage identified as rRNA superfamily VI. A considerable number of common genotypic and phenotypic features differentiate the genera Campylobacter and Arcobacter from the other members of this group. Therefore, we propose that the genera Campylobacter and Arcobacter should be included in a separate family, for which the name Campylobacteraceae is proposed.