- Volume 42, Issue 1, 1992

Nineteen gram-negative, aerobic, biodegradative isolates were identified by using a polyphasic taxonomic approach. The presence of the specific polyamine 2-hydroxyputrescine and the presence of a ubiquinone with eight isoprenoid units in the side chain (ubiquinone Q-8) allowed allocation of these organisms to the beta subclass of the Proteobacteria. On the basis of the results of additional characterization experiments (i.e., API 20NE tests, determinations of soluble protein patterns, and DNA-DNA hybridization experiments), we classified six isolates as either Comamonas testosteroni, Comamonas acidovorans, or Alcaligenes xylosoxidans subsp. denitrificans. By using the same criteria we allocated two additional isolates to the genus Alcaligenes. A comparison of a 16S rRNA fragment (positions 1220 to 1377; Escherichia coli nomenclature) indicated that the remaining isolates should be allocated as follows: One is a member of C. testosteroni and one is a member of Acidovorax facilis, as confirmed by the results of additional DNA-DNA hybridizations; two others probably belong to the family Alcaligenaceae; six are related to “Alcaligenes eutrophus”; and one, strain NRRL 12228, occupies an isolated position.

Helical organisms with novel ultrastructural characteristics were isolated from the intestinal mucosa of rats and mice. These bacteria were characterized by the presence of 9 to 11 periplasmic fibers which appeared as concentric helical ridges on the surface of each cell. The cells were motile with a rapid corkscrewlike motion and had bipolar tufts of 10 to 14 sheathed flagella. The bacteria were microaerophilic, nutritionally fastidious, and physiologically similar to Helicobacter species and Wolinella succinogenes but could be differentiated from these organisms by their unique cellular ultrastructure. Using 16S rRNA sequencing, we found that strain ST1T (T = type strain) was related to previously described Helicobacter species, “Flexispira rappini,” and W. succinogenes. The closest relatives of strain ST1T were Helicobacter mustelae and “F. rappini” (average similarity value, 96%). On the basis of phylogenetic data, strain ST1T (= ATCC 49282T) represents a new species of the genus Helicobacter, for which we propose the name Helicobacter muridarum.

The bacteriolytic activities of different group D streptococcal species on various media and substrates were studied. Our results showed that all of the enterococcal species which we tested had bacteriolytic activity on at least one of the media used, while the group D nonenterococcal species had no such activity. In addition, using culture media containing different additives and different pH values, we defined seven major groups of bacteriolytic activity (lyogroups), each of which overlapped with one species (four lyogroups), two species (two lyogroups), or four species (one lyogroup). The detection of enterococcal lyogroups proved to be as reliable for species identification as the conventional methods presently in use.

A comprehensive study of the intra- and intergeneric relationships of Aquaspirillum species in which we used DNA-rRNA hybridization is described. Radioactively labeled rRNAs from Aquaspirillum anulus, A. serpens biovar bengal, A. delicatum, A. gracile, A. metamorphum, A. sinuosum, and several other organisms were hybridized with filter-fixed DNAs from representative strains of the genus Aquaspirillum and many other possibly related genera of gram-negative bacteria. Our results show that the genus Aquaspirillum is very heterogeneous and should be restricted to A. serpens, the type species, including A. serpens biovar serpens and A. serpens biovar bengal. DNA-DNA hybridization data showed that A. serpens biovar bengal exhibits only 55 to 60% binding with A. serpens biovar serpens strains. A. fasciculus, the only rod-shaped organism of the genus, is the only other Aquaspirillum species located on the A. serpens rRNA branch. With differences in the midpoint temperatures of thermal denaturation curves of the DNA-rRNA hybrids of more than 6°C, differences in genomic DNA G+C contents of 11 to 16 mol%, and a phenotype which differs from the phenotypes of all other Aquaspirillum species, A. fasciculus is a member of a different genus, for which we propose the name Prolinoborus fasciculus gen. nov., comb. nov. A. anulus, A. aquaticum, A. autotrophicum, A. delicatum, A. dispar, A. giesbergeri, A. gracile, A. itersonii, A. metamorphum, A. peregrinum, A. polymorphum, A. psychrophilum, A. putridiconchylium, and A. sinuosum should also be removed from the genus Aquaspirillum on the basis of phylogenetic data; these organisms form at least 10 distinct groups that are intermingled phylogenetically with the gram-negative bacteria belonging to rRNA superfamilies III and IV. Although DNA-rRNA hybridization results clearly reveal that the genus Aquaspirillum is a very heterogeneous taxon, no phenotypic information is available to differentiate the various phylogenetic branches of the aquaspirilia. Moreover, most of the species descriptions were based on only one or a few strains. Therefore, we will not propose nomenclatural changes until (i) sufficient readily determinable phenotypic characteristics are available and (ii) more strains have been isolated and investigated.

The inter- and intrageneric relationships of the genus Vibrio were investigated by performing a comparative analysis of the 16S rRNAs of 10 species, including four pathogenic representatives. The results of immunological and 5S rRNA studies were confirmed in that the genus is a neighboring taxon of the family Enterobacteriaceae. With regard to the intrageneric structure, Vibrio alginolyticus, Vibrio campbellii, Vibrio natriegens, Vibrio harveyi, Vibrio proteolyticus, Vibrio parahaemolyticus, and Vibrio vulnificus form the core of the genus, while Vibrio (Listonella) anguillarum, Vibrio diazotrophicus, and Vibrio hollisae are placed on the outskirts of the genus. Variable regions around positions 80, 180, and 450 could be used as target sites for genus- and species-specific oligonucleotide probes and polymerase chain reaction primers to be used in molecular identification.

The Clavibacter sp. associated with annual ryegrass toxicity (ARGT) in Australia has been reported to differ from other Clavibacter spp. in serological properties, allozyme electrophoretic mobility, bacteriophage sensitivity, and adhesion to nematodes (Anguina spp.). To clarify its taxonomic position, eight strains of the ARGT Clavibacter sp. were compared with strains of Arthrobacter ilicis, Clavibacter iranicum, Clavibacter michiganense, Clavibacter rathayi, Clavibacter tritici, Curtobacterium flaccumfaciens, and Rhodococcus fascians. The ARGT strains were distinguishable from the other bacteria on the basis of the results of a range of biochemical tests. DNA base composition, growth factor requirements, and growth rates were also determined. A new species, Clavibacter toxicus, is proposed, with type strain CS14 (= ICMP 9525 = NCPPB 3552).

An analysis of 23 Listeria ivanovii strains in which we used multilocus enzyme electrophoresis at 18 enzyme loci showed that this bacterial species could be divided into two main genomic groups. The results of DNA-DNA hybridizations and rRNA gene restriction patterns confirmed this finding. The DNA homology data suggested that the two genomic groups represent two subspecies, L. ivanovii subsp. ivanovii and L. ivanovii subsp. londoniensis subsp. nov. The two subspecies can be distinguished biochemically on the basis of the ability to ferment ribose and N-acetyl-β-d-mannosamine. The type strain of L. ivanovii subsp. londoniensis is strain CLIP 12229 (=CIP 103466).

A new species, Brachybacterium nesterenkovii, is proposed for a group of coryneform bacteria that were isolated from milk products. These organisms have morphological, biochemical, and chemotaxonomic characteristics that are peculiar to the genus Brachybacterium. In contrast to strains of the only previously described species of the genus, Brachybacterium faecium, the representatives of the new species lack glycine in their peptidoglycan, although the peptidoglycan is of the same general type, and have large amounts of rhamnose in their cells. The strains of B. nesterenkovii exhibit no serological relationship with strains of B. faecium; in a numerical phenotypic analysis the two species were easily separated and formed clear-cut clusters. DNA-DNA hybridization between the type strains of B. faecium and B. nesterenkovii showed a level of homology of 22%. Strain 35 (= IMV Ac-752) is the type strain of B. nesterenkovii.

The partial 16S rRNA sequence of an unknown bacterium that was originally isolated from middle ear fluids of children with persistent otitis media was determined by reverse transcription. A comparison of this sequence with sequences from other gram-positive species having low guanine-plus-cytosine contents revealed that this bacterium represents a new line of descent, for which the name Alloiococcus otitis gen. nov., sp. nov., is proposed. The type strain is strain NCFB 2890.

We compared non-methane-, non-methanol-, and methylamine-utilizing bacteria, including Pseudomonas aminovorans, a tetramethylammonium-utilizing bacterium, and an N-N-dimethylformamide-utilizing bacterium. These bacteria are gram-negative, nonsporeforming, subpolarly flagellated, rod-shaped organisms. Reproduction occurs by budding. The DNA base compositions are 62 to 64 mol% guanine plus cytosine. The cellular fatty acids contain a large amount of C18:1 acid. The major hydroxy acid is 3-OH C12:0 acid. The major ubiquinone is ubiquinone Q-10. These bacteria are clearly separated from authentic Pseudomonas species (Pseudomonas fluorescens rRNA group) on the basis of utilization of methylamine, morphological and chemotaxonomic characteristics, DNA-DNA homology, and rRNA-DNA homology. They were divided into three subgroups on the basis of their physiological characteristics and DNA-DNA homology data. A new genus, Aminobacter, and three new species, Aminobacter aminovorans comb. nov., Aminobacter aganoensis sp. nov., and Aminobacter niigataensis sp. nov., are proposed. The type species of Aminobacter gen. nov. is A. aminovorans, with type strain JCM 7852. The type strain of A. aganoensis is strain TH-3 (= JCM 7854), and the type strain of A. niigataensis is strain DM-81 (= JCM 7853).

We determined the sequences for a 260-base segment amplified by the polymerase chain reaction (corresponding to positions 44 to 337 in the Escherichia coli 16S rRNA sequence) from seven strains of fast-growing soybean-nodulating rhizobia (including the type strains of Rhizobium fredii chemovar fredii, Rhizobium fredii chemovar siensis, Sinorhizobium fredii, and Sinorhizobium xinjiangensis) and broad-host-range Rhizobium sp. strain NGR 234. These sequences were compared with the corresponding previously published sequences of Rhizobium leguminosarum, Rhizobium meliloti, Agrobacterium tumefaciens, Azorhizobium caulinodans, and Bradyrhizobium japonicum. All of the sequences of the fast-growing soybean rhizobia, including strain NGR 234, were identical to the sequence of R. meliloti and similar to the sequence of R. leguminosarum. These results are discussed in relation to previous findings; we concluded that the fast-growing soybean-nodulating rhizobia belong in the genus Rhizobium and should be called Rhizobium fredii.

A new coagulase-negative species of the genus Staphylococcus, Staphylococcus muscae, is described on the basis of the results of a study of four strains that were isolated from flies. 16S rRNA sequences of the type strains of S. muscae, Staphylococcus schleiferi, and Staphylococcus sciuri were determined and used, together with the corresponding sequences of Staphylococcus aureus and Staphylococcus epidermidis, for a comparative analysis. The new species is characterized taxonomically; this species is differentiated from the other novobiocin-susceptible staphylococci by its physiological and biochemical activities, cell wall composition, and levels of genetic relatedness. The type strain of this species is strain MB4 (= CCM 4175).

Two pure cultures of bacteria isolated from soil samples collected in Schirmacher Oasis, Antarctica, conformed to the definition of the genus Sphingobacterium. They differed from all of the known species of Sphingobacterium in being psychrotrophic. The G+C contents of the DNA of the two strains were found to be 39.3 and 40.3 mol%, and DNA-DNA hybridization studies indicated 7% homology with S. multivorum and S. spiritivorum. The name Sphingobacterium antarcticus sp. nov. is proposed for the two Antarctic strains. The type strain is 4BY (MTCC 675), and it has been deposited with the Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh, India.

DNA-rRNA hybridizations, DNA-DNA hybridizations, polyacrylamide gel electrophoresis of whole-cell proteins, and a numerical analysis of carbon assimilation tests were carried out to determine the relationships among the phylogenetically misnamed phytopathogenic taxa Pseudomonas avenae, Pseudomonas rubrilineans, “Pseudomonas setariae,” Pseudomonas cattleyae, Pseudomonas pseudoalcaligenes subsp. citrulli, and Pseudomonas pseudoalcaligenes subsp. konjaci. These organisms are all members of the family Comamonadaceae, within which they constitute a separate rRNA branch. Only P. pseudoalcaligenes subsp. konjaci is situated on the lower part of this rRNA branch; all of the other taxa cluster very closely around the type strain of P. avenae. When they are compared phenotypically, all of the members of this rRNA branch can be differentiated from each other, and they are, as a group, most closely related to the genus Acidovorax. DNA-DNA hybridization experiments showed that these organisms constitute two genotypic groups. We propose that the generically misnamed phytopathogenic Pseudomonas species should be transferred to the genus Acidovorax as Acidovorax avenae and Acidovorax konjaci. Within Acidovorax avenae we distinguished the following three subspecies: Acidovorax avenae subsp. avenae, Acidovorax avenae subsp. cattleyae, and Acidovorax avenae subsp. citrulli. Emended descriptions of the new taxa are presented.

A novel intracellular pathogen morphologically similar to the ehrlichiae has been isolated in cell culture and identified as the cause of an epizootic disease of salmonid fish. Like the ehrlichiae, the salmonid pathogen, designated strain LF-89, replicates within membrane-bound cytoplasmic vacuoles in host cells. This agent is the first with characteristics of this type to be isolated from a fish. Analysis of the LF-89 16S rRNA indicated that, unlike the ehrlichiae, LF-89 is a gamma proteobacterium distantly related to Coxiella burnetii and perhaps Wolbachia persica. A new genus and species (Piscirickettsia salmonis gen. nov., sp. nov.) are proposed for this organism, with ATCC(R) VR 1361 as the type strain.

Antigenic and genomic relatedness among Ehrlichia risticii, E. sennetsu, and E. canis was analyzed by enzyme-linked immunosorbent assay, Western blotting (immunoblotting) and DNA-DNA hybridization. E. risticii and E. sennetsu were serologically related, and their Western blot antigen profiles were nearly identical. Two antigens of E. sennetsu corresponding to the 28- and 51-kDa antigens of E. risticii were apparently larger than the E. risticii antigens, and the 55-kDa antigen of E. risticii appeared to be unique to this species. The 110-, 70-, and 44-kDa antigens of these two species were identical, as determined by the use of monospecific antibodies. DNA homology between these two species was high. On the other hand, E. canis was antigenically least reactive with the antisera to E. risticii and E. sennetsu. However, a dog convalescent-stage E. canis antiserum recognized antigens in the other two species which were different from those recognized by their homologous antisera. Similarly, homology between the DNA of E. canis and the DNAs of the other two species was very minimal. These results indicate that E. risticii and E. sennetsu are closely related both at the genomic and antigenic levels and that the relationship of these two species with E. canis is minimal.

Two new species of aerobic, gram-negative, peritrichously flagellated or nonmotile marine bacteria usually forming star-shaped aggregates were isolated from northeastern Atlantic Ocean bottom sediments. These organisms resembled eight star-shaped-aggregate-forming bacterial species from the Baltic Sea originally ascribed to the genus Agrobacterium but not included on the Approved Lists of Bacterial Names because of their questionable relationships to true agrobacteria. These two sets of star-shaped-aggregate-forming bacteria were compared by means of phenotypic data, DNA base compositions, DNA-DNA relatedness, and one-dimensional electrophoretic analysis of low-molecular-weight RNAs (5S rRNA and tRNA). According to the results of genotyping, the northeastern Atlantic Ocean isolates and three of the Baltic Sea species formed a group of closely related bacteria that could not be excluded from the genus Agrobacterium with certainty. Until more genotypic data are available, these five marine species are regarded as a distinct subdivision of the genus Agrobacterium consisting of Agrobacterium atlanticum sp. nov. (type strain, 1480T = DSM 5823T), A. meteori sp. nov. (type strain, 1513T = DSM 58241), A. ferrugineum sp. nov. nom. rev. emend. (type strain, ATCC 25652T), A. gelatinovorum sp. nov. nom. rev. emend. (type strain, ATCC 25655T), and A. stellulatum sp. nov. nom. rev. emend, (type strain, ATCC 15215T). “A. aggregatum” proved to be a later subjective synonym of A. stellulatum, which had priority. The remaining four Baltic Sea species, “A. agile,” “A. kieliense,” “A. luteum,” and “A. sanguineum,” could not be placed in the new subdivision of Agrobacterium.

The ribosomal proteins from 17 type strains of species belonging to various actinomycete genera were compared by two-dimensional polyacrylamide gel electrophoresis. I detected a striking variability among certain ribosomal proteins (designated AT-L30 proteins) with respect to electrophoretic mobility in the first dimension. In contrast, such variability was not observed among ribosomal L30 proteins from other bacteria, such as Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus. Although actinomycete AT-L30 proteins from different taxa exhibited considerable heterogeneity in electrophoretic mobility, within each genus the proteins had a specific mobility characteristic. On the basis of this observation, the ribosomal AT-L30 proteins from 11 type strains of species belonging to the mycolic acid-containing genera Nocardia, Rhodococcus, Gordona, and Tsukamurella were analyzed. The relative electrophoretic mobilities of AT-L30 protein preparations from these strains, as determined by two-dimensional gel electrophoresis, revealed that the genera Nocardia, Rhodococcus, Gordona, and Tsukamurella can be sharply separated from each other. My results are consistent with the previously discussed view that each of these genera merits separate genus status.

We analyzed the ribosomal AT-L30 proteins from 11 type strains of species belonging to the genus Streptosporangium. The electrophoretic mobilities of the AT-L30 preparations from these strains, as determined by two-dimensional polyacrylamide gel electrophoresis, revealed that they could be divided into three groups. The first group contained Streptosporangium viridogriseum, S. viridogriseum subsp. kofuense, and S. albidum, while the second group contained S. roseum, S. album, S. vulgare, S. nondiastaticum, S. fragile, S. violaceochromogenes, and S. amethystogenes. S. corrugatum was a member of the third group. These groups were completely consistent with Nonomura’s previous classification, which was based on morphological criteria. The results of partial amino acid sequencing of AT-L30 preparations from several representative strains strongly supported the hypothesis that each of the three groups of the genus Streptosporangium merits separate generic status.