- Volume 43, Issue 4, 1993

Ureaplasma strains isolated from dogs (Canis familiaris) were characterized and compared with the type strains of five previously described species of the genus Ureaplasma, Ureaplasma urealyticum(isolated from humans), Ureaplasma diversum(isolated from cattle), Ureaplasma gallorale(isolated from chickens), Ureaplasma cati(isolated from cats), and Ureaplasma felinum(isolated from cats). The canine strains hydrolyzed urea but not arginine or glucose, were membrane bound, lacked a cell wall, passed through 450-nm-pore-size membrane filters, required cholesterol for growth, and formed minute colonies (diameter, 20 to 140 m) on agar medium. These canine ureaplasma strains have been reported to be members of four serovars. The four serovars of canine strains fell into a single group on the basis of their genomic properties, as determined by DNA-DNA hybridization. On the basis of these findings, we propose that ureaplasmas with these characteristics belong to a new species, Ureaplasma canigenitalium, with strain D6P-C (= ATCC 51252) as the type strain.

DNA relatedness studies (hydroxyapatite method) were done on 112 strains of citrobacteria. By using the recommended definition of a genomospecies 11 genomospecies were identified in the genus Citrobacter. These genomospecies were separable by their biochemical profiles. Citrobacter koseri (Citrobacter diversus) and Citrobacter amalonaticus proved to be homogeneous species, as previously described. C. amalonaticus biogroup 1, as described by Farmer et al. (J. Clin. Microbiol. 21:46-76, 1985), was shown to be a separate homogeneous species, which was named Citrobacter farmeri sp. nov. The Citrobacter freundii complex was quite heterogeneous. C. freundii sensu stricto, as represented by the type strain, contained only 9 of 66 strains in this complex. The remaining 57 strains were members of seven genomospecies. Genomospecies 5, containing 21 strains, was named Citrobacter youngae sp. nov. Genomospecies 6, containing 15 strains, was named Citrobacter braakii sp. nov. Genomospecies 7 and 8, each containing six strains, were named Citrobacter werkmanii sp. nov. and Citrobacter sedlakii sp. nov., respectively. Genomospecies 9, 10, and 11, each containing three strains, were not named.

The genetic relatedness of the three medically important Bordetella species was examined by macrorestriction digestion of DNA with the rarely cutting enzyme Xba I and resolution by pulsed-field gel electrophoresis. Our data showed that Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica produced species-specific macrorestriction profiles and that there was some variation between different isolates of the same species. Conserved bands at 130 and 155 kb occurred with B. pertussis(130 isolates tested), but the nine variable bands between 200 and 412 kb distinguished 21 types. The 10 isolates of B. parapertussis tested produced up to 11 bands at 118 to 375 kb, 4 of which were variable, giving three types. The eight B. bronchiseptica isolates examined produced up to 16 bands at 118 to 394 kb, 11 of which were variable, giving three types. The results of this work are compared with the results of previous DNA-DNA hybridization and multilocus enzyme electrophoresis studies which suggested that these three species are closely related and should be considered members of the same species.

The genera Halomonas and Deleya, which constitute the family Halomonadaceae, are difficult to differentiate on the basis of phenotypic and chemotaxonomic attributes. DNA-rRNA hybridization studies have indicated that some Halomonas spp. have the same level of relationship to the type species of the genus Deleya as some Deleya spp. A phylogenetic analysis of the 16S rRNA sequences of seven members of the Halomonadaceae indicated that the members of the genera Halomonas and Deleya do not form separate monophyletic subgroups, confirming the lack of any phylogenetic support for retention of these taxa as separate genera. A phylogenetic analysis of the 16S rRNA sequence of Halovibrio variabilis confirmed that this species belongs in the Halomonadaceae. All of the members of the Halomonadaceae examined and Halovibrio variabilis possess a cytosine residue at position 486 (Escherichia coli numbering), which is an extremely rare attribute among the prokaryotes and has been reported in only one other species, Listonella anguillarum. Several other signature characteristics which define this group in the gamma subclass of the Proteobacteria are identified. The Jukes-Cantor distances between members of the family Halomonadaceae, including Halovibrio variabilis, range from 0.086 to 0.000 (the levels of similarity between the 16S rRNA sequences range from 92.6 to 100%). The members of the genera Halomonas, Deleya, and Halovibrio form a monophyletic group and share common chemotaxonomic and phenotypic characteristics. Subgroups containing members of the genera Halomonas, Deleya, and Halovibrio cannot be resolved on the basis of phylogenetic, chemotaxonomic, or phenotypic data. Our data indicate that the members of the genera Halomonas, Deleya, and Halovibrio should be united in a single genus.

Using a method known as low-frequency restriction fragment analysis (LFRFA) (M. L. Beyazova and M. P. Lechevalier, Int. J. Syst. Bacteriol. 42:422-433, 1992), we determined the molecular weights of Ase I restriction fragments of Streptomyces DNAs by pulsed-field gel electrophoresis. The levels of similarity of fragment patterns among strains were determined by using the simple matching coefficient, and clustering was performed by using the unweighted pair group with mathematical average algorithm. A total of 59 strains representing eight species and the numerically classified taxon Streptomyces cyaneus group A18 (S. T. Williams, M. Goodfellow, G. Alderson, E. M. H. Wellington, P. H. A. Sneath, and M. J. Sackin, J. Gen. Microbiol. 129:1743-1813, 1983) were studied. Forty-two strains (six species) formed eight clusters at levels of similarity of more than 80%; 17 strains (including the entire S. cyaneus group) were unclustered. Cluster 1 contained all of the Streptomyces albus strains studied plus two strains of Streptomyces somaliensis and two strains of Streptomyces lavendulae. Cluster 2 contained 8 of the 12 Streptomyces fradiae strains examined plus one strain each of S. somaliensis and S. lavendulae. Cluster 3 was heterogeneous in terms of species. Cluster 4 contained two S. somaliensis strains; cluster 5 contained three S. fradiae strains; cluster 6 contained three Streptomyces rimosus strains; and clusters 7 and 8 contained seven and three Streptomyces ipomoea strains, respectively. The S. cyaneus group strains exhibited no clustering among themselves or with the other species examined. Some Streptomyces species which exhibited high levels of similarity (85 to 95%) in physiological tests (e.g., S. albus and the S. fradiae strains in cluster 2) exhibited high levels of similarity in the LFRFA (84 and 81%, respectively). Other taxa (S. cyaneus group) which exhibited equally high levels of physiological similarity (90%) appeared to be unrelated as determined by the LFRFA. Species with lower levels of physiological similarity (e.g., S. somaliensis[75%], S. lavendulae[63%], and S. rimosus[68%]) exhibited low levels of LFRFA similarity (75, 64, and 54%, respectively). High levels of DNA-DNA relatedness (<90%) (S. ipomoea) were reflected in high levels of similarity as determined by the LFRFA (75 to 100%); lower levels of DNA-DNA relatedness (ca. 70%) (S. cyaneus group) were reflected in low levels of LFRFA similarity (strains not clustered). We concluded that the presently used physiological tests reflect too small a portion of the genome to be universally useful in streptomycete species characterization. In contrast, high levels of DNA-DNA relatedness (<90%) and high LFRFA similarity values will probably both be valuable in species delineation in actinomycetes.

We isolated a methanogenic strain, designated strain SA (= DSM 7056), from an enrichment culture inoculated with a Japanese paddy field soil. Cells of this strain were strictly anaerobic, nonmotile, short rods and stained gram positive. The strain was able to use H2-CO2or formate as a methanogenic substrate. It required vitamins, but not acetate, for growth. Growth was fastest at 35 to 40°C. Methane was produced most rapidly at pH 6.0 to 7.5. The cellular lipid composition of strain SA was similar to that of M. arboriphilicus A2 (= DSM 2462). The G+C content of the DNA was 26.4 mol%. Strain SA had DNA-DNA hybridization values of more than 70% with M. arboriphilicus DH1T(= DSM 1125T). On the basis of phenotypic and genotypic characteristics, we identified strain SA as M. arboriphilicus. In the course of our identification work, the genetic heterogeneity of M. arboriphilicus was revealed by the results of DNA-DNA hybridization experiments. Although strain AZ (= DSM 744) should be classified as a member of a species distinct from the species containing the other four strains studied (DH1T, A2, DC [= DSM 1536], and SA), further phenotypic characterization will be required before a new species can be proposed.

The taxonomic relationships of strains belonging to Centers for Disease Control group M-5 were examined. Previous studies of rRNA cistron similarities placed this organism on the Neisseriaceae rRNA branch of rRNA superfamily III; the closest neighbors included the genus Neisseria and groups EF-4a and EF-4b. The group M-5 strains were characterized by a range of phenotypic tests, and their G+C contents and DNA-DNA relatedness levels were determined. In addition, a numerical taxonomic analysis of the whole-cell protein patterns (obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of group M-5 and related taxa was performed. The strains studied included 45 group M-5 strains, the type strains of six Neisseria species or subspecies, three group EF-4a reference strains, and three group EF-4b reference strains plus the type strain of the phenotypically similar organism Oligella urethralis. Our results showed that the group M-5 strains were members of a homogeneous taxon distinct from phylogenetically closely related taxa. The genomic divergence as revealed by levels of rRNA cistron similarity and phenotypic characteristics indicate that group M-5 can be considered a new species of the genus Neisseria. We therefore propose the new species Neisseria weaveri, with NCTC 12742 (= CCUG 4007 = ISL775/91 = LMG 5135) as the type strain. N. weaveri strains are strictly aerobic, gram-negative, nonmotile, rod-shaped organisms which are catalase and oxidase positive, nonsaccha-rolytic, and able to grow on MacConkey agar and do not reduce nitrate but generally reduce nitrite. The guanine-plus-cytosine contents of the DNAs of six representative strains were in the range from 50 to 51 mol%. Almost all 45 group M-5 strains were originally isolated from human wounds following dog bites.

The 16S rRNA sequences of seven representative Agrobacterium strains, eight representative Rhizobium strains, and the type strains of Azorhizobium caulinodans and Bradyrhizobium japonicum were determined. These strains included the type strains of Agrobacterium tumefaciens, Agrobacterium rhizogenes, Agrobacterium radiobacter, Agrobacterium vitis, Agrobacterium rubi, Rhizobium fredii, Rhizobium galegae, Rhizobium huakuii, Rhizobium leguminosarum, Rhizobium loti, Rhizobium meliloti, and Rhizobium tropici. A phylogenetic analysis showed that the 15 strains of Agrobacterium and Rhizobium species formed a compact phylogenetic cluster clearly separated from the other members of the alpha subclass of the Proteobacteria. However, Agrobacterium species and Rhizobium species are phylogenetically entwined with one another, and the two genera cannot be separated. In the Agrobacterium species, the strains of biovar 1, biovar 2, Agrobacterium rubi, and Agrobacterium vitis were clearly separated. The two biovars exhibited homogeneity in their phenotypic, chemotaxonomic, and phylogenetic characteristics, and two species should be established for the two biovars. We considered the nomenclature of the two biovars, and revised descriptions of Agrobacterium radiobacter(for the biovar 1 strains) and Agrobacterium rhizogenes(for the biovar 2 strains) are proposed. The name Agrobacterium tumefaciens is rejected because the type strain of this species was assigned to Agrobacterium radiobacter, and consequently the description of the genus Agrobacterium is revised.

A thermoacidophilic, obligately chemolithoautotrophic, aerobic, hydrogen-oxidizing bacterium, strain 3H-1T(T = type strain), was isolated from a solfataric field in Tsumagoi, Japan. This strain is a gram-negative, motile, non-spore-forming rod-shaped organism that requires elemental sulfur for growth by hydrogen oxidation. Type b, c, and o cytochromes are present. Carbon dioxide may be fixed via the reductive tricarboxylic acid cycle. The optimum temperature for growth is 65°C. The optimum pH for growth is 3.0 to 4.0. The guanine-plus-cytosine content of DNA is 35.0 mol%. A straight-chain saturated C18:0acid and straight-chain unsaturated C18:1and C20:1acids are the major components of the cellular fatty acids. 2-Methylthio-3-VI, VII-tetrahydromultiprenyl7-1,4-naphthoquinone (methionaquinone) is the major isoprenoid quinone. This strain is considered a member of a new species of the genus Hydrogenobacter, a genus of obligately chemolithoautotrophic, aerobic, hydrogen-oxidizing bacteria. The name Hydrogenobacter acidophilus sp. nov. is proposed for the organism. The type strain of this species is strain 3H-1 (= JCM 8795).

Polyamine profiles of 140 Xanthomonas strains were determined by high-performance liquid chromatography. The results revealed that there are two polyamine profiles within the genus Xanthomonas. The first profile, characterized by spermidine as the major polyamine, was shared by Xanthomonas albilineans, Xanthomonas axonopodis, Xanthomonas campestris, Xanthomonas fragariae, Xanthomonas oryzae, Xanthomonas populi, and some unclassified xanthomonads. The second profile was typical of Xanthomonas maltophilia strains and contained spermidine and cadaverine as the major polyamines.

A new Amycolatopsis species isolated from soil produces a new glycopeptide antibiotic related to vancomycin. Traditional taxonomic methods and contemporary fatty acid analysis techniques were used to establish the position of this species. The hyphae fragment extensively when the organism is cultured in liquid media. The organism is characterized by white aerial hyphae that bear long chains of cylindrical conidia. The reverse side is yellowish brown; a faint light brown soluble pigment is occasionally produced. The organism has a type IV cell wall (meso-diaminopimelic acid), a type A whole-cell sugar pattern, and a type PII phospholipid pattern. Mycolic acids are not present in whole-cell hydrolysates. The major menaquinone is MK-9(H4); there is also a minor amount of MK-8(H4). The name proposed for this new species is Amycolatopsis alba. The type strain is strain A83850 (= NRRL 18532).

A mycoplasma designated strain 4229T(T = type strain) was isolated in 1984 from the turbinate of a duck in France, and similar strains were isolated from geese in France and from a partridge in England. All of these strains were originally identified as Mycoplasma gallisepticum by immunofluorescence and growth inhibition tests, but subsequent serological and molecular studies indicated only a partial relationship to this species and DNA-DNA hybridization studies revealed only approximately 40 to 46% genetic homology with M. gallisepticum PG31T. In this study morphological, cultural, and physical investigations were carried out on strain 4229Tand partridge strain B2/85, after we first demonstrated the similarity between these organisms by performing a restriction enzyme analysis of their DNAs. Both strains had phenotypic properties very similar to M. gallisepticum properties, including the presence of an attachment organelle. As a result of these investigations, the organisms were assigned to the class Mollicutes, the order Mycoplasmatales, and the genus Mycoplasma. They fermented glucose, reduced triphenyl tetrazolium chloride aerobically and anaerobically, and exhibited hemadsorption and hemagglutination, but other biochemical tests were negative. Apart from a serological cross-reaction with M. gallisepticum, these organisms exhibited no significant relationship with any previously described Mycoplasma species as determined by growth inhibition or immunofluorescence tests or with a number of additional serovars and unclassified avian strains. This Mycoplasma taxon therefore appears to be a new species, for which we propose the name Mycoplasma imitans. The type strain is strain 4229 (= NCTC 11733 = ATCC 51306). The significance of the organism has not been fully investigated, but preliminary in vitro and in vivo studies have indicated that it may be pathogenic.

A novel halophilic archaeon, strain Ch2, was isolated from a marine solar saltern in Geelong, Australia. The fact that this organism had a dam-methylated genome suggested that it is closely related to the taxon that includes Halobacterium saccharovorum, Halobacterium sodomense, and Halobacterium trapanicum. A sequence analysis of the 16S rRNA gene (Ch2 has three copies of this gene) showed that Ch2 is phylogenetically equidistant from the genera Haloarcula and Haloferax and closely related to H. saccharovorum. The susceptibility of both Ch2 and H. saccharovorum to the recently isolated halophage HF2 supported the hypothesis that these two organisms are closely related.

Numerical taxonomic, DNA-DNA hybridization, and phospholipid fatty acid composition analyses were performed on an extensive range of methanotrophic strains, including reference strains and environmental isolates obtained from sites throughout eastern Australia. When the results of these studies were related to the results of a study based on genomic physicochemical properties, they clarified group I and II methanotroph genus and species interrelationships. The group I methanotrophs were found to be made up of three broadly phenotypically and genotypically homologous clusters of species. The first group I methanotroph cluster included the carotenoid-containing species Methylomonas methanica, Methylomonas fodinarum, and Methylomonas aurantiaca. These species represent the true members of the genus Methylomonas. The second group I methanotroph cluster was made up of two subclusters of strains. One subcluster included species not capable of producing resting cells and consisted of the species “Methylomonas agile,” “Methylomonas alba,” and Methylomonas pelagica. The other subcluster included species capable of forming desiccation-resistant cysts and included Methylococcus luteus, marine Methylomonas-like strains, and Methylococcus whittenburyi. Strains designated “Methylococcus ucrainicus” and Methylococcus vinelandii were found to be synonyms of Methylococcus whittenburyi, while Methylococcus bovis was a synonym of Methylococcus luteus. It is proposed that these subclusters represent a new genus, Methylobacter gen. nov. The species in the new genus are type species Methylobacter luteus comb. nov., Methylobacter agilis sp. nov., Methylobacter albus sp. nov., nom. rev., Methylobacter marinus sp. nov., Methylobacter pelagicus comb. nov., and Methylobacter whittenburyi comb. nov. The remaining group I methanotrophs included the moderately thermophilic species Methylococcus capsulatus and Methylococcus thermophilus and a group of unnamed strains closely related to Methylococcus capsulatus. It is proposed that these species represent the true members of the genus Methylococcus. The group II methanotrophs consisted of two closely related groups. The first group included budding, exospore-producing strains, while the second group included nonmotile, cyst-forming strains. These groups represent the genera Methylosinus and Methyocystis, which are revived here. The genus Methylosinus gen. nov., nom. rev. includes the species Methylosinus trichosporium sp. nov., nom. rev. and Methylosinus sporium sp. nov., nom. rev., while the genus Methylocystis gen. nov., nom. rev. includes the species Methylocystis parvus sp. nov., nom. rev. and Methylocystis echinoides sp. nov., nom. rev.

The sequences of the 16S rRNA genes of 20 Thermus isolates were determined to a high fidelity by using automated DNA sequencing and fluorescent-dye-labelled primers. The strains tested included members of the three validly named Thermus species and representatives of major taxonomic clusters defined previously for this genus. The parsimony method was used to reconstruct the phylogeny of the strains from the aligned sequences, and a bootstrap analysis revealed a number of well-supported clades. Our results are not consistent with groupings inferred from numerical taxonomy data but support the conjecture that the genus Thermus contains more species than the three currently recognized species.

Levels of DNA relatedness between strains isolated from root nodules of Phaseolus vulgaris and reference strains of different Rhizobium species were determined by performing DNA-DNA hybridization experiments (S1 nuclease method). The nine strains examined were members of three genomic groups previously delineated by a restriction fragment length polymorphism analysis among strains isolated from P. vulgaris at different sites in France. In agreement with the results of the restriction fragment length polymorphism analysis, three genomic species were found. We confirmed that one of these species corresponded to Rhizobium leguminosarum since the strain examined was 100% related to the type strain of this species. The other two species were new genomic species which were less than 21% related to reference strains belonging to other Rhizobium species, including Rhizobium etli and Rhizobium tropici, and were 18% related to each other. As determined by an analysis of partial 16S ribosomal DNA sequences, each of the genomic species was found to belong to a lineage independent from the lineages of previously described Rhizobium species. Nevertheless, they were included in the group formed by the fast-growing Rhizobium species. Both genomic species 1 and genomic species 2 contained a majority of strains which were capable of nodulating both P. vulgaris and Leucaena leucocephala, like R. tropici. However, they also contained strains with a nodulation phenotype restricted to P. vulgaris, like R. leguminosarum bv. phaseoli and R. etli bv. phaseoli. Our data are the first evidence that in Europe species other than R. leguminosarum nodulate P. vulgaris.

The phylogenetic position of the causative agent of septicemia anserum exsudativa, now most often referred to as [Moraxella] anatipestifer (brackets indicate a generically misnamed taxon) or “[Pasteurella] anatipestifer,” was established by performing rRNA cistron similarity studies. [Moraxella] anatipestifer belongs to rRNA superfamily V, together with the genera Flavobacterium, Cytophaga, Flexibacter, Weeksella, Capnocytophaga, and Sphingobacterium. The detailed structure of rRNA superfamily V, which now contains five major rRNA homology groups, is described. An analysis of various phenotypic parameters, including new data (cellular proteins and fatty acids) and previously published data (respiratory quinones, enzyme activities, and classical phenotypic features), revealed that [Moraxella] anatipestifer differs in many aspects from its closest relatives, Flavobacterium indologenes, Flavobacterium gleum, Flavobacterium indoltheticum, Flavobacterium balustinum, Flavobacterium meningosepticum, and Weeksella zoohelcum. The combined genotypic and phenotypic data indicate that this organism should be placed in a separate genus; the name Riemerella anatipestifer gen. nov., comb. nov. is proposed for this bacterium. The specific epithet anatipestifer is kept in order to avoid nomenclatural confusion. However, it should be emphasized that the illness caused by this organism is a septicemic disease which is not restricted to ducks.

DNA hybridization data (hydroxyapatite method, 50 to 70°C) indicate that Rickettsia prowazekii, the type species of the type genus of the family Rickettsiaceae, is substantially less closely related to Rochalimaea species than was previously thought. The levels of relatedness of Rickettsia prowazekii to Rochalimaea species and to Bartonella bacilliformis under optimal conditions for DNA reassociation were 0 to 14%, with 25.5% or greater divergence in related sequences. When stringent reassociation criteria were used, the levels of relatedness were 0 to 2%. The genera Bartonella and Rochalimaea are currently classified in different families (the Bartonellaceae and the Rickettsiaceae) in the order Rickettsiales. On the basis of DNA relatedness data, previous 16S rRNA sequence data, guanine-plus-cytosine contents, and phenotypic characteristics, neither Bartonella bacilliformis nor Rochalimaea species are closely related to other organisms currently classified in the order Rickettsiales. In fact, the closest relative of these organisms is Brucella abortus. It is therefore proposed that the family Bartonellaceae should be removed from the order Rickettsiales. Previous 16S rRNA sequence data and DNA hybridization data revealed high levels of relatedness between Bartonella bacilliformis and the four Rochalimaea species, indicating that these species are members of a single genus. It is proposed that the genus Rochalimaea should be united with the genus Bartonella in the family Bartonellaceae. The name Bartonella is retained as the genus name since it has nomenclatural priority over the name Rochalimaea. This means that new combinations for the Rochalimaea species must be created. Proposals are therefore made for the creation of Bartonella quintana comb. nov., Bartonella vinsonii comb. nov., Bartonella henselae comb. nov., and Bartonella elizabethae comb. nov.

Peptostreptococcus micros is often isolated from abscesses in several parts of the human body. The oral cavity is considered the natural habitat for the species, which has been implicated as a periodontal pathogen. In plaque samples from periodontitis patients we observed the presence of a rough morphotype of P. micros in addition to the previously recognized smooth morphotype. The rough morphotype has not been described previously. Both morphotypes are frequently isolated simultaneously from the same patient. In this paper strains of both morphotypes are described. The smooth morphotype, represented by the type strain, grew as small, dome-shaped, bright white, nonhemolytic colonies. The rough morphotype grew as equally white dry colonies which were hemolytic and had wrinkled edges. DNA-DNA reassociation studies revealed homology at the species level between the two morphotypes; in addition, no differences in physiological characteristics were observed when the organisms were tested with API-32A and API-ZYM kits. The rough cells had long, thin fibrillar structures outside the cell envelope when they were stained negatively for electron microscopy. In the smooth morphotype these structures were not present. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of whole-cell extracts were different for the two morphotypes. In xylene-water phase partition studies, the smooth morphotype was found to be hydrophobic, whereas the rough morphotype was found to be relatively hydrophilic. The distinct morphotypes were stable on blood agar; however, the rough morphotype changed to a nonfibrillar type with a smooth colony morphology after repeated subculturing in broth.