- Volume 45, Issue 2, 1995

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.