- Volume 44, Issue 3, 1994

We obtained 16S ribosomal DNA (rDNA) sequence data for strains belonging to 11 species of Proteobacteria, including the type strains of Kingella kingae, Neisseria lactamica, Neisseria meningitidis, Moraxella lacunata subsp. lacunata, [Neisseria] ovis, Moraxella catarrhalis, Moraxella osloensis, [Moraxella] phenylpyruvica, and Acinetobacter lwoffii, as well as strains of Neisseria subflava and Acinetobacter calcoaceticus. The data in a distance matrix constructed by comparing the sequences supported the proposal that the genera Acinetobacter and Moraxella and [N.] ovis should be excluded from the family Neisseriaceae. Our results are consistent with hybridization data which suggest that these excluded taxa should be part of a new family, the Moraxellaceae. The strains that we studied can be divided into the following five groups: (i) M. lacunata subsp. lacunata, [N] ovis, and M. catarrhalis; (ii) M. osloensis; (iii) [M.] phenylpyruvica; (iv) A. calcoaceticus and A. lwoffii; and (ν) N. meningitidis, N. subflava, N. lactamica, and K. kingae. We agree with the previous proposal that [N] ovis should be renamed Moraxella ovis, as this organism is closely related to Moraxella species and not to Neisseria species. The generically misnamed taxon [M.] phenylpyruvica belongs to the proposed family Moraxellaceae, but it is sufficiently different to warrant exclusion from the genus Moraxella. Further work needs to be done to investigate genetically similar species, such as Psychrobacter immobilis, before the true generic position of this organism can be determined. Automated 16S rDNA sequencing with the PCR allows workers to accurately determine phylogenetic relationships between groups of organisms. This method is quicker and less complex than nucleic acid hybridization, produces similar results, and has the advantage that 16S rDNA sequence information for different species can be directly compared.

The relationship between photosynthetic rhizobia that nodulate 10 Aeschynomene species (Aeschynomene afraspera, Aeschynomene denticulata, Aeschynomene evenia, Aeschynomene indica, Aeschynomene nilotica, Aeschynomene pratensis, Aeschynomene rudis, Aeschynomene scabra, Aeschynomene schimperi, and Aeschynomene sensitiva) and reference strains of the genera Bradyrhizobium, Rhizobium, and Azorhizobium was investigated by analyzing cellular fatty acid methyl esters (FAME) and 16S rRNA sequences. The members of each genus produced very distinct FAME patterns, and the photosynthetic rhizobia formed a subcluster in the Bradyrhizobium cluster. The absence of the cyc C19:0 type of fatty acid in all of the photosynthetic rhizobium strains isolated from 10 Aeschynomene species distinguished these microorganisms from other known rhizobia, including strain BTAi 1, a photosynthetic symbiont of A. indica. We sequenced a 264-base segment of the 16S rRNA genes of selected strains after amplification by the PCR and compared the results with previously published sequences for species of rhizobia and related photosynthetic bacteria. Photosynthetic strains IRBG 2 (from A. afraspera), IRBG 230 (from A. nilotica), and ORS 322 (from A. afraspera) had identical sequences but were distinct from strain BTAi (from A. indica) and from strain IRBG 231 (from A. denticulata), which is similar to the type strain (DNA homology group Ia) of Bradyrhizobium japonicum. Nonphotosynthetic strain IRBG 274 (from A. afraspera) was closely related to Bradyrhizobium elkanii (DNA homology group II). All of the photosynthetic rhizobia clearly fell into the Bradyrhizobium cluster. Although the results of the FAME and 16S rRNA analyses were in excellent agreement, our placement of the photosynthetic rhizobia is in apparent conflict with phenotypic data, as determined by numerical taxonomy (Ladha and So, Int. J. Syst. Bacteriol., in press) which placed the photosynthetic rhizobia in a coherent cluster that is as far from the genus Bradyrhizobium as the genera Rhizobium and Azorhizobium are. While the FAME and 16S rRNA data probably provide a more reliable indication of phylogeny, the degree of phenotypic divergence observed raises questions concerning the polyphasic approach to bacterial systematics.

Pulsed-field gel electrophoresis was used to examine the chromosomal polymorphisms existing within and between four closely related members of the Staphylococcus epidermidis species group, S. epidermidis, Staphylococcus caprae, Staphylococcus capitis subsp. capitis, and S. capitis subsp. ureolyticus. SmaI was chosen as the restriction endonuclease for this study because it generated only a few well-separated chromosomal fragments. Each of the species and subspecies showed distinct SmaI digest patterns. The strains examined in this study were collected over a 20-year period from various geographical locations. The results indicate that DNA fragment patterns are unique to each species and subspecies and represent a reasonably stable component in the chromosome structure. S. caprae and S. capitis demonstrated considerable conservation in chromosome structure as indicated by the large numbers of conserved SmaI digest fragments. The polymorphisms found within each species appear to be linked to the species’ character variability. The genome size of each Staphylococcus strain was extrapolated from the SmaI digest fragment pattern obtained by pulsed-field gel electrophoresis. The average genome size for 5. epidermidis is 2,364 ± 119 kb; for S. caprae strains from humans it is 2,600 ± 157 kb and for S. caprae strains from goats it is 2,493 ± 15 kb; for S. capitis subsp. capitis it is 2,456 ± 71 kb; and for S. capitis subsp. ureolyticus it is 2,276 ± 90 kb.

Two pseudomonad strains that produce a yellow cellular pigment, in addition to a diffusible fluorescent pigment on Kings medium B, were isolated from cankers on walnut trees. Biochemical properties, such as a positive oxidase reaction, a negative arginine dihydrolase reaction, and the production of a fluorescent pigment, in addition to the results of an extensive nutritional characterization study and DNA-DNA hybridization experiments, indicated that these strains belong to a new Pseudomonas rRNA group I species. This conclusion was supported by the results of a determination of the sequence of the PCR-amplified 16S rRNA gene and a comparison with the 16S rRNA genes of other bacterial species. The genomic DNAs of the strains had a base composition of 63 mol% G+C. The name Pseudomonas flavescens sp. nov. is proposed. Strain B62 (= NCPPB 3063) is the type strain of the species.

We sequenced nearly complete small-subunit rRNAs of 54 reference strains belonging to the genera Vibrio, Photobacterium, Aeromonas, and Plesiomonas. We then performed a phylogenetic analysis by comparing the sequences which we obtained with all other known sequences for bacteria belonging to the gamma subgroup of the Proteobacteria (thus providing a data base consisting of 70 sequences for the genera investigated), using methods such as neighbor joining, maximum likelihood, and maximum parsimony, as well as bootstrap, to assess the robustness of each topology. Our results confirmed that the family Vibrionaceae should include only Photobacterium and Vibrio species (but not Vibrio marinus); that Aeromonas species deserve family rank; and that Plesiomonas shigelloides is linked to the family Enterobacteriaceae. The genera Vibrio, Photobacterium, Aeromonas, and Plesiomonas, together with the family Enterobacteriaceae, the family Pasteurellaceae, and probably the genus Alteromonas, form a robust monophyletic unit within the gamma 3 subgroup of the Proteobacteria.

We analyzed the 16S ribosomal DNAs of three obligately aerobic, bacteriochlorophyll a-containing bacteria, “ “Roseococcus thiosulfatophilus”,” ““ Erythromicrobium ramosum,” “ and new isolate T4T (T = type strain), which was obtained from a marine cyanobacterial mat. ““ Roseococcus thiosulfatophilus” “ is a member of the α-1 subclass of the Proteobacteria and is moderately related to Rhodopila globiformis, Thiobacillus acidophilus, and Acidiphilium cryptum (level of sequence similarity, 90%). ““ Erythromicrobium ramosum” and isolate T4T are closely related to Erythrobacter longus and Porphyrobacter neustonensis (level of sequence similarity, 95%). These organisms are members of the α-4 subclass of the Proteobacteria. Strain T4T is a motile, red or orange bacterium. The major carotenoids are bacteriorubixanthinal and erythroxanthin sulfate. In vivo measurements revealed bacteriochlorophyll absorption maxima at 377, 590, 800, and 868 nm. Strain T4T grows in the presence of 5 to 96%o basis of its distinct phylogenetic position and phenotypic characteristics which are different from those of Erythrobacter longus, we propose that strain T4T should be placed in a new species of the genus Erythrobacter, Erythrobacter litoralis. The descriptions of ““Roseococcus thiosulfatophilus” and ““ Erythromicrobium ramosum” are emended.

Twelve strains of homofermentative lactobacilli and two strains of heterofermentative lactobacilli were isolated from kefir grains by using R-CW agar medium. The physiological and biochemical characteristics, DNA guanine-plus-cytosine contents, and levels of DNA-DNA relatedness of these isolates and previously described lactobacilli were compared. Our results indicated that two new species, Lactobacillus kefirgranum and Lactobacillus parakefir, could be distinguished. The type strain of L. kefirgranum sp. nov. is GCL 1701 (= JCM 8572), and the type strain of L. parakefir sp. nov. is GCL 1731 (= JCM 8573).

The phylogenetic relationships of 17 phytopathogenic mycoplasmalike organisms (MLOs) representing seven major taxonomic groups established on the basis of MLO 16S ribosomal DNA (rDNA) restriction patterns were examined by performing a sequence analysis of the 16S rDNA gene. The sequence data showed that the MLOs which we examined are members of a relatively homogeneous group that evolved monophyletically from a common ancestor. In agreement with results obtained previously with other MLOs, our results also revealed that the organisms are more closely related to Acholeplasma laidlawii and other members of the anaeroplasma clade than to any other mollicutes. A phylogenetic tree based on 16S rDNAs showed that the MLOs which we examined can be divided into the following five primary clusters: (i) the aster yellows strain cluster; (ii) the apple proliferation strain cluster; (iii) the western-X disease strain cluster; (iv) the sugarcane white leaf strain cluster; and (v) the elm yellows strain cluster. The aster yellows, western-X disease, and elm yellows strain clusters were divided into two subgroups each. MLOs whose 16S rDNA sequences have been determined previously by other workers can be placed in one of the five groups. In addition to the overall division based on 16S rDNA sequence homology data, the primary clusters and subgroups could be further defined by a number of positions in the 16S rDNAs that exhibited characteristic compositions, especially in the variable regions of the gene.

Fifty orange-pigmented, gram-negative, rod-shaped isolates were recovered from healthy and diseased turbot and from coastal waters (collected in Scotland). On the basis of the results of an examination of 125 phenotypic characteristics and the results of DNA-DNA and DNA-rRNA hybridization experiments, we concluded that these isolates are members of a new species in the genus Flavobacterium, for which the name Flavobacterium scophthalmum is proposed. The type strain is CCM 4109 (= LMG 13028).

Strains of a new species, Staphylococcus vitulus, were isolated from food and a variety of mammals. This species was recognized on the basis of the results of an analysis of genomic EcoRI fragments containing portions of the rRNA operons. The patterns of hybridized fragments obtained from strains belonging to the new taxon were sorted into a distinguishable cluster and were distinct from the Staphylococcus lentus and Staphylococcus sciuri patterns. The results of DNA-DNA hybridization reactions demonstrated that strains in this cluster were more closely related to S. lentus and S. sciuri than to other Staphylococcus species and yet were significantly different. While these strains had some of the phenotypic characteristics of the S. sciuri species group, the newly recognized taxon could be distinguished by its very small colonies on P agar, absence of alkaline phosphatase activity, and lack of acid production from L-arabinose, maltose, N-acetylglucosamine, D-mannose, and raffinose. The type strain of the new species is strain DD 756 (= ATCC 51145).

Rhizobial isolates that were obtained from both surface and deep soil samples in the Sahelian and Sudano-Guinean areas of Senegal (West Africa) under Acacia albida trees were compared with representative strains of known rhizobial species and genera. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins was used to determine the taxonomic positions of these organisms and the relationships between isolates obtained from the surface and isolates obtained from deep soil. Most of the isolates belonged to eight electrophoretic clusters containing representative strains of Bradyrhizobium japonicum, Bradyrhizobium elkanii, and Bradyrhizobium sp. Isolates were also characterized by the Biolog system, and the results were compared with the results obtained by SDS-PAGE of total proteins; the level of correlation was very low. DNA-rRNA hybridizations with 16S or 23S rRNA from Bradyrhizobium japonicum LMG 6138T (T = type strain) confirmed that most of the protein electrophoretic clusters belong in the Bradyrhizobium-Rhodopseudomonas rRNA complex. Sequencing of 16S rRNA genes showed that some of the A. albida-nodulating isolates belong to a separate lineage together with representatives of other protein electrophoretic clusters. Other isolates that belong to the same electrophoretic cluster as the type strain of Bradyrhizobium japonicum are considered members of the lineage represented by this type strain. The first lineage is as far removed from Bradyrhizobium japonicum as it is from the genus Afipia, Blastobacter denitrificans, and the genus Rhodopseudomonas. The possible relationship among electrophoretic group, geographic origin, and depth of isolation at a particular site is discussed.

A new species, Nocardiopsis halophila, is proposed. This species is represented by a single isolate obtained from a saline soil sample in Iraq. This organism grows in both synthetic and organic media containing concentrations of NaCl up to 20% (wt/vol) and is resistant to lysozyme. Whole-cell hydrolysates contain meso-diaminopimelic acid (cell wall type III) and no diagnostic sugars (sugar pattern C). The phospholipid type is type PHI. The predominant menaquinone is MK-10(H6,H8), and mycotic acids are absent. This organism forms abundant white aerial mycelia and long irregularly branched spore chains that have a zigzag appearance and fragment into spores of various lengths. The substrate mycelia are extensively branched and are rarely fragmented. These properties suggest that this bacterium should be placed in the genus Nocardiopsis. However, this organism differs from all previously described species of the genus Nocardiopsis in its ability to grow in the presence of 20% (wt/vol) NaCl and also in many other physiological and biochemical characteristics. Thus, we propose that this organism is a new species, Nocardiopsis halophila. Type strain IQ-H3 has been deposited in the Culture Collection of the Institute of Microbiology, Academia Sinica, Beijing, People’s Republic of China, as strain A.S.4.1195.

Three mycoplasma strains, designated GIHT (T = type strain), UIAT, and VIST, were isolated from the external ear canals of goats and were shown to be serologically distinct from each other and from previously described Acholeplasma, Entomoplasma, Mesoplasma, and Mycoplasma species. Using light and transmission electron microscopy, we showed that the cells of these organisms were small, pleomorphic, coccoid, nonmotile, and nonhelical and that each cell was surrounded by a single cytoplasmic membrane. There was no evidence of a cell wall, and the organisms grew freely in media containing penicillin at concentrations of 1,000 U/ml or more and thallous acetate (final concentration, 1:4,000) and produced the “fried-egg” morphology typical of most mollicutes. Growth occurred both aerobically and anaerobically (as determined by the GasPak method). The ability to catabolize glucose and mannose and the ability to hydrolyze arginine varied among the three strains. AH three strains required sterol for growth, and none of the strains hydrolyzed urea. The guanine-plus-cytosine contents of the DNAs of strains UIAT, VIST, and GIHT were determined to be 26.9, 27.0, and 26.6 mol%, respectively. Our data indicate that the three strains represent new Mycoplasma species, for which we propose the names Mycoplasma auris, Mycoplasma cottewii, and Mycoplasma yeatsii. The type strain of M. auris is UIA (= ATCC 51348 = NCTC 11731), the type strain of M. cottewii is VIS (= ATCC 51347 = NCTC 11732), and the type strain of M. yeatsii is GIH (= ATCC 51346 = NCTC 11730).

Three independently isolated polychlorophenol-degrading strains of bacteria were characterized on the basis of chemotaxonomic and nutritional characteristics. Previously, these strains were assigned to the species Rhodococcus chlorophenolicus, which was described on the basis of the properties of one of the strains, strain PCP-IT (T = type strain) (J. H. A. Apajalahti, P. Kärpänoja, and M. S. Salkinoja-Salonen, Int. J. Syst. Bacteriol 36:246–251, 1986). However, the results of analyses of mycolic acids suggested that these organisms should be transferred to the genus Mycobacterium as Mycobacterium chlorophenolicum. These bacteria have meso-diaminopimelic acid, arabinose, and galactose as cell wall constituents, mycolic acids containing 75 to 80 carbon atoms, and a predominant menaquinone with nine isoprenoid units and one hydrogenated double bond. The fatty acids include mainly straight-chain saturated and monounsaturated fatty acids with 10 to 18 carbon atoms and a large proportion of 10-methyloctadecanoic acid (tuberculostearic acid). The G+C contents of the DNAs of the three strains range from 67 to 69 mol%.

We determined the nucleotide sequence of a 16S rRNA gene of Rhodococcus chlorophenolicus PCP-IT (= DSM 43826T) (T = type strain). Sequence comparisons revealed that there was a close relationship between strain PCP-IT and strains belonging to the genus Mycobacterium. The sequence data were used to construct a phylogenetic tree, which showed that Mycobacterium chubuense is the closest relative of strain PCP-IT. We propose that strain PCP-IT should be transferred to the genus Mycobacterium and renamed Mycobacterium chlorophenolicum PCP-IT comb. nov.

The taxonomic positions of strains previously assigned to Pseudomonas diminuta and Pseudomonas vesicularis were investigated by a polyphasic approach. The results of DNA-rRNA hybridization studies indicated that these two species belong to a separate genus in the a subclass (rRNA superfamily IV) of the Proteobacteria, for which the name Brevundimonas is proposed. Genus delineation and species delineation were determined by comparing the results of numerical analyses of whole-cell protein patterns, fatty acid compositions, and phenotypic characteristics and by measuring DNA base ratios and degrees of DNA relatedness. Taxonomic characteristics of Brevundimonas diminuta and Brevundimonas vesicularis strains were compared with characteristics of reference strains belonging to the following phylogenetically related taxa: a group of organisms gathered in Enevold Falsen group 21, the genera Sphingomonas and Rhizomonas, and the generically misclassified organisms [Pseudomonas] echinoides and “[Pseudomonas] riboflavina.”

The taxonomic status of 16 collection strains of chickpea (Cicer arietinum L.) rhizobia which were previously determined to belong to two groups (groups A and B) were compared with reference strains belonging to different genera and species of the family Rhizobiaceae. We used the following taxonomic, phylogenetic, and phenotypic characteristics and approaches to study these organisms: DNA homology, guanine-plus-cytosine content, restriction fragment length polymorphism of the amplified 16S-intergenic spacer rRNA gene, partial 16S rRNA sequencing, and auxanographic tests performed with 147 carbon sources. Similar groups of chickpea strains were identified by the different approaches. The chickpea strains were found to belong to the genus Rhizobium regardless of the phylogenetic group to which they belonged (group A or B). All strains fell into a tight cluster which included Rhizobium loti and Rhizobium galegae, and the group B strains were closely related to R. loti. An analysis of partial 16S ribosomal DNA sequences revealed identical nucleotide sequences for the slowly growing strains and fast-growing strains that were used as representatives of groups A and B, respectively, and these organisms fell into the Rhizobium-Agrobacterium lineage. When the sequences of these organisms were compared with the partial sequences of Rhizobium huakuii and R. loti, one- and two-nucleotide mismatches were observed, respectively, indicating that the chickpea rhizobia are closely related to these two species. The DNA-DNA hybridization data revealed that the chickpea rhizobia exhibited low levels of homology (less than 17%) to previously described Rhizobium and Bradyrhizobium species. Moreover, when we compared chickpea strains to R. loti and R. huakuii, the most closely related species as determined by the partial 16S rRNA sequence analysis, the homology values ranged from 21 to 52% and the ∆Tm values were greater than 5°C (∆Tm is the difference between the denaturation temperatures of the heterologous and homologous duplexes). These results confirmed that the rhizobia that nodulate chickpeas cannot be assigned to a previously described species. Within the chickpea rhizobia, the DNA homology values obtained when members of groups A and B were compared were less than 38%, indicating that the group A and group B organisms belong to different species. Furthermore, these organisms can be distinguished from each other by the results of phenotypic tests. We propose that the group B chickpea rhizobia should be assigned to a new species, Rhizobium ciceri; UPM-Ca7 is the type strain of R. ciceri.

The 16S rRNA gene sequence of a previously undescribed LL-diaminopimelic acid-containing coryneform bacterium isolated from herbage was determined in order to clarify the taxonomic position of this organism. A comparative sequence analysis revealed that the bacterium represents a new line of descent within the genus Nocardioides. On the basis of the results of a phylogenetic analysis and the phenotypic distinctiveness of the organism, a new species, Nocardioides plantarum, is proposed. The type strain is NCIMB 12834.

The essential GroEL proteins represent a subset of molecular chaperones ubiquitously distributed among species of the eubacterial lineage, as well as in eukaryote organelles. We employed these highly conserved proteins to infer eubacterial phylogenies. GroEL from the species analyzed clustered in distinct groups in evolutionary trees drawn by either the distance or the parsimony method, which were in general agreement with those found by 16S rRNA comparisons (i.e., proteobacteria, chlamydiae, bacteroids, spirochetes, firmicutes [gram-positive bacteria], and cyanobacteria-chloroplasts). Moreover, the analysis indicated specific relationships between some of the aforementioned groups which appeared not to be clearly defined or controversial in rRNA-based phylogenetic studies. For instance, a monophyletic origin for the low-G+C and high-G+C subgroups among the firmicutes, as well as their specific relationship to the cyanobacteria-chloroplasts, was inferred. The general observations suggest that GroEL proteins provide valuable evolutionary tools for defining evolutionary relationships among the eubacterial lineage of life.