- Volume 44, Issue 2, 1994

During our taxonomic study of motile actinomycetes, soil isolate RA 335 was found to form a blue substrate mycelium and aerial mycelia with motile arthrospores and to have lysine as the cell wall diamino acid. Actinoplanes caeruleus IFO 13939T (T = type strain) and “Actinoplanes azureus” IFO 13993T are known to have the same characteristics. Therefore, the taxonomic position of these three strains was studied. Aerial mycelia of these strains fragmented during the growth cycle and produced motile spores arranged in chains within the mycelia. Sporangia were not observed. The strains contained menaquinone 9(H4), had guanine-plus-cytosine contents of 69.9 to 72.1 mol%, and had D-glutamic acid, D- and L-serine, glycine, L-alanine, and L-lysine as cell wall amino acids (type A3α). The taxonomic characteristics of these strains differ from those of the previously described motile actinomycetes. On the basis of morphological, physiological, and chemotaxonomic data and the results of DNA-DNA hybridization and comparative 16S rRNA studies, we propose a new genus, Couchioplanes, for these organisms. The type species is Couchioplanes caeruleus comb. nov. (type strain, IFO 13939), which is divided into two subspecies, Couchioplanes caeruleus subsp. caeruleus subsp. nov. (type strain, IFO 13939) for A. caeruleus IFO 13939T and strain RA 335 and Couchioplanes caeruleus subsp. azureus (type strain, IFO 13993) for “A. azureus” IFO 13993T.

A new species, Porphyromonas canoris, is proposed for black-pigmented asaccharolytic strains isolated from subgingival plaque samples from dogs with naturally occurring periodontal disease. This bacterium is an obligately anaerobic, nonmotile, non-spore-forming, gram-negative, rod-shaped organism. On laked rabbit blood or sheep blood agar plates, colonies are light brown to greenish brown after 2 to 4 days of incubation and dark brown after 14 days of incubation. Colonies on egg yolk agar and on nonhemolyzed sheep blood agar are orange. The cells do not grow in the presence of 20% bile and have a guanine-plus-cytosine content of 49 to 51 mol%. The type strain is VPB 4878 (= NCTC 12835). The average levels of DNA-DNA hybridization between P. canoris strains and other members of the genus Porphyromonas are as follows: Porphyromonas gingivalis ATCC 33277T (T = type strain), 6.5%; Porphyromonas gingivalis cat strain VPB 3492, 5%; Porphyromonas endodontalis ATCC 35406T, 1%; Porphyromonas salivosa NCTC 11362T, 5%; and Porphyromonas circumdentaria NCTC 12469T, 6%. The level of hybridization between P. canoris NCTC 12835T DNA and Porphyromonas asaccharolytica ATCC 25260T DNA is 3%. P. canoris cells produce major amounts of acetic, propionic, isovaleric, and succinic acids and minor amounts of isobutyric and butyric acids as end products of metabolism in cooked meat medium. The major cellular fatty acid is 13-methyltetradecanoic acid (iso-C15:0). Glutamate and malate dehydrogenases are present, as are glucose-6-phosphate dehydrogenase activity (65.7 nmol mg of protein-1 min-1) and 6-phosphogluconate dehydrogenase activity (63.0 nmol mg of protein-1 min-1). P. canoris cells do not agglutinate sheep erythrocytes but exhibit brick red fluorescence at 265 nm and produce catalase.

Clostridium hydroxybenzoicum JW/Z-1T (= ATCC 51151 = DSM 7310)) (T = type strain), isolated from freshwater pond sediment, is a nonmotile, gram type-positive, spore-forming, amino acid-utilizing, anaerobic rod. This bacterium produces two inducible enzymes that catalyze the decarboxylation of para-hydroxybenzoates. The phenols produced are not utilized. C. hydroxybenzoicum requires yeast extract for growth. Sugars are not utilized. Sodium ions and acetic acid stimulate growth. The optimal temperature and optimal pH for growth are 33 to 34°C and 7.2 to 8.2, respectively. The DNA base composition of the type strain is 35.5 mol% guanine plus cytosine, whereas the DNA base compositions of the type strains of Clostridium sticklandii and Clostridium aminovalericum are 33 and 33.5 mol% guanine plus cytosine, respectively, as determined by a chemical method. 16S rRNA sequence analysis groups strain JW/Z-1T most closely with Clostridium purinolyticum and Clostridium acidiurici (10.6 and 11 inferred changes per 100 bases, respectively). However, C. hydroxybenzoicum does not utilize uric acid, hypoxanthine, xanthine, adenine, or guanine. The cell wall type is Alα (L-Lys direct).

The microflora of sourdough preparations was investigated by examining the physiological characteristics, whole-cell protein patterns, and 16S rRNA sequences of Lactobacillus isolates. Strains isolated from sourdough were placed in the species Lactobacillus brevis, Lactobacillus sanfrancisco, and Lactobacillus reuteri. 16S rRNA sequences were determined for L. brevis, Lactobacillus fructivorans, Lactobacillus fermentum, L. sanfrancisco, and L. reuteri, and oligonucleotide probes for fast specific identification of these sourdough lactobacilli were deduced. The physiological characteristics, protein patterns, and 16S rRNA sequences of these organisms were compared with data for other sourdough lactobacilli and additional reference strains. Strains of a Lactobacillus species were isolated from rye sourdough; these strains may account for most of the flora in sourdough made from wheat or rye. These organisms were differentiated from other sourdough lactobacilli by their protein pattern, 16S rRNA sequence, G+C content, and physiological properties. The 16S rRNA sequence of this species was determined, and we constructed a phylogenetic tree which reflected the relationships of this species to other lactobacilli. This organism is closely related to L. reuteri. A new Lactobacillus species, Lactobacillus pontis, is proposed. The type strain is L. pontis LTH 2587 (= DSM 8475 = LMG 14187). We describe a general strategy in which a polyphasic approach was used to characterize a new species.

One species of the new genus Actinocorallia, Actinocorallia herbida sp. nov., is described. The genus Actinocorallia is characterized by well-developed, branched, nonfragmented substrate mycelia. On the tips of unique coralloid sporophores, which arise from the substrate mycelia, long chains of nonmotile spores (more than 30 spores per chain) are borne. On rare occasions formation of coremia is observed. The chemotaxonomic characteristics of this organism are as follows: the cell wall chemotype is type III (meso-diaminopimelic acid), the whole-cell sugar pattern is type C (no diagnostic sugar type), the fatty acid pattern is type 1a (straight-chain saturated acids and monounsaturated acids), the predominant isoprenoid quinones are MK-9(H4) and MK-9(H6), phospholipid type PII (phosphatidylethanolamine) is present as the diagnostic polar lipid, the N-acyl type of muramic acid in the cell wall is the acetyl type, and the guanine-plus-cytosine content of the DNA is 73 mol%. The type strain of A. herbida is strain AL-50780 (= IFO 15485).

Plant-associated bacteria were characterized and are discussed in relation to authentic members of the genus Pseudomonas sensu stricto. Bacteria belonging to Pseudomonas rRNA group II are separated clearly from members of the genus Pseudomonas sensu stricto (Pseudomonas fluorescens rRNA group) on the basis of plant association characteristics, chemotaxonomic characteristics, DNA-DNA hybridization data, rRNA-DNA hybridization data, and the sequences of 5S and 16S rRNAs. The transfer of Pseudomonas cepacia, Pseudomonas mallei, Pseudomonas pseudomallei, Pseudomonas caryophylli, Pseudomonas gladioli, Pseudomonas pickettii, and Pseudomonas solanacearum to the new genus Burkholderia is supported; we also propose that Pseudomonas plantarii and Pseudomonas glumae should be transferred to the genus Burkholderia. Isolate VA-1316T (T = type strain) was distinguished from Burkholderia species on the basis of physiological characteristics and DNA-DNA hybridization data. A new species, Burkholderia vandii sp. nov. is proposed for this organism; the type strain of B. vandii is VA-1316 (= JCM 7957).

A high degree of genetic diversity among 29 strains of Prevotella (Bacteroides) ruminicola from the rumen was revealed by comparing restriction fragment length polymorphisms in 16S rRNA genes, sodium dodecyl sulfate-polyacrylamide gel profiles of total-cell proteins, and G+C contents of chromosomal DNAs. In order to obtain information on phylogenetic relationships, the sequences of a 389-bp region of the 16S rRNA gene, including variable regions 4 and 5, were compared for 10 strains. These 10 strains formed a single group when their sequences were compared with 16S ribosomal DNA sequences from other species, including Bacteroides spp. from the human colon. On the other hand, the great genetic distances between many P. ruminicola strains, including P. ruminicola subsp. brevis B14 and GA33 and P. ruminicola 23T (T = type strain), support the hypothesis that these organisms should be reclassified into new species. We identified signature oligonucleotides based on 16S ribosomal DNA sequences that distinguished strains related to strains 23T, B14, GA33, and M384, as well as an oligonucleotide that specifically recognized all but one of the Bacteroides and Prevotella strains tested. On the basis of the priming activities of these signature oligonucleotides in PCR reactions and on other criteria, we concluded that 12 of the original 29 strains were related to strain 23T, 4 were related to strain B14, and 4 were related to strain GA33. While there are clear grounds for subdividing the species P. ruminicola on the basis of genotypic differences, it is appropriate to delay formal reclassification until further work on the phenotypic differentiation of the new groups is completed.

A number of Bacillus strains isolated from desert soil samples were shown to belong to a previously unidentified species, for which we propose the name Bacillus mojavensis. The type strain is RO-H-1 (= NRRL B-14698). On the basis of restriction digest data, B. mojavensis is most closely related to Bacillus amyloliquefaciens, Bacillus atrophaeus, and Bacillus subtilis. So far, B. mojavensis can be distinguished from B. subtilis only by differences in whole-cell fatty acid composition, divergence in DNA sequence, and resistance to genetic transformation between taxa (in addition to reduced genome relatedness values). Sequence divergence and sexual isolation may prove to be more useful than metabolic characteristics for delimiting cryptic Bacillus species.

A chemotaxonomic analysis of 14 Streptosporangium species revealed the phenotypic heterogeneity of this genus. The type species, Streptosporangium roseum, and 11 other species share similar isoprenoid quinones, fatty acids, and polar lipids. Two species, Streptosporangium albidum and Streptosporangium viridogriseum, including S. viridogriseum subsp. viridogriseum and S. viridogriseum subsp. kofuense, were characterized by a pattern of chemotaxonomic properties that was clearly distinct from the pattern obtained for the authentic Streptosporangium species. The results of a previous phylogenetic analysis of 16S ribosomal DNA and 5S rRNA indicated that the type species and its relatives are distantly related to members of the genus Streptomyces, whereas a member of the other group was found to be phylogenetically related to, but clearly distinct from, members of the family Pseudonocardiaceae. On the basis of these phenotypic and genomic differences, we propose the name Kutzneria gen. nov. for a new genus containing the previously misclassified Streptosporangium species and describe Kutzneria viridogrisea comb, nov., Kutzneria albida comb, nov., and Kutzneria kofuensis comb. nov. The type species of the new genus is Kutzneria viridogrisea, and the type strain is DSM 43850 (= ATCC 25242).

The partial 16S rRNA gene sequences of some coryneform bacteria isolated from middle-ear fluids from patients with otitis media were determined. A comparative analysis performed with rRNA sequences of other high-G+C-content gram-positive bacteria showed that the coryneform bacteria isolated from otitis media patients were only remotely related to members of previously described genera. On the basis of the results of the phylogenetic analysis and previously published biochemical and chemotaxonomic data, we propose that the strains isolated from middle-ear infections should be classified in a new genus, Turicella, as Turicella otitidis gen. nov., sp. nov. The type strain of T. otitidis is strain DSM 8821.

Two new Planomonospora species were isolated from soil by novel baiting techniques. Traditional taxonomic methods and fatty acid analysis techniques were used to establish the identities of these organisms. The names proposed for these new species are Planomonospora sphaerica, whose type strain is strain A51460 (= NRRL 18923), and Planomonospora alba, whose type strain is strain A82600 (= NRRL 18924). The descriptions of these taxa are based on single isolates.

The organism named “Erwinia nulandii” was isolated in 1979 from bean seeds and was described in 1981, but the name was never validated. The results of biochemical tests and membrane protein profile and DNA relatedness studies indicated that this name is synonymous with Erwinia persicinus, a validly published name for a species previously isolated from, but not shown to be pathogenic for, tomatoes, bananas, and cucumbers. Pathogenicity tests revealed that all E. persicinus strains, including an “E. nulandii” strain, were pathogenic for bean pods and seeds.

An analysis of the ribosomal AT-L30 proteins from 42 strains of 35 species belonging to the genera Streptomyces, Streptoverticillium, and Kitasatosporia and related genera revealed that all of the members of the genera Streptoverticillium and Kitasatosporia examined had the same sequence as Streptomyces exfoliatus or a highly homologous sequence and exhibited high levels of relatedness to Streptomyces lavendulae. These results strongly support the previous suggestion of Witt and Stackebrandt (D. Witt and E. Stackebrandt, Syst. Appl. Microbiol. 13:361–371, 1990) and Wellington et al. (E. M. H. Wellington, E. Stackebrandt, D. Sanders, J. Wolstrup, and N. O. G. Jorgensen, Int. J. Syst. Bacteriol. 42:156–160, 1992) that the genera Streptoverticillium and Kitasatosporia should be united with the genus Streptomyces on the basis of 16S rRNA data.

The 16S rRNAs of 15 species of actinomycetes belonging to the genera Actinokineospora and Saccharothrix and the family Pseudonocardiaceae, including Amycolatopsis, Amycolata, Pseudonocardia, Saccharomonospora, and Saccharopolyspora species, were sequenced by using reverse transcriptase. The sequences were analyzed along with the sequences of reference actinomycetes by using distance matrix and parsimony methods. The wall chemotype IV genus Actinokineospora was found to be closely related to species of the genus Saccharothrix which have chemotype III walls. Together, these two genera formed a clade which was closely related to members of the family Pseudonocardiaceae which have chemotype IV walls. However, the phylogenetic branching pattern did not unambiguously resolve whether the members of all three taxa should be placed in a single family. We suggest, therefore, that the genera Actinokineospora and Saccharothrix should remain outside the family Pseudonocardiaceae until additional sequence or phenotypic data are available to decide the issue. The sequences of species belonging to the genera Amycolata and Pseudonocardia were always recovered as a mixed group in phylogenetic trees, and we propose that these organisms should be classified in an emended genus Pseudonocardia. This proposal is strongly supported by previously published lipid, ribosomal protein, and ultrastructure data.

The genus Pseudoamycolata was proposed for actinomycetes which closely resembled Amycolata species in most phenotypic features but lacked a key chemotaxonomic marker, phosphatidylcholine. We sequenced the 16S ribosomal DNA of Pseudoamycolata halophobica and found that this organism is very closely related to the genus Pseudonocardia, which now contains all of the strains formerly classified as members of the genus Amycolata. In light of these data, we believe that the absence of a single chemical characteristic is not sufficient to justify forming a new genus, and we propose that Pseudoamycolata halophobica (Akimov et al. 1989) should be reclassified as Pseudonocardia halophobica. The type strain is strain DSM 43089.

To clarify the taxonomic relationships of the genera Rhizomonas and Sphingomonas, the 16S rRNA sequence of Rhizomonas suberifaciens IFO 15211T (T = type strain) was determined. A phylogenetic analysis of aligned 16S rRNA gene sequences revealed that eight species of the genus Sphingomonas and R. suberifaciens are closely related to Erythrobacter longus and Porphyrobacter neustonensis and, therefore, belong in the alpha-4 subclass of the Proteobacteria. Within this subclass, Sphingomonas species and R. suberifaciens are phylogenetically interrelated and comprise several subgroups. Our findings show that the genus and species definitions of these organisms are in need of revision.

The 16S rRNA genes from virulent (AB) and benign (B) biotype strains of Fusobacterium necrophorum, the causative agent of ovine foot abscess, were cloned and sequenced. Notwithstanding the distinct phenotypic differences between the AB and B biotypes, a phylogenetic analysis in which the 16S rRNA gene sequences were used revealed the close relationship of these taxa. Comparison of the virulent and benign biotypes of F. necrophorum may therefore be a legitimate way to identify key virulence factors useful in vaccine development.

Other workers have found that clinical isolates of Helicobacter pylori exhibit very extensive DNA sequence polymorphisms when they are examined by ribotyping or some other genomic sequence characterization technique. In fact, it is rare to find similar clones, much less identical ones, among isolates. We found that the levels of divergence between the 16S ribosomal DNA sequences of individual organisms and the consensus sequence of the five isolates which we examined ranged from 0.2 to 0.5%. In contrast, other workers have shown that levels of divergence between the 16S ribosomal DNA sequence of H. pylori and the 16S ribosomal DNA sequences of four other Helicobacter species range from 2.7 to 8.0%. Our results show that the H. pylori 16S ribosomal DNA is not very polymorphic and support the conclusion that H. pylori is a unique species.

Two new Capnocytophaga species, for which we propose the names Capnocytophaga haemolytica and Capnocytophaga granulosa, were isolated from supragingival dental plaque of adults. The phenotypic characteristics of these organisms were the same as those of the genus Capnocytophaga: gram-negative rods; CO2 requirement; gliding motility; catalase negative; oxidase negative; acids produced from D-glucose, D-maltose, D-mannose, and D-sucrose; and acetate and succinate are the major end products of glucose fermentation. In addition, the cellular fatty acid contents and menaquinones of both species were similar to the cellular fatty acid contents and menaquinones of other Capnocytophaga species. Nevertheless, the levels of DNA-DNA relatedness of the two new species to each other and to other Capnocytophaga species were less than 20%. Two notable characteristics of C. haemolytica are the presence of hemolytic activity and the lack of aminopeptidase activity. C. granulosa has granular inclusions in its cells and grows aerobically. The type strains of C. haemolytica and C. granulosa are A0404 (= JCM 8565) and B0611 (= JCM 8566), respectively.