- Volume 47, Issue 1, 1997
Volume 47, Issue 1, 1997
- Original Papers Relating To Systematic Bacteriology
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A Phylogenetic Analysis of Borrelia Burgdorferi Sensu Lato Based on Sequence Information from the hbb Gene, Coding for A Histone-Like Protein
More LessWe describe a phylogenetic investigation of Borrelia burgdorferi sensu lato, the causative agent of Lyme disease, based on a DNA sequence analysis of the hbb gene, which encodes protein HBb, a member of the family of histone-like proteins. Because of their intimate contact with the DNA molecule, these proteins are believed to be fairly conserved through evolution. In this study we proved that the hbb gene is suitable for phylogenetic inference in the genus Borrelia. The hbb gene, which is 327 bp long and encodes 108 amino acids, was sequenced for 39 strains, including 37 strains of B. burgdorferi sensu lato, 1 strain of Borrelia turicatae, and 1 strain of Borrelia parkeri. Genetic variability was determined at the sequence level by computational analysis. Briefly, 81 substitutions were scored at the DNA level. Only 25 of these substitutions were responsible for amino acid substitutions at the translational level. The signature region for bacterial histone-like proteins was found in hbb. Although variable at the nucleotide level, it was highly conserved at the deduced amino acid level. A phylogenetic tree for the genus Borrelia that was generated from multiple sequence alignments was consistent with previously published data derived from DNA-DNA hybridization and multilocus enzyme electrophoresis analyses. The subdivision of B. burgdorferi sensu lato into five species (B. burgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii, Borrelia japonica, and “Borrelia andersonii”) and at least four genomic groups (groups PotiB2, VS116, CA2, and DN127) was confirmed.
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Phylogenetic Analysis of Borrelia burgdorferi Sensu Stricto by Arbitrarily Primed PCR and Pulsed-Field Gel Electrophoresis
More LessBoirelia burgdorferi sensu lato is present all over the world. However, most species belonging to this complex have a limited distribution; the only exception is B. burgdorferi sensu stricto, which is encountered both in Europe and in the United States. The aim of this study was to clarify the way that B. burgdorferi sensu stricto migrated. To do this, we compared the genetic polymorphism of 33 B. burgdorferi sensu stricto strains isolated in Europe and 28 strains isolated in the United States by using both pulsed-field gel electrophoresis and arbitrarily primed PCR. Phylogenetic analysis of each of the two sets of data was performed by a genetic distance method and a parsimony method. The results were consistent and revealed that the American strains were more heterogeneous than the European strains. The hypothesis that B. burgdorferi sensu stricto was introduced to Europe from America rather than vice versa has to be considered.
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Systematic Study of the Genus Vogesella gen. nov. and Its Type Species, Vogesella indigofera comb. nov.
More LessA blue-pigmented colony that had a metallic copper-colored sheen was isolated in 1973 from a standard spread plate count preparation of oxidation pond sediment. Over the next 11 years, an additional 12 strains of blue-pigmented bacteria were isolated from freshwater samples and compared to several reference strains of bacteria. Morphological and biochemical tests revealed that these 13 isolates were very similar to [Pseudomonas] indigofera ATCC 19706T (T = type strain) and ATCC 14036. A numerical analysis (in which simple matching similarity coefficients were clustered by the unweighted pair group mathematical averaging method) of morphological and biochemical characteristics revealed 90.0% relatedness between the 13 isolates and [P.] indigofera ATCC 19706T and ATCC 14036 and 73.6% relatedness between the 13 isolates and a cluster containing Burkholderia cepacia ATCC 25416T, Janthinobacterium lividum ATCC 12473T, and the Pseudomonas species tested. A phylogenetic analysis, in which both 5S rRNA and 16S rRNA were used, also revealed that the 13 isolates were closely related to each other and to strains ATCC 19706T and ATCC 14036. In addition, both 5S rRNA and 16S rRNA analyses demonstrated that the isolates and strains ATCC 19706T and ATCC 14036 were members of the beta subdivision of the Proteobacteria and were closely related to Chromobacterium violaceum ATCC 12742T but sufficiently distinct to warrant placement in a new genus. Accordingly, we propose that the 13 isolates and strains ATCC 19706T and ATCC 14306 be placed in the genus Vogesella gen. nov., which is named in honor of Otto Voges, who first isolated and described this blue-pigmented eubacterium in 1893. We also propose that [P.] indigofera be renamed Vogesella indigofera comb. nov. and designated the type species of the genus; strain ATCC 19706 is the type strain of this species.
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Aerococcus urinae: Intraspecies Genetic and Phenotypic Relatedness
A number of Arococcus-like organisms were recently recognized as human pathogens. Five Aerococcus-like strains were proposed as members of the new species Aerococcus urinae (with type strain E2 [= NCTC 12142]) on the basis of the results of a 16S rRNA sequence analysis. The intraspecies phenotypic and genetic relatedness of 22 selected A. urinae strains was investigated, and a hitherto unrecognized esculin hydrolysis-positive biotype was identified. A total of 14 of the 15 more common esculin-negative strains exhibited very high DNA relatedness as determined by the hydroxyapatite method (the levels of relatedness were greater than 90% in 55 and 70°C reactions, with 1.5% or less divergence in related sequences). The DNA relatedness among the six esculin-positive strains was more heterogeneous, and two DNA hybridization subgroups were formed. Our results are compatible with the hypothesis that both biotypes are members of the single species A. urinae, which contains two or more genetic subspecies. The putative subspecies have not been formally proposed since they cannot be definitively differentiated. The inclusion of A. urinae in the genus Aerococcus is supported by the results of 16S rRNA sequencing. The rRNA sequence data also is compatible with placing both biotypes in a single species.
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The Spiroplasma Motility Inhibition Test, a New Method for Determining Intraspecific Variation among Colorado Potato Beetle Spiroplasmas
More LessThe Colorado potato beetle, Leptinotarsa decemlineata, is a major holarctic pest of solanaceous crops. Presumably, this insect spread from Solanum species in central America to the Mexican plateau, and this was followed by multiple invasions of North America and Europe. Attempts are being made to control this beetle by using a genetically modified spiroplasma that occurs naturally in its gut. In the current study, spiroplasmas isolated from beetles collected in North America and Poland exhibited serologic (spiroplasma motility inhibition test) and genomic (restriction fragment length polymorphism) profiles that suggest that there were multiple spiroplasma introductions. Two serovars were identified; one is found in northern North America and at high elevations in Poland, and the other is found in southern North America and at low elevations in Poland. The patterns of genovar distribution coincide with the serovar patterns. The existence of such biovars—intraspecific taxal units reflected by serologic and genomic differences—should be taken into consideration when taxonomies are developed and strains are chosen for biocontrol.
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Phylogenetic Analysis of Mycoplasmas Based on Hsp70 Sequences: Cloning of the dnaK (hsp70) Gene Region of Mycoplasma capricolum
More LessA 5.0-kb region containing the hsp70 (dnaK) gene was cloned from Mycoplasma capricolum and sequenced. In addition to the hsp70 gene, this sequence region also contained the complete sequences for the grpE and orfA genes and partial sequences for the clpB and dnaj genes. The order of the above gene sequences in the cloned fragment was found to be clpB-orfA-grpE-hsp70-dnaJ. which is similar to the order seen in various other gram-positive groups of bacteria. The Hsp70 homologs from two mycoplasma species, Mycoplasma capricolum and Mycoplasma genitalium. contain a number of sequence signatures, including the absence of a large insert in the N-terminal quadrant, that are characteristics of the homologs from gram-positive bacteria and archae-bacteria. A detailed phylogenetic analysis based on Hsp70 sequences was also performed. In neighbor-joining and parsimony trees based on Hsp70 sequences, both mycoplasma species branched with the low-G+C-content gram-positive group of bacteria (e.g., Lactobacillus and Erysipelothrix species) in 87% and 96% of the bootstrap replicates, respectively, indicating their close evolutionary relationship to this group. The phylogenetic trees based on Hsp70 sequences show a polyphyletic branching of archaebacteria with the gram-positive species, which is statistically strongly favored.
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Phylogenetic Analysis of the Genus Actinomyces Based on 16S rRNA Gene Sequences: Description of Arcanobacterium phocae sp. nov., Arcanobacterium bernardiae comb. nov., and Arcanobacterium pyogenes comb. nov.
More LessA systematic phylogenetic analysis of the genus Actinomyces was performed. The 16S rRNA gene sequences of 13 Actinomyces species, an unnamed Actinomyces strain (ATCC 49338), and an Actinomyces-like isolate from sea mammals were determined. Comparative sequence analysis with closely related taxa revealed phylogenetic diversity and internal structure within the genus Actinomyces. In addition, some members of other genera (viz., the genera Arcanobacterium, Mobiluncus, and Rothia) were shown to be phylogenetically intermixed with the Actinomyces species. It was evident from both distance and tree topology considerations that the genus Actinomyces is in urgent need of taxonomic revision and requires subdivision into several genera. Based on the results of the present study it is proposed that Actinomyces bernardiae and Actinomyces pyogenes be assigned to the genus Arcanobacterium as Arcanobacterium bernardiae comb. nov. and Arcanobacterium pyogenes comb. nov., respectively. In addition, a new species, Arcanobacterium phocae, is proposed for Actinomyces-like bacteria isolated from seals.
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Comparative Sequence Analyses of the Genes Coding for 16S rRNA of Lactobacillus casei-Related Taxa
The primary structures of the 16S rRNA genes of the type strains of Lactobacillus casei and related taxa were determined by PCR DNA-sequencing methods. The sequences of Lactobacillus casei, Lactobacillus zeae, Lactobacillus paracasei, and Lactobacillus rhamnosus were different. The K nuc values ranged from 0.0040 to 0.0126. On the basis of the K nuc values and the levels of DNA-DNA relatedness among the strains of these species, the L. casei-related taxa should be classified in the following three species: L. zeae, which includes the type strains of L. zeae and L. casei; a species that includes the strains of L. paracasei and L. casei ATCC 334; and L. rhamnosus.
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Vibrio scophthalmi sp. nov., a New Species from Turbot (Scophthalmus maximus)
More LessAbstractSix strains isolated from the intestines of juvenile turbot in a fish hatchery in the north of Spain were found to be phenotypically members of the genus Vibrio. However, the phenotypic traits of these organisms did not place them in any of the currently known Vibrio species. These isolates formed an homogeneous group in which the DNA-DNA similarity values (the differences between the thermal denaturation midpoints of the homologous and heterologous duplexes) with reference strain A089T (T = type strain) ranged from 0 to 1.7°C. The results of a 16S rRNA sequence analysis of A089T placed this strain in the genus Vibrio in the gamma subclass of the Proteobacteria. The closest relative is Vibrio aestuarianus, with a sequence similarity of 97.8%. This group of strains can be easily differentiated from the other Vibrio species by their clear phenotype. We propose the name Vibrio scophthalmi sp. nov. for these strains; the type strain is strain A089 (= CECT 4638).
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Occurrence of Lipoteichoic Acid in Oral Streptococci
More LessAbstractThe heterogeneous bacterial group known as oral streptococci was screened for the presence of cellular polyglycerolphosphate-containing lipoteichoic acid. This compound was detected in phenol extracts of lyophilized cells by an immunoassay in which polyglycerolphosphate-specific monoclonal antibody was used. Polyglycerolphosphate-containing lipoteichoic acid occurred in all 86 strains of oral streptococci examined except the Streptococcus mitis and Streptococcus oralis strains. This confirms the findings of Rosan (B. Rosan, Science 201:918–920, 1978) and Hamada et al. (S. Hamada, J. Mizuno, S. Kotani, and M. Torii, FEMS Microbiol. Lett. 8:93–96, 1980), is consistent with the results of the taxonomic study of oral streptococci performed by Kilian et al. (M. Kilian, L. Mikkelsen, and J. Henrichsen, Int. J. Syst. Bacteriol. 39:471–484, 1989), who emended the descriptions of Streptococcus sanguis, S. oralis, and S. mitis, and reflects the phylogenetic relationship among S. mitis, S. oralis, and Streptococcus pneumoniae.
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Treponema medium sp. nov., Isolated from Human Subgingival Dental Plaque
AbstractA new Treponema species, for which we propose the name Treponema medium, was isolated from subgingival plaque from an adult with periodontal disease. The morphological characteristics, differential biochemical characteristics, and protein profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of this organism are described. The guanine-plus-cytosine content of the DNA of T. medium is 51 mol%. The levels of DNA-DNA relatedness of the new species to other Treponema species, including Treponema denticola, Treponema vincentii, Treponema socranskii, Treponema pallidum, and Treponema phagedenis, are less than 30%. A phylogenetic analysis based on 16S rRNA sequences distinguished the new Treponema strain from strains belonging to previously described Treponema species. The type strain of T. medium is strain G7201.
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Haloarcula argentinensis sp. nov. and Haloarcula mukohataei sp. nov., Two New Extremely Halophilic Archaea Collected in Argentina
More LessAbstractStrains arg-1T (T = type strain) and arg-2T, two new strains of extremely halophilic archaea, were isolated from the soils of the Argentine salt flats. The taxonomic features of arg-1T were similar to, but distinct from, those of the type strain of Haloarcula vallismortis and other Haloarcula species. On the 16S rRNA phylogenetic tree, strain arg-1T formed a cluster together with Haloarcula species. Strain arg-2T differed in its glycolipid composition but still was more closely related to the genus Haloarcula than to other established genera. We propose that strain arg-1T be classified as a member of a new species, Haloarcula argentinensis, and that strain arg-2T be classified as a member of Haloarcula mukohataei sp. nov., although arg-2T may belong to a new genus or a subgenus of the genus Haloarcula. The type strain of H. argentinensis is strain arg-1 (= JCM 9737), and the type strain of H. mukohataei is strain arg-2 (= JCM 9738).
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Spiroplasma diabroticae sp. nov., from the Southern Corn Rootworm Beetle, Diabrotica undecimpunctata (Coleoptera: Chrysomelidae)
AbstractSpiroplasma strain DU-1T (T = type strain), which was isolated from hemolymph of the corn rootworm Diabrotica undecimpunctata (Coleoptera: Chrysomelidae), was serologically distinct from other spiroplasma species, groups, and subgroups. Cells of strain DU-1T were shown by light microscopy to be helical motile filaments. Electron microscopy revealed cells bounded by a single cytoplasmic membrane, with no evidence of a cell wall. The organism was not sensitive to 500 U of penicillin per ml. Strain DU-1T grew well in SM-1, M1D, and SP-4 liquid media, in broth supplemented with 1% bovine serum fraction or conventional horse serum, and under both aerobic and anaerobic conditions. This organism did not appear to have a sterol requirement for growth, as has been reported for several other Spiroplasma species or strains. Optimal growth occurred at 32°C, with a doubling time of 0.9 h; strain DU-1T multiplied at 10 to 41°C but failed to grow at 5 or 43°C. It produced acid from glucose but hydrolyzed neither arginine nor urea. The results of reciprocal serologic tests in which antigens or antisera to established Spiroplasma species, groups, subgroups, and putative groups were used indicated that strain DU-1T was serologically distinct. This organism has a DNA guanine-plus-cytosine content of 25 ± 1 mol% and a genome size of 1,350 kbp. Strain DU-1T is a member of a cluster of fast-growing insect-associated spiroplasmas, as determined by sequence analysis of 16S rRNA. On the basis of the results of this study and previously published data, strain DU-1 (= ATCC 43210) is designated the type strain of a new species, Spiroplasma diabroticae.
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Streptococcus difficile Is a Nonhemolytic Group B, Type Ib Streptococcus
More LessAbstractWhole-cell protein electrophoretic analysis of the type strain of Streptococcus difficile (LMG 15799) revealed that this organism was indistinguishable from Streptococcus agalactiae strains. Although LMG 15799T (T = type strain) was originally described as serologically untypeable, we found that this strain was a group B streptococcus belonging to the capsular polysaccharide antigen type Ib group. The biochemical reactivity of S. difficile, which differed from the biochemical reactivity of typical S. agalactiae strains mainly by being less versatile, is similar to the biochemical reactivity of other group B, type Ib streptococci isolated from Poikilothermic animals, such as fish and frogs.
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Mycobacterium hassiacum sp. nov., a New Rapidly Growing Thermophilic Mycobacterium
More LessAbstractA new rapidly growing, scotochromogenic mycobacterium was isolated from urine. This strain is thermophilic (it grows at 65°C), tolerates 5% NaCl, and was unable to utilize any of the sugars tested or citrate or to take up iron. The isolate splits benzamide, urea, nicotinamide, and pyrazinamide and is sensitive to streptomycin, ethambutol, cycloserine, ciprofloxacin, and clarithromycin but resistant to isoniazid, rifampin, and prothionamide. These characteristics clearly place this organism in a new mycobacterial species, which was confirmed by the unique 16S rRNA nucleotide sequence. The high level of similarity between this rapid grower and Mycobacterium xenopi is surprising. For this new rapidly growing scotochromogenic and thermophilic mycobacterium we propose the name Mycobacterium hassiacum sp. nov.
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Corynebacterium coyleae sp. nov., Isolated from Human Clinical Specimens
More LessAbstractOver a 5-year period, six isolates of a previously unknown nonlipophilic coryneform bacterium were isolated from human clinical specimens. The most characteristic phenotypic reactions of these isolates included slow fermentative acid production from glucose but no acid production from maltose and sucrose and a strongly positive CAMP reaction. Chemotaxonomic investigations revealed that meso-diaminopimelic acid and mycolic acids were present, that palmitic, oleic, and stearic acids were the predominant cellular fatty acids, and that the G+C content was 62 to 64 mol%, characteristics which are consistent with assignment to the genus Corynebacterium. Phenotypically, the unknown coryneform bacterium could be readily differentiated from all other Corynebacterium species. A 16S rRNA gene sequence analysis and quantitative DNA-DNA hybridization experiments demonstrated unambiguously that the unknown coryneform bacterium is a member of the genus Corynebacterium and is genotypically distinct from all other members of this genus. Based on phenotypic and genorypic findings, a new species, Corynebacterium coyleae sp. nov., is proposed. The type strain of C. coyleae is strain DSM 44184 (= CCUG 35014).
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A New Genus of the Order Actinomycetales, Spirilliplanes gen. nov., with Description of Spirilliplanes yamanashiensis sp. nov.
More LessAbstractActinomycete strain YU127-1T (T = type strain), which produces zoospores, was isolated from a soil sample. The aerial mycelium of this organism at maturity forms short chains of spores. The hyphae form coils, and sporangia are not observed. Strain YU127-1T contains glutamic acid, glucosamine, glycine, alanine, and meso-diaminopimelic acid in its cell wall (wall chemotype II), 3-O-methylmannose, mannose, xylose, and glucose as whole-cell sugars, menaquinone 10(H4), and glycolyl cell wall polysaccharides and has a guanine-plus-cytosine content of 69.0 mol%. Mycolic acids are absent. Phosphatidylinositol and phosphatidylethanolamine are diagnostic phospholipids. The chemotaxonomic data, except for the lack of arabinose in the whole-cell sugars, indicate that this strain belongs to the family Micromonosporaceae. The morphological and physiological characteristics and chemotaxonomic and phylogenetic data for this strain differ from those of the previously described actinomycetes. We therefore propose a new genus, Spirilliplanes, for this organism; the type species of the genus is Spirilliplanes yamanashiensis sp. nov., and the type strain of S. yamanashiensis is strain YU127-1 (= IFO 15828).
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Investigations of the Taxonomy and Systematics of Fusobacterium nucleatum Using Allozyme Electrophoresis
More LessAbstractFusobacterium nucleatum forms part of the resident microbiota in both oral and extraoral sites in humans and animals. It is also involved in infections in such sites. Despite the genetic heterogeneity within the species, it has been divided into five subspecies, the validities of which have been questioned. In the present study, 44 F. nucleatum isolates were examined at 21 enzyme loci by using the allozyme electrophoretic technique to establish an accurate genetic framework for taxonomic purposes. Three distinct genetic clusters were identified; one cluster consisted exclusively of extraoral isolates, another cluster consisted predominantly of human oral isolates, and the third cluster consisted of a single human oral isolate. Our results highlight the urgent need for extensive biochemical, immunological, and epidemiological studies to accurately define the systematics of the genus Fusobacterium based on the framework derived in this study by using 21 independent genetic characteristics.
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Restriction Fragment Length Polymorphism Analysis of PCR-Amplified 16S Ribosomal DNA for Rapid Identification of Saccharomonospora Strains
More LessAbstractTwenty-one strains of Saccharomonospora azurea, Saccharomonospora cyanea, Saccharomonospora glauca, Saccharomonospora viridis, and “Saccharomonospora caesia” were examined to evaluate the discriminatory value of 16S ribosomal DNA (rDNA) fingerprints. The 16S rDNAs were amplified by PCR by using oligonucleotide primers complementary to 16S rRNA genes. A restriction fragment length polymorphism (RFLP) analysis of the 16S rDNAs was performed with SmaI and MluI. The four validly described Saccharomonospora species could be differentiated on the basis of their characteristic 16S rDNA restriction patterns. The strains of “S. caesia” gave a restriction pattern identical to that of S. azurea K161T (T = type strain). This result was anticipated from the previous report that S. azurea K161T and the strains of “5. caesia” have identical 16S rRNA sequences. We found that purification of amplified 16S rDNA products following PCR was necessary for our RFLP analysis.
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Moraxella boevrei sp. nov., a New Moraxella Species Found in Goats
More LessSix Moraxella-like strains that formed a phenotypically homogeneous group were isolated from the nasal flora of healthy goats. Total genomic DNA-DNA hybridization, rRNA gene restriction pattern, DNA base composition, and genetic transformation studies were performed to determine the relationships of these bacteria to species belonging to the genus Moraxella and other fastidious gram-negative species. The new group of isolates was very homogeneous, as shown by rRNA gene restriction fragment length patterns (ribotyping), and these organisms displayed high relative binding ratios (RBRs) to each other in DNA-DNA hybridization experiments (RBRs, = 58%) but distinctly lower levels of DNA homology with all other species investigated. However, the RBRs obtained with species of the genus Moraxella were higher than the RBRs obtained with all other gram-negative strains examined. Although the new strains had most of the Moraxella bovis phenotypic characteristics except nitrate reduction, quantitative and qualitative genetic transformation data led to the conclusion that they belong to a distinct new cluster in the genus Moraxella. The results of this study, combined with the general morphological and phenotypic profiles of the new strains, are consistent with the creation of a new Moraxella species, for which the name Moraxella boevrei is proposed. Strain 88365 (= ATCC 700022 = CCUG 35435 = NCTC 12925 = CIP 104716) is the type strain of M. boevrei.
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Thermonema rossianum sp. nov., a New Thermophilic and Slightly Halophilic Species from Saline Hot Springs in Naples, Italy
More LessSix slightly halophilic, thermophilic bacterial strains were isolated from saline hot springs along the Bay of Naples, Italy. These strains produce bright yellow colonies and have a filamentous morphology and an optimum growth temperature of about 60°C. Lipid composition and 16S ribosomal DNA sequence analyses showed that these strains belong to the genus Thermonema, a member of the cytophaga-flavobacter-bacteroides phylum. Growth was observed in medium containing 1 to 3% NaCl. The DNA G+C content was 50.9 mol%. DNA-DNA hybridization studies showed that these strains represent a new species of the genus Thermonema. We propose that strain NR-27T (T = type strain) and the other strains represent a new species, Thermonema rossianum. Strain NR-27 (= DSM 10300) is the type strain of this species.
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A Proposal To Reclassify Nocardia pinensis Blackall et al. as Skermania piriformis gen. nov., comb. nov.
More LessAbstractThe type strain of Nocardia pinensis was the subject of chemotaxonomic and 16S ribosomal DNA sequencing studies. The resultant nucleotide sequence was aligned with the sequences of representatives of the genera Corynebacterium, Dietzia, Gordona, Mycobacterium, Nocardia, Rhodococcus, and Tsukamurella, and phylogenetic trees were generated by using the Fitch-Margoliash, maximum-parsimony, maximum-likelihood, and neighbor-joining methods. It was evident from the phylogenetic analyses that N. pinensis represents a distinct phyletic line that is most closely associated with the Gordona clade. This genealogical evidence, together with chemotaxonomic and phenotypic data derived from this and previous studies, indicates that N. pinensis merits generic status within the family Nocardiaceae. Therefore, we propose that N. pinensis Blackall et al. 1989 be reclassified as Skermania piniformis gen. nov., comb. nov. The type strain of Skermania piniformis cleaved an array of conjugated substrates based on the fluorophores 7-amino-4-methylcoumarin and 4-methylumbelliferone.
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Genome Organization of Pseudomonas stutzen and Resulting Taxonomic and Evolutionary Considerations
More LessAbstractIn order to determine the genome variability within Pseudomonas stutzeri, 20 strains representing the seven described genomovars and strain JM300 were analyzed by using various resolution levels of rare cutting enzymes. XbaI and SpeI fingerprints revealed a high degree of heterogeneity of restriction patterns that did not correlate with the division into genomovars. However, a fragment pattern comparison led to the establishment of several groups of clonal variants within genomovars. One circular chromosome ranging in size from 3.75 to 4.64 Mb constitutes the genome of P. stutzeri strains. The I-CeuI, PaI, and SwaI low-resolution map of P. stutzeri type strain CCUG 11256 shows the locations of 12 genes, including rrn operons and the origin of replication. I-CeuI digests of the 20 strains studied plus the positions of six genes allowed a comparison of the rrn backbone organization within genomovars; the four rrn operons seemed to be at similar locations with respect to the origin of replication, as did the rest of the genes. However, a comparison of I-CeuI cleavage maps of the genomovar reference strains revealed a diverse genome organization in the genomovars relative to rrn operons and gene locations. In most genomovars, rrn operons are not arranged around the origin of replication but are equally distributed on the chromosome. Strain JM300 does not belong to any described genomovar, as determined from the organization of its genome. Large chromosomal rearrangements seem to be responsible for the differences in superordinate genome structure and must have played an important role in P. stutzeri diversification and niche colonization. An ancestral chromosome is suggested, and some plausible pathways for the generation of the various genome structures are proposed.
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Desulfonatronovibrio hydrogenovorans gen. nov., sp. nov., an Alkaliphilic, Sulfate-Reducing Bacterium
More LessA new alkaliphilic, sulfate-reducing bacterium, strain Z-7935T (T = type strain), was isolated from a soda-depositing lake, Lake Magadi in Kenya. This organism is a motile vibrio which utilizes only hydrogen and formate as electron donors and sulfate, sulfite, and thiosulfate, but not sulfur, as electron acceptors. Thiosulfate is dismutated. Strain Z-7935T is an obligately sodium-dependent alkaliphile which grows in sodium carbonate medium and does not grow at pH 7; the maximum pH for growth is more than pH 10, and the optimum pH is 9.5 to 9.7. The optimum NaCl concentration for growth is 3% (wt/vol). The optimum temperature for growth is 37°C. The G+C content of the DNA is 48.6 mol%. 16S ribosomal DNA sequence analysis revealed that strain Z-7935T represents a new lineage with genus status in the delta subclass of the Proteobac-teria. The name Desulfonatronovibrio hydrogenovorans gen. nov., sp. nov. is proposed for this organism; the type strain of D. hydrogenovorans is strain Z-7935 (= DSM 9292).
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Anaerobaculum thermoterrenum gen. nov., sp. nov., a Novel, Thermophilic Bacterium Which Ferments Citrate
More LessA thermophilic anaerobic bacterium designated strain RWcit2T (T = type strain) was isolated from the production water of a petroleum reservoir. The cells of this organism are straight to slightly curved rods that are gram negative and nonmotile. Spore formation has not been demonstrated. Growth occurs at temperatures ranging from 28 to 60°C, with optimum growth occurring at 55°C, and at pH values ranging from 5.5 to 8.6, with optimum growth occurring between pH 7 and 7.6. Growth occurs in media containing 0 to 20 g of NaCl per liter, and optimum growth occurs in the presence of 10 g of NaCl per liter. Strain RWcit2T grows on a range of organic acids, including citrate, pyruvate, malate, fumarate, and tartrate; on protein extracts; and on a limited number of carbohydrates. Sulfur, thiosulfate, and cystine are reduced to hydrogen sulfide. Sulfate, sulfite, and nitrate are not reduced. The DNA base composition is 44 mol% G+C. The 16S ribosomal DNA sequence revealed that strain RWcit2T is a member of the domain Bacteria and forms a branch that is approximately equidistant from Dictyoglomus thermophilum and Thermoanaerobacter spp. (level of similarity, 82%). Strain RWcit2T cannot be placed in any previously described taxon based on its phylogenetic and physiological traits and is named Anaerobaculum thermoterrenum gen. nov., sp. nov.
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Schwartzia succinivorans gen. nov., sp. nov., Another Ruminai Bacterium Utilizing Succinate as the Sole Energy Source
More LessAbstractFour strains of gram-negative, anaerobic, non-spore-forming bacteria that were curved rods which were motile by means of flagella originating from the concave side of the cells and which fermented succinate quantitatively to propionate were isolated from high dilutions of rumen ingesta obtained from cows on pasture. The bacteria were asaccharolytic and not proteolytic and did not ferment amino acids or peptides. Succinate was the only substrate fermented. Rumen fluid together with yeast extract was required for good growth on succinate. Growth on succinate was enhanced in the presence of fumarate. The strains did not grow at 22°C, and growth at 45°C was in all cases less than growth at 39°C. The cellular fatty acid compositions of all four strains were determined. The DNA base composition was about 46 mol% G+C. The complete 16S ribosomal DNA sequence of the type strain (strain Sl-1) was determined, and the phylogenetic relationships were analyzed. The most closely related genera were the genera Selenomonas, Zymophilus, and Pectinatus, whereas the recently described succinate-fermenting organism Succiniclasticum ruminis was distantly related. The name proposed for these strains is Schwartzia succinivorans gen. nov., sp. nov.; the type strain is strain S1-1 (=DSM 10502). These organisms are common inhabitants of the rumina of cows on pasture.
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Stenotrophomonas africana sp. nov., an Opportunistic Human Pathogen in Africa
More LessAbstractA gram-negative bacterium was isolated from a cerebrospinal fluid sample from an HIV-seropositive Rwandan refugee with primary meningoencephalitis. This Marseille-Goma sample B isolate, strain MGB1 (T = type strain), was found to exhibit evolutionary homology with Stenotrophomonas maltophilia, as determined by a 16S rRNA gene sequence analysis, and this finding was reflected by similar phenotypic traits. MGBT could, however, be distinguished from the 5. maltophilia type strain by using a number of biochemical and physiological tests, and a genotypic analysis of the two strains in which DNA homology was used revealed only 35% homology between them. Furthermore, the antibiotic susceptibility of MGBT was restricted to netilmicin, ciprofloxacin, trimethoprim-sulfamethoxazole, and colimycin. On the basis of these results we propose that MGBT is a representative of a new species in the genus Stenotrophomonas, Stenotrophomonas africana.
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Clostridium pascui sp. nov., a New Glutamate-Fermenting Sporeformer from a Pasture in Pakistan
More LessAbstractFour strains of an obligately anaerobic spore-forming bacterium were isolated from soil samples from a donkey pasture in Pakistan. Comparative 16S rRNA sequence analysis demonstrated that the strains are members of phylogenetic cluster I of the genus Clostridium (Collins et al. 1994). The strains are mesophilic, nonsaccharolytic, and nonproteolytic, utilize glutamate and histidine, and produce indole. Acetate, butyrate, ethanol, hydrogen, and carbon dioxide are the products of fermentation. Although the strains phenotypically resemble the classical glutamate-fermenting Clostridia, such as Clostridium cochlearium, Clostridium tetanomor-phum, Clostridium tetani, and especially Clostridium malenominatum, they differ from these organisms in sugar utilization, cellular fatty acid composition, and cellular protein pattern and by a 16S rRNA sequence divergence value of approximately 4 to 8%. Phylogenetically, the strains are more closely related to Clostridium estertheticum (sequence divergence, approximately 5%) and Clostridium subterminale (sequence divergence, approximately 5%) but are phenotypically readily distinguished from these species. On the basis of phenotypic and genotypic criteria, we conclude that the four strains are members of a new species of the genus Clostridium, for which the name Clostridium pascui is proposed. The type strain is strain Cm19 (= DSM 10365).
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Identification of Two Pathogenic Avian Mycoplasmas as Strains of Mycoplasma pullorum
More LessAbstractTwo mycoplasma strains were isolated from dead turkey embryos. Growth properties, biochemical and serological characteristics, and protein profiles indicated that these strains were closely related to Mycoplasma pullorum CKKT (T = type strain). This was confirmed by 16S rRNA sequence analysis, and the phylogenetic position of M. pullorum was established. Pathogenicity studies showed that the two strains, as well as M. pullorum CKKT, induced a statistically significant level of mortality after inoculation into chicken embryos.
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Spirochetes from Digital Dermatitis Lesions in Cattle Are Closely Related to Treponemes Associated with Human Periodontitis
More LessAbstractDigital dermatitis (DD), first described in 1974 by Cheli and Mortellaro (R. Cheli and C. Mortellaro, p. 208–213, in Proceedings of the 8th International Conference on Diseases of Cattle, 1974), is a major problem in dairy cows and beef cattle causing significant economic losses worldwide. Lesions are typically found at the volar skin proximal to the heel bulbs. Microscopic examination of biopsies or touch preparations of these lesions revealed a variety of different bacterial morphotypes including significant numbers of spirochetes which often represent the predominant morphotype. We used comparative 16S rRNA sequence analysis to determine the diversity and phylogeny of these hitherto unclassified DD spirochetes. Results indicate that those lesions looked at so far contained at least five spirochetal phylotypes, all clustering within the genus Treponema. Phylotype DDKL-4 was nearly identical (99.4% similarity) to that of a nonpathogenic human treponeme, T. phagedenis. Two phylotypes DDKL-3 and DDKL-13 were closely related to those from treponemes commonly found in human periodontitis lesions, i.e., T. denticola and T. vincentii, exhibiting 95 and 98% similarity, respectively. The other two phylotypes, DDKL-12 and DDKL-20, had no close relatives to any cultivable treponemal species but clustered to previously described group IV oral treponemes. Preliminary analysis using in situ hybridization with fluorescently labeled oligonucleotide probes against smears from DD biopsies revealed that from all lesions analyzed so far, T. denticola-like spirochetes were detected in the highest proportion of all spirochetal morphotypes.
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Intra- and Intergeneric Relationships of Various Actinomycete Strains Based on the Acyl Types of the Muramyl Residue in Cell Wall Peptidoglycans Examined in a Glycolate Test
More LessAbstractA total of 106 actinomycete strains representing 40 genera were studied to gain a better understanding of their intra- and intergeneric relationships by examining the types of acyl groups on the muramyl residues of the peptidoglycans in a glycolate test. The glycolyl type was found in about 43% of the actinomycete strains examined. These strains were distributed in the following two characteristic taxa: the actinoplanetes and their relatives, including species of the genera Actinoplanes, Catellatospora, Couchioplanes, Dactylosporangium, Glycomyces, Micromonospora, and Pilimelia; and the nocardioforms and species of the related genera Gordona, Nocardia, Rhodococcus, Tsukamurella, and Mycobacterium. Only glycolyl type bacteria were found to be present among the strains of these genera tested, except for a few doubtful strains. In contrast, the acetyl type was found extensively in bacteria belonging to the families Pseudonocardiaceae, Streptomycetaceae, Streptosporangiaceae, and Thermomonosporaceae and other taxa. The pattern of acyl type distribution among the actinomycetes correlates very well with the cell wall chemotype sensu Lechevalier and Lechevalier; that is, the chemotype I and III genera were all acetyl type taxa, the chemotype II genera were glycolyl type taxa, and the chemotype IV genera were split into a glycolyl, mycolate-containing group and an acetyl, non-mycolate group. We discuss the idea that combination of both the acyl type system and the cell wall chemotype system should provide a more useful tool for taxonomy of members of the order Actinomycetales.
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Taxonomic Study of Aromatic-Degrading Bacteria from Deep-Terrestrial-Subsurface Sediments and Description of Sphingomonas aromaticivorans sp. nov., Sphingomonas subterranea sp. nov., and Sphingomonas stygia sp. nov.
AbstractPhylogenetic analyses of 16S rRNA gene sequences by distance matrix and parsimony methods indicated that six strains of bacteria isolated from deep saturated Atlantic coastal plain sediments were closely related to the genus Sphingomonas. Five of the strains clustered with, but were distinct from, Sphingomonas capsulata, whereas the sixth strain was most closely related to Blastobacter natatorius. The five strains that clustered with S. capsulata, all of which could degrade aromatic compounds, were gram-negative, non-spore-forming, non-motile, rod-shaped organisms that produced small, yellow colonies on complex media. Their G+C contents ranged from 60.0 to 65.4 mol%, and the predominant isoprenoid quinone was ubiquinone Q-10. All of the strains were aerobic and catalase positive. Indole, urease, and arginine dihydrolase were not produced. Gelatin was not liquified, and glucose was not fermented. Sphingolipids were present in all strains; 2OH14:0 was the major hydroxy fatty acid, and 18:1 was a major constituent of cellular lipids. Acid was produced oxidatively from pentoses, hexoses, and disaccharides, but not from polyalcohols and indole. All of these characteristics indicate that the five aromatic-degrading strains should be placed in the genus Sphingomonas as currently denned. Phylogenetic analysis of 16S rRNA gene sequences, DNA-DNA reassociation values, BOX-PCR genomic fingerprinting, differences in cellular lipid composition, and differences in physiological traits all indicated that the five strains represent three previously undescribed Sphingomonas species. Therefore, we propose the following new species: Sphingomonas aromaticivorans (type strain, SMCC F199), Sphingomonas subterranea (type strain, SMCC B0478), and Sphingomonas stygia (type strain, SMCC B0712).
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Discrimination of Streptomyces albidoflavus Strains Based on the Size and Number of 16S-23S Ribosomal DNA Intergenic Spacers
More LessAbstractIn an attempt to develop a rapid and accurate method for discrimination of streptomycetes at the strain level, 21 strains identified by fatty acid analysis as Streptomyces albidoflavus and the type strains of nine subjective synonyms of S. albidoflavus were selected for a full or partial 16S ribosomal DNA (rDNA) sequence analysis and an investigation of the 16S-23S rDNA intergenic spacer region. 16S rDNA sequence analysis showed that 27 of the strains exhibited 100% sequence similarity; these strains included the type strain of S. albidoflavus and the type strains of the subjective synonyms Streptomyces canescens, Streptomyces coelicolor, Streptomyces felleus, Streptomyces limosus, Streptomyces odorifer, and Streptomyces sampsonii. The type strains of the other subjective synonyms of S. albidoflavus (i.e., Streptomyces gougerotii, Streptomyces intermedius, and Streptomyces rutgersensis) were found to have levels of 16S rDNA sequence difference of 1.0 to 1.1% when they were compared to the type strain of 5. albidoflavus. In order to discriminate between the strains which had identical 16S rDNA sequences, the 16S-23S rDNA intergenic spacer regions were amplified and cloned, and the sequences of the spacer regions were determined for four S. albidoflavus strains, including the type strain. The 16S-23S rDNA intergenic spacer region was found to vary in length and sequence composition among the strains and within each strain. The sizes and numbers of 16S-23S rDNA intergenic spacer regions for the 27 strains with identical 16S rDNA sequences were determined by high-resolution electrophoresis of FAM-labeled PCR products and a subsequent size analysis with GeneScan 672 software. This was shown to be a useful method for discrimination of S. albidoflavus strains. Strains with the same 16S-23S rDNA intergenic spacer band pattern, as determined by high-resolution electrophoresis of FAM-labeled PCR products, could be further discriminated on the basis of the sequence composition of the spacer region.
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Notes; A Phylogenetic Analysis of Aerobic Polychlorinated Biphenyl-Degrading Bacteria
More LessSeveral bacterial isolates were characterized based on their abilities to degrade specific polychlorinated biphenyls (PCBs) and their 16S rRNA gene sequences. The members of one group of bacteria consisting of Alcaligenes species, including the PCB-degrading bacterium Alcaligenes eutrophus H850, had strong abilities to degrade a broad range of PCBs but not the di-para-chlorine-substituted congeners. The members of another group, which included the PCB-degrading bacterium originally classified as Corynebacterium sp. strain MB1, had strong abilities to degrade di-para-chlorine-substituted PCBs. These bacteria were most likely different members of Rhodococcus species.
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Reassessment of the Phylogenetic Position of Caulobacter subvibrioides
More LessDetermination of the 16S rRNA gene sequence of Caulobacter subvibrioides ATCC 15264T (T = type strain) confirmed that this species is a member of the alpha subclass of the Proteobacteria and showed that it is phylogenetically most closely related to the Caulobacter group comprising the species Caulobacter bacteroides, Caulobacter crescentus, and Brevundimonas (Pseudomonas) diminuta, for which 16S rRNA sequences of the type strains are currently available. The closest known relative of strain ATCC 15264T among these species is B. diminuta (level of direct pairwise sequence similarity, 95%). On the basis of its previously determined 16S rRNA sequence (accession number M83797), C. subvibrioides is most closely related to Sphingomonas adhaesiva in the alpha-4 subgroup (level of similarity, 97.7%). Analysis of the hydroxy fatty acids of C. subvibrioides ATCC 15264T showed that the 2-hydroxymyristic acid which is characteristic of the genus Sphingomonas was absent.
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DNA Similarities among Mosquito-Pathogenic and Nonpathogenic Strains of Bacillus sphaericus
More LessBacillus sphaericus strains isolated on the basis of pathogenicity for mosquito larvae and strains isolated on the basis of a reaction with a B. sphaericus DNA homology group IIA 16S rRNA probe were analyzed for DNA similarity. All of the pathogens belonged to homology group IIA, but this group also contained nonpathogens. It appears inappropriate to designate this homology group a species based solely upon pathogenicity.
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Transfer of the Bacteriochlorophyll b-Containing Phototrophic Bacteria Rhodopseudomonas viridis and Rhodopseudomonas sulfoviridis to the Genus Blastochloris gen. nov.
More LessAbstractThe phylogenetic positions of the bacteriochlorophyll (BChl) b-producing budding phototrophic bacteria Rhodopseudomonas viridis and Rhodopseudomonas sulfoviridis were studied on the basis of 16S rRNA gene sequence information. These bacteria formed a tight cluster with the genus Rhodoplanes as a sister group within the alpha-2 subgroup of the Proteobacteria. Genomic DNA-DNA hybridization assays showed that R. viridis and R. sulfoviridis were closely related but were different species. Creation of the genus Blastochloris gen. nov. is proposed to accommodate these BChl b-producing species of phototrophic bacteria.
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Reclassification of Cytophaga aprica (Lewin 1969) Reichenbach 1989 in Flammeovirga gen. nov. as Flammeovirga aprica comb. nov. and of Cytophaga diffluens (ex Stanier 1940; emend. Lewin 1969) Reichenbach 1989 in Persicobacter gen. nov. as Persicobacter diffluens comb. nov.
More LessAbstractPhylogenetically, Cytophaga aprica and Cytophaga diffluens occupy independent positions in the flavobacter-bacteroides phylum. Both of these organisms are gram-negative rods that are motile by gliding, chemoor-ganotrophic, and aerobic, degrade several kinds of biomacromolecules, and inhabit marine environments. Their major isoprenoid quinone is menaquinone 7. The G+C content of the DNA of C. aprica is 35 to 37 mol%, and the G+C content of the DNA of C. diffluens is 40 to 42 mol%. In addition to constituting an independent phylogenetic lineage, each species has a distinctive cellular polyamine constitution. C. aprica is characterized by possessing cadaverine as its major polyamine, and C. diffluens is characterized by possessing spermidine, in contrast to most species of the genera Cytophaga, Flavobacterium, and Flexibacter and related organisms, which possess homospermidine. Transfer of C. aprica to the genus Flammeovirga gen. nov. as Flammeovirga aprica comb. nov. and transfer of C. diffluens to the genus Persicobacter gen. nov. as Persicobacter diffluens comb. nov. are proposed.
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Comparison of the 16S/23S Ribosomal Intergenic Regions of “ Candidatus Liberobacter asiaticum” and “Candidatus Liberobacter africanum,” the Two Species Associated with Citrus Huanglongbing (Greening) Disease
More LessAbstract16S/23S intergenic spacer regions from the rRNA operons of two strains of ““ Candidatus Liberobacter asiaticum” and one strain of “Candidatus Liberobacter africanum” were cloned and sequenced. The intergenic spacers of the two “Candidatus L. asiaticum” strains studied are identical and contain the genes for isoleucine tRNA (tRNAIle) and alanine tRNA (tRNAAla) separated by 11 nucleotides. The intergenic spacer of the “Candidatus L. africanum” strain contains only one tRNA gene (tRNAAla). The level of homology between the intergenic spacers of the two liberobacter species is 79.46%. Ribosomal operons with 16S/23S spacer regions other than those studied might be present in the two ““ Candidatus Liberobacter” species.
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Transfer of Pseudomonas cissicola (Takimoto 1939) Burkholder 1948 to the Genus Xanthomonas
More LessAbstractThe type strain of Pseudomonas cissicola (Takimoto 1939) Burkholder 1948, strain ICMP 8561 (= NCPPB 2982), was examined. The strain has the determinative characteristics and the fatty acid profile of the genus Xanthomonas. The nutritional characteristics of the strain as determined by Biolog microplate screening were also consistent with identification as a Xanthomonas species. Some characteristics of the fatty acid profile and reactions to tests on the Biolog microplate were not consistent with characteristics of known Xanthomonas species, suggesting that the strain represents a previously unreported species.
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Confirmation of Strain VKM B-1733 as the Type Strain of Halorubrum distributum
A. Oren, M. Kamekura and A. VentosaAbstractIn a recent paper, Zvyagintseva et al. (I. S. Zvyagintseva, E. B. Kudryashova, and E. S. Bulygina, Microbiology [Engl. Trans. Mikrobiologiya] 65:352–354, 1996) proposed that strain 4p, deposited as VKM B-1739, should be the new type strain of Halobacterium (Halorubrum) distributum. This strain is sufficiently different from the validated type strain (strain 1m [= VKM B-1733]) to be classified in a different species. The proposed establishment of a neotype strain for Halorubrum distributum while the originally designated type strain is extant is in conflict with Rule 18c of the International Code of Nomenclature of Bacteria. To avoid all confusion, strain VKM B-1733 should remain the type strain of Halorubrum distributum, and a new species may be proposed to include VKM B-1739 and a number of related isolates.
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Proposed Minimal Standards for Description of New Taxa in the Order Halobacteriales
A. Oren, A. Ventosa and W. D. GrantAbstractIn accordance with Recommendation 30b of the International Code of Nomenclature of Bacteria, which calls for the development of minimal standards for describing new species, we propose minimal standards for description of new taxa in the order Halobacteriales. The minimal standards include information on the following characteristics: cell morphology; motility; pigmentation; the requirement for salt to prevent cell lysis; optimum NaCl and MgCl2 concentrations for growth and range of salt concentrations enabling growth; temperature and pH ranges for growth; anaerobic growth in the presence of nitrate or arginine; acid production from a range of carbohydrates; ability to grow on single carbon sources; catalase and oxidase tests; hydrolysis of starch, casein, and Tween 80; sensitivity to different antibiotics; and polar lipids. The placement of a new taxon should be consistent with phylogeny, which is usually based on 16S rRNA nucleotide sequence information, and with DNA-DNA hybridization data in the case of descriptions of new species. This proposal has been endorsed by the members of the Subcommittee on the Taxonomy of Halobacteriaceae of the International Committee on Systematic Bacteriology.
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