- Volume 46, Issue 1, 1996
Volume 46, Issue 1, 1996
- Original Papers Relating To Systematic Bacteriology
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Molecular Phylogeny of the Genus Frankia and Related Genera and Emendation of the Family Frankiaceae
The members of the actinomycete genus Frankia are nitrogen-fixing symbionts of many species of woody dicotyledonous plants belonging to eight families. Several strains isolated from diverse actinorhizal plants growing in different geographical areas were used in this study. The phylogenetic relationships of these organisms and uncharacterized microsymbionts that are recalcitrant to isolation in pure culture were determined by comparing complete 16S ribosomal DNA sequences. The resulting phylogenetic tree revealed that there was greater diversity among the Alnus-infective strains than among the strains that infect other host plants. The four main subdivisions of the genus Frankia revealed by this phylogenetic analysis are (i) a very large group comprising Frankia alni and related organisms (including Alnus rugosa Sp+ microsymbionts that are seldom isolated in pure culture), to which Casuarina-infective strains, a Myrica nagi microsymbiont, and other effective Alnus-infective strains are related; (ii) unisolated microsymbionts of Dryas, Coriaria, and Datisca species; (iii) Elaeagnus-infective strains; and (iv) “atypical” strains (a group which includes an Alnus-infective, non-nitrogen-fixing strain). Taxa that are related to this well-defined, coherent Frankia cluster are the genera Geodermatophilus, “Blastococcus,” Sporichthya, Acidothermus, and Actinoplanes. However, the two genera whose members have multilocular sporangia (the genera Frankia and Geodermatophilus) did not form a coherent group. For this reason, we propose that the family Frankiaceae should be emended so that the genera Geodermatophilus and “Blastococcus” are excluded and only the genus Frankia is retained.
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Analysis of the Phylogenetic Relationships of Strains of Burkholderia solanacearum, Pseudomonas syzygii, and the Blood Disease Bacterium of Banana Based on 16S rRNA Gene Sequences
More LessWe determined nearly complete 16S rRNA gene sequences for 19 isolates of Burkholderia solanacearum, three isolates of the blood disease bacterium of bananas, and two isolates of Pseudomonas syzygii, the cause of Sumatra disease of cloves. The dendrogram produced by comparing all of these sequences revealed that there were two divisions, which corresponded to the results obtained previously in a restriction fragment length polymorphism analysis (D. Cook, E. Barlow, and L. Sequeira. Mol. Plant Microbe Interact. 2:113–121, 1989) and a total 16S ribosomal DNA (rDNA) sequence analysis of four isolates representing four biovars of B. solanacearum (X. Li, M. Dorsch. T. Del Dot, L. I. Sly, E. Stackebrandt, and A. C. Hayward, J. Appl. Bacteriol. 74:324–329, 1993). Division 1 comprised biovars 3, 4, and 5 and an aberrant biovar 2 isolate (strain ACH0732), and division 2 included biovars 1, 2, and N2, the blood disease bacterium, and P. syzygii. Specific nucleotides at positions 458 to 460 (UUC) and 474 (A) characterized division 2, whereas in division 1 the nucleotides at these positions were ACU and U, respectively. However, strain ACH0732 had a U at position 458, as did division 2 isolates, and G instead of U at position 474. Division 2 consisted of two subdivisions; one subdivision contained two B. solanacearum isolates that originated from Indonesia, P. syzygii strains, and blood disease bacterium strains, and the other subdivision contained all of the other division 2 isolates. Within division 1, the level of 16S rDNA sequence similarity ranged from 99.8 to 100%, and within division 2, the levels of 16S rDNA sequence similarity ranged from 99.1 to 100%. The division 1 isolates exhibited an average level of 16S rDNA sequence similarity to division 2 isolates of 99.3% (range, 99.1 to 99.5%). The occurrence of consistent polymorphisms in the 16S rDNA sequences of B. solanacearum strains, in particular unique 16S rDNA sequence differences in aberrant biovar 2 isolate ACH0732, and the occurrence of the Indonesian subdivision of division 2 suggest that this group is a rapidly evolving (tachytelic) group.
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Transfer of “Pseudomonas riboflavina” (Foster 1944), a Gram-Negative, Motile Rod with Long-Chain 3-Hydroxy Fatty Acids, to Devosia riboflavina gen. nov., sp. nov., nom. rev.
More LessThe taxonomic position of “Pseudomonas riboflavina” was studied by 16S rRNA gene sequencing and chemotaxonomic methods. This organism is a gram-negative, strictly aerobic rod and has a DNA guanine-plus-cytosine content of 61.4 mol%: the major isoprenoid quinone is ubiquinone 10, and the unusual cellular fatty acids 3-hydroxytetracosenoic acid (3-OH 24:1) and 3-hydroxyhexacosenoic acid (3-OH 26:1) are the major 3-hydroxy cellular fatty acids. A phylogenetic analysis based on 16S rRNA sequences revealed that “P. riboflavina” IFO 13584T (T = type strain) occupies an independent position in the α subclass of the Proteobacteria. On the basis of our data, we propose that “P. riboflavina” IFO 13584T should be transferred to the genus Devosia gen. nov. as Devosia riboflavina sp. nov., nom. rev.
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Rhodococcus percolatus sp. nov., a Bacterium Degrading 2,4,6-Trichlorophenol
More LessA bacterial strain that was able to mineralize 2,4,6-trichlorophenol was isolated from a chlorophenol-fed percolator and was identified as a member of the genus Rhodococcus on the basis of chemotaxonomic characteristics and 16S RNA phylogenetic inference data. This organism (strain MBS1T [T = type strain]) exhibited a typical irregular rod-coccus cycle, and the cells had fimbria-like structures on their surfaces. The diagnostic cell wall amino acid was meso-diaminopimelic acid, and the sugars were arabinose and galactose; the mycolic acids contained 46 to 54 carbon atoms. The main menaquinone was MK-8(H2), and MK-9(H2) was a minor component. The cellular phospholipids were phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositolmannoside, phosphatidylglycerol, and diphosphatidylglycerol. Tuberculostearic acid was present. The whole-cell fatty acids were straight-chain acids with 14 to 18 C atoms. The G + C content of the DNA was 67.4 mol%. This organism grew on sucrose, pyruvate, and 2,4,6-trichlorophenol, and it oxidized a large number of carbon compounds, including catechol, 3-hydroxyphenylacetic acid, and phenol. It also exhibited β-galactosidase, urease, and 2-acetyl-lactate decarboxylase activities. On a phylogenetic tree that was based on 16S ribosomal DNA gene sequences strain MBS1T was found among the rhodococci on an independent branch. On the basis of the chemotaxonomic and phenotypic characteristics of strain MBS1T and its phylogenetic position we suggest that this bacterium should be placed in a new species, Rhodococcus percolatus; the specific epithet was chosen because the organism was isolated by using an enriched percolator. The type strain is strain MBS1.
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Eubacterium minutum sp. nov., Isolated from Human Periodontal Pockets
More LessWe describe Eubacterium minutum sp. nov., which was isolated from human periodontal pockets. This new species was established on the basis of DNA-DNA hybridization data. The guanine-plus-cytosine content of its DNA is 38 to 40 mol%. The results of differential biochemical and enzymatic tests are described. The type strain of this species is strain M-6.
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Characterization of Legionella Species by Numerical Analysis of Whole-Cell Protein Electrophoresis
More LessThe results of a computer-assisted whole-cell protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of 291 isolates and 74 reference strains belonging to all known species of the genus Legionella revealed that the majority of the species of this genus can be adequately identified by this method. The type strain of Legionella bozemanii did not cluster with the other strains of this species, and the only strain of Legionella geestiana available clustered with the strains of Legionella feeleii. When we performed a numerical analysis by omitting certain portions of the pattern containing dense bands, all of the species could be distinguished. Our results also show that the type strains of Legionella nautarum and Legionella londiniensis deposited in the National Collection of Type Cultures do not correspond to the type strains deposited in the American Type Culture Collection. We used the results of a fatty acid and ubiquinone composition analysis to complement the SDS-PAGE results for several strains whose identities as determined by indirect immunofluorescence were doubtful. Computer-assisted SDS-PAGE of whole-cell proteins can be used in the classification of Legionella species and to identify and screen large numbers of isolates for further, in-depth taxonomic studies of smaller numbers of strains.
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Emended Description of Buttiauxella agrestis with Recognition of Six New Species of Buttiauxella and Two New Species of Kluyvera: Buttiauxella ferragutiae sp. nov., Buttiauxella gaviniae sp. nov., Buttiauxella brennerae sp. nov., Buttiauxella izardii sp. nov., Buttiauxella noackiae sp. nov., Buttiauxella warmboldiae sp. nov., Kluyvera cochleae sp. nov., and Kluyvera georgiana sp. nov.
More LessA total of 219 strains belonging to the genera Buttiauxella and Kluyvera were studied; 171 of these strains were isolated from mollusks, mainly snails and slugs, obtained from around the world. On the basis of DNA-DNA hybridization data, the strains were grouped into 11 genomospecies. A total of 44 phenotypic characters were used to differentiate the genera Buttiauxella and Kluyvera at the genus level and to identify genomospecies. There were significantly higher phenotypic probability distances between the genomospecies in the genus Buttiauxella and the genomospecies in the genus Kluyvera than between the genomospecies in the same genus. Therefore, the existence of Buttiauxella and Kluyvera as different genera was confirmed. The existence of new species necessitated broadening the definitions of both genera. In two cases, two Buttiauxella species could not be quantitatively differentiated biochemically, and several other pairs of species could be separated only by the results of one biochemical test. Nonetheless, combinations of several characteristics were used to differentiate all of the species with levels of certainty ranging from log 10.79 to log 57.77 (calculated as probability distances). The following new species are proposed: Buttiauxella ferragutiae (type strain, ATCC 51602 [DSM 9390]), Buttiauxella gaviniae (type strain, ATCC 51604 [DSM 9393]), Buttiauxella brennerae (type strain, ATCC 51605 [DSM 9396]), Buttiauxella izardii (type strain, ATCC 51606 [DSM 9397]), Buttiauxella noackiae (type strain, ATCC 51607 [DSM 9401]), Buttiauxella warmboldiae (type strain, ATCC 51608 [DSM 9404]), Kluyvera cochleae (type strain, ATCC 51609 [DSM 9406]), and Kluyvera georgiana (type strain, ATCC 51603 [DSM 9409]).
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Genomic Diversity and Differentiation among Phytoplasma Strains in 16S rRNA Groups I (Aster Yellows and Related Phytoplasmas) and III (X-Disease and Related Phytoplasmas)
Conserved gene sequences, including 16S rRNA and ribosomal protein gene sequences, were used to evaluate genetic variations in phytoplasma strains belonging to 16S rRNA groups I (aster yellows and related phytoplasmas) and III (X-disease and related phytoplasmas). We used PCR to amplify the sequences of the 16S ribosomal DNA and a segment of the ribosomal protein gene operon (encoding the 3’ region of rps19, all of rpl22, and rps3) from diverse phytoplasma group I and III strains. Additional chromosomal gene sequences of group I strains were also amplified. The PCR products amplified from members of each group of phytoplasmas were compared by performing restriction fragment length polymorphism (RFLP) analyses. On the basis of the RFLP patterns observed and similarity coefficients derived from combined RFLP analyses, the phytoplasma strains belonging to groups I and III were placed in distinct 16S rRNA, ribosomal protein, and 16S rRNA-ribosomal protein subgroups. Analyses of two or more conserved gene sequences revealed that members of the two groups were more diverse than previously thought. Subgroup differentiation on the basis of our combined analyses of 16S rRNA and ribosomal protein gene sequences seemed to adequately reflect the levels of chromosomal homology determined by DNA-DNA hybridization assays. On the basis of unique RFLP profiles, we identified new, previously unclassified group I phytoplasma strains, including the organisms that are associated with Ipomoea obscura witches’-broom [subgroup 16SrI-F(rr-rp)], maize bushy stunt [subgroup 16SrI-I(rr-rp)], and Mexican periwinkle virescence [subgroup 16SrI-J(rr-rp)], and new, previously unclassified group III phytoplasma strains, including the organism that is associated with pecan bunch [subgroup 16SrIII-H(rr-rp)]. On the basis of the results of our analyses of 16S rRNA and ribosomal protein conserved gene sequences, we recognized 9 group I subgroups and eight group III subgroups. We propose that phytoplasma strains belonging to each group I and III subgroup should be distinguished taxonomically at a level equivalent to the subspecies level.
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Bacillus ehimensis sp. nov. and Bacillus chitinolyticus sp. nov., New Chitinolytic Members of the Genus Bacillus
More LessFive chitin-degrading bacteria were isolated from soil. These organisms were strictly aerobic and rod shaped, formed spores, contained menaquinone 7 as the major isoprenoid quinone and 12-methyltetradecanoic acid as the major cellular fatty acid, and had guanine-plus-cytosine contents of 51.3 to 54.9 mol%, characteristics which indicate that they belong to the genus Bacillus. These five strains were divided into two groups on the basis of physiological characteristics and the results of a DNA-DNA hybridization study. As low levels of DNA relatedness were found between our isolates and previously described Bacillus strains, we propose that our isolates should be classified in two new Bacillus species, Bacillus ehimensis and Bacillus chitinolyticus. The type strains of B. ehimensis and B. chitinolyticus are strains IFO 15659 and IFO 15660, respectively.
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Two Coryneform Bacteria Isolated from the Surface of French Gruyère and Beaufort Cheeses Are New Species of the Genus Brachybacterium: Brachybacterium alimentarium sp. nov. and Brachybacterium tyrofermentans sp. nov.r †
New species names, Brachybacterium alimentarium and Brachybacterium tyrofermentans, are proposed for two coryneform bacteria isolated from the surfaces of Gruyère and Beaufort cheeses. These two species are similar in their biochemical and chemotaxonomic characteristics but distinct from previously described bacteria. The most distinctive characteristics are the presence of meso-diaminopimelic acid-containing peptidoglycan with a d-GIU-d-ASP interpeptide bridge and the presence of erythritol teichoic acids that contain diaminoglucuronic acid (an uncommon substituent). The menaquinone pattern of these organisms is unique among coryneform bacteria. DNA-DNA hybridization experiments revealed that the level of hybridization between the two organisms is 15%, which indicates that they are distinct species. Despite the unique biochemical characteristics of these bacteria, a 16S rRNA sequence comparison revealed that they are unquestionably related to Brachybacterium faecium, Brachybacterium nesterenkovii, and Brachybacterium conglomeratum. DNA-DNA hybridization experiments performed with these three species, B. alimentarium, and B. tyrofermentans revealed that the levels of complementarity ranged from 11 to 38%, values that are similar to the values obtained for Brachybacterium strains described previously. With the inclusion of B. alimentarium and B. tyrofermentans the genus Brachybacterium becomes somewhat heterogeneous with respect to chemotaxonomic characteristics.
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Agromyces mediolanus sp. nov., nom. rev., comb. nov., a Species for “Corynebacterium mediolanum” Mamoli 1939 and for Some Aniline-Assimilating Bacteria Which Contain 2,4-Diaminobutyric Acid in the Cell Wall Peptidoglycan
More LessIn the course of identifying aniline-assimilating bacteria, researchers found some gram-positive strains that contain 2,4-diaminobutyric acid in their cell wall peptidoglycans and menaquinone 12 as the predominant menaquinone. “Corynebacterium mediolanum” and “Flavobacterium dehydrogenans” also are known to contain 2,4-diaminobutyric acid in their cell walls, as well as menaquinone 12, but the taxonomic position of these organisms has not been established previously. We found that the aniline-assimilating strains, together with “C. mediolanum” and “F. dehydrogenans,” belong to a single species of the genus Agromyces, as determined by phenotypic characteristics, DNA-DNA relatedness data, and 16S ribosomal DNA sequence similarity data. The name Agromyces mediolanus sp. nov., nom. rev., comb. nov., is proposed for these organisms. The type strain of A. mediolanus is strain JCM 3346 (= ATCC 14004 = NCIMB 7206).
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Phylogenetic Relationships of the Filamentous Sulfur Bacterium Thiothrix ramosa Based on 16S rRNA Sequence Analysis †
More LessThe phylogeny of Thiothrix ramosa based on 16S rRNA sequences was determined. This species is the first species in this genus that has been shown to be capable of autotrophic growth with reduced sulfur compounds as sole energy sources. T. ramosa forms a monophyletic clade with Thiothrix nivea, as determined by distance, parsimony, and maximum-likelihood methods. Both of these species clearly belong to the gamma subdivision of the Proteobacteria, where they are loosely associated with other sulfur-oxidizing chemoautotrophic organisms.
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Bacillus haloalkaliphilus sp. nov.
More LessTen obligately alkaliphilic, extremely halotolerant Bacillus isolates were studied and compared with strain WN13T (T = type strain), an earlier isolate provided by H. G. Trüper. All of these strains produced round, terminally located spores in swollen sporangia. DNA-DNA hybridization values (78 to 91%) and phenotypic similarity analyses revealed that 10 of the 11 strains formed a relatively homogeneous group, and although one strain (strain AH/6/1) could not be distinguished phenotypically, it exhibited hybridization values of only 46 to 47%. This group of strains is sufficiently different from all previously validly described Bacillus species in its morphological, physiological, and biochemical properties that a separate species is considered appropriate, for which the name Bacillus haloalkaliphilus sp. nov. is proposed.
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16S rRNA and 16S to 23S Internal Transcribed Spacer Sequence Analyses Reveal Inter- and Intraspecific Bifidobacterium Phylogeny
More LessIn the last few years many attempts have been made to differentiate more than 20 Bifidobacterium species. It has been recognized that identification of bifidobacterial species is problematic because of phenetic and genetic heterogeneities. In order to contribute to our understanding of Bifidobacterium taxonomy, we studied Bifidobacterium phylogeny by performing both 16S rRNA and 16S to 23S (16S-23S) internally transcribed spacer (ITS) sequence analyses. In this study, we determined 16S rRNA sequences of five Bifidobacterium strains representing four species, and compared them with the sequences available in the GenBank database, and used them to construct a distance tree and for a bootstrap analysis. Moreover, we determined the ITS sequences of 29 bifidobacterial strains representing 18 species and compared these sequences with each other. We constructed a phylogenetic tree based on these sequence data and compared this tree with the tree based on 16S rRNA sequence data. We found that the two trees were similar topologically, suggesting that the two types of molecules provided the same kind of phylogenetic information. However, while 16S rRNA sequences are a good tool to infer interspecific links, the 16S-23S rDNA spacer data allowed us to determine intraspecific relationships. Each of the strains was characterized by its own ITS sequence; hence, 16S-23S rRNA sequences are a good tool for strain identification. Moreover, a comparison of the ITS sequences allowed us to estimate that the maximum level of ITS divergence between strains belonging to the same species was 13%. Our data allowed us to confirm the validity of most of the Bifidobacterium species which we studied and to identify some classification errors. Finally, our results showed that Bifidobacterium strains have no tRNA genes in the 16S-23S spacer region.
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Serological and Molecular Characterization of Mesoplasma seiffertii Strains Isolated from Hematophagous Dipterans in France
Three strains of nonhelical mollicutes previously isolated in France from two different mosquitoes and one tabanid fly were designated strains Ar 2328 (isolated from Aedes detritus), Ar 2392 (isolated from Aedes caspius), and CP 13 (isolated from Chrysops pictus). All of these strains exhibited properties of the genus Mesoplasma, a recently described genus of non-sterol-requiring mollicutes isolated from plants and insects. The results of metabolism inhibition and growth inhibition tests revealed that these strains and Mesoplasma entomophilum TAC or Mesoplasma florum L1 were not serologically related, but all three dipteran strains reacted strongly with Mesoplasma seiffertii F7T (T = type strain) antibodies. Using metabolism inhibition and growth inhibition tests, we found that the dipteran strains were related to each other and to strain F7T but were not identical. We also found that they were able to multiply and persist in the central nervous systems of suckling mice inoculated intracerebrally, a property that makes their use as biological control agents for pest dipterans inadvisable. Scanning electron microscopy revealed marked differences in the morphologies of the colonies of the different strains on SP4 solid medium. The levels of DNA-DNA homology for strains Ar 2328, Ar 2392, CP 13, and F7T were more than 70%, indicating that these strains are closely related members of the same species, M. seiffertii. In addition, one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that each strain produced about 40 protein bands. This technique also revealed differences between strains. Using the coefficient of Smeath-Jacquart, we constructed a dendrogram that allowed us to estimate of the levels of relatedness of these four strains. The results which we obtained were confirmed by two-dimensional protein electrophoresis results.
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Description of Bacillus carboniphilus sp. nov.
We observed activation of growth with six aerobic spore-forming isolates on an otherwise nonpermissive medium when a carbon material, such as graphite or activated charcoal, was added to the medium (Bacto Antibiotic Medium 3). On the basis of the phenotypic characteristics and physiological and biochemical properties of these organisms and DNA homology data we concluded that they belong to a new Bacillus species, which is designated Bacillus carboniphilus. Strain Kasumi 6 (= JCM 9731) is the type strain of this new species.
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“Condidatus comitans,” a Bacterium Living in Coculture with Chondromyces crocatus (Myxobacteria)
More LessWe describe the phylogenetic position and some taxonomically relevant characteristics of a small pleomorphic gram-negative bacterium that was cocultured with some strains of the myxobacterium Chondromyces crocatus that were isolated from the same geographic and ecological habitat. A 16S ribosomal DNA analysis revealed that the companion was a member of the “Cytophaga-Flavobacterium-Bacteroides” complex and was most closely related to members of the genus Sphingobacterium. The results of a fatty acid analysis, an isoprenoid composition analysis, and a DNA G+C content analysis and the presence of sphingolipids confirmed that this bacterium is affiliated with the genus Sphingobacterium. As the companion bacterium survived for only a few generations on solid media and could not be maintained in pure culture, we assign to this novel taxon that lives in close association with the myxobacterium C. crocatus Candidatus status as “Candidatus comitans.”
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Characterization of a New Obligately Anaerobic Thermophile, Thermoanaerobacter wiegelii sp. nov.
More LessAn obligately anaerobic, extremely thermophilic Thermoanaerobacter species was isolated from a freshwater pool formed from a geothermally heated (56 to 69°C) water outlet in Government Gardens, Rotorua, New Zealand. This organism was a spore-forming, gram-negative, rod-shaped bacterium. Strain Rt8.B1T (= DSM 10319T) (T = type strain) fermented a wide variety of mono-, di-, and polysaccharides and produced ethanol, acetate, lactate, propionate, and hydrogen. Sugar alcohols were also fermented, but organic acids and amino acids were not utilized. On the basis of its morphological characteristics, DNA G+C content, obligately anaerobic, thermophilic, polysaccharolytic nature, and levels of 16S rRNA sequence homology, we propose that strain Rt8.B1T should be classified in the genus Thermoanaerobacter as a new species, Thermoanaerobacter wiegelii.
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Cutting a Gordian Knot: Emended Classification and Description of the Genus Flavobacterium, Emended Description of the Family Flavobacteriaceae, and Proposal of Flavobacterium hydatis nom. nov. (Basonym, Cytophaga aquatilis Strohl and Tait 1978)
More LessThe phylogenetic positions and G+C contents of most species belonging to the genera Flavobacterium, Cytophaga, and Flexibacter and several related taxa were determined. Most of the strains included in this study belong to rRNA superfamily V, as shown by DNA-rRNA hybridization data, but the three main genera are highly polyphyletic. Several so-called Cytophaga and Flexibacter species isolated from soil and freshwater cluster with the type species of the genus Flavobacterium, Flavobacterium aquatile, and with Flavobacterium branchiophilum. The fatty acid and protein profiles of members of this group of organisms were determined. We provide an emended description of the genus Flavobacterium and propose new combinations for the following 7 of the 10 validly described species included in this genus: Flavobacterium columnare, Flavobacterium flevense, Flavobacterium johnsoniae (we also correct the specific epithet of this taxon), Flavobacterium pectinovarum, Flavobacterium psychrophilum, Flavobacterium saccharophilum, and Flavobacterium succinicans. A new name, Flavobacterium hydatis, is proposed for [Cytophaga] aquatilis Strohl and Tait 1978. The emended genus Flavobacterium contains bacteria that have the following main characteristics: gram-negative rods that are motile by gliding, produce yellow colonies on agar, are chemoorganotrophs and aerobes, decompose several polysaccharides but not cellulose, and are widely distributed in soil and freshwater habitats. Three Flavobacterium species are pathogenic for fish. The G+C contents of Flavobacterium DNAs range from 32 to 37 mol%. An emended description of the family Flavobacteriaceae is also provided.
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Characterization of the SF Agent, an Ehrlichia sp. Isolated from the Fluke Stellantchasmus falcatus, by 16S rRNA Base Sequence, Serological, and Morphological Analyses
More LessThe organism designated the SF agent was originally isolated in Japan in 1962 from Stellantchasmus falcatus metacercaria parasitic on gray mullet fish. The SF agent resembles members of the genus Ehrlichia morphologically and exhibits weak antigenic cross-reactivity with Ehrlichia sennetsu. This organism causes mild clinical signs in dogs, but severe splenomegaly and lymphadenopathy in mice. This suggests that the SF agent may be similar to either Neorickettsia helminthoeca, an intracellular parasite of a fluke and the cause of salmon poisoning disease in dogs, or E. sennetsu, the causative agent of human sennetsu ehrlichiosis in Japan and Malaysia. In order to determine the phylogenetic relationship between the SF agent and other ehrlichial species, the 16S rRNA gene was amplified by the PCR and sequenced. The SF agent sequence was most closely related to the sequences of Ehrlichia risticii (level of sequence similarity, 99.1%), the causative agent of Potomac horse fever, and E. sennetsu (level of sequence similarity, 98.7%). The next most similar sequence was that of N. helminthoeca, but the level of sequence similarity was only 93.7%. E. sennetsu, E. risticii, the SF agent, and N. helminthoeca formed a distinct cluster that was separated from all other ehrlichial species. As determined by immunofluorescence labeling, antiserum against the SF agent cross-reacted strongly with E. sennetsu, E. risticii, and N. helminthoeca. When three genetically distinct ehrlichial isolates obtained from horses with Potomac horse fever were compared with the SF agent, we found that the SF agent was most closely related to Ohio isolate 081, followed by IllinoisT (T = type strain) and a Kentucky isolate. We observed strong antigenic cross-reactivities and similarities in Western blot (immunoblot) reaction profiles when we compared the SF agent, E. risticii, and E. sennetsu; however, weaker antigenic cross-reactivity was observed when the SF agent and N. helminthoeca were compared. Our results indicate that the SF agent is antigenically more closely related to E. risticii and E. sennetsu than to N. helminthoeca. The biological and antigenic characteristics and the 16S rRNA sequence data suggest that the SF agent is a new species that belongs to the genus Ehrlichia.
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Vibrio ichthyoenteri sp. nov., a Pathogen of Japanese Flounder (Paralichthys olivaceus) Larvae
More LessSeven similar strains which were pathogens of Japanese flounder (Paralichthys olivaceus) larvae with opaque intestines had characteristics of the genus Vibrio. These strains were divided into two genomic species (species 1 containing six strains, and species 2 containing one strain) on the basis of the results of DNA-DNA hybridization experiments in which the membrane filter method was used, and these two species could be differentiated from each other by the following characteristics: acid production from d-galactose and utilization of d-glucuronate and β-hydroxybutyrate. Strain F-2, the type strain of species 1, exhibited levels of DNA relatedness with 29 previously described Vibrio species of 5 to 18%. The flounder isolates belonging to species 1 were also differentiated from the previously described Vibrio species phenotypically by the following characteristics: they were nitrate reduction positive; each cell had a single polar flagellum; they did not produce arginine dihydrolase, chitinase, gelatinase, and lipase; they did not utilize d-cellobiose and citrate; and they did not grow at 35°C. The G+C contents of the DNAs of four species 1 strains were 43 to 44 mol%. The name Vibrio ichthyoenteri sp. nov. is proposed for genomic species 1. The type strain of V. ichthyoenteri is strain F-2 (= IFO 15847). Species 2 was also considered a new genomic species, but a species name is not proposed in this paper because only one strain is available and the phenotypic variability of the species is not known.
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Culture and Characteristics of Helicobacter bizzozeronii, a New Canine Gastric Helicobacter sp.
More LessOrganisms whose cells were large, tight spirals were isolated from gastric biopsies of dogs. Touch cytology samples from all of the dogs contained large spiral organisms. Characteristics of 10 strains are described. These organisms were 5 to 10 μm long by 0.3 μm wide, and each cell had 10 to 20 sheathed flagella at both ends of the cell. The cells did not have periplasmic fibrils. These organisms were microaerophilic and grew at 37 and 42°C but not at 25°C on brain heart infusion agar containing blood. They did not grow on brucella blood agar. They were catalase and oxidase positive, hydrolyzed urea but not hippurate, reduced nitrate, and were resistant to nalidixic acid but susceptible to cephalothin and metronidazole. In contrast to Helicobacter felis, they hydrolyzed indoxyl acetate. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles of all of the strains were similar, and the protein patterns of these organisms differed from those of other Helicobacter spp. Dot blot DNA-DNA hybridization experiments revealed that the new strains were closely related to each other but clearly different from H. felis, Helicobacter pylori, Helicobacter mustelae, and Campylobacter jejuni. The name Helicobacter bizzozeronii sp. nov. is proposed for these organisms. Our results suggest that other “uncultured” gastric helicobacters may be cultured if optimal culture conditions are found.
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Analysis of the Genetic Polymorphism of Borrelia burgdorferi Sensu Lato by Multilocus Enzyme Electrophoresis
More LessIn recent years, Borrelia burgdorferi sensu lato has been subdivided into three species, Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii, and a new species restricted to Japan, Borrelia japonica, has been isolated from Ixodes ovatus. In addition, members of several new genomic groups have been found in America and in Europe, suggesting that there are additional genospecies. In order to study the diversity of B. burgdorferi sensu lato, we analyzed 54 isolates cultured from humans and from different tick species and obtained from diverse geographic areas, including Europe, the United States, Japan, and the People’s Republic of China. In order to investigate the genetic relationship between microorganisms that are transmitted by soft ticks and microorganisms that cause Lyme disease, we also included three strains of relapsing fever spirochetes. The method which we used was multilocus enzyme electrophoresis; 12 genetic loci were characterized on the basis of the electrophoretic mobilities of their products, and 50 distinct allele profiles (electrophoretic types) were distinguished. The mean genetic diversity per locus was 0.747. A cluster analysis of a matrix of genetic distances for pairs of electrophoretic types revealed 11 divisions that were separated at genetic distances greater than 0.65. Five of these divisions corresponded to B. burgdorferi sensu stricto. B. garinii, B. afzelii, B. japonica, and the newly proposed species “Borrelia andersonii”. Our results also confirmed that there are two additional genomic groups in Europe and at least one additional group in the United States. The relapsing fever spirochetes were not clearly separated from the spirochetes associated with Lyme disease. In conclusion, we believe that the previously proposed subdivision of B. burgdorferi sensu lato into only four species should be reconsidered.
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Phylogeny of the Sphaerotilus-Leptothrix Group Inferred from Morphological Comparisons, Genomic Fingerprinting, and 16S Ribosomal DNA Sequence Analyses
More LessPhase-contrast light microscopy revealed that only one of eight cultivated strains belonging to the Sphaerotilus-Leptothrix group of sheathed bacteria actually produced a sheath in standard growth media. Two Sphaerotilus natans strains produced branched cells, but other morphological characteristics that were used to identify these bacteria were consistent with previously published descriptions. Genomic fingerprints, which were obtained by performing PCR amplification with primers corresponding to enterobacterial repetitive intergenic consensus sequences, were useful for distinguishing between the genera Sphaerotilus and Leptothrix, as well as among individual strains. The complete 16S ribosomal DNA (rDNA) sequences of two strains of “Leptothrix discophora” (strains SP-6 and SS-1) were determined. In addition, partial sequences (approximately 300 nucleotides) of one strain of Leptothrix cholodnii (strain LMG 7171), an unidentified Leptothrix strain (strain NC-1), and four strains of Sphaerotilus natans (strains ATCC 13338T [T = type strain], ATCC 15291, ATCC 29329, and ATCC 29330) were determined. We found that two of the S. natans strains (ATCC 15291 and ATCC 13338T), which differed in morphology and in their genomic fingerprints, had identical sequences in the 300-nucleotide region sequenced. Both parsimony and distance matrix methods were used to infer the evolutionary relationships of the eight strains in a comparison of the 16S rDNA sequences of these organisms with 16S rDNA sequences obtained from ribosomal sequence databases. All of the strains clustered in the Rubrivivax subdivision of the β subclass of the Proteobacteria, which confirmed previously published conclusions concerning selected individual strains. Additional analyses revealed that all of the S. natans strains clustered in one closely related group, while the Leptothrix strains clustered in two separate lineages that were approximately equidistant from the S. natans cluster. This finding suggests that the tentative species “L. discophora” needs to be more clearly defined and compared with other species belonging to the genus Leptothrix.
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Tolumonas auensis gen. nov., sp. nov., a Toluene-Producing Bacterium from Anoxic Sediments of a Freshwater Lake
More LessA new toluene-producing bacterium, strain TA 4T (T = type strain), was isolated from anoxic sediments of a freshwater lake. The individual cells of this organism were nonmotile, gram-negative rods that were 0.9 to 1.2 by 2.5 to 3.2 μm. The optimum temperature and pH for growth were 22°C and pH 7.2, respectively. The G+C content of the DNA was 49 mol%. Toluene was produced from phenylalanine, phenylpyruvate, phenyllactate, and phenylacetate, and phenol was produced from tyrosine. Both the presence of a carbon source and the presence of a toluene precursor were essential for initiation of toluene production. Bacterial growth occurred under oxic and anoxic conditions. Acetate, ethanol, and formate were the major fermentation products of the bacterium when it was grown on glucose. The major lipoquinones were ubiquinone 8 and menaquinone 8 under both oxic and anoxic growth conditions. On the basis of the results of a 16S ribosomal DNA sequence analysis, we concluded that this organism is a member of the γ subclass of the Proteobacteria, and we suggest the name Tolumonas auensis for this species.
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Phylogenetic Analysis of Butyrivibrio Strains Reveals Three Distinct Groups of Species within the Clostridium Subphylum of the Gram-Positive Bacteria
More LessThe phylogenetic positions of 40 Butyrivibrio strains were determined by performing a comparative sequence analysis of the 16S rRNA genes of these organisms. We found that all of the strains which we studied belong to cluster XIVa (M. D. Collins, P. A. Lawson, A. Willems. J. J. Cordoba, J. Fernandez-Garayzabal, P. Garcia, J. Cai, H. Hippe, and J. A. E. Farrow, Int. J. Syst. Bacteriol. 44:812-826, 1994) of the Clostridium subphylum of the gram-positive bacteria, which also includes several Clostridium, Coprococcus, Eubacterium, and Ruminococcus species. We also found that the Butyrivibrio strains which we examined were genotypically heterogeneous and exhibited 12 distinct rRNA sequence types. The 12 rRNA sequence types formed three distinct lineages in cluster XIVa, which were separate from each other and from all other species belonging to this cluster. One lineage consisted of strains which exhibited a single rRNA and corresponded to the species Butyrivibrio crossotus. The second lineage consisted of 12 strains designated Butyrivibrio fibrisolvens which exhibited seven distinct rRNA sequence types. The type strain of B. fibrisolvens was a member of this lineage, but its position was peripheral. The third lineage comprised 26 B. fibrisolvens strains which exhibited four distinct rRNA sequence types. Tree topology and sequence divergence considerations indicated that the three lineages correspond to three separate genera and that the genus Butyrivibrio should be restricted to the group that contains the type strain of B. fibrisolvens.
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16S rRNA Gene Sequence Analysis Relative to Genomovars of Pseudomonas stutzeri and Proposal of Pseudomonas balearica sp. nov.
More LessWe compared the 16S rRNA gene sequences of 14 strains of Pseudomonas stutzeri, including type strain CCUG 11256 and strain ZoBell (= ATCC 14405), which represented the seven P. stutzeri genomovars (DNA-DNA similarity groups) that have been described. Our sequence analysis revealed clusters which were highly correlated with genomovar clusters derived from DNA-DNA hybridization data. In addition, we identified signature nucleotide positions for each genomovar. We found that the 16S rRNA gene sequences of genomovar 6 strains SP1402T (T = type strain) and LS401 were different enough from the sequence of the type strain of P. stutzeri that these organisms should be placed in a new species, Pseudomonas balearica. The type strain of P. balearica is strain SP1402 (= DSM 6083).
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Serpulina pilosicoli sp. nov., the Agent of Porcine Intestinal Spirochetosis
Phenotypic and genetic traits of porcine intestinal spirochete strain P43/6/78T (= ATCC 51139T) (T = type strain), which is pathogenic and weakly beta-hemolytic, were determined in order to confirm the taxonomic position of this organism and its relationships to previously described species of intestinal spirochetes. In BHIS broth, P43/6/78T cells had a doubling time of 1 to 2 h and grew to a maximum cell density of 2 x 109 cells per ml at 37 to 42°C. They hydrolyzed hippurate, utilized d-glucose, d-fructose, sucrose, d-trehalose, d-galactose, d-mannose, maltose, N-acetyl-d-glucosamine, d-glucosamine, pyruvate, l-fucose, d-cellobiose, and d-ribose as growth substrates, and produced acetate, butyrate, ethanol, H2, and CO2 as metabolic products. They consumed substrate amounts of oxygen and had a G+C content (24.6 mol%) similar to that of Serpulina hyodysenteriae B78T (25.9 mol%). Phenotypic traits that could be used to distinguish strain P43/6/78T from S. hyodysenteriae and Serpulina innocens included its ultrastructural appearance (each strain P43/6/78T cell had 8 or 10 periplasmic flagella, with 4 or 5 flagella inserted at each end, and the cells were thinner and shorter and had more pointed ends than S. hyodysenteriae and S. innocens cells), its faster growth rate in liquid media, its hydrolysis of hippurate, its lack of β-glucosidase activity, and its metabolism of d-ribose. DNA-DNA relative reassociation experiments in which the S1 nuclease method was used revealed that P43/6/78T was related to, but was genetically distinct from, both S. hyodysenteriae B78T (level of sequence homology, 25 to 32%) and S. innocens B256T (level of sequence homology, 24 to 25%). These and previous results indicate that intestinal spirochete strain P43/6/78T represents a distinct Serpulina species. Therefore, we propose that strain P43/6/78 should be designated as the type strain of a new species, Serpulina pilosicoli.
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Genomic Variability of Staphylococcus aureus and the Other Coagulase-Positive Staphylococcus Species Estimated by Macrorestriction Analysis Using Pulsed-Field Gel Electrophoresis
More LessThe genomic DNAs of 95 culture collection and hospital Staphylococcus aureus subsp. aureus strains of various origins, as well as the genomic DNAs of other coagulase-positive Staphylococcus species, were cleaved with restriction endonuclease Sma I and subjected to pulsed-field gel electrophoresis. The levels of similarity of the Sma I restriction patterns of the S. aureus subsp. aureus strains varied from 30 to 100%, which is considered characteristic of this species; thus, these organisms belonged to the same species restriction group. Within this range of similarity values 13 S. aureus intraspecies restriction groups were identified, and each group consisted of strains whose levels of similarity ranged from 65 to 100%. S. aureus subsp. aureus CCM 885T (T = type strain) belonged to the major intraspecies restriction group that comprised 39% of the S. aureus strains which we studied. The strains of the other coagulase-positive staphylococci, including Staphylococcus aureus subsp. anaerobius, Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus delphini, and Staphylococcus schleiferi subsp. coagulans, clustered with their type strains in separate restriction groups. S. aureus subsp. aureus exhibited almost no similarity to these species. We found 44-kb Sma I fragments in all of the S. aureus subsp. aureus and S. aureus subsp. anaerobius strains studied, and these fragments are considered characteristic of the species S. aureus. The high level of homology of these fragments was confirmed by the results of DNA hybridization experiments in which we used representatives of individual intraspecies restriction groups. Of the other staphylococci studied, only Staphylococcus epidermidis and one strain of S. hyicus contained these fragments. However, the levels of homology between these fragments and the fragments of S. aureus were found to be very low.
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Characterization and Identification of Marine Alteromonas nigrifaciens Strains and Emendation of the Description
Nine nonpigmented strains of gram-negative, aerobic, marine bacteria with polar flagella were isolated from the mussels Crenomytilus grayanus and Patinopecten jessoensis. These organisms were conspecific and exhibited relatively high levels of genetic relatedness (61 to 100%). The G+C contents of the DNAs of these strains were 38.5 to 40.2 mol%. The strains isolated from mussels were phenotypically distinct from previously described Alteromonas species that have similar DNA G+C contents (Alteromonas haloplanktis, Alteromonas tetraodonis, Alteromonas atlantica, and Alteromonas carrageenovora), and their DNAs exhibited only 12 to 41% similarity with the DNAs of the type strains of these species. DNA-DNA hybridization data revealed that the levels relatedness between the strains which we studied and the type strain of Alteromonas nigrifaciens were significant (66 to 70%). Production of a melanin-like pigment, which is characteristic of A. nigrifaciens, was observed only in tyrosine-containing media. The strains isolated from mussels were identified as A. nigrifaciens. We present an emended description of A. nigrifaciens that includes several phenotypic and chemotaxonomic characteristics.
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Spiroplasma diminutum sp. nov., from Culex annulus Mosquitoes Collected in Taiwan
Initially, strain CUAS-1T (T = type strain), which was isolated from a frozen triturate of Culex annulus mosquitoes collected in Taiwan, was thought to be a member of spiroplasma group VII. This placement was based on the spiroplasma deformation test titer observed when strain CUAS-1T spiroplasmas were tested with Spiroplasma monobiae MQ-1T antiserum. The results of subsequent reciprocal spiroplasma deformation, metabolism inhibition, and growth inhibition tests clearly revealed that strain CUAS-1T is not serologically related to previously described spiroplasma groups (groups I to XXIV) and thus is a representative of a new group, group XXV. Strain CUAS-1T was characterized by using the minimal standards for mollicute species descriptions. During logarithmic-phase growth, strain CUAS-1T cells are characteristically very short helices with 1.5 to 2 helical turns (1 to 2μm), highly motile, and bounded by a single trilaminar membrane and form granular colonies with satellites when the organism is grown aerobically on MID medium containing 1.6% agar. Growth in M1D broth occurs at temperatures ranging from 10 to 37°C, and the optimum temperature is 30°C. Substrate utilization tests revealed that cholesterol is required for growth, that glucose is hydrolyzed, and that arginine is not hydrolyzed both in the presence and in the absence of glucose. The genome of strain CUAS-1T is 1,080 kbp long, and the guanine-plus-cytosine content is 26 ± 1 mol%. On the basis of the results of our studies we propose that strain CUAS-1T (group XXV) should be placed in a new species, Spiroplasma diminutum. Strain CUAS-1 (= ATCC 49235) is the type strain of S. diminutum.
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Agrococcus jenensis gen. nov., sp. nov., a New Genus of Actinomycetes with Diaminobutyric Acid in the Cell Wall
More LessTwo strains of a new gram-positive coryneform bacterium isolated from soil and from a sandstone surface are described. Strain 2002-39/1T (T = type strain) is a coccoid, nonmotile, non-acid-fast, microaerophilic organism. The menaquinones of this strain are MK-12 and MK-11, and the main components of the whole-cell sugars are glucose and rhamnose. No mycolic acids are present. The G+C content of the DNA is 74 mol%. Comparative 16S ribosomal DNA studies and a cell wall analysis revealed that this strain represents a new genus belonging to the group of actinomycetes that have diaminobutyric acid in their peptidoglycans. The second strain, strain ST54, which was isolated from a sandstone surface, had the same characteristic features as strain 2002-39/1T. The name Agrococcus jenensis gen. nov., sp. nov., is proposed for these organisms. The type strain is strain 2002-39/1, which has been deposited in the German Collection of Microorganisms and Cell Cultures as strain DSM 9580.
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Phylogenetic Relationships among Rhizobium Species Nodulating the Common Bean (Phaseolus vulgaris L.)
More LessThe phylogenetic relationships among Rhizobium species that nodulate Phaseolus vulgaris (common bean) were determined by directly sequencing the amplified 16S ribosomal DNA genes of these organisms. The bean strains formed four separate clusters. One cluster was composed of Rhizobium leguminosarum bv. trifolii, R. leguminosarum bv. viciae, and R. leguminosarum bv. phaseoli. Two other clusters comprised Rhizobium etli and Rhizobium tropici, and the fourth cluster contained a single bean-nodulating strain. Data for species identification were obtained from DNA-DNA reassociation experiments. The levels of DNA relatedness among strains belonging to the three biovars of R. leguminosarum ranged from 58 to 67%. The levels of DNA relatedness between R. leguminosarum bv. phaseoli and R. etli and R. tropici ranged from 43 to 45% and 13 to 16%, respectively. The levels of DNA relatedness between the strain belonging to the fourth cluster and strains of the other three Rhizobium species that nodulate beans were less than 10%.
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Analyses of the Genomes of Chlamydial Isolates from Ruminants and Pigs Support the Adoption of the New Species Chlamydia pecorum
Analysis of the genomic DNAs of chlamydial isolates from sheep, cattle, and pigs was performed by Southern blot hybridization and by restriction endonuclease (RE) profiling of DNA amplified by PCR. The hybridization probes were derived from whole genomic DNA, the major outer membrane protein (MOMP) gene, the 16S rRNA gene, and an avian Chlamydia psittaci isolate plasmid. The PCR analysis used targets in the MOMP gene, the 16S rRNA gene, and the 60-kDa cysteine-rich protein gene. Together, the results showed that although there was considerable heterogeneity in the DNA sequence in the MOMP gene region, all the isolates had the same underlying total genomic RE profiles and yielded identical RE profiles for the rRNA and 60-kDa-protein gene regions. Most of the isolates were found to hybridize with the plasmid probe. Comparison of the MOMP sequence of one of the isolates (P787) with that of a known Chlamydia pecorum strain together with the results of the RE analyses allowed the conclusion that the isolates should all be classified within this new species.
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Sutterella wadsworthensis gen. nov., sp. nov., Bile-Resistant Microaerophilic Campylobacter gracilis-Like Clinical Isolates
Campylobacter gracilis (formerly Bacteroides gracilis) is an asaccharolytic, nitrate-positive, urease-negative organism that requires formate and fumarate or hydrogen as a growth additive and may pit agar media. Clinical isolates that were obtained primarily from appendiceal and peritoneal fluid specimens and initially were identified in our laboratory as B. gracilis were later found to include “unusual” strains that could be distinguished by biochemical and genetic criteria. These unusual C. gracilis strains were bile resistant, could not reduce tetrazolium chloride under aerobic conditions if formate and fumarate were added to the medium, and could grow in the presence of 2 or 6% oxygen if no blood was added to the medium. C. gracilis, other campylobacters, and the unusual strains produced distinctive dehydrogenase patterns when gels were incubated anaerobically. A cellular fatty acid analysis revealed that the cluster formed by the unusual organisms was distinct from the (separate) clusters formed by C. gracilis, Bacteroides ureolyticus, and other Campylobacter species. 16S rRNA sequence data indicated that these organisms are not related phylogenetically to either C. gracilis or other Campylobacter species; the most closely related taxa as determined by rRNA sequence analysis were unrelated aerobes (members of the genera Bordetella, Alcaligenes, Rhodocyclus, and Comamonas). DNA homology data confirmed that these taxa are separate groups. Our data indicate that the unusual organisms are members of a new genus and new species, for which we propose the name Sutterella wadsworthensis. The type strain of S. wadsworthensis is strain WAL 9799 (= ATCC 51579).
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Nocardia pseudobrasiliensis sp. nov., a New Species of Nocardia Which Groups Bacterial Strains Previously Identified as Nocardia brasiliensis and Associated with Invasive Diseases
We studied five strains of a new Nocardia taxon recently identified among Nocardia brasiliensis strains associated with invasive diseases (R. J. Wallace, Jr., B. A. Brown, Z. Blacklock, R. Ulrich, K. Jost, J. M. Brown, M. M. McNeil, G. Onyi, V. A. Steingrube, and J. Gibson, J. Clin. Microbiol. 33:1528-1533, 1995) to determine their taxonomic status. Several characteristics of these organisms, including the presence of chemotype IV cell walls, nocardomycolic acids, a predominant menaquinone similar to that of Nocardia asteroides ATCC 19247T (T = type strain), and G+C contents ranging from 67 to 68 mol%, are characteristics of the genus Nocardia. Phylogenies based on small-subunit ribosomal DNA sequences clearly confirmed that all five strains belong to the genus Nocardia and occur on a single branch that is clearly distinct from N. brasiliensis. This branch forms a clade with Nocardia vaccinii, Nocardia nova, Nocardia otitidiscaviarum, and Nocardia seriolae. The five new strains exhibited high levels of DNA relatedness with each other, as determined by DNA-DNA hybridization experiments (S1 nuclease procedure), but not with N. brasiliensis strains or with strains of the four phylogenetically related Nocardia species mentioned above. The five new strains differ from N. brasiliensis in the following characteristics: mycolic acid pattern, decomposition of adenine, nitrate reduction, and antimicrobial agent susceptibilities. Therefore, we propose that these strains belong to a new species, Nocardia pseudobrasiliensis. The type strain is strain ATCC 51512, which was isolated from a leg abscess on a patient suffering from ulcerative colitis.
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Fervidobacterium gondwanense sp. nov., a New Thermophilic Anaerobic Bacterium Isolated from Nonvolcanically Heated Geothermal Waters of the Great Artesian Basin of Australia
More LessA new thermophilic, carbohydrate-fermenting, obligately anaerobic bacterial species was isolated from a runoff channel formed from flowing bore water from the geothermally heated aquifer of the Great Artesian Basin of Australia. The cells of this organism were nonsporulating, motile, gram negative, and rod shaped and generally occurred singly or in pairs. The optimum temperature for growth was 65 to 68°C, and no growth occurred at temperatures below 44°C or above 80°C. Growth was inhibited by 10 μg of lysozyme per ml, 10 μg of penicillin per ml, 10 μg of tetracycline per ml, 10 μg of phosphomycin per ml, 10 μg of vancomycin per ml, and NaCl concentrations greater than 0.2%. The optimum pH for growth was 7.0, and no growth occurred at pH 5.5 or 8.5. The DNA base composition was 35 mol% guanine plus cytosine, as determined by thermal denaturation. The end products of glucose fermentation were lactate, acetate, ethanol, CO2, and H2. Sulfur, but not thiosulfate, sulfite, or sulfate, was reduced to sulfide. Phase-contrast microscopy of whole cells and an electron microscopic examination of thin sections of cells revealed the presence of single terminal spheroids, a trait common in members of the genus Fervidobacterium. However, a phylogenetic analysis of the 16S rRNA sequence revealed that the new organism could not be assigned to either of the two previously described Fervidobacterium species. On the basis of these observations, we propose that the new organism should be designated a new Fervido-bacterium species, Fervidobacterium gondwanense. The type strain of this species is strain AB39 (= Australian Collection of Microorganisms strain ACM 5017).
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Reclassification of Paenibacillus (formerly Bacillus) pulvifaciens (Nakamura 1984) Ash et al. 1994, a Later Subjective Synonym of Paenibacillus (formerly Bacillus) larvae (White 1906) Ash et al. 1994, as a Subspecies of P. larvae, with Emended Descriptions of P. larvae as P. larvae subsp. larvae and P. larvae subsp. pulvifaciens
A polyphasic taxonomic study of four strains of Paenibacillus larvae and four strains of Paenibacillus pulvifaciens (including duplicates of both type strains) supported the reclassification of both former Bacillus species into one species, P. larvae. Our conclusions were based on morphological and Analytab Products (API) tests, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins, gas chromatography of methylated fatty acids, pyrolysis mass spectrometry, DNA-DNA binding, and the following genomic fingerprinting methods: amplified ribosomal DNA restriction analysis, random amplified polymorphic DNA analysis, and AFLP analysis. The last method is a novel high-resolution DNA fingerprinting technique based on the selective amplification of restriction fragments. Despite more than 90% DNA relatedness between the strains studied, SDS-PAGE of whole-cell proteins, biochemical tests, and DNA fingerprinting (AFLP) distinguished between the P. larvae and P. pulvifaciens strains at the subspecies level. Taking this evidence along with differences in pathogenicity, we propose to reclassify the honeybee pathogens P. larvae and P. pulvifaciens as P. larvae subsp. larvae and P. larvae subsp. pulvifaciens. An emended description of the species and descriptions of the subspecies are given. The type strains are P. larvae subsp. larvae ATCC 9545 (LMG 9820) and P. larvae subsp. pulvifaciens NRRL B-3685 (LMG 6911 and ATCC 13537).
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Semantide- and Chemotaxonomy-Based Analyses of Some Problematic Phenotypic Clusters of Slowly Growing Mycobacteria, a Cooperative Study of the International Working Group on Mycobacterial Taxonomy
During previous cooperative numerical taxonomic studies of slowly growing mycobacteria, the International Working Group on Mycobacterial Taxonomy described a number of strains whose taxonomic status was ambiguous. A new study of DNA, RNA, and proteins from 66 of these organisms was performed to correlate their properties with phenotypic clustering behavior; the results of this study permitted 51 of the strains studied to be assigned to known species. The methods used to characterize the semantides included nucleotide sequencing and assessment of levels of semantide relatedness by affinity binding techniques, including whole DNA-DNA hybridization, probe hybridization, and antibody binding. There was good overall agreement between the phenotypic and chemotaxonomic clusters and the groups of organisms identified by semantide analyses. Our results supported the conclusion that we should continue to rely on polyphasic taxonomy to provide satisfactory systematic resolution of members of the genus Mycobacterium. We identified no single 16S rRNA interstrain nucleotide sequence difference value that unequivocally defined species boundaries. DNA-DNA hybridization remains the gold standard, but common resources are needed to permit DNA-DNA hybridization analyses to be made available to laboratories that are not prepared to use this technology. One of the large novel clusters which we studied corresponds to the recently described species Mycobacterium interjectum, a pathogen that resembles the nonpathogen Mycobacterium gordonae phenotypically. We also identified strains that appear to represent ribovars of Mycobacterium intracellulare which do not react with the commercial diagnostic probes that are currently used for identification of this species. Other branches or clusters consisted of too few strains to permit a decision about their taxonomic status to be made.
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Utilization of Fatty Acid Methyl Esters for the Differentiation of New Xanthomonas Species
L. VAUTERIN, P. YANG and J. SWINGSA database of the fatty acid profiles of the genus Xanthomonas containing the profiles of more than 1,200 authentic xanthomonad strains (P. Yang, L. Vauterin, M. Vancanneyt, J. Swings, and K. Kersters, Syst. Appl. Microbiol. 16:47–71, 1993) was reevaluated to provide data for descriptions and rapid identification of new Xanthomonas species. A total of 1,061 strains in the fatty acid database were grouped into the new species described in the classification of Vauterin et al. (L. Vauterin, B. Hoste, K. Kersters, and J. Swings, Int. J. Syst. Bacteriol. 45:472–489, 1995), and the average fatty acid profiles of the species were determined to obtain a representative fatty acid profile for each species. Within each species, the relative variation in each fatty acid was calculated to determine the potential of fatty acid data for discriminating between species. The fatty acid content of each Xanthomonas species and the relative variation in each fatty acid provide additional data for species descriptions. With the exception of Xanthomonas axonopodis in particular and Xanthomonas arboricola, Xanthomonas hortorum, and Xanthomonas campestris to lesser extents, most Xanthomonas species produce characteristic fatty acid profiles that can be used to differentiate them from other species.
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Spirochaeta alkalica sp. nov., Spirochaeta africana sp. nov., and Spirochaeta asiatica sp. nov., Alkaliphilic Anaerobes from the Continental Soda Lakes in Central Asia and the East African Rift
More LessDuring a study of microbial communities in athalassic bodies of water, three new species within the genus Spirochaeta were described. These are alkaliphilic Spirochaeta alkalica sp. nov. Z-7491 (DSM 8900) and halophilic S. africana sp. nov. Z-7692 (DSM 8902) from the soda-depositing Lake Magadi in Central Africa and haloalkaliphilic S. asiatica sp. nov. Z-7591 (DSM 8901) from Lake Khatyn, Central Asia. These mesophilic spirochetes develop at pHs of >9 as anaerobic saccharolytic dissipotrophs. The DNA base compositions (moles percent G+C) of the strains were as follows: S. alkalica Z-7491, 57.1; S. africana Z-7692, 56.1; and S. asiatica Z-7591, 49.2. The optimum growth parameters (temperature, pH, and NaCl concentration [percent, wt/vol], respectively) were as follows: for S. alkalica Z-7491, 35°C, 9.2, and 5 to 7%; for S. africana Z-7692, 35°C, 9.3, and 5 to 7%; and for S. asiatica Z-7591, 35°C, 8.9, and 3 to 6%. The products of glucose fermentation were acetate, hydrogen, ethanol, and lactate, in different proportions, for S. alkalica and S. africana; for S. asiatica, they were acetate, ethanol, and lactate. S. asiatica is strictly anaerobic, while S. alkalica and S. africana are rather aerotolerant. All three species group within the radiation of the majority of the species of the genus Spirochaeta. Studies of the genes encoding 16S rRNA indicate a possible fanning out of the phylogenetic tree of spirochetes.
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Emended Description of Thermosipho africanus as a Carbohydrate-Fermenting Species Using Thiosulfate as an Electron Acceptor
More LessWe found that Thermosipho africanus was able to ferment d-glucose, d-ribose, Maltose, and starch, while d-galactose, fructose, and sucrose were utilized poorly. Acetate, H2, and CO2, as well as small amounts of ethanol and lactate, were end products of glucose metabolism in this organism. The presence of thiosulfate as an electron acceptor greatly improved growth and increased acetate production from the sugars. The genus Thermosipho is the only genus in the order Thermotogales that has been described as a non-carbohydrate fermenter. We propose that the description of the genus Thermosipho be emended because the only species in this genus, T. africanus, is a carbohydrate fermenter that is able to utilize thiosulfate as an electron acceptor.
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Arbitrarily Primed PCR Analysis of Mycoplasma hyopneumoniae Field Isolates Demonstrates Genetic Heterogeneity
More LessMycoplasma hyopneumoniae is the primary agent of mycoplasmal pneumonia in swine. In this study we performed an arbitrarily primed PCR (AP-PCR) analysis, in which low-stringency amplification with a single primer was used, to investigate genetic variability in M. hyopneumoniae strains and field isolates. We performed preliminary experiments to examine the efficacy of 40 different 10-mer oligonucleotides for priming an AP-PCR with M. hyopneumoniae JT (T = type strain) chromosomal DNA. On the basis of our results, we selected primers OPA-3, OPA-17, and OPB-10 for use in an analysis performed with 23 field isolates. The most informative results were obtained with primer OPA-3. A total of 21 of 23 clinical isolates produced multiband patterns with this primer, while 2 isolates failed to produce any detectable bands. Our data show that M. hyopneumoniae is genetically diverse and that M. hyopneumoniae strains can be divided into at least six epidemiological subgroups on the basis of AP-PCR results.
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Species-Specific Sequences at the omp2 Locus of Brucella Type Strains
More LessA DNA sequence analysis of the omp2 locus of Brucella type strains revealed nucleotide differences that can be used for species identification. We developed specific probes which were used to verify the observed differences among the type strains following PCR amplification of portions of the omp2 locus.
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Classification of the Genus Mobiluncus Based on Comparative Partial 16S rRNA Gene Analysis
More LessOn the basis of partial 16S rRNA gene sequences and the results of Southern blot analyses, we confirmed the division of the genus Mobiluncus into the species Mobiluncus curtisii and Mobiluncus mulieris. Division of M. curtisii into M. curtisii subsp. curtisii and M. curtisii subsp. holmesii was not supported by our data.
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Reclassification of Lactobacillus casei subsp. casei ATCC 393 and Lactobacillus rhamnosus ATCC 15820 as Lactobacillus zeae nom. rev., Designation of ATCC 334 as the Neotype of L. casei subsp. casei, and Rejection of the Name Lactobacillus paracasei
More LessThe type strain of Lactobacillus casei subsp. casei (ATCC 393) exhibits low levels of DNA homology with other strains of L. casei subsp. casei (8 to 46%) and strains of Lactobacillus paracasei (30 to 50%), but exhibits a level of DNA similarity of 80% with Lactobacillus rhamnosus ATCC 15820, the original type strain of “Lactobacterium zeae” Kuznetsov 1959. Strains ATCC 393T (T = type strain) and ATCC 15820T are members of one protein profile cluster that is separate from the other Lactobacillus spp. The randomly amplified polymorphic DNA PCR profile of strain ATCC 393T is also different from the profiles obtained for the other species. L. casei ATCC 334T is genetically closely related to L. casei subsp. casei strains (71 to 97%) and L. paracasei strains (71 to 91%), is a member of the same protein profile cluster as these organisms, and shares several DNA amplicons with L. paracasei strains. On the basis of these results, we propose that L. casei subsp. casei ATCC 393T L. rhamnosus ATCC 15820 should be reclassified as members of Lactobacillus zeae nom. rev. (type strain, ATCC 15820), that strain ATCC 334 should be designated the neotype strain of L. casei subsp. casei, and that the name L. paracasei should be rejected.
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Phylogenetic Analysis of Fusobacterium prausnitzii Based upon the 16S rRNA Gene Sequence and PCR Confirmation
More LessIn order to develop a PCR method to detect Fusobacterium prausnitzii in human feces and to clarify the phylogenetic position of this species, its 16S rRNA gene sequence was determined. The sequence described in this paper is different from the 16S rRNA gene sequence of F. prausnitzii in the GenBank database (accession number M58682). A PCR assay based on the new sequence is specific for F. prausnitzii, and the results of this assay confirmed that F. prausnitzii is the most common species in human feces. However, a PCR assay based on the original GenBank sequence was negative when it was performed with two strains of F. prausnitzii obtained from the American Type Culture Collection. A phylogenetic tree based on the new 16S rRNA gene sequence was constructed. On this tree F. prausnitzii was not a member of the Fusobacterium group but was closer to some Eubacterium spp. and located between Clostridium “clusters III and IV” (M. D. Collins, P. A. Lawson, A. Willems, J. J. Cordoba, J. Fernandez-Garayzabal, P. Garcia, J. Cai, H. Hippe, and J. A. E. Farrow, Int. J. Syst. Bacteriol. 44:812–826, 1994).
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“Candidatus Microthrix parvicella,” a Filamentous Bacterium from Activated Sludge Sewage Treatment Plants
“Candidatus Microthrix parvicella” is a filamentous bacterium that grows with great difficulty in cultures from the mixed liquor of activated sludge sewage treatment plants. It is gram positive, and the ultrastructure of its cell walls has been determined to be of the gram-positive type by electron microscopical examination. Phylogenetically, it is a deep-branching member of the subphylum actinomycetes within the gram-positive phylum of the domain Bacteria. As for phenotypic features, it is known that the organism contains polyphosphate inclusions and that it is catalase positive. In mixed cultures in activated sludge plants and in pure culture in the laboratory, it has a characteristic and distinctive winding filamentous morphology, with filaments hundreds of micrometers long.
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Halobacterium salinarum nom. corrig., a Name To Replace Halobacterium salinarium (Elazari-Volcani) and To Include Halobacterium halobium and Halobacterium cutirubrum
A. VENTOSA and A. ORENThe specific epithet of Halobacterium salinarium is a grammatically incorrect form for the genitive of salinae (salt works). Therefore, we propose that the name of the type species of the genus Halobacterium should be changed to Halobacterium salinarum nom. corrig. This species includes strains often designated Halobacterium halobium or Halobacterium cutirubrum.
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The Phylogeny of Methanopyrus kandleri
More LessThe phylogenetic position of Methanopyrus kandleri has been difficult to determine because reconstructions of phylogenetic trees based on rRNA sequences have been ambiguous. The most probable trees determined by most algorithms place the genus Methanopyrus at the base of a group that includes the halobacteria and the methanogens and their relatives, although occasionally some algorithms place this genus near the eocytes (the hyperthermophilic, sulfur-metabolizing prokaryotes), suggesting that it may belong to this lineage. In order to resolve the phylogeny of the genus Methanopyrus, we determined the sequence of an informative region of elongation factor 1-alpha that contains an 11-amino-acid insertion in eocytes and eukaryotes which is replaced by a 4-amino-acid insertion in methanogens, halobacteria, and eubacteria. On the basis of the results of our elongation factor 1-alpha gene analysis, we concluded that the genus Methanopyrus diverged from the eocyte branch before the eukaryotic and eocyte lineages separated and therefore is not an eocyte.
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