- Volume 46, Issue 1, 1996

We 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.

The 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.

A 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.

We 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.

The 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.

A 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]).

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.

Five 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.

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.

In 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).

The 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.

Ten 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.

In 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.

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.

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.

We 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.”

An 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.

The 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.

The 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.