- Volume 45, Issue 3, 1995

The elevation of the two genotypes of Prevotella intermedia to species rank as P. intermedia and Prevotella nigrescens has increased the need for reliable differentiation between the two taxa. In this study, 53 strains, including strains whose species affiliations were known as well as fresh dental plaque isolates, were subjected to a multilocus enzyme electrophoretic analysis, DNA analyses in which we used whole genomic DNA, rRNA sequences, and an oligonucleotide specific for the former P. intermedia genotype II (P. nigrescens) as probes, and a sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of soluble cellular proteins. All of these tests consistently separated the strains into the same two distinct groups corresponding to P. intermedia and P. nigrescens, confirming that the two species constitute two distinct populations of bacteria. Each of the tests used independently provided reliable identification to the species level. A previously reported heterogeneity in the pattern of human immunoglobulin A1 (IgA1) degradation was not confirmed. No differences between species were observed. All of the strains induced total degradation of IgA1 within 48 h, a property that may be a virulence factor in periodontal disease development. The enzymes responsible for IgA1 degradation were not inactivated by the physiological proteinase inhibitors α2-macroglobulin and α1-proteinase inhibitor.

The 16S rRNA gene sequence of the type strain of Tissierella praeacuta (formerly Bacteroides praeacutus) was determined by PCR direct sequencing. A comparative sequence analysis showed that T. praeacuta is a member of the Clostridium subphylum of the gram-positive bacteria and has a close phylogenetic affinity with the species that form Clostridium cluster XII (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). Although T. praeacuta is gram negative and does not produce endospores, 16S rRNA sequence data showed that it is closely related genealogically (level of sequence similarity, 99.9%) to Clostridium hastiforme. On the basis of our results and the results of previous studies, a second species of Tissierella, Tissierella creatinini sp. nov., is described.

Bacillus infernus sp. nov. was isolated from ca. 2,700 m below the land surface in the Taylorsville Triassic Basin in Virginia. B. infernus was a strict anaerobe that grew on formate or lactate with Fe(III), MnO2, trimethylamine oxide, or nitrate (reduced to nitrite) as an electron acceptor, and it also grew fermentatively on glucose. Type strain TH-23 and five reference strains were gram-positive rods that were thermophilic (growth occurred at 61°C), halotolerant (good growth occurred in the presence of Na+ concentrations up to 0.6 M), and very slightly alkaliphilic (good growth occurred at pH 7.3 to 7.8). A phylogenetic analysis of its 16S rRNA indicated that B. infernus should be classified as a new species of the genus Bacillus. B. infernus is the only strictly anaerobic species in the genus Bacillus.

Witches’-broom disease of small-fruited acid lime (WBDL) is a severe disease caused by a mycoplasmalike organism (MLO) in the Sultanate of Oman and the United Arab Emirates. The WBDL MLO was characterized by studying its genome size, the sequences of its 16S ribosomal DNA and the 16S-23S ribosomal DNA spacer region, and hybridization profiles obtained by using WBDL MLO-specific probes. The size of the WBDL MLO genome is 720 kbp. Genomic similarities with the MLOs of sunhemp, sesame, and alfalfa phyllodies were demonstrated, and we found that the WBDL MLO belongs to the sunhemp phyllody phylogenetic subgroup.

Nine moderately alkalitolerant thermophilic bacteria with similar properties were isolated from water and soil samples obtained from Yellowstone National Park. These Gram-type-positive, rod-shaped bacteria produce cells with primary branches. The cells are peritrichous and exhibit only slight tumbling motility. At 60°C the pH range for growth is 6.9 to 10.3, and the optimum pH is 8.5. At pH 8.5 the temperature range for growth is 34 to 66°C, with an optimum temperature of 57°C. The strains are mainly proteolytic. The fermentation products from yeast extract are acetate, CO2, and H2. Fumarate added to minimal medium containing yeast extract is stoichiometrically converted to succinate, indicating that it is used as an alternative electron acceptor. The DNA G+C content is 33 to 34 mol%. On the basis of its unique properties, such as branch formation, growth at alkaline pH values at elevated temperatures, and the relative distance of its 16S rRNA sequence from those of other known bacteria, we propose that strain JW/YL-138T (T = type strain) and eight similar strains represent a new genus and species, Anaerobranca horikoshii. Strain JW/YL-138 is designated the type strain of the type species, A. horikoshii, which was named in honor of Koki Horikoshi, a pioneer in the field of alkaliphilic bacteria.

Four strains isolated from European eels in Valencia, Spain, were found to constitute a DNA relatedness group which is 0 to 50% related to the 13 species and DNA group 11 of the genus Aeromonas. Phenotypically, these strains have all of the properties that define the genus Aeromonas. However, they differ from the previously described Aeromonas species by three or more properties. The strains are positive for motility, growth at 37°C, indole production, and arginine dihydrolase activity. They exhibit negative reactions in tests for growth at 42°C and in thiosulfate-citrate-bile salts-sucrose medium (Oxoid), Simmons citrate tests, and tests for lysine and ornithine decarboxylase activities. They produce acid from salicin but not from l-arabinose, d-cellobiose, or lactose. All four strains hydrolyze esculin and arbutin but not elastin. They use l-serine as a sole carbon and energy source but cannot utilize l-arabinose, l-arginine, d-gluconate, or l-glutamine. The strains are resistant to ampicillin. The guanine-plus-cytosine content of the DNA is 59.4 to 60.8 mol%. The name Aeromonas encheleia sp. nov. is proposed for these strains; strain S181 (= CECT 4342) is the type strain. This new species is generally not pathogenic for eels or mice.

Eight phenotypically homogenous Moraxella-like strains were isolated from the nasal flora of healthy goats. Total genomic DNA-DNA hybridization, DNA base composition determination, and genetic transformation studies were performed to determine the relationships of these bacteria to the classical moraxellae. The eight new isolates exhibited very high levels of genetic affinity to Moraxella bovis, as shown by quantitative and qualitative genetic transformation data, and exhibited high DNA-DNA relative binding ratios to each other (63% or more) but lower levels of DNA homology with all of the other species investigated, including the closely related classical moraxellae. Our results, combined with the general morphologic and phenotypic profiles of these organisms, indicate that they should be classified with the classical moraxellae, and we propose the name Moraxella caprae for them. Strain 8897 (= CCUG 33297 = NCTC 12877) is the type strain of M. caprae.

A comprehensive DNA-DNA hybridization study was performed by using 183 strains of the genus Xanthomonas. This genus was shown to comprise 20 DNA homology groups which are considered genomic species. Four groups corresponded to the previously described species Xanthomonas albilineans, Xanthomonas fragariae, Xanthomonas oryzae, and Xanthomonas populi. The previously described species Xanthomonas campestris was heterogeneous and was divided into 16 DNA homology groups. One of these groups exhibited a high level of DNA homology with Xanthomonas axonopodis. The 62 pathovars represented in this study were allocated to appropriate species. Our results, together with previous taxonomic data, supported a comprehensive revision of the classification of the genus Xanthomonas. The species X. albilineans, X. fragariae, X. oryzae, and X. populi are not affected. The type species of the genus, X. campestris (Pammel 1895) Dowson 1939, is emended to include only the pathovars obtained from crucifers (i.e., X. campestris pv. aberrans, X. campestris pv. armoraciae, X. campestris pv. barbareae, X. campestris pv. campestris, X. campestris pv. incanae, and X. campestris pv. raphani). X. axonopodis Starr and Garces 1950 is emended to include 34 former X. campestris pathovars. The following species names are proposed: Xanthomonas arboricola sp. nov., including X. arboricola pv. corylina, X. arboricola pv. juglandis, X. arboricola pv. poinsettiicola (type C strains of the former X. campestris pathovar), X. arboricola pv. populi, and X. arboricola pv. pruni; Xanthomonas bromi sp. nov. for strains isolated from bromegrass; Xanthomonas cassavae (ex Wiehe and Dowson 1953) sp. nov., nom. rev.; Xanthomonas codiaei sp. nov., including type B strains of the former taxon X. campestris pv. poinsettiicola; Xanthomonas cucurbitae (ex Bryan 1926) sp. nov., nom. rev.; Xanthomonas hortorum sp. nov., including X. hortorum pv. hederae, X. hortorum pv. pelargonii, and X. hortorum pv. vitians; Xanthomonas hyacinthi (ex Wakker 1883) sp. nov., nom. rev.; Xanthomonas melonis sp. nov.; Xanthomonas pisi (ex Goto and Okabe 1958) sp. nov., nom. rev.; Xanthomonas sacchari sp. nov. for strains isolated from diseased sugarcane in Guadeloupe; Xanthomonas theicola sp. nov.; Xanthomonas translucens (ex Jones, Johnson, and Reddy 1917) sp. nov., nom. rev., including X. translucens pv. arrhenatheri, X. translucens pv. cerealis, X. translucens pv. graminis, X. translucens pv. hordei, X. translucens pv. phlei, X. translucens pv. phleipratensis, X. translucens pv. poae, X. translucens pv. secalis, X. translucens pv. translucens, and X. translucens pv. undulosa; Xanthomonas vasicola sp. nov., including X. vasicola pv. holcicola and X. vasicola pv. vasculorum (type B strains of the former taxon X. campestris pv. vasculorum); and Xanthomonas vesicatoria (ex Doidge 1920) sp. nov., nom. rev., which includes the type B strains of the former taxon X. campestris pv. vesicatoria. Differentiating characteristics were determined for the new species on the basis of metabolic activity on a range of carbon substrates by using the Biolog GN microplate system.

A new cellulolytic anaerobic clostridium was isolated from the intestinal tract of pigs. The single isolate was a gram-positive, motile rod, formed terminal to subterminal swollen sporangia, and required a fermentable carbohydrate for growth. Cellulose, cellobiose, maltose, starch, and glycogen supported growth, but glucose and fructose did not. The major end products from the fermentation of cellobiose were butyrate and formate; minor amounts of hydrogen and ethanol were also formed. Ruminal fluid (15%) or yeast extract (1%) was required for good growth. The optimum temperature for growth was 39 to 42°C, and the optimum pH was 6.8 to 7.2. Cell lysis occurred rapidly once stationary growth was reached. A 16S rRNA sequence analysis showed that the strain was related to a group of gram-positive anaerobes that includes Clostridium oroticum and the cellulolytic species Clostridium polysaccharolyticum and Clostridium populeti. The DNA base composition of the isolate is 38 mol% G+C. We propose the name Clostridium herbivorans for this organism; strain 54408 (= ATCC 49925) is the type strain.

Aerobic, thermophilic, gram-negative bacteria obtained from Yellowstone National Park that were placed in the genus Thermus on the basis of phenotypic data were examined by chemotaxonomic techniques to determine their peptidoglycan compositions, their respiratory quinones, their mean DNA base compositions, and their levels of DNA-DNA homology as determined by both the filter hybridization and reassociation rate methods. These isolates from hot springs included Thermus aquaticus strains and strains of a new genospecies. We propose the name Thermus brockianus for this new genospecies; strain YS38 is the type strain of this taxon. A collection of 10 strains, including the type strain of “Thermus thermophilus,” which were isolated from widely separated geothermal sites, exhibited high levels of DNA-DNA homology with each other and had similar physiological properties. Therefore, we propose that the species Thermus thermophilus (Oshima and Imahori) should be reestablished, with strain HB8 as the type strain.

To understand the phylogeny and taxonomy of eight new toluene-degrading denitrifying isolates, we performed a 16S rRNA sequence analysis and a gas chromatographic analysis of their cellular fatty acids and examined some of their biochemical and physiological features. These isolates had 16S rRNA sequence signatures identical to those of members of the beta subclass of the Proteobacteria. The levels of similarity were as follows: 97.9 to 99.9% among the new isolates; 91.2 to 92.4% between the new isolates and Azoarcus sp. strain S5b2; 95.3 to 96.2% between the new isolates and Azoarcus sp. strain BH72; and 94.8 to 95.3% between the new isolates and Azoarcus indigens VB32T (T = type strain). Phylogenetic trees constructed by using the distance matrix, maximum-parsimony, and maximum-likelihood methods showed that our eight denitrifying isolates form a phylogenetically coherent cluster which represents a sister lineage of the previously described Azoarcus species. Furthermore, the fatty acid profiles, the cell morphology, and several physiological and nutritional characteristics of the eight isolates and the previously described members of the genus Azoarcus were also similar. In contrast to the previously described members of the genus Azoarcus, the eight new isolates were capable of degrading toluene under denitrifying conditions. We concluded that these toluene-degrading denitrifiers are members of a new species of the novel nitrogen-fixing genus Azoarcus. We propose the name Azoarcus tolulyticus for these toluene-degrading denitrifying isolates and designate strain Tol-4 the type strain.

The ribosomal AT-L30 proteins from 81 species of the genus Streptomyces as listed by Williams et al. in Bergey’s Manual of Systematic Bacteriology were analyzed. My results provided further evidence that the genus Streptomyces is well circumscribed. On the basis of levels of AT-L30 N-terminal amino acid sequence homology, the strains were classified into four groups (groups I to IV) and a nongrouped category, whose members contained amino acid sequences characteristic of each species. A phylogenetic tree constructed on the basis of the levels of similarity of the amino acid sequences revealed the existence of six clusters within the genus. The first cluster contains the members of groups I and II together with several other species; the second cluster contains the members of groups III and IV and several other species; the third cluster contains Streptomyces ramulosus and Streptomyces ochraceiscleroticus; the fourth cluster contains only Streptomyces rimosus; the fifth cluster contains Streptomyces aurantiacus and Streptomyces tubercidicus; and the sixth cluster contains Streptomyces albus and Streptomyces sulphureus. Considerable agreement between the results of the AT-L30 analyses and the results of numerical phenetic classification was found, although there were numerous disagreements in details. For example, four groups (groups I to IV) defined by the AT-L30 analysis data did not correlate with the aggregate groups defined by numerical classification. In general, but not always, the species classified in a particular cluster in the numerical classification system had the same or similar AT-L30 terminal amino acid sequences. The AT-L30 analysis data were more consistent with the 16S rRNA analysis data than with the numerical classification data, indicating that there was a good correlation between the four groups defined by AT-L30 analysis data and the aggregate groups defined by 16S rRNA analysis data. I stress that discrepancies between results of phenetic analyses and results of phylogenetic analyses should be taxonomically significant and can be resolved by other taxonomic approaches, such as DNA relatedness analysis.

Taxonomic characteristics of seven bacterial strains which were isolated from soil and hydrolyze resistant curdlan were studied. These bacteria were aerobic, spore-forming rods, contained menaquinone 7 as a major quinone, contained anteiso-C15:0 and iso-C16:0 as major cellular fatty acids, had guanine-plus-cytosine contents of 50 to 52 mol%, and could be divided into two groups on the basis of physiological and chemotaxonomic characteristics and DNA-DNA hybridization data. We propose the following two new species: Bacillus curdlanolyticus for strains YK9, YK121, YK161, YK201, and YK203, with type strain YK9 (= IFO 15724); and Bacillus kobensis for strains YK205 and YK207, with type strain YK205 (= IFO 15729).

Chemotaxonomic and genomic 16S ribosomal DNA sequence analyses of two isolates obtained from two different clinical materials clearly delineated a new species of the genus Tsukamurella. This new species can be identified by its 16S ribosomal DNA similarity values, as well as its physiological characteristics. The name Tsukamurella inchonensis sp. nov. is proposed for these isolates, which are represented by strain IMMIB D-771T (= DSM 44067T) (T = type strain). This strain exhibits only 45% DNA relatedness to Tsukamurella paurometabola.

The Hyphomicrobium strains which we examined in this study are gram negative and facultatively methylotrophic and utilize methanol and monomethylamine, but not methane, by the serine pathway with activated formaldehyde incorporation. The cellular fatty acids include a large amount of straight-chain unsaturated C18:1 acid, and the hydroxy fatty acids include large amounts of 3-OH C14:0 and 3-OH C16:0 hydroxy acids. The major ubiquinone is ubiquinone Q-9. However, the DNA base compositions of these Hyphomicrobium strains are heterogeneous, ranging from 58 to 66 mol% guanine plus cytosine. Strains TK 0415T (T = type strain), TK 0414, TK 0416, and TK 0441 were distinguished from other Hyphomicrobium strains on the basis of physiological characteristics and DNA-DNA hybridization data. A new species, Hyphomicrobium denitrificans, is proposed for these organisms; the type strain of H. denitrificans is TK 0415 (= DSM 1869 = NCIB 11706 = Attwood and Harder strain X).

A new Corynebacterium species, Corynebacterium argentoratense was isolated from the throats of four human patients. It is characterized by the presence of chemotype IV, a cell wall, corynomycolic acids, and a G+C content ranging from 60 to 61 mol%. Strains belonging to this species exhibit high levels of DNA relatedness as determined by DNA-DNA hybridization experiments (S1 nuclease procedure) but no close DNA relatedness with related Corynebacterium species. Phytogenies based on comparative analyses of nearly complete small-subunit rDNA sequences confirmed the inclusion of this new species within the genus Corynebacterium and grouped it in a cluster with C. diphtheriae, C. ulcerans, C. pseudotuberculosis, and C. kutscheri. PCR experiments revealed an absence of the gene coding for diphtheria toxin. This new species can be identified by its mycolic acid pattern, fermentation of sugars, and enzymatic activities. Strain IBS B10697 (CIP 104296) is the type strain of C. argentoratense.

The taxonomic position of Gordona sp. strain DSM 44015T, isolated from the packing material of a biofilter used for biological odor abatement of animal rendering emissions, has been clarified by a polyphasic study comprising chemotaxonomic, sequencing, and phenotypic results. The strain possesses a wall chemotype IV, MK-9 (H2), as the predominant menaquinone; relatively long-chain mycolic acids (54 to 62 carbon atoms); and straight-chain, saturated, and monounsaturated fatty acids with considerable amounts of tuberculostearic acid. The polar lipids include phosphatidylethanolamine, and the G+C content of the DNA is 69 mol%. Similarity values for genes encoding 16S rRNA indicate that Gordona sp. strain DSM 44015T represents a new species within the genus Gordona for which the name Gordona hydrophobica is proposed.

A number of mycobacterial strains with similar growth characteristics, metabolic properties, and lipid compositions, which were previously placed in the Helsinki group (E. Brander, E. Jantzen, R. Huttunen, A. Juntunen, and M.-L. Katila, J. Clin. Microbiol. 30:1972-1975, 1992), were characterized by performing 16S rRNA gene sequencing. Of the 14 strains studied, 9 had a unique, previously undescribed sequence in the variable region of 16S rRNA. These nine strains, all of which were isolated from respiratory tract specimens, were nonpigmented and grew at 25 C to 45°C, reaching full colony size after 2 to 3 weeks. They produced arylsulfatase, nicotinamidase, and pyrazinamidase and were negative for Tween 80 hydrolysis, catalase, urease, and nitrate reductase activities, and niacin. Their glycolipid patterns were identical. A mycolic acid analysis performed by using thin-layer chromatography showed that these organisms contained alpha-mycolates, ketomycolates, and carboxy mycolates. Gas-liquid chromatography revealed that 2-eicosanol was the major alcohol and hexacosanoic acid was the major mycolic acid cleavage product. On the basis of their growth, biochemical, and lipid characteristics and their unique 16S rRNA sequence, we propose that these organisms should be assigned to a new species, Mycobacterium branderi. Comparative 16S rRNA sequencing revealed that this new species is closely related to Mycobacterium celatum, Mycobacterium cookii, and Mycobacterium xenopi. Strains 52157T (T = type strain) and 43548 have been deposited in the American Type Culture Collection as strains ATCC 51789 and ATCC 51788, respectively.

Representatives of the family Methanosarcinaceae were analyzed phylogenetically by comparing partial sequences of their methyl-coenzyme M reductase (mcrI) genes. A 490-bp fragment from the A subunit of the gene was selected, amplified by the PCR, cloned, and sequenced for each of 25 strains belonging to the Methanosarcinaceae. The sequences obtained were aligned with the corresponding portions of five previously published sequences, and all of the sequences were compared to determine phylogenetic distances by Fitch distance matrix methods. We prepared analogous trees based on 16S rRNA sequences; these trees corresponded closely to the mcrI trees, although the mcrI sequences of pairs of organisms had 3.01 ± 0.541 times more changes than the respective pairs of 16S rRNA sequences, suggesting that the mcrI fragment evolved about three times more rapidly than the 16S rRNA gene. The qualitative similarity of the mcrI and 16S rRNA trees suggests that transfer of genetic information between dissimilar organisms has not significantly affected these sequences, although we found inconsistencies between some mcrI distances that we measured and previously published DNA reassociation data. It is unlikely that multiple mcrI isogenes were present in the organisms that we examined, because we found no major discrepancies in multiple determinations of mcrI sequences from the same organism. Our primers for the PCR also match analogous sites in the previously published mcrII sequences, but all of the sequences that we obtained from members of the Methanosarcinaceae were more closely related to mcrI sequences than to mcrII sequences, suggesting that members of the Methanosarcinaceae do not have distinct mcrII genes.