- Volume 45, Issue 1, 1995

A moderately thermophilic, facultatively anaerobic, amylolytic bacterium was isolated from palm wine, a tropical alcoholic beverage that was sampled in Senegal. The cells were gram positive, catalase positive, non-spore forming, rod shaped, and slightly motile with peritrichous flagella. The strain which we examined did not possess cytochrome and produced l-(+)-lactate, acetate, ethanol, and formate but not hydrogen during carbohydrate fermentation. Growth occurred at pH values ranging from 5.4 to 8.5, and optimum growth occurred at around pH 7.0. The optimum temperature for growth was around 50°C, and the upper temperature limit for growth was 58°C. The guanine-plus-cytosine content of the DNA was 38.8 ± 0.2 mol%. A sequence analysis of the 16S rRNA gene revealed that the new organism is closely related phylogenetically to members of genus Bacillus. Despite the lack of spores, we propose that on the basis of phylogenetic characteristics, the new isolate should be classified as a new Bacillus species, Bacillus thermoamylovorans. The type strain is strain DKP (= Collection of Institut Pasteur CNCM 1-1378).

Polyphosphate-accumulating bacteria that were previously isolated from activated sludge and exhibited high phosphate removal activity were studied taxonomically and phylogenetically. These organisms were gram-positive, coccus-shaped, aerobic chemoorganotrophs that had a strictly respiratory type of metabolism in which oxygen was a terminal electron acceptor. They accumulated large amounts of polyphosphate under aerobic conditions. The major quinone was menaquinone MK-9(H4). The cell wall peptidoglycan contained ll-diaminopimelic acid. The guanine-plus-cytosine content of the DNA was 67.9 mol%. Our isolates were similar phenotypically and chemotaxonomically to Luteococcus japonicus, which was proposed recently as a new genus and species. However, our isolates differed from L. japonicus in cellular fatty acid composition and some other traits. A phylogenetic analysis based on 16S rRNA sequences showed that our isolate differ from the genus Luteococcus and other genera belonging to the high-G+C-content gram-positive group. Accordingly, we concluded that our strain NM-1T (T = type strain) should be assigned to a new genus and species, for which we propose the name Microlunatus phosphovorus.

Eight strains of mollicutes were isolated from pooled suspensions prepared from western black-legged ticks (Ixodes pacificus) collected in Oregon. Morphologic examination by electron and dark-field microscopic techniques showed that each strain consisted of a mixture of motile, tightly coiled helical cells, small coccoid cells with diameters ranging from 300 to 500 nm, and pleomorphic, straight or branched filamentous forms. All cellular forms were surrounded by a single cytoplasmic membrane, and there was no evidence of a cell wall. The organisms were filterable and fastidious in their growth requirements. The optimum temperature for growth was 30°C, but multiplication occurred at temperatures ranging from 23 to 32°C. The strains catabolized glucose but did not hydrolyze arginine or urea. The genome size of strain Y32T (T = type strain) was 2,220 kbp, and the DNA base composition (guanine-plus-cytosine content) of this organism was 25 ± 1 mol%. The eight isolates were serologically related to each other but were not related to 37 other type or representative strains belonging to the genus Spiroplasma. Strain Y32 (= ATCC 33835) is the type strain of Spiroplasma ixodetis sp. nov.

Mycoplasma sp. strain G145T (T = type strain) was isolated from a goat’s abscessed ankle. Strain G145T required cholesterol or serum for growth and possessed characteristics similar to those of other members of the genus Mycoplasma. This strain was serologically distinct from previously described Mycoplasma species and from a group of currently unnamed strains thought to belong to the genus Mycoplasma. Strain G145T hydrolyzed arginine, but did not hydrolyze urea or ferment glucose. The guanine-plus-cytosine content of the DNA was 29.6 mol%. We propose that strain G145 (= ATCC 27948) is the type strain of a new species, for which we propose the name Mycoplasma adleri.

Sequencing of the 16S ribosomal DNAs (rDNA) of two strains of Rhodococcus maris was performed to determine the relationship of this species to other mycolic acid-containing actinomycetes. For this purpose we also determined the 16S rDNA sequences for the type species of the genus Rhodococcus, Rhodococcus rhodochrous, and for Mycobacterium chlorophenolicum (formerly Rhodococcus chlorophenolicus), Rhodococcus erythropolis, Gordona bronchialis, and Gordona terrae, for which only partial sequence data have been available previously. The sequences of the two strains of R. maris were identical. The results of a distance matrix analysis indicated that R. maris is not a member of the genus Rhodococcus but is located between members of the genus Corynebacterium and members of the Rhodococcus-Nocardia-Mycobacterium-Gordona-Tsukamurella cluster. The finding that R. maris is phylogenetically isolated is supported by the presence of N-acetyl residues in the glycan moiety of the peptidoglycan and the lack of phosphatidylinositol and phosphatidylinositol mannosides, characteristics which distinguish this taxon from related taxa. On the basis of our results and previous findings, we propose that R. maris should be reclassified in a new genus, Dietzia. The type species is Dietzia maris comb. nov.

A polyphasic taxonomic study that included DNA-rRNA hybridizations, DNA-DNA hybridizations, DNA base ratio determinations, whole-cell protein and fatty acid analyses, and an examination of classical phenotypic characteristics was performed in order to classify human and veterinary isolates that resemble Bordetella avium. Twelve poultry isolates and two human isolates were assigned to a new species, for which we propose the name Bordetella hinzii. The position of this organism in the family Alcaligenaceae and various genotypic, phenotypic, and chemotaxonomic characteristics are described.

Because of the very similar physiological properties and base sequences of the 16S rRNAs of Bacillus cereus, Bacillus mycoides, and Bacillus thuringiensis, some taxonomists question the validity of separating these organisms into distinct species. DNA relatedness studies based on spectrophotometrically measured renaturation rates were carried out to determine the taxonomic relationships of the three species. A study of 58 strains revealed that the levels of relatedness between B. cereus and B. mycoides and between B. cereus and B. thuringiensis ranged from 22 to 44% and from 59 to 69%, respectively. On the basis of the moderately high levels of DNA relatedness which we determined, B. cereus and B. thuringiensis appeared to be genetically related but taxonomically distinct entities. The B. mycoides group was genetically distantly related to the B. cereus group and represented a separate taxon. Furthermore, our data indicated that the B. mycoides group consists of two genetically distinct groups, each of which represents a distinct species. In addition to rhizoidal colonial morphology and lack of motility, the B. mycoides group could be distinguished from B. cereus by differences in fatty acid profiles and acetanilide-producing activities.

Three isolates of a previously undescribed Dermatophilus sp. obtained from chelonids (two strains obtained from turtles and one strain obtained from a tortoise) were compared with 30 Dermatophilus congolensis isolates obtained from Australian mammals. The microscopic appearance, the colony morphology, and most biochemical test results for the chelonid isolates were characteristic of the genus Dermatophilus. Our isolates differed from the mammalian D. congolensis isolates in a number of cultural characteristics, including faster growth at 27°C than at 37°C, formation of two hemolysis zones around colonies on blood agar at 37°C in the presence of 10% CO2, poor motility, and production of a distinctive odor. The DNA restriction enzyme digestion and protein electrophoresis patterns of our strains were distinct. The electrophoretic mobilities of 11 enzymes differed from the mobilities observed with D. congolensis strains. A monoclonal antibody to a surface antigen of an ovine isolate did not react with zoospores or filaments of the chelonid isolates. Biochemical differences between our isolates and D. congolensis included the ability of the chelonid isolates to reduce nitrate to nitrite and the fact that the chelonid isolates exhibit collagenase activity in vitro. We propose that the chelonid isolates should be placed in a new species, Dermatophilus chelonae. Strain W16, which was isolated from a nose scab on a snapping turtle, is the type strain; a culture of this strain has been deposited in the American Type Culture Collection as strain ATCC 51576.

Biochemical, chemotaxonomic, and molecular methods were used to establish the precise taxonomic position of the Centers for Disease Control (CDC) coryneform group 2 bacteria. The results of a comparative 16S rRNA sequence analysis demonstrated that the CDC coryneform group 2 bacteria constitute a distinct species within the genus Actinomyces. Actinomyces pyogenes was found to be the closest genealogical relative of the CDC coryneform group 2 bacteria, although these taxa were readily distinguished from each other and other Actinomyces spp. by using phenotypic criteria. On the basis of our findings we propose the name Actinomyces bernardiae sp. nov. for the CDC coryneform group 2 bacteria. The type strain is DSM 9152 (CCUG 33419).

Campylobacter hyoilei sp. nov. is the name proposed for an organism formerly described as strain RMIT 32AT (T = type strain) and a group of similar bacteria isolated from intestinal lesions of pigs with proliferative enteritis. The phenotypic characteristics of these organisms indicated that they are closely related to each other and are not strains of other Campylobacter spp. commonly isolated from pigs. The results of probing of ClaI-, EcoRV-, or Bg/II-cleaved genomic DNAs from C. hyoilei strains with a radiolabeled DNA probe that distinguishes between Campylobacter jejuni and Campylobacter coli indicated that C. hyoilei and C. coli are closely related. However, the 16S rRNA sequence of the reference strain of C. hyoilei, RMIT 32AT, was four bases different from the 16S rRNA sequence of C. jejuni CCUG 11284T and five bases different from the 16S rRNA sequence of C. jejuni subsp. doylei CCUG 24567T, suggesting that C. hyoilei is more closely related to C. jejuni than to C. coli. Hybridization between DNA from C. hyoilei type strain RMIT 32A and DNAs from selected type and reference strains of other Campylobacter species and subspecies, including C. jejuni, C. jejuni subsp. doylei, C. coli, Campylobacter mucosalis, and Campylobacter hyointestinalis, as well as the other C. hyoilei strains (the RMIT 32AT-like isolates), revealed that high levels of DNA hybridization (>70%) occurred only between the reference strain and other strains of C. hyoilei.

Ten strains of thermophilic actinomycetes were isolated from waste and mushroom composts, as well as from the air of compost plants and a refuse incineration plant in Germany. These organisms produce white aerial mycelia and form hyphae with so-called pseudosporangia that fragment into rod-like structures. The organisms have type III cell walls (meso-diaminopimelic acid and whole-cell sugar type C), the phospholipid type is type PII, and mycolic acids are not present. The major menaquinone is MK-9(H4), and the fatty acids are mainly iso- and anteiso-branched fatty acids, hydroxy fatty acids, and 10-methyl-branched fatty acids. The guanine-plus-cytosine content of the DNA is 69 to 73 mol%. The chemotaxonomic markers (especially whole-cell sugar type C) and 16S ribosomal DNA sequence data indicated that these organisms represent a new genus of the order Actinomycetales, for which the name Thermocrispum is proposed. On the basis of phylogenetic and phenotypic data, this new genus is closely related to members of the family Pseudonocardiaceae and related taxa and contains two species: Thermocrispum municipale sp. nov. and Thermocrispum agreste sp. nov. The type species of the genus is T. municipale, with type strain MKD 35 (= DSM 44069), and the type strain of T. agreste is CHB 77 (= DSM 44070).

A spirochete which infects short-tailed shrews (Blarina brevicauda) and white-footed mice (Peromyscus leucopus) has been shown previously to be ultrastructurally and serologically distinct from other spirochetes. Two of the original isolates from Connecticut and Minnesota and 16 new isolates obtained from shrews captured in Minnesota were characterized phenotypically and genetically in this study. A comparative analysis of the 16S rRNA sequences of two shrew isolates and one mouse isolate and the 16S rRNA sequences of 16 other spirochetes and Escherichia coli revealed that these organisms exhibited low levels of similarity (range of similarity values, 73.9 to 77.8%; average level of similarity, 74.7%). The shrew and mouse isolates which we examined formed a deeply branching subgroup that was clearly distinct from the other genera of spirochetes examined. These and other results indicated that the new spirochetes represent a unique taxon in the order Spirochaetales. Accordingly, we propose that they should be classified as members of a new genus, Brevinema. The three strains of Brevinema which we examined had 16S rRNA sequences that were nearly identical. We also compared these isolates by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fatty acid and enzyme analyses, restriction endonuclease analysis, and Southern hybridization and found that the levels of genetic and phenotypic homogeneity among the strains were very high. We concluded that the isolates which we examined were members of a single species, for which we propose the name Brevinema andersonii. The type strain of Brevinema andersonii is CT11616 (= ATCC 43811). The guanine-plus-cytosine content of the DNA of this species was determined to be 34 to 36 mol% by the thermal denaturation method.

A thermophilic sulfate-reducing bacterium, Desulfacinum infernum, is described. This bacterium was isolated from produced formation water from a North Sea petroleum reservoir. In liquid culture, the cells are oval, 1.5 by 2.5 to 3 μm, nonmotile and gram negative. Spore formation has not been observed. Growth occurs at temperatures ranging from 40 to 65°C, with optimum growth occurring at 60°C, and at levels of salinity ranging from 0 to 50 g of NaCl per liter, with optimum growth occurring in the presence of 10 g of NaCl per liter. D. infernum grows on a range of organic acids, including formate, acetate, butyrate, and palmitate, and alcohols. D. infernum can grow autotrophically with H2. A vitamin supplement is required for growth. Sulfite and thiosulfate are used as electron acceptors. Sulfur and nitrate are not reduced. The DNA base composition is 64 mol% G+C. Phylogenetically, D. infernum clusters with members of the delta subdivision of the Proteobacteria. Its closest relatives are Syntrophobacter wolinii (level of similarity, 90.6%) and Desulfomonile tiedjei (level of similarity, 87.1%).

DNA-DNA hybridization studies were performed with members of the genus Porphyromonas. Porphyromonas salivosa NCTC11632T (T = type strain) exhibited an average level of intraspecies DNA-DNA hybridization with VPB 3313 and VPB 3444 of 95% and an average level of DNA-DNA hybridization with Bacteroides macacae ATCC 33141T of 81%. However, while the cat strains and monkey strain which I studied exhibited sufficient hybridization to be considered members of a single species, the tight DNA hybridization clustering and phenotypic differences suggested that the biovars isolated from cats and monkeys are distinct and can be recognized by colonial and growth characteristics, lipase activity, sorbitol utilization, proteinase patterns, and whole-cell protein profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. As B. macacae is a senior synonym of P. salivosa, Porphyromonas macacae comb. nov. is proposed.

Taxonomic studies of 26 putative Bacillus brevis strains (12 strains formerly assigned to groups 2, 4 through 7, and B and 14 ungrouped strains) were carried out. These organisms were placed in five distinct groups on the basis of phenotypic characteristics, the results of DNA base composition and reassociation analyses, and the results of cellular fatty acid and isoprenoid quinone composition analyses. Group 2, which contained three strains, was identified as Bacillus choshinensis, and group 4, which contained one strain, was identified as Bacillus parabrevis. The three remaining unidentified groups were clearly distinguishable phenotypically and genetically from the type strains of B. brevis and the recently described species Bacillus agri, Bacillus centrosporus, Bacillus migulanus, Bacillus choshinensis, and Bacillus parabrevis. Therefore, the names Bacillus reuszeri sp. nov., Bacillus formosus sp. nov., nom. rev., and Bacillus borstelensis sp. nov., nom. rev. are proposed for groups 5 (3 strains), 6 (3 strains), and 7 (16 strains), respectively. The S-layer proteins found in the three new species were immunologically and genetically similar to the S-layer proteins found previously in B. brevis, B. agri, B. centrosporus, B. choshinensis, and B. parabrevis. The G+C content of NRRL NRS-1206, the type strain of B. reuszeri sp. nov., was 46.5 mol%; the G+C content of NRRL NRS-863, the type strain of B. formosus sp. nov., nom. rev., was 47.2 mol%; and the G+C content of NRRL NRS-818, the type strain of B. borstelensis sp. nov., nom. rev., was 51.3 mol%.

Investigation of fatty acid and mycolic acid properties of certain members of the genera Nocardia and Rhodococcus indicated identical spectra for certain pairs of organisms. Binary 16S rDNA sequence homology for the pairs Rhodococcus rhodochrous and R. roseus, Nocardia calcarea and R. erythropolis, and N. restricta and R. equi ranged between 99.2 and 99.9% for each of these pairs of species. Corresponding DNA-DNA similarity values, obtained by the spectrophotometric method, were significantly higher than 70%. Because certain chemotaxonomic properties and the majority of published phenotypic characters support the high degree of relatedness, we propose to transfer R. roseus to R. rhodochrous. The transfer of N. calcarea to R. erythropolis, and the transfer of Nocardia restricta to Rhodococcus equi (M. Goodfellow, p. 1472–1481, in P. H. A. Sneath et al., ed., Bergey’s Manual of Systematic Bacteriology, vol. 2, 1986) is confirmed.

A mycoplasma strain designated ISM1499 was used to develop a mycoplasma genetic system (G. G. Mahairas and F. C. Minion, J. Bacteriol. 171:1775–1780, 1989; G. G. Mahairas, C. Jian, and F. C. Minion, Gene 93:61–65, 1990), but phenotypic inconsistencies led to the conclusion that this organism had been classified incorrectly as a member of the species Mycoplasma pulmonis. Studies were initiated to determine the proper taxonomic position of ISM1499, and on the basis of the results of our genetic analysis, this strain was assigned to the Acholeplasma oculi strain cluster. The base composition of strain ISM1499 was identical to the base composition of A oculi 19L, but not to the base composition of Acholeplasma laidlawii PG8 (28.3 and 30.7 mol% G+C, respectively). The taxonomic position of ISM1499 was examined by performing a parsimony analysis with 16S ribosomal DNA sequence data, and the results were compared with previous phylogenetic reconstructions. Our results indicated that ISM1499 is more closely related phylogenetically to A. oculi 19L than to A. laidlawii PG8 and JA1. Heterogeneity in the 16S ribosomal DNA sequences of A. oculi 19L and ISM1499 and in the 16S ribosomal DNA sequences of A. laidlawii PG8 and JA1 may indicate that unusual dissimilarities occur in the 16S ribosomal DNA sequences of members of the genus Acholeplasma.

The phylogenetic relationships of the genera belonging to the family Pseudonocardiaceae were examined by a novel approach, amino acid sequencing of ribosomal AT-L30 proteins. The results of partial amino acid sequencing of AT-L30 preparations revealed that the members of the family Pseudonocardiaceae are divided into four clusters; the first cluster contains the genus Actinopolyspora, the second cluster contains the genus Saccharopolyspora, the third cluster contains the genus Amycolatopsis, and the fourth cluster contains the genera Amycolata, Pseudonocardia, Saccharomonospora, and Kibdelosporangium, indicating a close phylogenetic relationship between the genera Amycolata and Pseudonocardia. The genus Actinokineospora is closely related to the genus Saccharothrix, and these two genera formed a cluster separate from the clusters for the genera of the Pseudonocardiaceae. These results agree in almost all respects with previous 16S rRNA sequencing work by Embley et al. (T. M. Embley, J. Smida, and E. Stackebrandt, Syst. Appl. Microbiol. 11:44–52, 1988) and Warwick et al. (S. Warwick, T. Bowen, H. McVeigh, and T. M. Embley, Int. J. Syst. Bacteriol. 44:293–299, 1994), thus supporting the proposal of Warwick et al. that the genera Amycolata and Pseudonocardia should be combined in an emended genus. Pseudonocardia. However, a discrepancy was found between the present study and that of Warwick et al. In the present study, the Nocardia-Rhodococcus group and the Saccharothrix-Actinokineospora group were both recovered within the clade for the family Pseudonocardiaceae.

Three heterotrophic bacterial strains were isolated from different locations in Puget Sound, Washington, by using biphenyl as the principal carbon source. These strains grow by using a limited number of organic compounds, including the aromatic hydrocarbons naphthalene, phenanthrene, anthracene, and toluene, as sole carbon sources. These aerobic, gram-negative rods are motile by means of single polar flagella. Their 16S rRNA sequences indicate that they are all members of the γ subdivision of the Proteobacteria. Their closest known relatives are the genera Methylobacter and Methylomonas (genera of methane-oxidizing bacteria), uncultured sulfur-oxidizing symbionts found in marine invertebrates, and clone FL5 containing 16S ribosomal DNA amplified from an environmental source. However, the Puget Sound bacteria do not use methane or methanol as a carbon source and do not oxidize reduced sulfur compounds. Furthermore, a 16S rRNA base similarity comparison revealed that these bacteria are sufficiently different from other bacteria to justify establishment of a new genus. On the basis of the information summarized above, we describe a new genus and species, Cycloclasticus pugetii, for these bacteria; strain PS-1 is the type strain of C. pugetii.