- Volume 46, Issue 2, 1996

We characterized a microbial strain that was isolated from a hot spring at a geothermal area in Hakone, Japan. This isolate, whose lobed-shaped cells were about 1.0 μ in diameter, was a facultative chemolithoautotroph that required aerobic conditions for growth. The optimum pH was 3.0 (pH range, 1.0 to 4.0), and the optimum temperature was 70°C (temperature range, 50 to 80°C). Lithotrophically, this strain grew on elemental sulfur and reduced sulfur compounds. The G+C content of the genomic DNA was 38.4 mol%. This organism contained calditoglycerocaldarchaeol, which is characteristic of members of the Sulfolobaceae. The levels of 16S rRNA sequence similarity between the new isolate and Sulfolobus acidocaldarius, Sulfolobus solfataricus, and Sulfolobus shibatae were less than 89.8%. Unlike S. acidocaldarius, S. solfataricus, and S. shibatae, the new isolate utilized sugars and amino acids poorly as sole carbon sources, and the levels of DNA-DNA hybridization between the new isolate and these Sulfolobus species were very low. Phenotypically, the new isolate was also distinct from the obligately lithotrophic organism Sulfolobus metallicus. We concluded that the new organism belongs to a new Sulfolobus species, for which we propose the name Sulfolobus hakonensis.

The intrageneric structure of the genus Micromonospora was determined by analyzing the 16S ribosomal DNAs of the type strains of all but one of the 15 validly described species and four subspecies and 19 other Micromonospora strains, most of which represented invalidly described species. One of the 19 other Micromonospora strains grouped outside a phylogenetically tight genus Micromonospora, which appeared to be closely related to other genera belonging to the family Micromonosporaceae. The level of sequence similarity for the 16S ribosomal DNAs of most of the type strains was 98%. Values in the same range were also found when the sequences of the type strains and most of the other Micromonospora strains were compared. Whether the invalidly described species actually represent taxa that are worthy of species status should be investigated by performing DNA reassociation studies and thoroughly comparing physiological and chemotaxonomic properties.

Six moderately acidophilic, thermophilic bacterial strains with similar properties were isolated from geothermally heated water and sediment samples collected in New Zealand. These Gram stain-negative but Gram type-positive, rod-shaped bacteria formed oval terminal endospores. The cells were peritrichously flagellated and exhibited tumbling motility. At 60°C the pH range for growth was 3.8 to 6.8, and the optimum pH was 5.2 when the organisms were grown with xylose. At pH 5.2 the temperature range for growth was 35 to 66°C, and the optimum temperature was 60 to 63°C. The fermentation products from glucose or xylose were ethanol, acetate, lactate, CO2, and H2. The DNA G+C content was 34.5 to 35 mol%. On the basis of properties such as formation of elemental sulfur from thiosulfate, growth at acidic pH values at elevated temperatures, and the results of a 16S rRNA sequence comparison performed with previously validly published species belonging to the genus Thermoanaerobacterium, we propose that strain JW/SL-NZ613T (T = type strain) and five similar strains isolated from samples collected in New Zealand represent a new species, Thermoanaerobacterium aotearoense. Strain JW/SL-NZ613T (= DSM 10170) is the type strain of this species.

The organism described in this paper, strain ST90T (T = type strain), is a thermophilic, spore-forming, rod-shaped sulfate reducer that was isolated from North Sea oil reservoir formation water. In cultivation the following substances were used as electron donors and carbon sources: H2-CO2, lactate, pyruvate, ethanol, propanol, butanol, and C3 to C10 and C14 to C17 carboxylic acids. Sulfate was used as the electron acceptor in these reactions. Lactate was incompletely oxidized. Sulfite and thiosulfate were also used as electron acceptors. In the absence of an electron acceptor, the organism grew syntrophically on propionate together with a hydrogenothrophic methanogen. The optimum conditions for growth on lactate and sulfate were 62°C, pH 6.7, and 50 to 200 mM NaCI. The G+C content was 56 mol%, as determined by high-performance liquid chromatography and 57 mol% as determined by thermal denaturation. Spore formation was observed when the organism was grown on butyrate or propanol as a substrate and at low pH values. On the basis of differences in G+C content and phenotypic and immunological characteristics when the organism was compared with other thermophilic Desulfotomaculum species, we propose that strain ST90T is a member of a new species, Desulfotomaculum thermocisternum. D. thermocisternum can be quickly identified and distinguished from closely related Desulfotomaculum species by immunoblotting.

We examined aerobic, thermophilic, gram-negative bacteria that were isolated from hot springs in Portugal and were identified as Thermus strains and placed in phenetic groups on the basis of their phenotypic characteristics. We determined the composition of the peptidoglycan, identified the respiratory quinones, and determined the mean base composition of the DNA, and the levels of DNA-DNA homology were determined by both the filter hybridization and reassociation rate methods. Thermus aquaticus, Thermus brockianus, and Thermus filiformis were not detected in this collection of organisms, although three Thermus thermophilus strains were identified. We propose that the isolates that belonged to phenetic clusters E and F are members of a new species, Thermus oshimai; the type strain of T. oshimai is strain SPS17.

Thiobacillus thermosulfatus ATCC 51520T (T = type strain) was isolated from sewage sludge samples enriched with elemental sulfur. The cells of this organism were gram negative, rod shaped, motile, facultatively autotrophic, and strictly aerobic and contained polyphosphate inclusions and polyhedral bodies. During growth on thiosulfate, the following intermediates were produced: tetrathionate, trithionate, and sulfate, and the pH was lowered from neutrality to around 2.5. Autotrophic growth was observed at pH values between 4.3 and 7.8 and at temperatures of 34 to 65°C; optimum growth occurred at pH 5.2 to 5.6 and 50 to 52.5°C. Ubiquinone Q8 was present in the respiratory chain. The DNA contained 61 ± 1 mol% G+C. No denitrification was observed under autotrophic and heterotrophic conditions. The cells produced a glycocalyx during growth in the presence of S0. As determined by a 16S rRNA gene sequence analysis, T. thermosulfatus is a distinct species that belongs to the beta subdivision of the Proteobacteria and is closely related phylogenetically to Thiobacillus perometabolis. The GenBank accession number for the complete 16S rRNA gene sequence of T. thermosulfatus is U27839.

We determined nearly complete flagellin gene sequences for Borrelia burgdorferi sensu lato isolates (11 isolates obtained from Ixodes persulcatus ticks and patients in Hokkaido, Japan, and 1 European isolate) representing six different restriction fragment length polymorphism (RFLP) ribotype groups following cloning of the PCR-amplified genes. These sequences were aligned with those of representatives of the three Borrelia species associated with Lyme disease, and a phylogenetic tree was construced by the Clustal method. On the Lyme disease borrelia portion of the tree, the species were clearly delineated into three different phylogenetic groups, in complete agreement with the division of B. burgdorferi sensu lato into three species. A phylogenetic analysis revealed that the representatives of RFLP ribotype groups IV, V, and VI clustered tightly with each other and belonged on the same branch as Borrelia garinii. We used the criteria that are currently used to delineate bacterial species and determined the levels of DNA relatedness for these Borrelia isolates. For the RFLP ribotype group IV, V, and VI isolates, the levels of DNA relatedness ranged from 79 to 88%, and the levels of relatedness to the reference strain of B. garinii ranged from 70 to 80%. The levels of DNA relatedness of the RFLP ribotype group IV, V, and VI isolates to the representatives of other species associated with Lyme disease ranged from 53 to 66%. All of these findings indicate that the RFLP ribotype group IV, V, and VI isolates should be included in the species B. garinii.

A new aerobic, obligately chemolithoautotrophic, thermophilic, sulfur-oxidizing bacterium, Thermothrix azorensis, was isolated from a hot spring on Sao Miguel Island in the Azores. The cells of this organism are gram negative, nonsporulating, and rod shaped. Filament formation appears to occur as a response to nonoptimal growth condition. Growth occurs at 63 to 86°C, and the optimum temperature is 76 to 78°C. The optimum pH range for growth is 7.0 to 7.5. The G+C content of the DNA of our isolate is 39.7 mol%. This isolate uses thiosulfate, tetrathionate, hydrogen sulfide, and elemental sulfur as energy sources. Of particular interest are the absence of Calvin cycle enzymes and the initial appearance of sulfide during the lag phase of growth of aerobic cultures grown on elemental sulfur. The subsequent formation of thiosulfate is followed by oxidation of the thiosulfate to sulfate. T. azorensis differs from the only other Thermothrix species that has been described, Thermothrix thiopara, by having higher optimum and maximum growth temperatures, by being an obligate chemolithoautotroph, and by its close but separate position on a 16S rRNA sequence-based phylogenetic tree. Our T. azorensis isolate has been deposited in the American Type Culture Collection as strain ATCC 51754T (T = type strain).

Chemotaxonomic and 16S ribosomal DNA sequence analyses of an isolate from the sputum of a patient with a mycobacterial lung infection clearly delineated a new species of the genus Tsukamurella. This new species can be defined on the basis of genotypic and phenotypic data. The name Tsukamurella pulmonis sp. nov. is proposed for this organism; the type strain is IMMIB d-1321T (= DSM 44142T). This isolate shows 44.2 and 36.2% DNA relatedness to Tsukamurella paurometabola DSM 20162T (T = type strain) and Tsukamurella inchonensis DSM 44067T, respectively.

Organisms with the typical characteristics of mycoplasmas were isolated from the genital tracts of female elephants. The results of growth inhibition tests, metabolic inhibition tests, indirect immunofluorescence tests, and immunobinding assays showed that the isolated mycoplasmas were identical and distinct from previously described Mycoplasma, Entomoplasma, Mesoplasma, and Acholeplasma species. These organisms represent a new species, for which the name Mycoplasma elephantis is proposed. M. elephantis ferments glucose, fructose, maltose, mannose, and sucrose, produces films and spots, does not hydrolyze arginine, esculin, and urea, does not reduce methylene blue, tetrazolium chloride, and potassium tellurite, does not possess phosphatase activity, and reduces resazurin. It lyses avian, ovine, and guinea pig erythrocytes. It does not adsorb erythrocytes. Cholesterol or serum is required for growth. The optimum growth temperature is 37°C. The G+C content of the DNA is 24.0 mol%. The type strain of M. elephantis is E42 (= ATCC 51980).

Strain DCB-2T (T = type strain) (T. Madsen and D. Licht, Appl. Environ. Microbiol. 58:2874–2878, 1992) is an anaerobic, spore-forming bacterium that is capable of reductive dechlorination of chlorophenols. The cells of this strain are rod shaped and 3.3 to 6 μm long by 0.6 to 0.7 μm wide and occur singly and in pairs. Short chains are formed. Spores are terminal. This bacterium is motile, and each cell has one or two terminal flagella. Cells in the exponential and stationary phases are gram negative. This organism does not hydrolyze gelatin and is indole positive and catalase negative, and the guanine-plus-cytosine content of its cellular DNA is 47 mol%. The optimum temperature for growth is 37°C. Only pyruvate and tryptophan are used as substrates. Pyruvate and 2,4,6-trichlorophenol are converted to acetate, CO2, and 4-chlorophenol by strain DCB-2T. When grown on pyruvate, this bacterium produces sulfide if thiosulfate or sulfite is added as an electron acceptor. Fe(III) is reduced to Fe(II), but Mn(IV) is not reduced. Sulfate is not reduced to sulfide in the presence of pyruvate or other carbon sources typically used by sulfate-reducing bacteria. Cytochrome c is present, but desulfoviridin is not. DCB-2T reductively dechlorinates 3-chloro-4-hydroxyphenylacetate to 4-hydroxyphenylacetate and conserves energy from the reaction. 16S rRNA sequencing revealed that strain DCB-2T clusters with the Clostridium-Bacillus subphylum and groups with Desulfitobacterium dehalogenans and Desulfotomaculum orientis. Desulfotomaculum orientis does not dechlorinate 2,4,6-trichlorophenol. On the basis of the phylogenetic and physiological differences and similarities of strain DCB-2T, Desulfitobacterium dehalogenans, and Desulfotomaculum orientis, we concluded that DCB-2T belongs to the genus Desulfitobacterium. We propose that strain DCB-2 is the type strain of Desulfitobacterium hafniense sp. nov.

Two Lactobacillus strains isolated from rye sourdough most closely resemble Lactobacillus oris on the basis of their physiological, morphological, and chemotaxonomic characteristics. A 16S ribosomal DNA sequence analysis showed that these two strains clustered with, but were separate from, L. oris, Lactobacillus vaginalis, and Lactobacillus pontis. The results of DNA-DNA hybridization experiments indicated that the new two isolates represent a new Lactobacillus species, for which we propose the name Lactobacillus panis; strain DSM 6035T is the type strain of this species.

Strain 30AT (T = type strain), which was isolated from an anaerobic bioreactor fed on waste from a potato starch factory in De Krim, The Netherlands, is a nonmotile, gram-positive, anaerobic, rod-shaped organism that is able to degrade various amino acids, including alanine, leucine, isoleucine, valine, serine, and threonine. Acetate is required as an electron acceptor for the utilization of alanine, valine, leucine, and isoleucine. Other growth substrates, including pyruvate, α-ketobutyrate, α-ketoisocaproate, α-keto-3-methylvalerate, α-ketoisovalerate, and peptone, are intermediates in amino acid catabolism. Strain 30AT utilizes neither the branched-chain amino acids nor alanine via interspecies hydrogen transfer with methanogenic and sulfate-reducing bacteria or via the Stickland reaction with proline or glycine as an electron acceptor. No growth occurs with the following electron acceptors: fumarate, nitrate, nitrite, sulfite, sulfate, and oxygen. Yeast extract is required for growth. Sugars are not degraded. The optimal temperature and optimal pH for growth are 39 to 43°C and 6.4 to 7.6, respectively. The results of a 16S rRNA sequence analysis phylogenetically placed strain 30AT in Clostridium group I (genus Clostridium sensu stricto), where it forms a new and distinct line of descent.

One strain of a thermophilic, slightly halotolerant bacterium was isolated from a thermally polluted industrial runoff near Salisbury, United Kingdom. This organism, strain PRD-1T (T = type strain), for which we propose the name Rubrobacter xylanophilus sp. nov., produces short gram-positive rods and coccoid cells and forms pink colonies. The optimum growth temperature is approximately 60°C. Unusual internal branched-chain fatty acids (namely, 12-methylhexadecanoic acid and 14-methyloctadecanoic acid) make up the major acyl chains of the lipids. The results of our 16S rRNA sequence comparisons showed that strain PRD-1T is related to Rubrobacter radiotolerans and that these two organisms form a deep evolutionary line of descent within the gram-positive Bacteria.

A bacterium isolated from a polluted stream, capable of metabolizing biphenyl, naphthalene, phenanthrene, and higher-molecular-weight polycyclic aromatic hydrocarbons (D. Gibson, V. Mahadevan, D. Jerina, H. Yagi, and H. Yeh, Science 189:295–297, 1975), was previously identified as Beijerinckia sp. strain B1. In this investigation, 16S rRNA gene sequencing, biochemical tests, fatty acid methyl ester analysis, polyacrylamide gel electrophoresis of protein, and DNA-DNA hybridization were used to determine the taxonomic relationship of Beijerinckia sp. strain B1. The sequence of the 16S rRNA gene of B1 was identical to that of Sphingomonas yanoikuyae ATCC 51230T. The biochemical tests, fatty acid analysis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of soluble proteins of strain B1 showed results similar to those of S. yanoikuyae. DNA-DNA hybridization indicated that B1 and S. yanoikuyae ATCC 51230T are 75% homologous at the DNA level. We propose that Beijerinckia sp. strain B1 be reclassified as S. yanoikuyae.

Five Bacillus strains isolated from Death Valley soil were shown to belong to a previously unidentified species, for which we propose the name Bacillus vallismortis. The type strain is strain DV1-F-3 (= NRRL B-14890). On the basis of previously published restriction digestion data, B. vallismortis is most closely related to Bacillus subtilis. At this time B. vallismortis can be distinguished from B. subtilis only by differences in whole-cell fatty acid compositions. DNA sequences, and levels of reassociation of genomic DNA.

The complete 16S rRNA sequences of 12 strains of Streptomyces spp., including potato scab pathogens, were determined. Among the strains of Streptomyces scabies that were isolated from diverse geographical areas and differed in melanoid pigment production, either no difference or one difference in sequence was observed. The sequence of S. scabies was most similar to the sequences of Streptomyces diastatochromogenes, Streptomyces bottropensis, and Streptomyces neyagawaensis, which belong to the Diastatochromogenes group. The levels of similarity of the 16S rRNA sequences of Streptomyces acidiscabies and S. scabies were almost the same as the levels of similarity between S. acidiscabies and other Streptomyces strains. Streptomyces sp. strain 91-Sy-13, which was isolated in Japan from potato scab and belongs to a distinct species on the basis of phenotypic characteristics and DNA relatedness, exhibited lower levels of 16S rRNA sequence similarity with other potato scab pathogens, as well as other Streptomyces species. The phylogenetic tree constructed on the basis of 16S rRNA sequence data showed that the Streptomyces spp. that cause potato scab composed unique branches. The results of our phylogenetic analysis based on complete 16S rRNA sequences confirmed the lack of close relationships among Streptomyces spp. that cause potato scab. Our findings suggest that potato scab is caused by phylogenetically diverse Streptomyces species and that the pathogenicities of these organisms developed independently.

A taxonomic characterization was carried out on strains of the bacteria that cause the brown ring disease of clams. On the basis of their phenotypic and genotypic characteristics, these strains can be considered to constitute a new taxonomic unit, distinct from other Vibrio species. The guanine-plus-cytosine content of the strains ranged between 42.9 and 45.5 mol% (43.2 mol% for the proposed type strain). DNA-DNA hybridization studies showed 100% intragroup relatedness, but levels of genetic relatedness to the reference strains of different Vibrio species tested ranged between 15 and 58%. The strains have all the properties characteristic of the genus Vibrio and can be clearly differentiated from other species of this genus by their growth at 4°C and their negative responses for growth at 30°C and in 6% NaCl, arginine dehydrolase, lysine decarboxylase, ornithine decarboxylase, and Voges-Proskauer reaction. The name Vibrio tapetis is proposed for the new species; strain B1090 (CECT 4600) is the type strain.

Different Rhizobium species may be identified by using polymorphisms in their glutamine synthetases (GSII) but not by their GSI profiles. We analyzed the GSs of various Rhizobium tropici and Rhizobium etli strains (which are capable of nodulating and fixing nitrogen in Phaseolus vulgaris beans), as well as strains of other species included for comparison. The GS polymorphisms were determined by identifying variations in native enzyme mobility (revealed by GS activity staining) and in the isoelectric points of the monomers (revealed by immunodetection with antibodies against the GS proteins) by using gel electrophoresis. Restriction fragment length polymorphism patterns obtained by hybridizing an internal fragment of the GSII gene obtained from R. etli with total fragmented DNAs from different strains clearly distinguished the different groups. GSII is a novel and useful marker for Rhizobium groups and species, and GSII data support R. tropici and R. etli as bona fide species.