- Volume 48, Issue 1, 1998

Medicago ruthenica [(L.) Ledebour] is native to Inner Mongolia where rhizosphere samples were collected for the isolation of 106 rhizobial cultures. Besides nodulating the original trap host, the isolates formed nitrogen-fixing symbioses with Phaseolus vulgaris. Only half of the isolates nodulated alfalfa (Medicago sativa). but these did not form nitrogen-fixing symbioses. Rhizobium tropici also formed nitrogen-fixing symbioses with Medicago ruthenica. A total of 56 distinctive muitilocus electrophoretic types (ETs) were identified among 94 of the 106 isolates which were analysed for variation in electrophoretic mobility of 12 enzyme loci. One isolate (USDA 1920) possessed a unique ET, while the ETs of the other isolates formed two weakly divergent subgroups approximately equal in size. It was concluded from small subunit rRNA gene sequences of eight isolates of Medicago ruthenica that they belonged to the genus Rhizobium and not to the genus Sinorhizobium which is more commonly associated with Medicago. Genomic similarity, determined from DNA hybridization analysis, between USDA 1920 and the strain representing the remaining isolates (USDA 1844) was lower than 20%. Based upon these observations it was concluded that at least three genomic species of rhizobia form nitrogen-fixing symbioses with Medicago ruthenica. One of these genomic species is R. tropici, another is represented by the single isolate USDA 1920 and the name Rhizobium mongolense is proposed for the third genomic species represented by USDA 1844.

Two extremely thermophilic archaea, designated W-12 and P-4, were isolated from a geothermal vent in the tidal zone of Whale Island, New Zealand, and from geothermally heated bottom deposits of the Bay of Plenty, New Zealand, respectively. Cells of isolate W-12 are irregular cocci, 0·3--1·2 pm in diameter, motile with polar flagella. The cell envelope consists of one layer of subunits with a major protein of M r 75000. Cells produce protrusions of different kinds: prostheca-like, chains of bubbles, or network of fimbriae. Cells of isolate P-4 are regular cocci, 0·7--1·0 µm in diameter, motile with polar flagella. The cell envelope consists of two layers of subunits; its major protein has an M r of 56000. Both organisms are obligate anaerobes, fermenting peptides in the case of strain W-12, or peptides and starch in the case of P-4. Elemental sulfur is required for growth and is reduced to hydrogen sulfide. The optimal growth temperature of the new isolates is in the range 80-88 °C. The optimal growth pH is 6·5-7·2. The G+C content of the DNA of strain W-12 is 50·6 mol%, and of strain P-4 is 53·3 mol%. Based on physiological characteristics, 16S rDNA sequence comparison and DNA base composition, the new isolates were considered to be members of the genus Thermococcus. The low level of DNA-DNA hybridization with the type strains of other Thermococcus species confirms the novel species status of the new isolates. The new isolates are described as Thermococcus gorgonarius sp. nov., with type strain W-12 (= DSM 10395T), and Thermococcus pacificus sp. nov., with type strain P-4 (= DSM 10394T).

Recently, a new procedure was developed which allowed for the first time the isolation of a hyperthermophilic archaeum tracked by 16S rRNA analysis from a terrestrial hot solfataric spring (‘Obsidian Pool’, Yellowstone National Park, WY, USA). This novel isolate is characterized here. Cells are round cocci with a diameter of 0·2-0·8 µm, occurring singly, in pairs, short chains and in grape-like aggregates. The aggregates exhibit a weak bluish-green fluorescence under UV radiation at 420 nm. The new isolate is an anaerobic obligate heterotroph, using preferentially yeast extract for growth. The metabolic products include CO2, H2, acetate and isovalerate. Growth is observed between 65 and 90 °C (optimum: 85 °C), from pH 5·0 to 7·0 (optimum: 6·5) and up to 0·7% NaCI. The apparent activation energy for growth is about 149 kJ mol−1. Elemental sulfur or hydrogen inhibits growth. The core lipids consist mainly of acyclic and cyclic glycerol diphytanyl tetraethers. The cell envelope contains a cytoplasmic membrane covered by an amorphous layer of unknown composition; there is no evidence for a regularly arrayed surface-layer protein. The G+C content is 46 mol%. On the basis of 16S rRNA sequence comparisons in combination with morphological, physiological and biochemical properties, the isolate represents a new genus within the Desulfurococcaceae, which has been named Thermosphaera. The type species is Thermosphaera aggregans, the type strain is isolate M11TLT (= DSM 11486T).

Thirty-three previously non-typable faintly pigmented Gram-negative anaerobic bacterial isolates, biochemically most closely related to Prevotella intermedia and Prevotella nigrescens, were analysed for enzymic reactions, cellular fatty acid (CFA) composition, electrophoretic mobility of malate and glutamate dehydrogenases, hybridization with P. intermedia and P. nigrescens species-specific oligonucleotide probes and, for genetic heterogeneity, by arbitrarily primed PCR (AP-PCR). P. intermedia ATCC 25611T and P. nigrescens ATCC 33563T were run in parallel for comparison. Twenty-nine isolates originated from the normal oral flora of 18 subjects (including five mother-child pairs), and four isolates from various infections. Except for a negative lipase reaction, enzymic profiles of the test isolates were similar to those of P. intermedia and P. nigrescens. Clustering of CFAs, electrophoretic mobility patterns, hybridization with DNA probes for P. intermedia and P. nigrescens, and AP-PCR band patterns of the test isolates differed from those of the type strains of P. intermedia and P. nigrescens, suggesting the existence, in humans, of a new anaerobic species of pigmented, moderately saccharolytic, indole-positive Gram-negative rods.

Complete 16S rRNA gene sequences of three representative strains of anaerobic, Gram-negative, pigmented, moderately saccharolytic, indole-positive bacteria isolated from the oral cavity of humans were determined. According to comparative analyses of the rRNA sequence data, this organism represents a previously unknown species within the genus Prevotella. In addition, 22 representative strains and 21 reference strains (including 11 Prevotella intermedia and 10 Prevotella nigrescens strains) were subjected to multilocus enzyme electrophoretic analysis. The strains were consistently separated into three clearly distinct groups, corresponding to their previous entities. On the basis of the present phylogenetic results that confirmed our biochemical and genetic data, we propose a new species, Prevotella pallens. The type strain is NCTC 13042 (= AHN 10371).

The generic position of an aerobic, Gram-positive, non-acid-alcohol-fast actinomycete was determined following isolation of the PCR-amplified 16S rRNA genes and alignment of the resultant sequence with corresponding sequences from representatives of the family Pseudonocardiaceae. The assignment of the organism to the genus Saccharopolyspora was strongly supported by chemotaxonomic and morphological data. The strain was distinguished from representatives of validly described Saccharopolyspora species by a number of phenotypic properties. It is proposed that the organism, strain AS4.198T, be classified in the genus Saccharopolyspora as Saccharopolyspora spinosporotrichia sp. nov.

Four moderately thermophilic, carboxydotrophic streptomycetes were the subject of a comparative taxonomic investigation designed to establish their taxonomic relationships. Almost complete sequences of the 16S rRNA genes of the test strains were determined following the isolation and direct sequencing of the amplified genes. The resultant nucleotide sequences were aligned with the sequences of previously studied streptomycetes, and phylogenetic trees generated by using the neighbour-joining, Fitch-Margoliash, maximum-likelihood and maximum-parsimony methods. It was evident from the phylogenetic analyses that strains AT50, AT51 and AT52 were most closely related to Streptomyces thermodiastaticus DSM 40573T and strain AT37 to Streptomyces glaucescens DSM 40716 and Streptomyces pseudogriseolus NRRL 3985. Random DNA amplification profiles clearly distinguished strains AT50, AT51 and AT52 from Streptomyces thermodiastaticus and from strain AT37. The molecular systematic evidence, together with phenotypic data derived from this and previous studies, indicate that the test strains merit species status within the genus Streptomyces. The name Streptomyces thermocarboxydovorans sp. nov. is proposed for strains AT50, AT51 and AT52 (type strain) and Streptomyces thermocarboxydus sp. nov. for strain AT37.

Over a five-year period, four strains of a yellowish-pigmented coryneform bacterium were received for identification by the Culture Collection of the University of Géteborg. All strains had been isolated from normally sterile human body fluids. Initial biochemical characterization revealed that all four isolates were very similar, with weak pyrazinamidase and urease activities, as well as slow fermentative acid production from glucose as the most significant phenotypic features which differentiated the strains from all other presently defined corynebacteria. Chemotaxonomic investigations demonstrated that the strains belonged to the genus Corynebacterium. SDS-PAGE of whole-cell proteins suggested that all four strains were representatives of the same species. Comparative 16S rRNA gene sequence analysis unambiguously demonstrated that the four strains were genealogically related and represent a new subline within the genus Corynebacterium for which the designation Corynebacterium falsenii sp. nov. is proposed. The type strain of Corynebacterium falsenii is CCUG 33651.

Thirty-three strains previously classified into 11 species in the bacterial family Moraxellaceae were subjected to phylogenetic analysis based on 16S rRNA sequences. The family Moraxellaceae formed a distinct clade consisting of four phylogenetic groups as judged from branch lengths, bootstrap values and signature nucleotides. Group I contained the classical moraxellae and strains of the coccal moraxellae, previously known as Branhamella, with 16S rRNA similarity of ≥95%. A further division of group I into five tentative clusters is discussed. Group II consisted of two strains representing Moraxella atlantae and Moraxella osloensis. These strains were only distantly related to each other (93.4%) and also to the other members of the Moraxellaceae (≤93%). Therefore, reasons for reclassification of these species into separate and new genera are discussed. Group III harboured strains of the genus Psychrobacter and strain 752/52 of [Moraxella] phenylpyruvica. This strain of [M.] phenylpyruvica formed an early branch from the group III line of descent. Interestingly, a distant relationship was found between Psychrobacter phenylpyruvicus strain ATCC 23333T (formerly classified as [M.] phenylpyruvica) and [M.] phenylpyruvica strain 752/52, exhibiting less than 96% nucleotide similarity between their 16S rRNA sequences. The establishment of a new genus for [M.] phenylpyruvica strain 752/52 is therefore suggested. Group IV contained only two strains of the genus Acinetobacter. Strategies for the development of diagnostic probes and distinctive sequences for 16S rRNA-based species-specific assays within group I are suggested. Although these findings add to the classificatory placements within the Moraxellaceae, analysis of a more comprehensive selection of strains is still needed to obtain a complete classification system within this family.

An anaerobic, extremely thermophilic xylanolytic, non-spore-forming bacterium was isolated from a sediment sample taken from Owens Lake, California, and designated strain OLT (T = type strain). Strain OLT had a Gram-negative reaction and occurred as short rods which sometimes formed long chains containing a few coccoid cells. It grew at 50--80 °C, with an optimum at 75 °C. The pH range for growth was 5·5--9·0 with an optimum at about pH 7·5. When grown on glucose at optimal conditions, its doubling time was 7·3 h. In addition to glucose, the isolate utilized sucrose, xylose, fructose, ribose, xylan, starch, pectin and cellulose. Yeast extract stimulated growth on carbohydrates but was not obligately required. The end products from glucose fermentation were lactate, acetate, ethanol, H2 and CO2. The G+C content of strain OLT was 36·6 mol%. The 16S rDNA sequence analysis indicated that strain OLT was a member of the subdivision containing Gram-positive bacteria with DNA G+C content of less than 55 mol% and clustered with members of the genus Caldicellulosiruptor. Because strain OLT is phylogenetically and phenotypically different from other members of this genus, it is proposed to designate this isolate Caldicellulosiruptor owensensis sp. nov. Strain OLT is the type strain (=ATCC700167T).

Twelve strains named Bacillus pantothenticus, at least 29 Bacillus strains representing 16 species belonging to rRNA groups 1 and 2, one Bacillus dipsosauri strain, and 38 strains of Amphibacillus, Aneurinibacillus, Brevibacillus, Halobacillus, Paenibacillus, Sporosarcina and Marinococcus, were characterized genotypically using amplified rDNA restriction analysis (ARDRA), and phenotypically using routine diagnostic characters comprising 61 biochemical tests in the API System and 15 observations of vegetative cell and sporangial morphology. The B. pantothenticus strains were also characterized by fatty acid methyl ester analysis and SDS-PAGE of whole-cell proteins. ARDRA revealed that strains of B. pantothenticus formed a cluster quite separate from other species in rRNA group 1, supporting the recognition of the former as a separate genus, for which the name Virgibacillus is proposed. The polyphasic data also indicate the presence of an as yet undescribed new species within this genus. The species Virgibacillus pantothenticus and related organisms comprising this new genus can be distinguished from members of Bacillus rRNA group 1 (Bacillus sensu stricto), and from members of Paenibacillus and other aerobic endospore-forming bacteria by routine phenotypic tests.

Genomic diversity in 21 strains of Bacillus cereus and 10 strains of Bacillus licheniformis was investigated by random amplified polymorphic DNA (RAPD) analysis, which samples the whole genome, and by two PCR fingerprinting techniques sampling the hypervariable spacers between the conserved 16S and 23S rRNA genes of the rRNA gene operon (ITS-PCR) and regions between tRNA genes (tDNA-PCR). RAPD analysis showed a remarkable diversity among strains of B. cereus that was not observed with the rRNA and tRNA intergenic-spacer-targeted PCR, where all the strains showed practically identical fingerprints. A wide variability among the B. cereus strains was also observed in the plasmid profiles, suggesting that the genetic diversity within B. cereus species can arise from plasmid transfer. One contribution to the diversity detected by RAPD analysis was determined by the presence of large extrachromosomal elements that were amplified during RAPD analysis as shown by Southern hybridization experiments. In contrast to the strains of B. cereus, the 10 strains of B. licheniformis were grouped into two clusters which were the same with all the methods employed. The 16S rRNA genes were identical in all 10 strains when examined using single strand conformation polymorphism analysis after digestion with Alul and Rsal. From these data we hypothesize two different evolutionary schemes for the two species.

One hundred reference strains representing all species belonging to the different phylogenetic lineages of the viridans streptococci were examined by means of one-dimensional whole-organism protein electrophoresis. For most of the species examined, multiple strains characterized by DNA-DNA hybridization were included and, wherever described, representatives of different biochemical variants were analysed. Most species were clearly differentiated. The data support the viewpoint that members of the Streptococcus anginosus group constitute a single species and indicate that Streptococcus mitis biovar 2 is a heterogeneous taxon comprising strains from several streptococcal species.

Four PCR-based DNA fingerprinting techniques were compared for their ability to identify at the species level a heterogeneous collection of isolates belonging to the six valid Listeria species. 16S rDNA-RFLP analysis identified all species and 16S rDNA-SSCP analysis identified almost all species. Also, isolates with unusual biochemical characteristics and/or unusual antigenic composition could be identified correctly. rRNA-intracistronic length polymorphism analysis suffered from high intraspecific variability, a limited number of fragments per profile, and small length differences between the spacers of different species. tRNA-intergenic length polymorphism analysis resulted in identification of all isolates but one, when fluorescent DNA capillary electrophoresis was used such that fragment length differences of 1 bp could be resolved. The four techniques yielded comparable results relevant to the taxonomy of Listeria. They all indicate a high degree of genetic relatedness between L. innocua and L. welshimeri, homogeneity of L. grayi, distinct but clear relatedness of L. grayi to the other Listeria species, a clear distinction between the two subspecies of L. ivanovii, and a clear distinction between Listeria isolates and isolates from closely related taxa or from species which are phenotypically difficult to distinguish from Listeria. New sequence determination of the 16S rRNA gene was necessary to obtain sequences in accordance with the findings of 16S rDNA-RFLP analysis.

Fifty-one Rahnella aquatilis and R. aquatilis-like strains from water, snails and human sources were characterized by routine biochemical tests, carbon source utilization tests, DNA relatedness (hydroxyapatite method) and 16S rRNA sequencing. The results of the genetic methods indicated that the strains comprised three closely related species within the genus Rahnella. It was not possible to differentiate R. aquatilis from the two newly recognized species. The new species were therefore given the vernacular names Rahnella genomospecies 2 and Rahnella genomospecies 3.

The genus Ammoniphilus is proposed for aerobic endospore-forming Gram-variable rod-shaped bacteria, which are ammonium-dependent, obligately oxalotrophic and haloalkalitolerant, oxidase- and catalase-positive, mesophilic and motile by peritrichous flagella. Cell wall contained two electron-dense layers. The external layer consists of a chain of electron-dense granules morphologically resembling the cellulosomes of Clostridium thermocellum. Two species are described, Ammoniphilus oxalaticus gen. nov., sp. nov. and Ammoniphilus oxalivorans gen. nov., sp. nov. The type strains of these species are strains RAOx-1 (= DSM 11538) and RAOx-FS (= DSM 11537), respectively. Ammoniphilus strains were isolated from the rhizosphere of sorrel (Rumex acetosa) and from decaying wood. The strains require a high concentration of ammonium ions and use oxalate as the sole organic source of carbon and energy for growth; no growth factors were required. Growth occurred at pH 6.8--9.5. The optimum temperature and pH for growth were 28--30 °C and 8.0--8.5. All strains grew in a saturated solution of ammonium oxalate, and tolerated 3% NaCl. Whole-cell hydrolysates contain meso-diaminopimelic acid and glucose. The menaquinone of the strains was MK 7, and the major cellular fatty acids were 12-methyl tetradecanoic, cis-hexadec-9-enoic and hexadecanoic acids. The G+C content of the DNA was 45--46 mol% for A. oxalaticus and 42 mol% for A. oxalivorans. The almost complete 16S rDNA sequence of three strains of the two species of Ammoniphilus shows that the genus falls into the radiation of the Clostridium-Bacillus subphylum of Gram-positive bacteria. The closest phylogenetic neighbour of Ammoniphilus is Oxalophagus oxalicus. The DNA-DNA hybridization value between strains RAOx-1 and RAOx-FS was 39.7%.

Sixteen heparinase-producing isolates, related to Sphingobacterium heparinum, were grouped into three major clusters by SDS-PAGE and DNA-rRNA hybridizations. Based on a polyphasic approach, it was shown that isolates of two of these clusters and S. heparinum species belong to a new genus for which the name Pedobacter is proposed. The genus consists of Pedobacter heparinus comb. nov. (formerly Sphingobacterium heparinum), which is the type species, Pedobacter piscium comb. nov. (formerly Sphingobacterium piscium), Pedobacter africanus sp. nov. and Pedobacter saltans sp. nov. and four as-yet-unnamed DNA hybridization groups. All the previously named taxa can be discriminated by phenotypic features, but have strong overall similarities with representatives of the genus Sphingobacterium and the misclassified species [Flexibacter] canadensis. All these organisms constitute a separate rRNA branch in rRNA superfamily V for which the family Sphingobacteriaceae fam. nov. is proposed.

The taxonomic relationship between several Shewanella putrefaciens isolates from the Baltic Sea and reference strains of this species is presented in this study. Results from DNA-DNA hybridization using a newly developed nonradioactive detection system and from 165 rRNA gene sequencing demonstrated that S. putrefaciens is a heterogeneous species containing more than a single genomic group. The genomic group II was phylogenetically, genotypically and phenotypically distant enough from the species type strain to be classified as a single species within the genus Shewanella. Therefore, we propose to reclassify Owen’s genomic group II as Shewanella baltica sp. nov. with the type strain NCTC 10735.

The 16S rDNAs from 38 strains of the genus Nocardioides, two Aeromicrobium species and Terrabacter tumescens were directly sequenced and then analysed. The mean nucleotide similarity value between the type strains of validly described Nocardioides species was 96.1±3.0%. The mean nucleotide similarity value between the type strains of validly described Nocardioides species and the two Aeromicrobium species was 93.7±1.4%. T. tumescens was distantly related to the genera Nocardioides and Aeromicrobium. The mean intraspecific nucleotide similarity value of 16S rDNA sequences from Nocardioides albus was 99.5±0.5%. The mean intraspecific nucleotide similarity of 16S rDNA sequences from Nocardioides simplex was 100%, except for N. simplex strains ATCC 13260, ATCC 19565 and ATCC 19566, which were shown not to be members of the genus Nocardioides. ‘Nocardioides flavus’ strains IFO 14396T and IFO 14397, and ‘Nocardioides fulvus’ JCM 3335T showed a 16S rDNA similarity value of 100% with Nocardioides luteus KCTC 9575T and Nocardioides albus JCM 5854. ‘N. fulvus’ IFO 14399 shared its highest 16S rDNA similarity with Nocardioides sp. ATCC 39419 at 99%. ‘N. fulvus’ IFO 14399 and Nocardioides sp. ATCC 39419 formed a phylogenetic lineage distinct from the genera Nocardioides and Aeromicrobium. ‘Nocardioides thermolilacinus’ strains IFO 14335T and IFO 14336 displayed a close relationship to the genus Streptomyces. From 16S rDNA sequence analyses, it is considered that some strains that have been attributed to the genus Nocardioides should be taxonomically re-evaluated.