- Volume 51, Issue 5, 2001

Thirty-one heavy-metal-resistant bacteria isolated from industrial biotopes were subjected to polyphasic characterization, including 16S rDNA sequence analysis, DNA-DNA hybridizations, biochemical tests, whole-cell protein and fatty-acid analyses. All strains were shown to belong to the Ralstonia branch of the beta-Proteobacteria. Whole-cell protein profiles and DNA-DNA hybridizations revealed two clearly distinct groups, showing low similarity to known Ralstonia species. These two groups, of 8 and 17 isolates, were assigned to two new species, for which the names Ralstonia campinensis sp. nov. and Ralstonia metallidurans sp. nov. are proposed. The type strains are WS2T (= LMG 19282T = CCUG 44526T) and CH34T (= LMG 1195T = DSM 2839T), respectively. Six isolates were allocated to Ralstonia basilensis, which presently contains only the type strain; an emendation of the latter species description is therefore proposed.

The taxonomic status of a soil isolate, provisionally assigned to the genus Nocardia, was established following a polyphasic study. An almost complete sequence of the 16S rDNA of the strain was determined and aligned with available sequences for nocardiae and phylogenetic trees were inferred using four tree-making algorithms. The organism was associated consistently with the type strains of Nocardia brasiliensis and Nocardia farcinica in the neighbour-joining analysis, albeit with low bootstrap values. It was also found to have chemotaxonomic and morphological properties consistent with its assignment to the genus Nocardia. The organism was readily separated from representatives of other Nocardia species by a broad range of phenotypic properties. The name proposed for the novel species is Nocardia beijingensis sp. nov. The type strain is AS4.1521T (= IFO 16342T = JCM 10666T).

A thermophilic, anaerobic, chemo-organotrophic bacterium, designated MV1087T, was isolated from a deep-sea hydrothermal chimney sample collected from the Mid-Atlantic Ridge. The cells were straight, motile and stained gram-negative. Growth was observed from 45 to 65 degrees C, with an optimum around 65 degrees C. No growth was observed at 40 or 70 degrees C. Growth was observed from pH 5.5 to 9.0 and the optimum pH was around 7. The salinity range for growth was 10-100 g sea salt l(-1) (corresponding to 6.5-65 g NaCl l(-1)) with an optimum at 30 g sea salt l(-1) (20 g NaCl l(-1)). Strain MV1087T was heterotrophic, able to ferment proteinaceous substrates, such as brain/heart infusion and gluten, and carbohydrates, such as glucose, xylan and starch. The DNA G+C content was 27 mol%. Phylogenetic analyses using 16S rDNA sequences indicated that strain MV1087T belonged to cluster XII of the Clostridium subphylum. Due to its phenotypic and genotypic characteristics, isolate MV1087T is proposed as a novel species of a new genus, Caloranaerobacter azorensis gen. nov., sp. nov. The type strain is MV1087T (= CNCM I-2543T = DSM 13643T).

The diversity of organisms present in the subgingival pockets of patients with periodontitis and acute necrotizing ulcerative gingivitis (ANUG) were examined previously. The 16S rRNA genes of subgingival plaque bacteria were amplified using PCR with a universal forward primer and a spirochaete-selective reverse primer. The amplified DNA was cloned into Escherichia coli. In one subject with ANUG, 70 clones were sequenced. Seventy-five per cent of the clones were spirochaetal, as expected. Twelve of the remaining clones fell into two clusters that represent novel phylotypes in the family Coriobacteriaceae. The first novel phylotype was most closely related to Atopobium rimae (98% similarity). The phylotype probably represents a novel Atopobium species, but will not be named until cultivable strains are obtained. The second novel phylotype was only 91% similar to described Atopobium species and 84% similar to Coriobacterium glomerans. The 16S rRNA sequences of the type strain of Lactobacillus uli and a strain representing the Moores' Eubacterium group D52 were determined as part of on ongoing sequence analysis of oral bacteria. The sequence for L. uli was more than 99.8% similar to sequences for the second clone phylotype. It therefore appears that the second clone phylotype and L. uli represent the same species. The sequence for the Eubacterium D52 strain was 95.6% similar to that of L. uli. The G+C content of the DNA of L. uli and Eubacterium D52 is 63-64 mol %. These organisms are thus distinct from the neighbouring genus Atopobium, which has a DNA G+C content of 35-46 mol%. A new genus, Olsenella gen. nov., is proposed for these two species on the basis of phenotypic characteristics and 16S rRNA sequence analysis to include Olsenella uli comb. nov. and Olsenella profusa sp. nov.

Two strictly anaerobic, dissimilatory ferric-iron-reducing bacteria, strains Dfr1T and Dfr2T, were isolated from freshwater mud samples with ferrihydrite as electron acceptor. Both strains also grew by reducing Mn(IV), S0 and fumarate. Electron donors used by strains Dfr1T and Dfr2T for growth with ferric iron as electron acceptor included hydrogen, formate, acetate, pyruvate, succinate, fumarate and ethanol. An affiliation with the family Geobacteraceae was revealed by comparative analysis of 165 rRNA gene sequences. Strains Dfr1T and Dfr2T shared 92.5% sequence identity and their closest known relative was Geobacter sulfurreducens, with approximately 93% sequence identity. Cultures and colonies of strains Dfr1T and Dfr2T were intensely red in colour, due to the presence of c-type cytochromes. On the basis of physiological and phylogenetic data, strain Dfr1T (= DSM 12179T = OCM 796T) is described as Geobacter bremensis sp. nov. and strain Dfr2T (= DSM 12255T = OCM 797T) as Geobacter pelophilus sp. nov.

Four motile spored strains were isolated from soil samples collected in Japan. The cultures formed long, narrow sporangia on short sporangiophores directly on the substrate mycelium. The sporangia develop singly or in clusters above the surface of the substrate. Each sporangium contains a single row of six or more motile spores. Glutamic acid, glucosamine, glycine, alanine and 3-OH-diaminopimelic acid are present in the cell wall; the whole-cell sugars are 3-O-methylmannose, rhamnose, mannose, arabinose, galactose, xylose and glucose; and the predominant menaquinones are 10(H4), 10(H6) and 10(H8). The diagnostic phospholipid is phosphatidylethanolamine. The acyl type of the muramic acid is glycolyl. The G+C content is 71 mol%. Mycolic acids are absent. The chemotaxonomic data indicate that these strains belong to the family Micromonosporaceae. Analysis of 165 rDNA sequences suggested that these organisms fall into a distinct clade within the family Micromonosporaceae for which a new genus, Virgosporangium gen. nov., is proposed containing the species Virgosporangium ochraceum sp. nov. (strains YU655-43T, YU793-41 and YU794-41) and Virgosporangium aurantiacum sp. nov. (strain YU438-5T).

A survey was performed for Lyme disease borrelia in the southern part of China, in Zhejiang, Sichuan and Anhui provinces, along the Yangtze River valley, in May of 1997 and 1998. Twenty isolates from Ixodes granulatus, Ixodes ovatus, Apodemus agrarius and Niviventer confucianus were obtained. These isolates were characterized by RFLP of the 5S-23S rDNA intergenic spacer, sequence analysis of the intergenic spacer, 16S rDNA and flagellin gene, DNA-DNA hybridization analysis, SDS-PAGE and Western blotting with mAbs. Six isolates from A. agrarius, five from I. granulatus collected in Zhejiang province and one from N. confucianus in Sichuan province were highly similar to strains 10MT and 5MT isolated in Korea and classified as Borrelia valaisiana. Four isolates from A. agrarius and I. granulatus collected in Zhejiang province generated unique RFLP patterns and phylogenetic analysis of the 16S rDNA and flagellin gene sequences suggested that the isolates should be classified as B. valaisiana. Furthermore, three isolates (CMN1a, CNM2, CMN3T) from N. confucianus captured in Sichuan province and one (CWO1) from I. ovatus in Anhui province showed lower 165 rDNA sequence similarity (less than 99.0%) to sequences of previously described Lyme disease-related Borrelia species. DNA-DNA hybridization results revealed that strains CMN3T and CMN1a were clearly distinct from all other known Lyme disease Borrelia species. Electron microscope observation showed the spirochaetes to be morphologically similar to those of Borrelia, but the cells contained only four periplasmic flagella inserted at each end of the spirochaetes. Based on these results, a new Borrelia species, Borrelia sinica sp. nov., is proposed. Strain CMN3T is the type strain of this new species.

Two novel haloalkaliphilic archaea, strains C231T and C42, were isolated from a soda lake in China. Cells of the two strains were rod-shaped and gram-negative and colonies were bright red. They required at least 2.5 M NaCl for growth, with an optimum at 3.5 M NaCl, and grew over a pH range from 8.0 to 10.5, with an optimum at pH 8.5. Hypotonic treatment with less than 1.5 M NaCl caused cell lysis. They had similar polar lipid compositions, possessing the diphytanyl (C20:C20) and phytanyl-sesterterpanyl (C20:C25) diether derivatives of phosphatidylglycerol and phosphatidylglycerophosphate methyl ester and a minor phospholipid, PL1. No glycolipids were detected. Comparison of 16S rDNA sequences and morphological features placed them in the genus Natronobacterium. Detailed phenotypic characterization and DNA-DNA hybridization studies revealed that the two strains belong to a new species in the genus Natronobacterium, for which the name Natronobacterium nitratireducens sp. nov. is proposed. The type strain is C231T (= AS 1.1980T = JCM 10879T).

The taxonomy of strain KF46FT, which was isolated previously after an aerobic enrichment with the azo compound 1-(4'-carboxyphenylazo)-2-naphthol as the sole source of energy and carbon, was investigated by a polyphasic approach. The organism contained a quinone system with ubiquinone Q-8 and 2-hydroxyputrescine and putrescine as the major polyamines, suggesting that strain KF46FT belonged to the beta-subclass of the Proteobacteria. The polar lipid profile consisted mainly of phosphatidylethanolamine and minor amounts of phosphatidylglycerol and diphosphatidylglycerol. Sequencing of the 16S rRNA gene supported its placement in the family Comamonadaceae, but the sequence similarities to the most closely related species of the genera Hydrogenophaga, Acidovorax, Comamonas and Xylophilus were only in the range 95.0 to 96.1%. Different methods for the construction of phylogenetic trees showed the separate position of strain KF46FT 'between' the genera Hydrogenophaga, Variovorax, Comamonas and Xylophilus. Analysis of the fatty acids revealed an unusual profile, with the presence of 8:0 3-OH, 10:0 3-OH, 16:1 2-OH, 16:0 2-OH and 18:1 2-OH in addition to 17:0 cyclo, which is unique among the previously described genera of the family Comamonadaceae. Thus, a new taxon is proposed for strain KF46FT, with the name Xenophilus azovorans gen. nov., sp. nov.

The form species concept for the Cyanobacteria was evaluated using a comprehensive set of Nostoc samples that were collected during the past two centuries, from all continents, including regions from the Tropics to the Poles. Phylogenies were constructed based upon the conserved regions of tRNALeu (UAA) group I intron DNA sequences. Thirty-four forms contained a tRNALeu (UAA) intron of 284 nt. These 284-nt introns contained 200 nt of conserved sequence that, in most cases, shared 100% sequence identity, they had three variable regions (I, II and III) amounting to 84 nt, contained no hypervariable region and formed a discrete cluster in phylogenetic analysis. These forms represented 31 independent populations in both hemispheres and constitute examples of form species Nostoc commune. Multiple introns were obtained from several of the populations. Ten populations contained introns of 287-340 nt with a hypervariable region, 8 to 59 nt in length, located between variable regions I and II. Alignments identified 15 examples where 5'-AAAAUCC-3' occurred at the hypervariable region-variable region II boundary; this sequence is identical to the conserved sequence at the 3' intron-exon boundary (splice site) within the tRNALeu (UAA) gene. The possibility that hypervariable regions were removed from the primary intron through secondary splicing was tested in vitro but proved to be negative under the experimental conditions used. Shared morphologies of genetically different strains, dissimilar morphologies in strains that share identical genetic markers, incorrect naming of culture collection strains and genetic drift in cultured strains emphasize that the successful delineation of cyanobacterial species requires the application of multiple taxonomic criteria.

A novel thermophilic, hydrogen-oxidizing bacterium, VM1T, has been isolated from a marine hydrothermal area of Vulcano Island, Italy. Cells of the strain were gram-negative rods, 2-4 microm long and 1-1.5 microm wide with four to seven monopolarly inserted flagella. Cells grew chemolithoautotrophically under an atmosphere of H2/CO2 (80:20) in the presence of low concentrations of O2 (optimum 1-2%). Carbohydrates and peptide substrates were not utilized, neither for energy generation nor as a source of cellular carbon. Growth of VM1T occurred between 45 and 80 degrees C with an optimum at 65 degrees C. Growth was observed between pH 5 and 7. NaCl stimulated growth in the range 0.5-6% with an optimum at 2-3%. Hydrogen could not be replaced by elemental sulfur or thiosulfate as electron donors. Nitrate and sulfate were not used as electron acceptors. The major respiratory lipoquinone was a new menathioquinone. Analysis of the fatty acids of VM1T revealed straight-chain saturated C18:0 and the unsaturated C18:1omega9c and C20:1omega9c as major components. The G+C content of the total DNA was 43 mol%. Phylogenetic analysis placed strain VM1T near the members of the genera Hydrogenobacter, Thermocrinis and Aquifex on a separate deep-branching phylogenetic lineage. Therefore, it is proposed that strain VM1T (= DSM 12046T = JCM 10974T) represents a novel species within a new genus, for which the name Hydrogenothermus marinus gen. nov., sp. nov., is proposed. In addition, it is shown that Calderobacterium hydrogenophilum should be transferred to the genus Hydrogenobacter; the name Hydrogenobacter hydrogenophilus comb. nov. (DSM 2913T = JCM 8158T) is proposed for this organism. Furthermore, on the basis of 16S rRNA sequence analysis, Hydrogenobacter acidophilus is only distantly related to Hydrogenobacter species. Owing to this finding and its growth at low pH, the name Hydrogenobaculum acidophilum gen. nov., comb. nov., is proposed for Hydrogenobacter acidophilus. The type strain is JCM 8795T (= DSM 11251T).

Rhodopseudomonas acidophila has unique properties among the phototrophic alpha-Proteobacteria and is quite distinct from the type species of Rhodopseudomonas, Rhodopseudomonas palustris. Therefore, the transfer of Rhodopseudomonas acidophila to Rhodoblastus acidophilus gen. nov., comb. nov., is proposed. This proposal is in accordance with other taxonomic reclassifications proposed previously and fully reflects the phylogenetic distance from Rhodopseudomonas palustris.

The taxonomic position of Pseudomonas strain K24, which was isolated previously after an aerobic enrichment with the azo compound 1-(4'-carboxyphenylazo)-4-naphthol as the sole source of carbon and energy, was investigated. The detection of a quinone system with ubiquinone Q-8 as the predominant compound and a polyamine pattern with putrescine and 2-hydroxyputrescine as the major polyamines present suggested that strain K24T belongs to the beta-subclass of the Proteobacteria. This was supported by sequencing the 16S rRNA gene, which demonstrated about 95-96% sequence similarity to different species of the genera Achromobacter, Alcaligenes and Bordetella. This suggested that strain K24T is a member of the family Alcaligenaceae. The G+C content of the DNA was 68.5 mol %. Different methods for the construction of phylogenetic dendrograms placed strain K24T separate from the genera Alcaligenes, Achromobacter and Bordetella. Analysis of the fatty acids demonstrated the presence of 10:0 3-OH and high concentrations of summed feature 7 (18:1omega7c, 18:1omega9t and/or 18:1omega12t) and 19:0 cycloomega8c, which is unique among previously described species of the genera Alcaligenes, Achromobacter and Bordetella. On the basis of the low 16S rRNA sequence similarities, the composition of the fatty acid profile and unique phenotypic properties, a new genus and species is proposed for strain K24T with the name Pigmentiphaga kullae gen. nov., sp. nov.

A novel methanogen, designated strain N2F9704T, was isolated from an aquaculture fish pond near Wang-gong, Taiwan. The cells were irregular cocci, non-motile, 1.2-2.0 microm in diameter and stained gram-negative. Cells of strain N2F9704T lysed easily by SDS treatment (0.1 g l(-1)) and the S-layer protein had an Mr of 137000. The catabolic substrates used included formate and H2+CO2, but not acetate, methanol, trimethylamine or secondary alcohols. The optimal growth parameters for strain N2F9704T were pH 6.5, 37 degrees C with 0.5% NaCl. Trace amounts of tungstate not only promoted growth but also extended the range of growth conditions. Analysis of the 16S rDNA sequence revealed a phylogenetic relationship to Methanofollis species and the name Methanofollis aquaemaris sp. nov. is therefore proposed for strain N2F9704T (= OCM 746T = CCRC 16166T). Additionally, the strain was infected with a novel coccus-shaped, enveloped virus with a diameter of 200 nm.

Spiral bacteria were isolated from the intestines of laboratory mice during a study examining the presence of Helicobacter species and other spiral organisms naturally infecting mice maintained at four different animal facilities in Sydney, Australia. One group of 17 isolates, cultured from mice from three of the four facilities, were found to be helicobacters but did not fall within any of the 18 currently recognized species. These isolates were unusual in that they only grew anaerobically at 37 degrees C and were incapable of growth under microaerobic conditions. Like Helicobacter rodentium, isolates possessed single, bipolar, unsheathed flagella and were urease-negative. They were positive for oxidase and reduced nitrate to nitrite but did not hydrolyse hippurate or indoxyl acetate, grew on charcoal agar and were resistant to cephalothin. 16S rDNA sequences from four strains were determined and found to be identical to one another. H. rodentium was the most closely related species in terms of 16S rDNA sequence similarity (98.2%). Numerical analysis of whole-cell proteins by SDS-PAGE for nine isolates was carried out with a comparison to all known Helicobacter species, including newly determined profiles from three H. rodentium strains. The new isolates were clearly differentiated from H. rodentium and other Helicobacter spp. On the basis of this data, including genetic, biochemical and protein analysis, it is proposed that these isolates belong to Helicobacter ganmani sp. nov. (type strain CMRI H02T = CCUG 43526T = CIP 106846T).

The taxonomic status of two recently described phenetically distinctive groups within the genus Acinetobacter, designated phenon 1 and phenon 2, was investigated further. The study collection included 51 strains, mainly of clinical origin, from different European countries with properties of either phenon 1 (29 strains) or phenon 2 (22 strains). DNA-DNA hybridization studies and DNA polymorphism analysis by AFLP revealed that these phenons represented two new genomic species. Furthermore, 16S rRNA gene sequence analysis of three representatives of each phenon showed that they formed two distinct lineages within the genus Acinetobacter. The two phenons could be distinguished from each other and from all hitherto-described Acinetobacter (genomic) species by specific phenotypic features and amplified rDNA restriction analysis patterns. The names Acinetobacter ursingii sp. nov. (type strain LUH 3792T = NIPH 137T = LMG 19575T = CNCTC 6735T) and Acinetobacter schindleri sp. nov. (type strain LUH 5832T = NIPH 1034T = LMG 19576T = CNCTC 6736T) are proposed for phenon 1 and phenon 2, respectively. Clinical and epidemiological data indicate that A. ursingii has the capacity to cause bloodstream infections in hospitalized patients.

Two hyperthermophilic bacteria, strains RKU-1T and RKU-10T, which grew optimally at 80 degrees C, were isolated from the production fluid of the Kubiki oil reservoir in Niigata, Japan. They were strictly anaerobic, rod-shaped fermentative heterotrophs. Based on the presence of an outer sheath-like structure (toga) and 16S rDNA sequences, they were shown to belong to the genus Thermotoga. Cells of strain RKU-1T were 2-7 microm by 0.7-1.0 microm, with flagella. They grew at 47-88 degrees C on yeast extract, peptone, glucose, fructose, ribose, arabinose, sucrose, lactose, maltose, starch and cellulose as sole carbon sources. Cells of strain RKU-10T were 2-7 microm by 0.8-1.2 microm, with flagella. They grew at 48-86 degrees C on yeast extract, peptone, glucose, galactose, fructose, mannitol, ribose, arabinose, sucrose, lactose, maltose and starch as sole carbon sources. While strains RKU-1T and RKU-10T reduced elemental sulfur to hydrogen sulfide, their final cell yields and specific growth rates decreased in the presence of elemental sulfur. Thiosulfate also inhibited growth of strain RKU-1T but not strain RKU-10T. The G+C contents of the DNA from strains RKU-1T and RKU-10T were 46.8 and 46.1 mol%. Phenotypic characteristics and 165 rDNA sequences of the isolates were similar to those of Thermotoga maritima and Thermotoga neapolitana, both being hyperthermophilic bacteria isolated from hydrothermal fields. However, the isolates differed from these species in their minimum growth temperatures, utilization of some sugars, sensitivity to rifampicin and the effects of elemental sulfur and thiosulfate on growth. The low levels (less than 31%) of DNA reassociation between any two of these hyperthermophilic Thermotoga strains indicated that the isolates were novel species. Analysis of the gyrB gene sequences supported the view that the isolates were genotypically different from these reference species. The isolates were named Thermotoga petrophila sp. nov., with type strain RKU-1T (= DSM 13995T = JCM 10881T), and Thermotoga naphthophila sp. nov., with type strain RKU-10T (= DSM 13996T = JCM 10882T).

A novel sulfate-reducing bacterium, strain Mb1PaT, was isolated from the sediments of a freshwater floodplain lake. Cells of strain Mb1PaT were rod-shaped, 1-1.3 microm wide and 2.6-3 microm long, motile and Gram-negative. The bacterium grew on straight-chain carboxylic acids with 4-17 carbon atoms. Electron donors with an even number of carbon atoms were oxidized to acetate and electron donors with an odd number of carbon atoms were oxidized to acetate and propionate. No other compounds were found to be used as electron donors. No growth occurred in the absence of sulfate. The optimum temperature for growth was between 25 and 30 degrees C and the maximum temperature for growth was 32 degrees C. Strain Mb1PaT grew very slowly in medium with 5 g NaCl l(-1) with optimum growth occurring with up to 1.0 g NaCl l(-1). Analysis of the 16S rRNA gene showed that strain Mb1PaT belonged to the delta-subclass of the Proteobacteria, was a member of the family Desulfobacteraceae, but lacked similarity with any currently described representatives. The combined phylogenetic analysis and physiological data indicate that strain Mb1PaT represents a new genus and the name Desulforegula conservatrix is proposed. The type strain is Mb1PaT (= DSM 13527T = ATCC BAA-134T).

A phylogenetic analysis was conducted upon ten strains of the psychrophobic yeast species Arxiozyma telluris using nuclear rDNA (18S and 26S) and mitochondrial cytochrome-c oxidase subunit II (COX2) gene sequences. Strains examined included those described originally as Candida slooffii, Torulopsis bovina (= Candida bovina) and Torulopsis pintolopesii (= Candida pintolopesii), which are all currently accepted as synonyms of Arxiozyma telluris. Comparative 18S rDNA sequence analysis showed that these strains formed a genealogically highly related group, which was phylogenetically distinct from any other ascomycetous species studied. The results showed that A. telluris, as currently described, appears to be composed of a complex of closely related but nevertheless separate taxa. rDNA and COX2 gene sequence data revealed that CBS 1787T, the type strain of C. pintolopesii, the currently recognized asexual form (anamorph) of A. telluris, along with strains CBS 2676 and CBS 2985 formed a distinct taxon that is phylogenetically separate from A. telluris. Similarly, the sequence data also showed that C. slooffii is a distinct taxon and support the reinstatement of this species. However, with regard to the relationship between the type strains of A. telluris (CBS 2685T) and C bovina (CBS 2760T), discrepancies were observed between the rDNA and COX2 sequence datasets, and these results are discussed in more detail.