- Volume 50, Issue 2, 2000

Two isolates of Gram-positive cocci (Ben 109T and Ben 110) which could accumulate polyphosphate and were microscopically similar in appearance to so-called 'G-bacteria', appearing as tetrads, were isolated from samples of activated sludge biomass by micromanipulation and grown in axenic culture. On the basis of their phenotypic and chemotaxonomic characters and 16S rDNA sequences, these isolates, together with strain T1-X7T isolated and described previously in Japan, belong to a new genus. These isolates are phylogenetically different from Tessaracoccus bendigoensis, Friedmanniella spumicola and Friedmanniella capsulata, Gram-positive cocci isolated previously in this laboratory. They are characterized by type A1 gamma peptidoglycan, with meso-diaminopimelic acid as the diagnostic diamino acid. The main cellular fatty acid of Ben 109T, Ben 110 and T1-X7T is 14-methylpentadecanoic acid (i-C16:0). The major menaquinones of Ben 109T are MK-8(H4), with MK-8(H2) and MK-8 in trace amounts. In Ben 110 MK-8(H4) and MK-6(H4) are the major menaquinones, while T1-X7T has MK-8(H4), MK-7(H4) and MK-6(H4) as its menaquinones. All three contain phosphatidylinositol, phosphatidylglycerol and diphosphatidylglycerol as their polar lipids. These properties, together with 16S rDNA sequence data, suggest that they all belong to a single new genus for which the name Tetrasphaera gen. nov. is proposed. However, the lipid, cellular fatty acid profiles and DNA-DNA similarity data suggest that Ben 109T and Ben 110 are sufficiently different from T1-X7T to represent a different species of the genus Tetrasphaera. Strain T1-X7T represents the type species Tetrasphaera japonica sp. nov. of this new genus, and strains Ben 109T and Ben 110 belong to the other species, Tetrasphaera australiensis sp. nov.

A 1341 bp sequence of the 16S rDNA of an undescribed species of Pasteuria that parasitizes the soybean cyst nematode, Heterodera glycines, was determined and then compared with a homologous sequence of Pasteuria ramosa, a parasite of cladoceran water fleas of the family Daphnidae. The two Pasteuria sequences, which diverged from each other by a dissimilarity index of 7%, also were compared with the 16S rDNA sequences of 30 other bacterial species to determine the phylogenetic position of the genus Pasteuria among the Gram-positive eubacteria. Phylogenetic analyses using maximum-likelihood, maximum-parsimony and neighbour-joining methods showed that the Heterodera glycines-infecting Pasteuria and its sister species, P. ramosa, form a distinct line of descent within the Alicyclobacillus group of the Bacillaceae. These results are consistent with the view that the genus Pasteuria is a deeply rooted member of the Clostridium-Bacillus-Streptococcus branch of the Gram-positive eubacteria, neither related to the actinomycetes nor closely related to true endospore-forming bacteria.

Strain DVT, a halotolerant, Gram-negative, facultatively anaerobic bacterium, was isolated from a hypersaline pond located in Death Valley, California. The cells were non-spore-forming, motile, curved rods (1.0-1.8 x 0.5-0.6 microns) and occurred singly, in pairs or rarely in chains. Strain DVT was oxidase-, catalase-, Voges-Proskauer-, amylase-, gelatinase- and lipase-positive and indole-negative. Nitrate, sulfate and fumarate were not used as electron acceptors. Carbohydrates served as energy sources both aerobically and anaerobically. Strain DVT grew optimally at 37 degrees C (temperature range 20-50 degrees C) with 2.5% NaCl (NaCl range 0-12.5%) and pH 7.3 (pH range of 5.5-8.5) in a glucose/yeast extract medium with a doubling time of 20 min (aerobically) or 41 min (anaerobically). The end products of glucose fermentation were ethanol, isobutyrate, propionate, lactate, formate and CO2. Strain DVT was resistant to penicillin, D-cycloserine, streptomycin and tetracycline (200 micrograms ml-1). The G + C content was 50 mol%. 16S rRNA gene sequence analysis indicated that it was closely related to Salinivibrio costicola (97.7%) and this was confirmed by DNA-DNA hybridization (93% relatedness). However, phenotypic characteristics such as halotolerance, gas production, growth at 50 degrees C, antibiotic resistance, sugar-utilization spectrum and phylogenetic signatures are sufficiently different from Salinivibrio costicola to warrant designating strain DVT as a new subspecies of Salinivibrio costicola, Salinivibrio costicola subsp. vallismortis subsp. nov. (= DSM 8285T).

A psychrotolerant Clostridium species was isolated from vacuum-packed, temperature-abused raw lamb. Colonies of this micro-organism on sheep-blood agar were circular with an entire margin, grey-white, translucent and beta-haemolytic. Cells were single, tapered, motile rods. Elliptical subterminal spores were produced in the late stationary growth phase. Spores did not cause swelling of the maternal cells. The micro-organism was obligately anaerobic. In peptone yeast extract glucose starch (PYGS) broth at pH 7.0, the micro-organism grew optimally between 25.5 and 30.0 degrees C. The temperature range for growth was 2.5-32.2 degrees C. At 26 degrees C, the micro-organism grew optimally at pH 6.8 to 7.0. The pH range for anaerobic growth was 4.7-9.1. The micro-organism was saccharoclastic, hydrolysed starch and degraded xylan. The fermentation products formed in PYGS broth were acetate, formate, lactate, ethanol, butyrate, butanol, hydrogen and carbon dioxide. The G + C content of the DNA was 38.4 mol%. Phylogenetic analyses indicated that the strain belongs to cluster XIVa of the genus Clostridium (sensu Collins et al. 1994). The new strain differed from phylogenetically related clostridia in terms of cellular fatty acid composition, soluble protein profiles and phenotypic properties. On the basis of phenotypic and genotypic characterization data, the strain was assigned to a new species, namely Clostridium algidixylanolyticum. The type strain is strain SPL73T (= DSM 12273T).

A new group of anaerobic, Gram-positive, high G + C (56-60 mol%) bacteria was isolated from the bovine rumen. Of four strains characterized, all were non-motile and none produced spores. The isolates did not produce indole or H2S and did not hydrolyse gelatin. Cells of each strain exhibited similar rod-shaped morphology (0.5-1.0 x 1.0-1.5 microns) although bulbous ends were sometimes present. None of the four strains were able to grow via oxidation of a variety of potentially fermentable substrates but rather obtained energy for growth via anaerobic respiration processes, oxidizing hydrogen, formate or lactate for reduction of various oxidized nitrogen compounds. Trimethylamine oxide and DMSO were also used as electron acceptor. All four strains shared greater than 99% 16S rRNA gene sequence identity. The closest match found between the 16S rRNA gene sequence of all four strains, NPOH1T, NPOH2, NPOH3 and MAJ1, to sequences available in GenBank was that of Coriobacterium glomerans (86% sequence similarity), a phenotypically dissimilar anaerobe within the class Actinobacteria. To accommodate these bacteria the creation of a new genus and species, Denitrobacterium detoxificans, for placement within the family Coriobacteriaceae is proposed. The type strain, NPOH1T (ATCC 700546T), grew equally well over a narrow range of incubation temperatures tested (32-39 degrees C).

A novel, mesophilic, Gram-negative bacterium was isolated from an anaerobic digestor for municipal wastewater. The bacterium degraded adipate in the presence of sulfate, sulfite, thiosulfate and elemental sulfur. (E)-2-Hexenedioate accumulated transiently in the degradation of adipate. (E)-2-Hexenedioate, (E)-3-hexenedioate, pyruvate, lactate, C1-C12 straight-chain fatty acids and C2-C10 straight-chain primary alcohols were also utilized as electron donors. 3-Phenylpropionate was oxidized to benzoate. The G + C content of the DNA was 60 mol%. 16S rDNA sequence analysis revealed that the new isolate clustered with species of the genus Syntrophobacter and Desulforhabdus amnigenus. Strain TsuAS1T resembles Desulforhabdus amnigenus DSM 10338T with respect to the ability to utilize acetate as an electron donor and the inability to utilize propionate without sulfate in co-culture with Methanospirillum hungatei DSM 864. Strains TsuAS1T and DSM 10338T form a 'non-syntrophic subcluster' within the genus Syntrophobacter. Desulfovirga adipica gen. nov., sp. nov. is proposed for the newly isolated bacterium, with strain TsuAS1T (= DSM 12016T) as the type strain.

The strictly anaerobic, Gram-negative, spore-forming bacterium strain WoGl3T had been enriched and isolated in mineral medium with glutarate as the sole source of energy and organic carbon. Glutarate was fermented to a mixture of butyrate, isobutyrate, CO2 and small amounts of acetate. Strain WoGl3T grew only with the dicarboxylates glutarate, methylsuccinate and succinate. 16S rDNA sequence analysis revealed an affiliation of strain WoGl3T to the family Syntrophomonadaceae. This monophyletic group is comprised of strain WoGl3T and the genera Syntrophomonas, Syntrophospora and Thermosyntropha, within the phylum of Gram-positive bacteria with a low DNA G + C content. Overall intra-group 16S rRNA sequence similarities of 89.2-93.9% document a separate phylogenetic status for strain WoGl3T. Strain WoGl3T (= DSM 6652T) is described as the type strain of a new species within a new genus, Pelospora glutarica gen. nov., sp. nov.

The taxonomic structure of members of the genus Azoarcus sensu lato was reassessed in a polyphasic approach. Two species, Azoarcus communis and Azoarcus indigens, three unnamed species containing diazotrophs associated with Kallar grass roots (groups C, D) and a group of strains (E) isolated from fungi were analysed. They were compared by PAGE analyses of cellular proteins, genomic fingerprints, morphological and nutritional features to new isolates from rice roots. All strains within groups C, D and E containing 5-12 isolates showed group-specific cell and colony morphology and carbon source utilization patterns, with exception of the obligately microaerobic strain BS20-3, a member of group C. All strains, with this exception, also had almost indistinguishable electrophoretic protein patterns and genomic fingerprints generated with tDNA-directed primers, suggesting they belong to the same species. Phylogenetic analyses of almost complete 16S rDNA sequences carried out with three different algorithms (neighbour-joining, maximum-likelihood, parsimony) revealed that Azoarcus sensu lato is not monophyletic. Groups C, D and E formed three distinct lineages located between the Azoarcus/Thauera and the Rhodocyclus clusters. Phylogenetic distances between groups C, D and E were as large as between other genera (93-94% sequence similarity). This suggested they have the rank of three different genera. Since it was possible to differentiate them from each other and other related bacteria by phenotypic features, three new genera with one type species each are proposed: Azovibrio restrictus gen. nov., sp. nov., Azospira oryzae gen. nov., sp. nov. and Azonexus fungiphilus gen. nov., sp. nov.

The placement of Actinomyces humiferus within the genus Actinomyces has always been controversial. A humiferus differs from typical members of the genus both phenotypically and in possessing a relatively high DNA G + C content. Comparative 16S rRNA gene sequencing has shown that A. humiferus is related only distantly to other species of the genus Actinomyces and is, in fact, a member of the genus Cellulomonas. On the basis of phylogenetic evidence, it is proposed that A. humiferus be reclassified in the genus Cellulomonas as Cellulomonas humilata nom. corrig., comb. nov.

The genus Xanthomonas contains a large number of strains, which have been characterized by a variety of phenotypic and genotypic classification methods. The Xanthomonas collection constitutes one of the largest groups of bacteria that have been characterized phylogenetically by DNA-DNA homology studies and genomic fingerprinting. Presently, a total genomic DNA-DNA homology value of 70% represents an internationally accepted criterion to define bacterial species levels. However, the complexity of DNA-DNA reassociation kinetics methods precludes the rapid analysis of large numbers of bacterial isolates, which is imperative for molecular microbial diversity studies. Therefore, the aim of this study was to compare more facile PCR-based genomic fingerprinting techniques, such as repetitive-sequence-based (rep)-PCR and AFLP genomic fingerprinting, to DNA-DNA hybridization studies. Using three different primer sets, rep-PCR genomic fingerprint patterns were generated for 178 Xanthomonas strains, belonging to all 20 previously defined DNA-DNA homology groups, and one Stenotrophomonas maltophilia strain. In addition, AFLP genomic fingerprints were produced for a subset of 80 Xanthomonas strains belonging to the 20 DNA-DNA homology groups and for the S. maltophilia strain. Similarity values derived from rep-PCR- and AFLP-generated fingerprinting analyses were calculated and used to determine the correlation between rep-PCR- or AFLP-derived relationships and DNA-DNA homology values. A high correlation was observed, suggesting that genomic fingerprinting techniques truly reveal genotypic and phylogenetic relationships of organisms. On the basis of these studies, we propose that genomic fingerprinting techniques such as rep-PCR and AFLP can be used as rapid, highly discriminatory screening techniques to determine the taxonomic diversity and phylogenetic structure of bacterial populations.

A new genus, Mogibacterium, is proposed for anaerobic, non-spore-forming, Gram-positive, rod-shaped bacteria which have been isolated from the periodontal pockets of adult human patients with periodontal disease and infected root canals. The novel isolates, strains D2-18T, BA11a-f and D5-2T, were inert in most of the conventional biochemical tests and phenotypically resemble asaccharolytic Eubacterium species. The protein profiles of whole cells on SDS-PAGE gels and Western immunoblotting reaction analysis distinguished these organisms from type strains belonging to the previously described Eubacterium species. The G + C content of the DNA is 45-46 mol% for Mogibacterium pumilum and 46 mol% for Mogibacterium vescum. The levels of DNA-DNA relatedness of these new species to other Eubacterium species, including Eubacterium limosum, Eubacterium brachy, Eubacterium lentum, Eubacterium nodatum, Eubacterium saphenum, and the more recently proposed Eubacterium minutum and Eubacterium exiguum (reclassified as Slackia exigua), are less than 2%. The DNA-DNA hybridization value between M. pumilum and M. vescum was 30%. Eubacterium timidum exhibited DNA homologies with Mogibacterium species which were low (17 and 18%) but clearly higher than with all the other Eubacterium species. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the closest phylogenetic neighbour of Mogibacterium species was E. timidum, and that these three species represent a novel lineage distinct from the previously described genera of Gram-positive, rod-shaped bacteria. On the basis of phenotypic characteristics and 16S rRNA gene sequence comparisons, it is also proposed that E. timidum is transferred to the genus Mogibacterium gen. nov. as Mogibacterium timidum gen. nov., comb. nov. (type strain ATCC 33093T).

A total of 101 staphylococcal strains were ribotyped using EcoRI and HindIII as restriction enzymes and plasmid pBA2 as the rDNA probe. Isolates from 10 newly described staphylococcal taxa were among those examined. All the ribotypes were added to our database, Staph DB, which now contains the sizes of the bands of 135 EcoRI and 120 HindIII ribotypes from 408 strains belonging to 42 staphylococcal taxa. The relatedness of ribotypes was evaluated by using the Dice coefficient. The ribotypes, and thus the strains, were clustered by the unweighted pair group method with averages (UPGMA). Separation into clusters correlated well with the delineation of the staphylococcal species but not with that of the different subspecies. No discrimination was possible between Staphylococcus vitulinus and Staphylococcus pulvereri. Ecovar-specific groups were evident within Staphylococcus intermedius and Staphylococcus hyicus. The data increase the usefulness of rRNA gene restriction site polymorphism analysis for staphylococcal taxonomy.

A Gram-positive rod was isolated from a commercial grape wine undergoing a sluggish/stuck alcoholic fermentation. The organism produced DL-lactic acid from glucose without gas formation, produced dextran from sucrose, hydrolysed aesculin and fermented galactose, D-glucose, D-fructose, D-mannose, L-sorbose, rhamnose, mannitol, sorbitol, methyl alpha-D-glucoside, N-acetylglucosamine, amygdalin, salicin, cellobiose, maltose, sucrose, trehalose and beta-gentiobiose. 16S rRNA gene sequence analysis revealed that the isolate was phylogenetically a member of the genus Lactobacillus and formed a distinct subline within the Lactobacillus casei cluster of species. On the basis of phenotypic and phylogenetic evidence, Lactobacillus nagelii sp. nov. ATCC 700692T is proposed as a new species.

Five strains of 'Candidatus Nostocoida limicola' were isolated by micromanipulation from two activated sludge plants. Two (Ben17 and Ben18) were from Sunbury, Victoria, Australia, and three (Ben67, Ver1 and Ver2) were from Verona, Italy. The near complete 16S rDNA sequences were determined for five strains and the phylogenetic location of this important bulking filament in the actinomycete subphylum is reported for the first time. Phylogenetically, the Ben strains formed one group with 99.4% 16S rDNA similarity, and the Ver strains formed another with 99.9% 16S rDNA similarity. The mean similarity between the two groups was 97.4%. By 16S rDNA comparison, the closest relative to all strains was Terrabacter sp. strain DPO1361 (95.0-95.5% identical). On R2A medium, all strains generally grew as short filaments or clumps of cocci, whereas on glucose sulfide (GS) medium, all grew as irregular twisting filaments comprising Gram-positive and Gram-negative cells, which is close to their in situ morphology. Polyphosphate was stored either as granules (R2A) or throughout the trichomes (GS). None of the strains could grow without added nitrogen, reduce nitrate to nitrogen gas or grow anaerobically, whereas all could grow at 15-30 degrees C, produce catalase and reduce nitrate to nitrite. All were inactive in the Hugh & Leifson test. This paper describes 'Candidatus Nostocoida limicola'.

Whereas poly-beta-hydroxybutyrate (PHB) production by Pseudomonas species is rare, synthesis of medium-chain-length poly-beta-hydroxyalkanoates (mcl-PHAs) other than PHB, has been observed in fluorescent and non-fluorescent species. Contrary to original reports, Pseudomonas corrugata and Pseudomonas ficuserectae accumulate mcl-PHAs and not PHB. The taxonomic implications of these characteristics are discussed.

A Gram-positive, cold-adapted, aerobic, spherical actinobacterium (strain T2T) with a quite low cardinal growth temperature was isolated from Chinese Great-Wall station in Antarctica. Sequence comparisons of the 16S rDNA indicated the isolate to be a phylogenetic member of the genus Micrococcus, family Micrococcaceae, in which it represents a novel lineage. The phylogenetic distinctness of the isolate with respect to the type strains Micrococcus luteus and Micrococcus lylae was supported by DNA-DNA similarity values of less than 40%. Chemotaxonomic properties supported the placement of the isolate in the genus Micrococcus. The diagnostic diamino acid of the cell-wall peptidoglycan is lysine. The predominant menaquinones are MK-8 and MK-8(H2). The G + C content of the DNA of the isolate is 66.4 mol%. Genotypic, morphological and physiological characteristics were used to describe a new species of Micrococcus, for which the name Micrococcus antarcticus is proposed. The type strain is T2T (= AS 1.2372T).

A diverse range of fermentative bacteria have been isolated from the commercial salterns of Salin-de-Giraud (Camargue, France). One of these isolates, strain SG 3902T, has many of the morphological and physiological characteristics of the genus Orenia, as was confirmed by a phylogenetic study based on 16S rRNA gene sequencing. The closest species is Orenia marismortui, with a similarity of only 95.1%. However, strain SG 3902T, unlike O. marismortui, does not ferment mannose, glycogen or starch. The G + C contents of the DNA also differ significantly, being 29.6 mol% for O. marismortui and 33.7 mol% for strain SG 3902T. On the basis of these physiological and genetic differences, it is proposed that strain SG 3902T should be considered as a representative of a new species belonging to the genus Orenia, under the name Orenia salinaria sp. nov. The type strain is SG 3902T (= ATCC 700911).

A phylogenetic analysis was performed on a red-pigmented, radiation-resistant, Gram-negative, rod-shaped organism originating from irradiated pork. Comparative 16S rRNA gene sequencing showed the bacterium was a member of the Cytophaga-Flavobacterium-Bacteroides line of descent and represents a new subline within the genus Hymenobacter. A new species, Hymenobacter actinosclerus, is described for this novel radiation-resistant bacterium. The type strain of Hymenobacter actinosclerus is CCUG 39621T.

A bacterial isolate with an optimum growth temperature of about 55 degrees C was recovered on a medium composed of one part Kligler's iron agar and four parts of Thermus Agar from the host spring at São Pedro do Sul in central Portugal. Phylogenetic analyses using the 16S rRNA gene sequence of strain SPS-1037T indicated that the new organism represented a new genus and species of beta-Proteobacteria. The major fatty acids of strain SPS-1037T are C16:0 and C17:0. Ubiquinone 8 is the major respiratory quinone, and the major polar lipids are phosphatidylethanolamine and phosphatidylglycerol. The new isolate is aerobic and chemolithoheterotrophic. Thiosulfate and tetrathionate were oxidized to sulfate. The growth yield of the organism was improved by the addition of thiosulfate to media containing organic carbon sources, but the organism did not grow autotrophically under the conditions examined. Heterotrophic growth of strain SPS-1037T occurs on amino acids and organic acids, but this organism does not assimilate carbohydrates. On the basis of the phylogenetic analyses, and physiological and biochemical characteristics, it is proposed that strain SPS-1037T represents a new genus and a new species for which the name Tepidimonas ignava is proposed.