- Volume 51, Issue 2, 2001

A spore-forming, halophilic bacterium was isolated from surface sediment located on the beach of Palaeochori Bay near to a shallow water hydrothermal vent area, Milos, Greece. The bacterium, designated SH 714T, consisted of motile, strictly aerobic rods which contained an Orn-D-Glu type murein and a G+C content of 35 mol%. Thin sections showed a cell wall typical for Gram-positive bacteria; the peptidoglycan layer, however, was very thin. The Gram-reaction of the organism was negative. Comparative 16S rRNA gene sequencing demonstrated that the isolate represents a new line of descent within the spore-forming rods branching at the periphery of the rRNA group 1 Bacillus (Bacillus sensu stricto). The nearest phylogenetic neighbours of the unknown bacterium were Bacillus haloalkaliphilus, Marinococcus albus and Halobacillus species. Based on phylogenetic and phenotypic evidence it is proposed that the unknown bacterium be classified as Filobacillus milensis gen. nov., sp. nov. The type strain is SH 714T (= DSM 13259T = ATCC 700960T).

Five hydrocarbon-oxidizing strains were isolated from formation waters of oilfields in Russia, Kazakhstan and China. These strains were moderately thermophilic, neutrophilic, motile, spore-forming rods, aerobic or facultatively anaerobic. The G+C content of their DNA ranged from 49.7 to 52.3 mol%. The major isoprenoid quinone was menaquinone-7; cellular fatty acid profiles consisted of significant amounts of iso-15:0, iso-16:0 and iso-17:0 fatty acids (61.7-86.8% of the total). Based on data from 16S rDNA analysis and DNA-DNA hybridization, the subsurface isolates could be divided into two groups, one of which consisted of strains UT and X and the other of which consisted of strains K, Sam and 34T. The new strains exhibited a close phylogenetic relationship to thermophilic bacilli of 'Group 5' of Ash et al. [Ash, C., Farrow, J. A. E., Wallbanks, S. & Collins, M. D. (1991). Lett Appl Microbiol 13, 202-206] and a set of corresponding signature positions of 16S rRNA. Comparative analysis of the 16S rDNA sequences and fatty acid compositions of the novel isolates and established species of thermophilic bacilli indicated that the subsurface strains represent two new species within a new genus, for which the names Geobacillus subterraneus gen. nov., sp. nov., and Geobacillus uzenensis sp. nov. are proposed. It is also proposed that Bacillus stearothermophilus, Bacillus thermoleovorans, Bacillus thermocatenulatus, Bacillus kaustophilus, Bacillus thermoglucosidasius and Bacillus thermodenitrificans be transferred to this new genus, with Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) as the type species.

A polyphasic taxonomic study was performed on the type strain of Bacillus thermosphaericus DSM 10633T and three related soil isolates. On the basis of phenotypic characteristics, chemotaxonomic profiles and phylogenetic data a new genus, Ureibacillus gen. nov., is proposed for the strains in the Bacillus thermosphaericus cluster. Strains of this cluster fall into two DNA-DNA similarity groups: while one group contains the type strain of Ureibacillus thermosphaericus comb. nov. and a single soil isolate, the other contains two soil isolates. The two groups differed in the composition of isoprenoid quinones and some phenotypic properties. These data support the description of a novel species of Ureibacillus for which the name Ureibacillus terrenus is proposed. The type strain of this new species is TH9AT (= DSM 12654T = LMG 19470T).

A novel thermoalkaliphilic, obligately anaerobic bacterium was isolated from a humid soil sample of a hot inlet of Lake Bogoria, Kenya. The newly isolated strain grows optimally at pH 9.5 and 50-55 degrees C and its growth range is pH 6.0-10.5 and 30-65 degrees C. Unlike the already known thermoalkaliphiles, the strain grows heterotrophically on a variety of mono- and polysaccharides (glucose, ribose, mannose, fructose, sucrose, maltose, starch, pullulan, xylan and cellulose) and on proteinaceous substrates such as yeast extract, peptone and tryptone. No dissimilatory sulfate reduction was observed, whereas thiosulfate was found to enhance growth when glucose or starch were used as substrates. Under optimal conditions, the doubling time is 48 min. Sodium ions are necessary for growth, with an optimal concentration of 230 mM (1% NaCl, w/v) at pH 9.5. The rod-shaped cells are motile in the exponential growth phase under optimal growth conditions. Despite the Gram-negative staining and negative KOH assay, the strain is a Gram-positive organism, having an atypically thin cell wall. A sheath-like structure occurs at the cell separation area and parts of a surface layer-like structure were also observed. Based on physiological properties and molecular biological analysis, the strain falls within the radiation of the clostridia and represents a new species of Anaerobranca within the Clostridium/Bacillus subphylum of the Gram-positive bacteria. Strain LBS3T (= DSM 13577T) is named Anaerobranca gottschalkii sp. nov. and is designated as the type strain.

A novel actinomycete was isolated from soil in the tropical rainforest of Singapore. The cells of this actinomycete are highly pleomorphic. In the early stages of growth, most cells are of irregular squarish shape and varied sizes. Cells remain attached after cell division, often forming chains or aggregates of a few cells. Cells at the end of a chain tend to elongate. With prolonged cultivation, cells show different degrees of elongation and enlargement, producing branched hyphae of uneven thickness. At the periphery of the colony, long hyphae form, which are divided into alternating segments of elongated cells and chains of squarish cells. This actinomycete is considerably salt-tolerant, able to grow in the presence of 15% NaCl. Chemotaxonomically, it contains LL-diaminopimelic acid (DAP) in the cell wall, type PI phospholipids and MK-9(H6) as the predominant menaquinone. 16S rDNA sequence analysis assigned this actinomycete to the family Nocardioidaceae, but its 16S rDNA shared no more than 91.2% sequence similarity with other members of the family. Based on phenotypic, chemotaxonomic and phylogenetic evidence, it is proposed that this actinomycete be classified as a new species in a new genus, Actinopolymorpha singaporensis gen. nov., sp. nov.

Phenotypic and phylogenetic studies were performed on an unknown, Gram-positive, catalase-negative coccus isolated from human blood. Comparative 16S rRNA gene sequencing demonstrated that the organism represents a new subline within the genus Aerococcus. The unknown bacterium was readily distinguished from the three currently recognized Aerococcus species, Aerococcus christensenii, Aerococcus urinae and Aerococcus viridans, by biochemical tests and electrophoretic analysis of whole-cell proteins. On the basis of phylogenetic and phenotypic evidence, it is proposed that this unknown bacterium from blood be classified as Aerococcus sanguicola sp. nov. The type strain of Aerococcus sanguicola is CCUG 43001T (= CIP 106533T).

A novel thermophilic hydrogen-oxidizing bacterium, Hydrogenophilus hirschii Yel5aT (= DSM 11420T = JCM 10831T) has been isolated from the Angel Terrace Spring, Yellowstone National Park. The isolate was rod-shaped (1.0-1.5 x 0.8 microm) with a polarly inserted flagellum. Cells grew chemolithoautotrophically under an atmosphere of H2 and CO2 (80:20) in the presence of low concentrations of O2 (optimum 2.5%). Organotrophic growth occurred on complex organic substrates such as yeast extract and peptone and on organic acids. Carbohydrates and amino acids were not utilized. The strain grew between 50 and 67 degrees C; optimal growth occurred at a temperature of 63 degrees C. The pH optimum was 6.5. NaCl inhibited growth at concentrations higher than 1.5%. The major respiratory lipoquinone was ubiquinone-8. Analysis of fatty acids of Yel5aT revealed a straight-chain saturated C16:0 as the major component followed by cyclo C17:0 and cyclo C19:0. The G+C content of total DNA was 61 mol%. Phylogenetic analysis placed the strain in the beta-proteobacteria. The 16S rDNA sequence of strain Yel5aT was related to that of Hydrogenophilus thermoluteolus. To our knowledge, Hydrogenophilus hirschii is the most thermophilic micro-organism found within the proteobacteria that grows in the temperature range 50-68 degrees C.

Strain CT, a non-motile, mesophilic, hydrogenotrophic, methanogenic bacterium, was isolated from an anaerobic digester used for the treatment of raw cassava-peel waste in Congo. The cells were rods, 0.4-0.5 x 2-10 microm in size, and stained Gram-positive. Hydrogen and carbon dioxide were the only substrates that supported growth and methane production. Methane production, but not growth, occurred with CO2 in the presence of either 2-propanol, 2-butanol or cyclopentanol as hydrogen donors. The temperature range for growth was 25-50 degrees C, the optimum being between 37 and 42 degrees C. The optimum pH for growth was 7.2; consistent growth and methane production were not observed below pH 5.9 or above pH 8.2. The doubling time under optimal growth conditions was 7.5 h. The DNA base composition was 39.5 mol% G+C. On the basis of 16S rRNA gene sequence analysis and phenotypic characteristics, the isolate is proposed as a new species of the genus Methanobacterium, namely Methanobacterium congolense sp. nov. The type strain is strain CT (= DSM 7095T = OCM 779T).

A thermophilic, anaerobic, chemo-organotrophic sulfur-reducing bacterium, designated MV1075T, was isolated from a deep-sea hydrothermal chimney sample collected on the Mid-Atlantic Ridge. Cells were rod-shaped with a sheath-like outer structure, motile with polar flagella and stained Gram-negative. They appeared singly, in pairs or in short chains. The temperature range for growth was 25-65 degrees C, with an optimum at 55 degrees C. Growth was observed from pH 5 to pH 9, and the optimum pH was around 7. The salinity range for growth was 15-70 g sea salt l(-1) (corresponding to 10-45 g NaCl l(-1)), with an optimum at 30 g l(-1) (20 g NaCl l(-1)). The isolate was able to grow on a broad spectrum of carbohydrates or complex proteinaceous substrates. Sulfur was not necessary for growth. Growth was inhibited by H2, but, in presence of sulfur, this inhibition was removed and H2S was produced. The G+C content of the genomic DNA was 29 mol %. Phylogenetic analyses of the 16S rRNA gene located the strain within the order Thermotogales, in the domain Bacteria. On the basis of 16S rDNA sequence comparisons, in combination with morphological and physiological characteristics, it is proposed that the isolate should be described as a novel species of a new genus, Marinitoga gen. nov., of which Marinitoga camini sp. nov. is the type species. The type strain is MV1075T (= CNCM 1-2413T = DSM 13578T).

Nodularia, a member of the order Nostocales, is a bloom-forming filamentous cyanobacterium that possesses the ability to form toxic blooms. The toxin produced by Nodularia, nodularin, is a hepatotoxin, similar in structure to the heptapeptide toxin microcystin. Twenty-one strains of Nodularia, representing the species Nodularia spumigena, Nodularia harveyana and Nodularia sphaerocarpa, were analysed for toxin production by protein phosphatase inhibition assay and sequenced over the 16S rDNA region. Phylogenetic analysis of Nodularia 16S rDNA sequences found that Nodularia clustered into two main groups. An N. spumigena cluster was distinct from the benthic species N. harveyana and N. sphaerocarpa. There was no distinction between strains isolated from globally diverse locations. Nodularin-producing species were restricted to the single, evolutionally distinct cluster of N. spumigena. This observation has enabled the design of a specific 16S rRNA PCR for the rapid detection of nodularin-producing strains. Alignment of 16S rDNA sequences from toxic and non-toxic Nodularia with other members of the cyanobacteria allowed the design of both Nodularia generic and toxic N. spumigena-specific primers.

Toxic and non-toxic cyanobacterial strains from Anabaena, Aphanizomenon, Calothrix, Cylindrospermum, Nostoc, Microcystis, Planktothrix (Oscillatoria agardhii), Oscillatoria and Synechococcus genera were examined by RFLP of PCR-amplified 16S rRNA genes and 16S rRNA gene sequencing. With both methods, high 16S rRNA gene similarity was found among planktic, anatoxin-a-producing Anabaena and non-toxic Aphanizomenon, microcystin-producing and non-toxic Microcystis, and microcystin-producing and non-toxic Planktothrix strains of different geographical origins. The respective sequence similarities were 99.9-100%, 94.2-99.9% and 99.3-100%. Thus the morphological characteristics (e.g. Anabaena and Aphanizomenon), the physiological (toxicity) characteristics or the geographical origins did not reflect the level of 16S rRNA gene relatedness of the closely related strains studied. In addition, cyanobacterial strains were fingerprinted with repetitive extragenic palindromic (REP)- and enterobacterial repetitive intergenic consensus (ERIC)-PCR. All the strains except two identical pairs of Microcystis strains had different band profiles. The overall grouping of the trees from the 16S rRNA gene and the REP- and ERIC-PCR analyses was similar. Based on the 16S rRNA gene sequence analysis, four major clades were formed. (i) The clade containing filamentous heterocystous cyanobacteria was divided into three discrete groups of Anabaena/Aphanizomenon, Anabaena/Cylindrospermum/ Nodularia/Nostoc and Calothrix strains. The three other clades contained (ii) filamentous non-heterocystous Planktothrix, (iii) unicellular non-heterocystous Microcystis and (iv) Synechococcus strains.

Previous studies on the ubiquity and diversity of microbial (per)chlorate reduction resulted in the isolation of 20 new strains of dissimilatory (per)chlorate-reducing bacteria. Phylogenetic analysis revealed that all of the isolates were members of the Proteobacteria with representatives in the alpha-, beta- and gamma-subclasses. The majority of the new isolates were located in the beta-subclass and were closely related to each other and to the phototrophic Rhodocyclus species. Here an in-depth analysis of these organisms which form two distinct monophyletic groups within the Rhodocyclus assemblage is presented. Two new genera, Dechloromonas and Dechlorosoma, are proposed for these beta-subclass lineages which represent the predominant (per)chlorate-reducing bacteria in the environment. The type species and strains for these new genera are Dechloromonas agitata strain CKBT and Dechlorosoma suillum strain PST, respectively.

Seven spore-forming, nitrogen-fixing bacterial isolates from spruce forest humus in Finland were studied using the polyphasic approach. PCR amplification of 16S rRNA gene fragment with specific primers showed that the isolates were members of Paenibacillus. Levels of 16S rDNA similarity between the isolates were 97.3-100.0% and those between the isolates and other Paenibacillus species were 90.3-96.5%. The highest similarities were observed with Paenibacillus azotofixans and Paenibacillus durus. Ribotyping with EcoRI and PvuII restriction showed a high diversity in the Paenibacillus species and distinguished the isolates from these closely related species. The main whole-cell fatty acids were anteiso-C15:0 (33-48%), straight-chain C14:0 (7-21%) and C16:0 (9-20%), and iso-C15:0 (6-15%). Electron microscopy revealed a unique striped morphology of the spore surfaces. Based on phylogenetic inference and phenotypic and chemotaxonomic characteristics, these isolates are proposed as a new species, Paenibacillus borealis sp. nov., the type strain of which is KK19T (= DSM 13188T = CCUG 43137T).

Strains isolated independently from two patients could be recognized as Ralstonia pickettii biovar 3/'thomasii'. The 16S rDNA sequences of these strains and two other strains of R. pickettii biovar 3/'thomasii' clustered at less than 98% similarity versus all other described Ralstonia species and at less than 97 % versus the two other R. pickettii biovars. The separate species status of R. pickettii biovar 3/'thomasii' was confirmed by DNA-DNA hybridization, indicating less than 60% DNA homology with the R. pickettii biovars Va-1 and Va-2 and with two as-yet unclassified but biochemically similar Ralstonia strains. Phenotypically, this Ralstonia species can be distinguished from all described Ralstonia species by its acidification of D-arabitol and mannitol and by its lack of nitrate reduction and of alkalinization of tartrate and from two as-yet unclassified Ralstonia strains only by its lack of nitrate reduction. The name Ralstonia mannitolytica sp. nov. is proposed, reflecting the characteristic acidification of mannitol. Resistance to desferrioxamine is another difference from R. pickettii and Ralstonia solanacearum. Although several nosocomial outbreaks have been associated with R. mannitolytica, life-threatening infections have not yet been reported, possibly due to misidentification as Pseudomonas fluorescens or Burkholderia cepacia. In at least one of the two cases reported here, the R. mannitolytica isolate was found to be clinically relevant, causing recurrent nosocomial meningitis, with an infected implanted catheter as the source. The type strain of R. mannitolytica is NCIMB 10805T (= LMG 6866T), which was isolated during the first described outbreak as 'Pseudomonas thomasii' at St Thomas' Hospital, London, UK, in 1971.

Five bacterial strains were isolated from tropical flowers collected in Thailand and Indonesia by the enrichment culture approach for acetic acid bacteria. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolates were located within the cluster of the genus Asaia. The isolates constituted a group separate from Asaia bogorensis on the basis of DNA relatedness values. Their DNA G+C contents were 58.6-59.7 mol%, with a range of 1.1 mol%, which were slightly lower than that of A. bogorensis (59.3-61.0 mol%), the type species of the genus Asaia. The isolates had morphological, physiological and biochemical characteristics similar to A. bogorensis strains, but the isolates did not produce acid from dulcitol. On the basis of the results obtained, the name Asaia siamensis sp. nov. is proposed for these isolates. Strain S60-1T, isolated from a flower of crown flower (dok rak, Calotropis gigantea) collected in Bangkok, Thailand, was designated the type strain ( = NRIC 0323T = JCM 10715T = IFO 16457T).

Forty-three strains of obligately chemolithoautotrophic sulfur-oxidizing bacteria were isolated from highly alkaline soda lakes in south-east Siberia (Russia) and in Kenya using a specific enrichment procedure at pH 10. The main difference between the novel isolates and known sulfur bacteria was their potential to grow and oxidize sulfur compounds at pH 10 and higher. The isolates fell into two groups that were substantially different from each other physiologically and genetically. Most of the Siberian isolates belonged to the group with a low DNA G+C content (48.0-51.2 mol%). They were characterized by a high growth rate, a low growth yield, a high cytochrome content, and high rates of oxidation of sulfide and thiosulfate. This group included 18 isolates with a DNA homology of more than 40%, and it is described here as a new genus, Thioalkalimicrobium, with two species Thioalkalimicrobium aerophilum (type species) and Thioalkalimicrobium sibericum. The other isolates, mainly from Kenyan soda lakes, fell into a group with a high DNA G+C content (61.0-65.6 mol%). In general, this group was characterized by a low growth rate, a high molar growth yield and low, but relatively equal, rates of oxidation of thiosulfate, sulfide, elemental sulfur and polythionates. The group included 25 isolates with a DNA homology of more than 30%. It was less compact than Thioalkalimicrobium, containing haloalkalophilic, carotenoid-producing, nitrate-reducing and facultatively anaerobic denitrifying strains. These bacteria are proposed to be assigned to a new genus, Thioalkalivibrio, with three species Thioalkalivibrio versutus (type species), Thioalkalivibrio denitrificans and Thioalkalivibrio nitratis. Phylogenetic analysis revealed that both groups belong to the gamma-Proteobacteria. The Thioalkalimicrobium species were closely affiliated with the neutrophilic chemolithoautotrophic sulfur bacteria of the genus Thiomicrospira, forming a new alkaliphilic lineage in this cluster. In contrast, Thioalkalivibrio was not related to any known chemolithoautotrophic taxa, but was distantly associated with anaerobic purple sulfur bacteria of the genus Ectothiorhodospira.

Recent studies on the diversity and ubiquity of Fe(III)-reducing organisms in different environments led to the isolation and identification of four new dissimilatory Fe(III)-reducers (strains H-2T, 172T, TACP-2T and TACP-5). All four isolates are non-motile, Gram-negative, freshwater, mesophilic, strict anaerobes with morphology identical to that of Geobacter metallireducens strain GS-15T. Analysis of the 16S rRNA sequences indicated that the new isolates belong to the genus Geobacter, in the delta-Proteobacteria. Significant differences in phenotypic characteristics, DNA-DNA homology and G+C content indicated that the four isolates represent three new species of the genus. The names Geobacter hydrogenophilus sp. nov. (strain H-2T), Geobacter chapellei sp. nov. (strain 172T) and Geobacter grbiciae sp. nov. (strains TACP-2T and TACP-5) are proposed. Geobacter hydrogenophilus and Geobacter chapellei were isolated from a petroleum-contaminated aquifer and a pristine, deep, subsurface aquifer, respectively. Geobacter grbiciae was isolated from aquatic sediments. All of the isolates can obtain energy for growth by coupling the oxidation of acetate to the reduction of Fe(III). The four isolates also coupled Fe(III) reduction to the oxidation of other simple, volatile fatty acids. In addition, Geobacter hydrogenophilus and Geobacter grbiciae were able to oxidize aromatic compounds such as benzoate, whilst Geobacter grbiciae was also able to use the monoaromatic hydrocarbon toluene.

The taxonomic relationships of Azoarcus and Thauera isolates in the beta-subclass of the Proteobacteria capable of degrading fluoro-, chloro- or bromobenzoate under denitrifying conditions were analysed. A detailed classification of these strains was performed using a polyphasic approach, which included studies on morphology, phenotypic characterization, fatty acid analysis, 16S rRNA gene sequence analysis, 16S rRNA gene mapping (ribotyping) and DNA-DNA hybridization. The analyses of fatty acids and 16S rRNA gene sequencing differentiated strains 2FB2, 2FB6 and 4FB10 as new members of the genus Azoarcus and strains 4FB1, 4FB2, 3CB2, 3CB3 and 3BB1 as new members of the genus Thauera. DNA-DNA hybridization experiments established that strains 2FB2, 2FB6 and 4FB10 belong to the species Azoarcus tolulyticus. Strains 3CB2 and 3CB3 were assigned to the species Thauera aromatica on the basis of DNA-DNA hybridization and ribotyping experiments. Strains 4FB1, 4FB2 and 3BB1 showed close relatedness with strain 3CB-1T, previously described as T. aromatica genomovar chlorobenzoica. This group of strains is clearly differentiated from the species T. aromatica on the basis of 16S rRNA sequence analysis, DNA homology and ribotyping analysis. Strains 3CB-1T, 4FB1, 4FB2 and 3BB1 are proposed as members of the new species Thauera chlorobenzoica sp. nov., strain 3CB-1T (= ATCC 700723T) being the type strain.

A nonylphenol-assimilating bacterium isolated at a sewage-treatment plant in Tokyo was studied phenotypically, genotypically and phylogenetically. Analysis of the 16S rDNA sequence, the G+C content of the DNA (63 mol%) and the isoprenoid quinone composition, as well as the presence of sphingoglycolipid and the whole-cell fatty acid profile, revealed that the isolate is a member of the genus Sphingomonas. However, the sequence similarity of the 16S rDNA with that of known Sphingomonas spp. was found to be at most 96%, implying that the isolate is distinctive. Furthermore, the results of DNA-DNA hybridization experiments and its physiological characteristics clearly indicated that the isolate represents a new Sphingomonas species, for which the name Sphingomonas cloacae is proposed; strain S-3T (= JCM 10874T = IAM 14885) is the type strain.