- Volume 50, Issue 3, 2000

A new genus, Methylocella, and a new species, Methylocella palustris, are proposed for three strains of methane-oxidizing bacteria isolated from acidic Sphagnum peat bogs. These bacteria are aerobic, Gram-negative, colourless, non-motile, straight and curved rods that utilize the serine pathway for carbon assimilation, multiply by normal cell division and contain intracellular poly-beta-hydroxybutyrate granules (one at each pole). These strains use methane and methanol as sole sources of carbon and energy and are moderately acidophilic organisms with growth between pH 4.5 and pH 7.0, the optimum being at pH 5.0-5.5. The temperature range for growth is 10-28 degrees C with the optimum at 15-20 degrees C. The intracytoplasmic membrane system is different from those of type I and II methanotrophs. Cells contain an extensive periplasmic space and a vesicular membrane system connected to the cytoplasmic membrane. The strains grew only on media with a low salt content (0.2-0.5 g l(-1)). All three strains were found to possess soluble methane monooxygenase and are able to fix atmospheric nitrogen via an oxygen-sensitive nitrogenase. No products were observed in a PCR with particulate methane monooxygenase-targeted primers; hybridization with a pmoA probe was also negative. The major phospholipid fatty acids are 18:1 acids. The G+C content of the DNA is 61.2 mol%. The three strains share identical 16S rRNA gene sequences and represent a novel lineage of methane-oxidizing bacteria within the alpha-subclass of the class Proteobacteria and are only moderately related to type II methanotrophs of the Methylocystis-Methylosinus group. The three strains are most closely related to the acidophilic heterotrophic bacterium Beijerinckia indica subsp. indica (96.5% 16S rDNA sequence similarity). Collectively, these strains comprise a new species and genus Methylocella palustris gen. nov., sp. nov.; strain KT (= ATCC 700799T) is the type strain.

Unknown Eubacterium-like organisms VPI 12708 and five strains (Y-1113, I-10, M-18, TH-82 and 36S) had high bile acid 7alpha-dehydroxylating activity; the unknown Clostridium-like organisms TN-271T and TN-272 also had the same activity. Analysis of their 16S rDNA sequences demonstrated that all strains belong to cluster XIVa of the genus Clostridium (Collins et al., 1994). Strain VPI 12708 and five other strains (Y-1113, I-10, M-18, TH-82 and 36S) formed a single cluster and strains TN-271T and TN-272 formed another single cluster. Clostridium scindens JCM 6567T was the most closely related species for two clusters in the phylogenetic tree. Values for DNA-DNA similarities among C. scindens JCM 6567T, strain VPI 12708 and the other five strains were greater than 70%, showing that these micro-organisms were a single species. Therefore, we identified strain VPI 12708 and the five other strains as C. scindens. In addition, DNA-DNA similarities among C. scindens JCM 6567T, strain TN-271T and strain N-272 revealed that strains TN-271T and TN-272 were distinct from C. scindens JCM 6567T. On the basis of phylogenetic analysis and DNA-DNA similarity data, it was concluded that strains TN-271T and TN-272 are members of a new species of the genus Clostridium, for which the name Clostridium hylemonae is proposed. The type strain is strain TN-271T (= JCM 10539T).

Five strains of anaerobic non-sporing Gram-positive bacilli isolated from advanced periodontitis (four strains) and a dentoalveolar abscess (one strain) that did not correspond to existing species were subjected to phenotypic and genetic characterization. Following 16S rDNA sequence analysis, they were found to constitute a novel branch of the low G+C Gram-positive division of the phylogenetic tree related to Erysipelothrix rhusiopathiae and Holdemania filiformis. A new genus Bulleidia, and the species Bulleidia extructa, are proposed. Growth of B. extructa in broth media was poor but was enhanced by the addition of fructose, glucose or maltose together with Tween 80. Glucose and maltose were fermented and arginine was hydrolysed. Acetate, lactate and trace amounts of succinate were the end products of glucose fermentation. The G+C content of the DNA of the type strain is 38 mol%. The type strain of Bulleidia extructa is DSM 13220T.

During the course of isolating and identifying purple non-sulfur bacteria, one nitrate-reducing strain was isolated which did not fit the description of any other purple non-sulfur bacterium known to date. The isolate had rod-shaped cells that contained lamellar intracytoplasmic membranes and produced red cultures. Absorption maxima of photosynthetically grown cell homogenates were at 376, 471, 503, 540, 591, 805 and 878 nm. The new isolate grew anaerobically in the light or aerobically in the dark. Various organic compounds served as carbon sources and electron donors. The predominant quinone was ubiquinone 10, the predominant fatty acid was 18:1omega7c. The polar lipids comprised diphosphatidyl glycerol, phosphatidyl glycerol, phosphatidyl ethanolamine and phosphatidyl choline. Analysis of the 16S rDNA gene sequences revealed that the new isolate was closely related to Rhodopseudomonas palustris. A DNA-DNA- hybridization study differentiated the new isolate and Rhodopseudomonas palustris at the species level. Therefore, the name Rhodopseudomonas rhenobacensis sp. nov. is proposed for the new isolate.

Two newly described species of mesophilic, cellulose-degrading, aerobic bacteria were isolated from forest humus soils along the southern border of the Caspian Sea. Cellulomonas persica and Cellulomonas iranensis are proposed as new specific epithets based on comparative sequence analyses of 16S rDNA, DNA-DNA hybridization and phenotypic characteristics. Formal species descriptions are provided.

An isolate of an acidophilic archaeon, strain YT, was obtained from a bioleaching pilot plant. The organism oxidizes ferrous iron as the sole energy source and fixes inorganic carbon as the sole carbon source. The optimal pH for growth is 1.7, although growth is observed in the range pH 1.3 to 2.2. The cells are pleomorphic and without a cell wall. 16S rRNA gene sequence analysis showed this strain to cluster phylogenetically within the order 'Thermoplasmales' sensu Woese, although with only 89.9 and 87.2% sequence identity, respectively, to its closest relatives, Picrophilus oshimae and Thermoplasma acidophilum. Other principal differences from described species of the 'Thermoplasmales' are autotrophy (strain YT is obligately autotrophic), the absence of lipid components typical of the ' Thermoplasmales' (no detectable tetraethers) and a lower temperature range for growth (growth of strain YT occurs between 15 and 45 degrees C). None of the sugars, amino acids, organic acids or other organic compounds tested was utilized as a carbon source. On the basis of the information described above, the name Ferroplasma acidiphilum gen. nov., sp. nov. is proposed for strain YT within a new family, the Ferroplasmaceae fam. nov. Strain YT is the type and only strain of F. acidiphilum. This is the first report of an autotrophic, ferrous-iron-oxidizing, cell-wall-lacking archaeon.

Heterogeneity in the 16S-23S rDNA spacer of uncultured 'Tropheryma whippelii' has previously been reported. In this study, the hypervariable insertion in the 23S rDNA domain III characteristic for actinobacteria was analysed. The finding of a unique sequence virtually identical among the subtypes supports the classification of 'T. whippelii' among the actinobacteria and the concept of three subtypes rather than three subspecies, and provides an alternative target for diagnostic assays.

A collection of 106 Photobacterium damselae subsp. piscicida strains and 19 Photobacterium damselae subsp. damselae strains, including reference and type strains, were genetically characterized using AFLP. The total genomic DNA of each bacterial strain was digested using restriction endonucleases HindIII and TaqI. Using numerical analysis, six clusters were recognized. The largest cluster (n = 106) contained the majority of the strains tested and consisted exclusively of Photobacterium damselae subsp. piscicida. The Photobacterium damselae subsp. damselae strains fell outside this cluster. DNA-DNA hybridization experiments showed 77% DNA binding between the two subspecies, indicating a close genetic relationship. This clearly demonstrates the applicability of AFLP in studying the taxonomic position of Photobacterium damselae subsp. piscicida. In addition, AFLP proved to be a useful genotypic technique for epidemiological surveys of the pathogen, since it was able to discriminate between Mediterranean and Japanese Photobacterium damselae subsp. piscicida isolates.

Bacillus globisporus and Bacillus psychrophilus are one among many pairs of ecologically distinct taxa that are distinguished by very few nucleotide differences in 16S rRNA gene sequence. This study has investigated whether the lack of divergence in 16S rRNA between such species stems from the unusually slow rate of evolution of this molecule, or whether other factors might be preventing neutral sequence divergence at 16S rRNA as well as every other gene. B. globisporus and B. psychrophilus were each surveyed for restriction-site variation in two protein-coding genes. These species were easily distinguished as separate DNA sequence clusters for each gene. The limited ability of 16S rRNA to distinguish these species is therefore a consequence of the extremely slow rate of 16S rRNA evolution. The present results, and previous results involving two Mycobacterium species, demonstrate that there exist closely related species which have diverged long enough to have formed clearly separate sequence clusters for protein-coding genes, but not for 16S rRNA. These results support an earlier argument that sequence clustering in protein-coding genes could be a primary criterion for discovering and identifying ecologically distinct groups, and classifying them as separate species.

The cellular fatty acid composition of five of the six genera of unicellular cyanobacteria in subsection II, Pleurocapsales (Dermocarpa, Xenococcus, Dermocarpella, Myxosarcina and the Pleurocapsa assemblage) contained high proportions of saturated straight-chain fatty acids (26-41% of the total) and unsaturated straight chains (40-67%). Isomers of 16:1 were the main monounsaturated acid component (11-59%). Polyunsaturated acids were present at trace levels (0-1% or less) in Xenococcus and Myxosarcina, at concentrations of less than 7% in Dermocarpa, Dermocarpella, Pleurocapsa and CCMP 1489, and at high concentrations (35% or more) in Chroococcidiopsis. Chroococcidiopsis was also different in terms of the percentage of 16:1 isomers (10-12%) compared to other genera (30-59%), and in terms of total 16-carbon and 18-carbon fatty acids. In general, the composition and heterogeneity of fatty acids in the order Pleurocapsales was similar to that reported for the unicellular cyanobacteria of subsection I, order Chroococcales.

Two novel strains of Propionigenium maris able to reductively debrominate 2,4,6-tribromophenol (TBP) to monobromophenols were isolated from marine hemichordate and polychaete burrows. These two strains, DSL-1 and ML-1, were anaerobic, non-motile rods that stained Gram-negative and required 0.05% yeast extract for growth. Strain DSL-1 fermented pyruvate and succinate to predominantly butyrate and strain ML-1 fermented glucose and succinate primarily to propionate. No inorganic terminal electron acceptors were identified. The pH and temperature optima for growth were 7.6 and 30 degrees C for strain DSL-1 and 7.0 and 32 degrees C for strain ML-1, respectively; doubling times for strains DSL-1 and ML-1 were 0.32 h and 0.30 h, respectively. Both strains required 2-3% (w/v) NaCl for optimal growth. Morphological and physiological features, as well as the results of 16S rDNA sequence analysis, showed these to be new strains of Propionigenium maris. Because they differ from the P. maris type strain (DSM 9537T) in a number of respects, including their ability to rapidly debrominate di- and tribromophenols, and in their specific habitats, the species description is amended to include these ecologically important properties.

An investigation was undertaken of the genetic diversity of Nodularia strains from the Baltic Sea and from Australian waters, together with the proposed type strain of Nodularia spumigena. The Nodularia strains were characterized by using a polyphasic approach, including RFLP of PCR-amplified 16S rRNA genes, 16S rRNA gene sequencing, Southern blotting of total DNA, repetitive extragenic palindromic- and enterobacterial repetitive intergenic consensus-PCR, ribotyping and phenotypic tests. With genotypic methods, the Nodularia strains were grouped into two clusters. The genetic groupings were supported by one phenotypic property: the ability to produce nodularin. In contrast, the cell sizes of the strains were not different in the two genetic clusters. 16S rRNA gene sequences indicated that all the Nodularia strains were closely related, despite their different origins. According to this study, two genotypes of Nodularia exist in the Baltic Sea. On the basis of the taxonomic definitions of Komarek et al. (Algol Stud 68, 1-25, 1993), the non-toxic type without gas vesicles fits the description of Nodularia sphaerocarpa, whereas the toxic type with gas vesicles resembles the species N. spumigena and Nodularia baltica.

16S rRNA phylogenetic analysis of a number of yellow- and orange-pigmented strains isolated from a variety of Antarctic habitats including sea ice, lakewater and cyanobacterial mats indicated a close relationship to the genus Flavobacterium but distinct from known Flavobacterium species. Phenotypic properties, DNA G+C content and whole-cell fatty acid profiles of the Antarctic strains were consistent with those of the genus Flavobacterium. DNA-DNA hybridization analysis indicated the presence of two distinct and novel genospecies each isolated from a different Antarctic habitat. From polyphasic taxonomic data it is proposed that the two groups represent new species with the following proposed names: Flavobacterium gillisiae (ACAM 601T) and Flavobacterium tegetincola (ACAM 602T). In addition polyphasic analysis of the species '[Cytophaga] xantha' (Inoue and Komagata 1976), isolated from Antarctic mud, indicated it was a distinct member of the genus Flavobacterium and was thus revived as Flavobacterium xanthum. Phylogenetic and fatty acid analyses also indicate that the species [Flavobacterium] salegens (Dobson et al. 1993), from Organic Lake, Antarctica, is misclassified at the genus level. It is proposed that this species belongs to a new genus, Salegentibacter salegens gen. nov., comb. nov.

An extremely halophilic Archaeon belonging to the order Halobacteriales was isolated from the solar salterns of Cabo Rojo, Puerto Rico. The organism, designated strain PR5T, is rod-shaped, non-motile and requires at least 12% (w/v) NaCl to grow. The strain is highly thermotolerant: its temperature optimum is 50 degrees C and growth is possible up to 60 degrees C. Polar lipid analysis revealed the presence of the bis-sulfated glycolipid S2-DGD-1 as sole glycolipid and the absence of the glycerol diether analogue of phosphatidylglycerosulfate. Both C20,C20 and C20,C25 core lipids are present. The G+C content of the DNA is 63.3 mol%. According to 16S rDNA sequence data, strain PR5T is closely related to the representatives of the genera Haloterrigena and Natrinema, but on the basis of its phenotypic properties, 16S rDNA sequence and DNA-DNA hybridization studies, strain PR5T cannot be assigned to any of the recognized species within these genera. On the basis of its polar lipid composition, the isolate has been assigned to the genus Haloterrigena. The creation of a new species, Haloterrigena thermotolerans, is therefore proposed to accommodate this isolate. The type strain is strain PR5T (= DSM 11552T = ATCC 700275T).

A collection of 267 consecutively isolated Streptococcus anginosus strains was screened for the prevalence of previously described 'motile' strains by hybridization with oligonucleotide probes in a reverse line blot assay. The motile strains represented 101 (37.8%) of the S. anginosus strains. The vast majority of these strains fermented mannitol and raffinose, whereas most other S. anginosus strains did not (P<0.001). Most (83/101) of the motile strains were recovered from the urogenital tract (including five strains from neonatal surveillance cultures) and only a minority (36) of them were associated with infection-related samples (P<0.001). Strains that hybridized with the same oligonucleotide probes as the type strain S. anginosus ATCC 33397T (= NCTC 10713T) were designated ATCC-like strains. They accounted for 74 (27.7%) of the strains examined, were commonly distributed among the different body parts and were significantly more associated with infection-related samples. Three other hybridization patterns were recognized in the reverse line blot assay, ribogroup I (n = 51), ribogroup II (n = 21) and ribogroup III (n = 19). Ribogroup II strains were significantly more frequently recovered from the abdominal cavity and were associated with infection-related samples. Ribogroup I included the majority of the S. anginosus strains that carried Lancefield group C. Comparison of the nearly complete 16S rRNA sequence of two representative strains of each ribogroup revealed that all five ribogroups were closely related (>97% sequence similarities), and that most sequence divergences between the ribogroups occurred in the 1024-1064 bp region of the 16S rRNA gene. The present data confirm the heterogeneity within the S. anginosus species.

The almost complete 16S rDNA sequences of the type strains of eight validly described species and two invalid species of the genus Thermoactinomyces were determined and phylogenetically analysed. The validly described Thermoactinomyces species formed phylogenetic lineages related to the family Bacillaceae, as shown previously. However, the available strains of 'Thermoactinomyces glaucus' and 'Thermoactinomyces monosporus' exhibited their closest phylogenetic affinities not to the genus Thermoactinomyces but to the genus Saccharomonospora. Some Thermoactinomyces species were shown to be closely related from 16S rDNA sequence analysis. Particularly, Thermoactinomyces vulgaris KCTC 9076T and Thermoactinomyces candidus KCTC 9557T had the same 16S rDNA sequences and Thermoactinomyces thalpophilus KCTC 9789T and Thermoactinomyces sacchari KCTC 9790T showed 16S rDNA similarity value of almost 100%. From phylogenetic analysis based on 16S rDNA sequences, it is suggested that the genus Thermoactinomyces should be taxonomically re-evaluated using other useful taxonomic markers.

During the collection of airborne bacteria in a museum in England some bacterial strains were isolated which due to their fatty acid profiles were clearly identified as members of the genus Staphylococcus. As fatty acid compositions of coagulase-negative staphylococci are very similar, differing only in quantities but not in qualities, further identification at the species level without a fatty acid database was not achieved. Investigation of the isolates using the Staph ID 32 API system resulted in an identification of the isolates as Staphylococcus epidermidis (probabilities of 79.7-95.5%). For further genotypic characterization of these isolates, some Staphylococcus epidermidis strains from different sources and the type strains of Staphylococcus aureus, Staphylococcus capitis, Staphylococcus epidermidis, Staphylococcus gallinarum, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus warneri and Staphylococcus xylosus were subjected to repetitive-sequence PCR, including enterobacterial repetitive intergenic consensus (ERIC) PCR, BOX-PCR and repetitive extragenic palindromic unit sequence (REP) PCR. ERIC- and BOX-PCR yielded a species-specific banding pattern for all Staphylococcus epidermidis strains. Furthermore, all staphylococcal reference strains investigated exhibited distinct banding patterns, clearly distinguishable from that of Staphylococcus epidermidis. No species-specific banding patterns could be observed after REP-PCR. As species identification of coagulase-negative staphylococci by fatty acid analyses and biochemical tests is known to be difficult ERIC- and BOX-PCR seem to be excellent tools for the identification of Staphylococcus epidermidis isolates.

The present study was aimed at reducing the time and labour used to perform DNA-DNA hybridizations for classification of bacteria at the species level. A micro-well-format DNA hybridization method was developed and validated. DNA extractions were performed by a small-scale method and DNA was sheared mechanically into fragments of between 400 and 700 bases. The hybridization conditions were calibrated according to DNA similarities obtained by the spectrophotometric method using strains within the family Pasteurellaceae. Optimal conditions were obtained with 300 ng DNA added per well and bound by covalent attachment to NucleoLink. Hybridization was performed with 500 ng DNA, 5% (w/w) of which was labelled with photo-activatable biotin (competitive hybridization) for 2.5 h at 65 degrees C in 2 x SSC followed by stringent washing with 2 x SSC at the same temperature. The criteria for acceptance of results were a maximum of 15% standard deviation, calculated as a percentage of the mean for four replicate micro-wells, and that DNA similarities were not significantly different in at least two independent experiments. The relationship between DNA similarities obtained by the micro-well method (y) and by the spectrophotometric method (x) was y = 0.534x+30.6, when these criteria had been applied to 23 pairs of strains of Actinobacillus species, avian [Pasteurella] haemolytica-like bacteria and Mannheimia species. The correlation (Pearson) between DNA similarities obtained by interchange of strains used for covalent binding and hybridization was 0.794. Significantly lower DNA similarities were observed by the spectrophotometric compared with the micro-well method for three pairs of hybridizations. After removal of these data, the relationship between DNA similarities obtained by the micro-well and spectrophotometric methods improved to y = 0.855x + 11.0. It was found that the accuracy and precision of the micro-well method was at the same level as that of the spectrophotometric method, but the labour and analysis time were reduced significantly. The use of hybridization in the micro-well format will allow DNA-DNA hybridizations to be carried out between all strains selected for a particular taxonomic study, in order to construct complete data matrices and improve species definition.