- Volume 48, Issue 2, 1998

A halophilic endospore-forming, sulfate-reducing bacterium was isolated from an oilfield brine in France. The strain, designated SEBR 3139, was composed of long, straight to curved rods. It grew in 1–14% NaCI with an optimum at 6%. On the basis of morphological, physiological and phylogenetical characteristics, strain SEBR 3139 should be classified in the genus Desulfotomaculum. However, it is sufficiently different from the hitherto described Desulfotomaculum species to be considered as a new species. Strain SEBR 3139T(= DSM 11559T) represents the first moderate halophilic species of the genus Desulfotomaculum. The name Desulfotomaculum halophilum sp. nov. is proposed.

During screening for biosurfactant-producing, n-alkane-degrading marine bacteria, six heterotrophic bacterial strains were isolated from enriched mixed cultures, obtained from sea water/sediment samples collected near the Isle of Borkum (North Sea), using Mihagol-S (C14,15-n-alkanes) as principal carbon source. These Gram-negative, aerobic, rod-shaped bacteria use a limited number of organic compounds, including aliphatic hydrocarbons, volatile fatty acids, and pyruvate and its methyl ether. During cultivation on n-alkanes as sole source of carbon and energy, all strains produced both extracellular and cell-bound surface-active glucose lipids which reduced the surface tension of water from 72 to 29 mNm-1(16). This novel class of glycolipids was found to be produced only by these strains. The 16S rRNA gene sequence analysis showed that these strains are all members of the γ-subclass of the Proteobacteria. Their phospholipid ester-linked fatty acid composition was shown to be similar to that of members of the genus Halomonas, although they did not demonstrate a close phylogenetic relationship to any previously described species. On the basis of the information summarized above, a new genus and species, Alcanivorax borkumensis, is described to include these bacteria. Strain SK2Tis the type strain of A. borkumensis.

PCR-RFLP with nine restriction enzymes was applied to the 16S and 23S rRNA genes of 42 rhizobial and agrobacterial strains to determine the phylogenetic position of Rhizobium galegae and increase the understanding of the evolution of ribosomal operons. The strains were selected based on previous phylogenetic studies. PCR primers were designed so that they amplified a 2·3 kb fragment of the 23S rRNA gene (excluding the B8 loop). Universal primers rD1 and fD1 were used to amplify the full-length 16S rRNA. The RFLP analysis resulted in 27 and 32 different restriction patterns for 16S and 23S, respectively. The RFLP patterns were transformed to genetic distances and dendrograms were constructed from the data using the unweighted pair group method with averages. The shapes of the dendrograms derived from the analysis of the 16S and 23S rRNA genes correlated well, with only a few strains having different positions. The 23S tree generally had deeper branching than the 16S tree, allowing better discrimination between species and strains. The combined data from the two analyses described 36 genotypes. The eight R. galegae strains formed a homogeneous cluster in all dendrograms. The RFLP analysis was confirmed by partial sequence analysis of the 16S rRNA gene (the first 800 bp), which correlated well with full-length 16S rRNA sequence analysis. The 16S data placed R. galegae near the genus Agrobacterium with Agrobacterium vitis as its nearest neighbour, whereas in the 23S and the combined dendrograms it showed closer affinity to the genus Rhizobium.

Deep subterranean granitic aquifers have not been explored regarding methanogens until now. Three autotrophic methane-producing Archaea were isolated from deep granitic groundwater at depths of 68, 409 and 420 m. These organisms were non-motile, small, thin rods, 0·1-0·15 μm in diameter, and they could use hydrogen and carbon dioxide or formate as substrates for growth and methanogenesis. One of the isolates, denoted A8p, was studied in detail. It grew with a doubling time of 2·5 h under optimal conditions (20–40 °C, pH 7·8–8·8 and 0·2–1·2 M NaCI). Strain A8p is eurythermic as it grew between 3·6 and 45 °C. It was resistant to up to 20 mg bacitracin I-1. The G+C content was 54·5 mol%, as determined by thermal denaturation. Phylogenetic studies based upon 16S rRNA gene sequence comparisons placed the isolate A8p in the genus Methanobacterium. Phenotypic and phylogenetic characters indicate that the alkaliphilic, halotolerant strain A8p represents a new species. We propose the name Methanobacterium subterraneum for this species, and strain A8p (= DSM 11074T) is the type strain.

A collection of strains isolated from root nodules of Acacia species in Senegal was analysed previously by electrophoresis of total cell protein, auxanographic tests, rRNA-DNA hybridization, 16S rRNA gene sequencing, DNA base composition and DNA-DNA hybridization [de Lajudie, P., Willems, A., Pot, B. & 7 other authors (1994). Int J Syst Bacteriol 44, 715–733]. Strains from Acacia were shown to belong to two groups, Sinorhizobium terangae, and a so-called gel electrophoretic cluster U, which also included some reference strains from Brazil. Further taxonomic characterization of this group using the same techniques plus repetitive extragenic palindromic-PCR and nodulation tests is presented in this paper. Reference strains from Sudan and a number of new rhizobia isolated from nodules of Acacia Senegal, Acacia tortilis subsp. raddiana and Prosopis juliflora in Senegal were included. As a result of this polyphasic approach, the creation of a new species, Mesorhizobium plurifarium, is proposed for a genotypically and phenotypically distinct group corresponding to the former cluster U and containing strains isolated from Acacia, Leucaena, Prosopis and Chamaecrista in West Africa (Senegal), East Africa (Sudan) and South America (Brazil). The type strain of Mesorhizobium plurifarium ORS 1032 has been deposited in the LMG collection as LMG 11892.

Several Gram-positive, non-spore-forming and non-motile bacteria consisting of pairs or chains of cocci were isolated during an investigation of the bacterial flora of the caecum of donkeys. Physiological and metabolic data indicated that the strains belong to the genus Enterococcus; phenotypic traits of these organisms were not consistent with any of the currently known Enterococcus species. 16S rRNA gene sequence analysis placed these strains in the genus Enterococcus. Their closest relatives are Enterococcus avium, Enterococcus faecium and Enterococcus pseudoavium with a sequence similarity of 97·4%. This group of strains can be differentiated from the other Enterococcus spp. by their phenotypic characteristics: strains do not grow in 6·5% NaCI; they do not produce acid from mannitol, sorbitol, sorbose, sucrose, raffinose, ribose and tagatose; they produce acid from d-xylose; they are able to utilize pyruvate; and they present a negative reaction on arginine. The name Enterococcus asini sp. nov. is proposed for these strains; the type strain is AS2T(= DSM 11492T).

An approach to characterizing uncultivated bacteria which combines a PFGE procedure for obtaining purified full-length chromosomes with PCR amplification is described. Isolated chromosomes from uncultivated organisms provide a specifically identifiable source material for hybridization, amplification and cloning. The availability of purified chromosomes for DNA amplification should aid in examining the diversity of microbial populations and should also reduce the possibility of forming hybrid DNA artifact molecules. The approach is illustrated by isolating the chromosome of the uncultivated agent of rodent Grey Lung disease and using the purified chromosomes to amplify and directly sequence the evolutionarily conserved 16S rRNA gene. The Grey Lung agent (GLA) contains a 650 kb chromosome and is shown to be a Mycoplasma sp. located phylogenetically in the hominis group of mycoplasmas. If a simple genomic lesion(s) is responsible for the unculturability of GLA, it is conceivable that complementation with DNA from a close relative could permit growth on artificial media.

Bacillus popilliae and Bacillus lentimorbus, causative agents of milky disease in Japanese beetle and related scarab larvae, have hitherto been differentiated based upon a small number of phenotypic characteristics, but they have not previously been examined at the molecular level. In this study 34 isolates of these bacteria were examined for DNA similarity and by random amplified polymorphic DNA (RAPD) analysis. Two distinct but related similarity groups were identified: the first contained strains of B. popilliae and the second contained strains of B. lentimorbus. Two strains distinct from but related to B. popilliae may represent a subspecies. Some strains received as B. popilliae were found to be most closely related to B. lentimorbus and some received as B. lentimorbus were found to be most closely related to B. popilliae. RAPD analysis confirmed the DNA similarity results. Paraspore formation, previously believed to be a characteristic unique to B. popilliae, was found to occur among a sub-group of B. lentimorbus strains. Growth in media supplemented with 2% NaCI was found to be a somewhat less reliable characteristic in distinguishing the species than vancomycin resistance, the latter being present only in B. popilliae.

An HPLC procedure which separates d-and l-amino acid isomers was applied to an analysis of peptidoglycan of 2,4-diaminobutyric acid (DAB)-containing actinomycetes. The cell wall peptidoglycans of species of the genera Agromyces, Clavibacter and Rathayibacter contain DAB and have been differentiated principally by their menaquinone profile. These peptidoglycans are known to be identical in structure, all being of the B2γ type, possessing both d-and l-DAB. The type strains of all the subspecies of Clavibacter michiganensis have d-and l-DAB in almost equal proportions in their cell wall peptidoglycan as previously reported. In contrast, the type strains of Clavibacter toxicus and all valid species of the genera Agromyces and Rathayibacter contain the l-isomer of DAB almost exclusively. This characteristic is in good agreement with phylogenetic analyses based on 16S rDNA sequences and menaquinone profiles. On the basis of these data, the transfer of Clavibacter toxicus to the genus Rathayibacter as Rathayibacter toxicus comb. nov. is proposed. The isomer profile of DAB is shown to be a good taxonomic marker to differentiate these genera.

Almost complete 16S rRNA sequences from seven Thermomonospora strains, Thermomonospora curvata, Thermomonospora formosensis, Thermomonospora fusca, Thermomonospora mesophila, Thermomonospora chromogena, Thermomonospora alba and Thermomonospora mesouviformis (a synonym of Thermomonospora alba) were determined and subjected to phylogenetic analysis together with the sequences from all the representative members of the suborder Streptosporangineae. On the basis of phylogenetic, chemotaxonomic and phenotypic evidence, the transfer is proposed of Thermomonospora formosensis to the genus Actinomadura as Actinomadura formosensis comb. nov., Thermomonospora mesophila to the genus Microbispora as Microbispora mesophila comb. nov., and Thermomonospora fusca and Thermomonospora alba to a new genus, Thermobifida gen. nov., which belongs to the family Nocardiopsaceae, as Thermobifida fusca comb. nov. and Thermobifida alba comb. nov. Thermobifida alba is designated the type species of the genus. The transfer is also proposed of all species of the Microtetraspora pusilla group, which were transferred from Actinomadura, to a new genus, Nonomuria gen. nov., as Nonomuria africana comb, nov., Nonomuria angiospora comb. nov., Nonomuria fastidiosa comb. nov., Nonomuria ferruginea comb. nov., Nonomuria flexuosa comb. nov., Nonomuria helvata comb. nov., Nonomuria polychroma comb. nov., Nonomuria pusilla comb. nov., Nonomuria recticatena comb. nov., Nonomuria roseola comb. nov., Nonomuria roseoviolacea comb. nov., Nonomuria rubra comb. nov., Nonomuria salmonea comb. nov., Nonomuria spiralis comb. nov. and Nonomuria turkmeniaca comb. nov. Nonomuria pusilla is designated the type species of the genus.

Three strains of creatinine-hydrolysing bacteria isolated from human urine were characterized taxonomically. They were aerobic, non-spore-forming, Gram-positive rods with the peptidoglycan of the cell wall containing lysine. MK-8 and MK-9 were found to be the major types of menaquinone. The G+C content of the DNA was 66–67 mol%. The 16S rRNA sequence of one strain (GIFU 12498) was determined and aligned with other high-G+C-content Grampositive rods from different genera. Following phylogenetic analysis, this strain was placed in the genus Arthrobacter. Arthrobacter protophormiae was the most closely related species in the phylogenetic tree, and this species also showed the highest sequence homology value (97%) with GIFU 12498. However, DNA-DNA hybridization indicated that GIFU 12498 did not belong to A. protophormiae (33·8±13·5% chromosomal similarity). The three urine strains belonged to one species because they shared more than 95% DNA-DNA similarity. It is proposed that these strains are placed in the genus Arthrobacter as a new species, Arthrobacter creatinolyticus sp. nov. The type strain of A. creatinolyticus is GIFU 12498, which has been deposited in the Japan Collection of Microorganisms (JCM) with the accession number JCM 10102.

Twenty-four strains isolated mainly from infected respiratory tracts of pigeons were characterized by an integrated genotypic and phenotypic approach. An extensive biochemical examination using conventional tests and several API microtest systems indicated that all isolates formed a phenotypically homogeneous taxon with a DNA G+C content between 42 and 43 mol%. Whole-cell protein and fatty acid analysis revealed an unexpected heterogeneity which was confirmed by DNA-DNA hybridizations. Four main genotypic sub-groups (genomovars) were delineated. 16S rDNA sequence analysis of a representative strain indicated that this taxon belongs to the beta-subclass of the Proteobacteria with Taylorella equigenitalis as its closest neighbour (about 94·8% similarity). A comparison of phenotypic and genotypic characteristics of both taxa suggested that the pigeon isolates represented a novel genus for which the name Pelistega is proposed. In the absence of differential phenotypic characteristics between the genomovars, it was preferred to include all of the isolates into a single species, Pelistega europaea, and strain LMG 10982 was selected as the type strain. The latter strain belongs to fatty acid cluster I and protein electrophoretic sub-group 1, which comprise 13 and 5 isolates, respectively. It is not unlikely that the name P. europaea will be restricted in the future to organisms belonging to fatty acid cluster I, or even to protein electrophoretic sub-group 1, upon discovery of differential diagnostic features.

Seven bacterial strains capable of oxidizing methyl sulfides were isolated from experimental biofilters filled with tree-bark compost. The isolates could be divided into two groups according to their method of methyl sulfide degradation. Four isolates could use only dimethyl disulfide as the sole source of energy and three strains were able to use dimethyl sulfide and dimethyl disulfide. Oxidation of the methyl sulfides by both groups led to the stoichiometric formation of sulfate. Chemotaxonomic, morphological, physiological and phylogenetic properties identified all isolates as members of the genus Pseudonocardia. The absence of phosphatidylcholine from the polar lipid pattern, as well as results of 165 rDNA analyses, led to the proposal of two new species, Pseudonocardia asaccharolytica sp. nov. and Pseudonocardia sulfidoxydans sp. nov. The type strains are P. asaccharolytica DSM 44247Tand P. sulfidoxydans DSM 44248T. With respect to the characteristic polar lipid pattern and the ability to oxidize sulfides, an emended description of the genus Pseudonocardia is proposed.

Three spherical thermoacidophilic archaea (strains TA-1T, TA–13, TA–14) were obtained from acidic hot springs located in Ohwaku Valley, Hakone, Japan. All the isolates are facultatively anaerobic, and grew optimally at around 85 °C pH 2·0. Isolate TA-1Twas characterized further. The G+C content of DNA from TA-1T is 33 mol%. Although these properties resemble those of the genus Acidianus, the sequence of the 165 rRNA gene from strain TA-1Twas more similar to that of species of Stygiolobus than of Acidianus. DNA-DNA hybridization experiments also indicated that strain TA-1Tis clearly distinguished phylogenetically from the members of Acidianus, Sulfolobus and Metallosphaera. On the basis of the distinct physiological and molecular properties, we describe the new strains as members of the new genus Sulfurisphaera. The type species of the genus is Sulfurisphaera ohwakuensis, and the type strain of the species is TA-1T(= IFO 151 SI1).

Strain Tab4cTa helical prokaryote that was isolated from the body of a Haematopota sp. fly collected in Champchevrier, Indre-et-Loire, Touraine, France, was found to be a member of the class Mollicutes. The cells of strain Tab4cTwere small, motile helices that were devoid of a cell wall. The organism passed through filters with mean pore diameters as small as 0.20 mm. Strain Tab4cTgrew rapidly in liquid SP-4 medium at both 30 and 37 °C. The organism fermented glucose but did not hdrolyse arginine or urea, and did not require serum for growth. In preliminary electrophoretic analyses, the cell protein patterns of strain Tab4cTwere distinct from those of 14 other spiroplasmas found in mosquitoes, deer flies and horse flies from Europe and the Far-East. In reciprocal metabolism inhibition and deformation serological tests, employing antigens and antisera representative of spiroplasma groups l-XXXIII (including all sub-groups), plus ungrouped strains BARC 1901 and BARC 2649, no serological relationship with Tab4cTwas found. The G+C content of the DNA of strain Tab4cTwas about 25·1 mol% and its genome size was 1.305 kbp. It is proposed that spiroplasma strain Tab4cTbe assigned to group XVII (presently vacant) and that strain (ATCC 700271T) is the type strain of a new species, Spiroplasma turonicum.

Four strains of a hitherto-unknown catalase-positive, facultatively anaerobic Corynebacterium species were isolated from the milk of sheep affected by subclinical mastitis. The most characteristic phenotypic reactions of the four strains were their weak fermentative acid production from glucose, their failure to produce acid from mannitol, xylose, sucrose and maltose, and a strong CAMP reaction with Staphylococcus aureus. Chemotaxonomic investigations revealed the presence of a cell wall based on mesodiaminopimelic acid and short-chain mycotic acids, which is consistent with the genus Corynebacterium, A comparative 16S rRNA gene sequence analysis confirmed that the organisms from sheep were members of the genus Corynebacterium, where they formed a distinct subline, exhibiting >4% sequence divergence with other known Corynebacterium species. Based on both phenotypic and phylogenetic findings, a new species, Corynebacterium camporealensis, is proposed. The type strain of Corynebacterium camporealensis is CECT 4897 (= CRS-51T).

A mesophilic, Gram-negative, rod-shaped, marine, propionate-oxidizing sulfate reducer (strain M16T) was isolated from a water-oil separation system on a North Sea oil platform. The optimum conditions for growth were 31 °C, pH 6.8-7.2 and 1.5-2.0 %(w/v) NaCI and 0.1-0.3% (w/v) MgCl26H2O in the medium. The growth yield with sulfate was 4.6 g cell biomass (mol propionate oxidized)-1. Strain M16Tis nutritionally related to members of the genus Desulfobulbus, but differs in that it has no vitamin requirement and is able to utilize fumarate and malate as carbon and energy sources. Hydrogenase activity measured as hydrogen uptake was mainly membrane-bound and varied with the growth substrate. Highest activity [28 μmol min-1(mg protein)-1] was found in cells grown with hydrogen and lowest [50 nmol min-1(mg protein)-1] in cells grown with propionate as electron donors for sulfate reduction. Desulforubidin, menaquinone-5(H2) and cytochromes of the c- and b-type were present. The fatty acid pattern was similar to that found for Desulfobulbus propionicus. The DNA base composition was 50.6 mol% G+C. Strain M16Tis equidistantly related to D. propionicus and Desulfobulbus elongatus with 96.1 % 16S rDNA similarity. On the basis of differences in genotypic, phenotypic and immunological characteristics, strain M16T(= DSM 8777T) is proposed as the type strain of a new species, Desulfobulbus rhabdoformis.

Two strains of moderately thermophilic bacteria, which reduce elemental sulfur to hydrogen sulfide, were isolated from volcanic sources in Kamchatka. Strain K-119Twas obtained from a thermophilic microbial community associated with Thermothrix thiopara, and strain U–8Twas isolated from a cyanobacterial mat inhabiting a sulfide-rich hot spring. Cells of both strains are short Gram-negative rods, motile with one polar flagellum (strain K–119T) or non-motile (strain U–8T). Both strains are obligate anaerobes, have temperature optima of 54–55 °C and pH optima of 6.9-7.2. Molecular hydrogen, acetate, fumarate, malate, pyruvate, lactate and long-chain saturated fatty acids served as growth substrates for both species; strain U-8Twas also able to grow on propionate. All substrates were oxidized completely, H2S and CO2 being the only metabolic products. Elemental sulfur was obligately required for growth of strain K–119T, whereas strain U-8Twas able to grow also with thiosulfate as electron acceptor and on pyruvate without an external electron acceptor. The DNA G+C contents of strains K–119Tand U–8Twere 31.6 and 32.2 mol%, respectively. Phenotypic features and the results of 16S rRNA sequencing indicate the affiliation of the new isolates to the genus Desulfurella. The DNA-DNA hybridization with Desulfurella acetivorans was 40% for strain K–119Tand 55% for strain U-8T; the DNA-DNA hybridization between the new isolates was 32%. Based on the results of morphological, physiological and phylogenetic studies the following two new species are proposed: Desulfurella kamchatkensis sp. nov. with the type strain K–119T(= DSM 10409T) and Desulfurella propionica sp. nov. with the type strain U–8T(=DSM 10410T).

Five strains were isolated from moribund scallop (Pecten maximus) larvae over 5 years (1990–1995) during outbreaks of disease in a hatchery (Argenton, Brittany, France). Their pathogenic activity on scallop larvae was previously demonstrated by experimental exposure. The phenotypic and genotypic features of the strains were identical. The G+C content of the strains was in the range 39–41 mol%. DNA–DNA hybridization showed a minimum of 73% intragroup relatedness. Phylogenetic analysis of small-subunit rRNA sequences confirmed that these strains should be affiliated within the family Vibrionaceae and that they are closely related to Vibrio tapetis and Vibrio splendidus. Phenotypic and genotypic analyses revealed that the isolates were distinct from these two vibrios and so constitute a new species in the genus Vibrio. They utilized only a limited number of organic substrates as sole carbon sources, including betaine and rhamnose, but did not utilize glucose and fructose. In addition, their responses were negative for indole, acetoin, decarboxylase and dihydrolase production. The name Vibrio pectenicida is proposed for the new species; strain A365 is the type strain (= CIP 105190T).

A strain of a previously undescribed non-lipophilic coryneform bacterium was isolated from pleural fluids of a patient with chronic renal failure, stroke and pneumonia. Slow fermentative acid production from glucose, maltose and sucrose, and strong N-acetyl-β-glucosaminidase activity were the most characteristic features of the bacterium. Chemotaxonomic characterization unambiguously indicated that the organism belonged to the genus Corynebacterium. The results of comparative 16S rRNA gene sequence analysis revealed that the isolate represented a new species within the genus, for which the name Corynebacterium thomssenii sp. nov. is proposed. The type strain is DSM 44276.