- Volume 48, Issue 4, 1998

Many thermophilic actinomycetes were isolated from samples collected from a hot-spring, lake and soil in Yunnan, China. Chemical and molecular classification of four selected strains of thermophilic Streptomyces with an upper limited growth temperature of 65-68 °C and autolytic characteristics was carried out. A new species, Streptomyces thermogriseus sp. nov. is described. The type strain is Y-14046T (= CCTCC AA97014T).

Comparative studies of morphology, physiology, biochemistry and chemical composition of cells, and phylogenetic analysis based on 16S rRNA gene sequences were carried out with strains XJ-54Tand XJ-58 of the genus Saccharomonospora and type strains of related genera. The results indicated that the two strains are different from known members of the genus Saccharomonospora. A new species with the name Saccharomonospora xinjiangensis sp. nov. is proposed. The new species is characterized by the presence of longitudinal pairs of spores on both the aerial and the vegetative hyphae and contains phosphatidyl ethanolamine, phosphatidyl choline and unknown glucosamine-containing phospholipids, and the major menaquinones MK-9(H2), MK-9(H4) and MK-7(H4).

Two Lactobacillus strains were isolated from sour cassava starch fermentation. The cells were Gram-positive, catalase-negative, non-spore-forming, non-motile rods. They produced only l(+)lactate and were homofermentative. Growth occurred at pH values of 5·0-7·0 and optimum growth occurred at pH 6·0. Growth was positive at 15 and 45 °C. The DNA G+C content was 48·4±0·2 mol%. Sequence analysis of the 16S rRNA gene revealed that strains OND 32T and YAM 1 clustered with, but were separate from Lactobacillus casei- related taxa. Protein pattern and sequence analyses of the 16S rRNA gene confirmed that the two new isolates represent a new Lactobacillus species, for which the name Lactobacillus manihotivorans is proposed; strain OND 32T is the type strain of this species.

Strain INSLUZT(= DSM 4273T) was isolated from a biogas-producing bioreactor treating wastewater of a palm oil mill on North-Sumatra (Indonesia). Cells of strain INSLUZT were highly irregularly coccoid, 1·25–2·0 in diameter, had a cell envelope consisting of the cytoplasmic membrane and an S-layer of hexagonally arranged glycoprotein subunits with an M r of 120 000, and were flagellated (motility was not observed). Cells were mesophilic and grew most rapidly at 40 °C on H2/CO2, formate, 2-propanol/CO2, 2-butanol/CO2 and cyclopentanol/CO2 to give methane. Tungstate promoted growth on H2/CO2 with acetate as the solely required organic medium supplement. The G+C content of DNA was 59 mol% (T m method) and 59·5 mol% (HPLC method). 16S rDNA analysis revealed a phylogenetic relationship to Methanoculleus species; the name Methanoculleus palmolei sp. nov. is therefore proposed for strain INSLUZT (= DSM 4273T).

A Gram-positive, aerobic, spore-forming actinomycete strain, HR1-2T, was isolated from a peat bog near Gifhorn, Lower Saxony, Germany. Comparative analysis of the 16S rDNA sequence indicated that HR1-2Twas phylogenetically related to members of the family Micromonosporaceae, branching adjacent to Spirilliplanes yamanashiensis, Couchioplanes caeruleus, Catenuloplanes japonicus and members of the genus Micromonospora. The affiliation to the family was supported by the presence of family-specific 16S rDNA signature nucleotides, DNA G+C content of 70 mol%, peptidoglycan of type A1γ′ (directly crossed-linked, presence of glycine, alanine, glutamic acid and meso-diaminopimelic acid in the peptide side-chain), menaquinone MK-9(H4) as the major respiratory lipoquinone, polar lipid composition PII (phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylserine and phosphatidylinositolmannosides) and a glycolyl type of muramic acid. It differed from genera of the family by the lack of arabinose in whole-cell sugars and a unique nucleotide signature stretch between positions 1132 and 1143 (Escherichia coli numbering), 5’ CAAUUCGGUUG 3’. Morphologically strain HR1-2T resembles Micromonospora species but can be distinguished from them by the lack of arabinose in whole-cell sugars, the presence of 10-methyl C17:0 fatty acids and a distinct 16S rDNA sequence. Based on the unique combination of morphological, chemotaxonomic and phylogenetic properties a new genus, Verrucosispora gen. nov., is proposed. The type species of this genus is Verrucosispora gifhornensis sp. nov., and the type strain of V. gifhornensis is strain HR1-2T(= DSM 44337T).

Sequences of the 16S rDNA from all available type strains of Chromatium species have been determined and were compared to those of other Chromatiaceae, a few selected Ectothiorhodospiraceae and Escherichia coli. The clear separation of Ectothiorhodospiraceae and Chromatiaceae is confirmed. Most significantly the sequence comparison revealed a genetic divergence between Chromatium species originated from freshwater sources and those of truly marine and halophilic nature. Major phylogenetic branches of the Chromatiaceae contain (i) marine and halophilic species, (ii) freshwater Chromatium species together with Thiocystis species and (iii) species of the genera Thiocapsa and Amoebobacter as recently reclassif ied [Guyoneaud, R. & 6 other authors (1998). Int J Syst Bacteriol 48, 957–964], namely Thiocapsa roseopersicina, Thiocapsa pendens (formerly Amoebobacter pendens). Thiocapsa rosea (formerly Amoebobacter roseus), Amoebobacter purpureus and Thiolamprovum pedioforme (formerly Amoebobacter pedioformis). The genetic relationships between the species and groups are not in congruence with the current classification of the Chromatiaceae and a reclassification is proposed on the basis of 16S rDNA sequence similarity supported by selected phenotypic properties. The proposed changes include the transfers of Chromatium minus and Chromatium violascens to Thiocystis minor comb. nov. and Thiocystis violascens comb, nov., of Chromatium vinosum, Chromatium minutissimum and Chromatium warmingii to the new genus Allochromatium as Allochromatium vinosum comb. nov., Allochromatium minutissimum comb. nov., and Allochromatium warmingii comb. nov., of Chromatium tepidum to the new genus Thermochromatium as Thermochromatium tepidum comb, nov., of Chromatium salexigens and Chromatium glycolicum to the new genus Halochromatium as Halochromatium salexigens comb. nov. and Halochromatium glycolicum comb, nov., of Chromatium gracile and Chromatium purpuratum to the new genus as Marichromatium gracile comb. nov. and Marichromatium purpuratum comb, nov., of Thiocapsa pfennigii to Thiococcus pfennigii gen. nom. rev., of Thiocapsa halophila to the new genus Thiohalocapsa as Thiohalocapsa halophila comb, nov., and of Chromatium buderi to the new genus Isochromatium as Isochromatium buderi comb. nov.

The taxonomic position of Photobacterium damselae subsp. piscicida, the causative agent of fish pasteurellosis, is controversial as this organism has also been described as ‘pasteurella piscicida’. To clarify the taxonomic position of the pathogen, a total of 113 P. damselae subsp. piscicida strains and 20 P. damselae subsp. damselae strains, isolated from different geographical areas and from the main affected fish species, were analysed using 129 morphological and biochemical tests, including the commercial API 20E and API CH50 test systems. For comparison, the type strains of other Photobacterium species (i.e. Photobacterium leiognathi and Photobacterium angustum) were included in the analyses. The results were statistically analysed by unweighted pair group average clustering and the distance between the different clusters was expressed as the percentage disagreement. The analyses showed that, based on morphological and biochemical identification tests, P. damselae subsp. piscicida is related to other Photobacterium species. However, it is clearly distinguishable from P. damselae subsp. damselae and no phenotypic evidence was found to include P. damselae subsp. piscicida as a subspecies in the species P. damselae.

RFLP analyses of 16S rDNA nested PCR products from 34 phytoplasma strains with 17 restriction enzymes delineated distinct pattern types. Based on similarity coefficients derived from RFLP analyses, the 34 representative phytoplasma strains were differentiated into 14 major groups (termed 16Sr groups) and 32 sub-groups. The similarity coefficients of RFLP patterns between distinct groups were 90% or below. By including additional groups and sub-groups from which RFLP analyses were not performed but for which 16S rDNA sequence data were available to predict restriction sites, a total of 14 groups and 41 sub-groups were proposed. By combined RFLP analyses of 16S rRNA and ribosomal protein gene sequences, thus far, a total of 46 subgroups have been recognized. The phytoplasma 16Sr groups were consistent with the phylogenetic groups (subclades) defined by phylogenetic analysis of near-full-length 16S rRNA gene sequences, indicating that the RFLP-based groups are phylogenetically valid. The approach using RFLP analyses of PCR-amplified 16S rDNA (and ribosomal protein gene sequences) provides a simple, reliable and rapid means for differentiation and classification of unknown phytoplasmas.

As part of a general survey of the biodiversity and inherent ecophysiology of bacteria associated with coastal Antarctic sea-ice diatom assemblages, eight strains were identified by 16S rRNA sequence analysis as belonging to the genus Colwellia. The isolates were non-pigmented, curved rod-like cells which exhibited psychrophilic and facultative anaerobic growth and possessed an absolute requirement for sea water. One isolate was able to form gas vesicles. All strains synthesized the ω3 polyunsaturated fatty acid (PUFA) docosahexaenoic acid (22:6ω3, DHA) (0·7–8·0% of total fatty acids). Previously, DHA has only been detected in strains isolated from deep-sea benthic and faunal habitats and is associated with enhanced survival in permanently cold habitats. The G+C content of the DNA from the Antarctic Colwellia strains ranged from 35 to 42 mol% and DNA-DNA hybridization analyses indicated that the isolates formed five genospecies, including the species Colwellia psychrerythraea (ACAM 550T). 16S rRNA sequence analysis indicated that the strains formed a cluster in the γ-subclass of the Proteobacteria with Colwellia psychrerythraea. Sequence similarities ranged from 95·2 to 100% between the various Antarctic Colwellia isolates. Phenotypic characterization confirmed distinct differences between the different genospecies. These studies indicate that the DHA-producing Antarctic isolates consist of five different Colwellia species: Colwellia psychrerythraea and four novel species with the proposed names Colwellia demingiae sp. nov. (ACAM 459T), Colwellia psychrotropica sp. nov. (ACAM 179T), Colwellia rossensis sp. nov. (ACAM 608T) and Colwellia hornerae sp. nov. (ACAM 607T).

Thermococcus strains TYST and TYT isolated from the Guaymas Basin hydrothermal vent site and previously described were compared by DNA-DNA hybridization analysis with the closest Thermococcus species in terms of physiology and nutritional aspects. On the basis of the new data and taking into consideration the molecular, physiological and morphological traits published previously, it is proposed that strains TYT and TYST should be classified as new species named Thermococcus aggregans sp. nov. and Thermococcus guaymasensis sp. nov., respectively. The type strain of T. aggregans is strain TYT (= DSM 10597T) and the type strain of T. guaymasensis is strain TYST (= DSM 11113T).

A phylogenetic analysis of 69 halobacterial 16S rRNA gene sequences has been carried out, integrating data from new isolates, previously described halobacteria and cloned sequences from uncultivated halobacteria. Halobacterium halobium NCIMB 777, Halobacterium trapanicum NCIMB 784 and Halobacterium salinarium NCIMB 786, together with several other strains (strains T5.7, L11 and Halobacterium trapanicum NCIMB 767) constitute a distinct lineage with at least 98·2% sequence similarity. These strains have been incorrectly assigned to the genus Halobacterium. Therefore, based on a variety of taxonomic criteria, it is proposed that Halobacterium salinarium NCIMB 786 is renamed as Natrinema pellirubrum nom. nov., the type species of the new genus Natrinema gen. nov., and that Halobacterium halobium NCIMB 777 and Halobacterium trapanicum NCIMB 784 are renamed as a single species, Natrinema pallidum nom. nov. It was notable that halobacteria closely related to the proposed new genus have been isolated from relatively low-salt environments.

A mollicute (strain BARC 318T) isolated from gut tissue of a green tiger beetle (Coleoptera: Cicindelidae) was found by dark-field microscopy to consist of non-helical, non-motile, pleomorphic coccoid forms of various sizes. In ultrastructural studies, individual cells varied in diameter from 300 to 1200 nm, were surrounded by a cytoplasmic membrane and showed no evidence of cell wall. The organisms were readily filterable through membrane filters with mean pore diameters of 450 and 300 nm, with unusually large numbers of organisms filterable through 200 nm pore membrane filters. Growth occurred over a temperature range of 15–32 °C with optimum growth at 30 °C. The organism fermented glucose and hydrolysed arginine but did not hydrolyse urea. Strain BARC 318T was insensitive to 500 U penicillin ml–1 and required serum or cholesterol for growth. It was serologically distinct from all currently described sterol-requiring, fermentative Mycoplasma species and from 12 non-sterol-requiring Mesoplasma species, 13 non-sterol-requiring Acholeplasma species and 5 previously described sterol-requiring Entomoplasma species. Strain BARC 318T was shown to have a G+C content of 34 mol% and a genome size of 870 kbp. The 16S rDNA sequence of strain BARC 318T was compared to 16S rDNA sequences of several other Entomoplasma species and to other representative species of the genera Spiroplasma and Mycoplasma, and to other members of the class Mollicutes. These comparisons indicated that strain BARC 318T had close phylogenetic relationships to other Entomoplasma species. On the basis of these findings and other similarities in morphology, growth and temperature requirements and genomic features, the organism was assigned to the genus Entomoplasma. Strain BARC 318T (ATCC 51999T) is designated the type strain of Entomoplasma freundtii sp. nov.

A dark-green-pigmented marine bacterium, previously designated D2, which produces components that are inhibitory to common marine fouling organisms has been characterized and assessed for taxonomic assignment. Based on direct double-stranded sequencing of the 16S rRNA gene, D2T was found to show the highest similarity (93%) to members of the genus Pseudoalteromonas. The G+C content of D2T is 42 mol%, and it is a facultatively anaerobic rod and oxidase-positive. D2T is motile by a sheathed polar flagellum, exhibited non-fermentative metabolism and required sodium ions for growth. The strain was not capable of using citrate, fructose, sucrose, sorbitol and glycerol but it utilizes mannose and maltose and hydrolyses gelatin. The molecular evidence, together with phenotypic characteristics, showed that this bacterium which produces an antifouling agent constitutes a new species of the genus Pseudoalteromonas. The name Pseudoalteromonas tunicata is proposed for this bacterium, and the type strain is D2T (= CCUG 26757T).

A group of pigmented, psychrophilic, strictly aerobic chemoheterotrophs isolated from sea-ice cores collected from coastal areas of eastern Antarctica was found to represent a novel 16S rRNA lineage within the gamma subclass of the Proteobacteria, adjacent to the genus Alteromonas. The isolates are motile. Gram-negative, rod-shaped cells, which are psychrophilic and slightly halophilic, and possess an absolute requirement for seawater. Differences in phenotypic characteristics and DNA–DNA hybridization indicated the isolates formed two distinct taxa which have DNA G+C contents of 44–46 mol% and 40 mol%, respectively. Whole-cell fatty acid profiles of the isolates were however very similar and included 16:1ω7c, 18:1ω7c, 16:0 and 17:1ω8c as the major fatty acid components. Overall, sufficient differences exist to distinguish the sea-ice strains from currently recognized bacterial genera. It is proposed the sea-ice strains represent a new genus, Glaciecola, which contains two species, Glaciecola punicea gen. nov., sp. nov. (ACAM 611T) and Glaciecola pallidula gen. nov., sp. nov. (ACAM 615T).

Sequences of the RNase P RNA gene were investigated for the phylogenetic analysis of the genus Saccharomonospora. Aligned nucleotide sequences, determined from the PCR-amplified RNase P RNA gene of representative strains of the genus Saccharomonospora, displayed 94·2±1·3% interspecific variances. The intraspecific similarity value was 99·7–100% in all species tested. Saccharomonospora azurea K161Tand “Saccharomonospora caesia” K76Tdisplayed identical RNase P RNA gene sequences in the region that was determined and Saccharomonospora sp. K180 showed sequences distinct from validly described species with a similarity value of 94·6±1·0%. The phylogenetic trees constructed by aligning the sequences either within the genus Saccharomonospora or with other Gram-positive bacteria were similar to the ones derived using sequences of the 16S rDNA gene. Advantageous features of this gene for application as a molecular phyletic marker are discussed.

The species Streptococcus dysgalactiae was proposed to accommodate a heterogeneous group of streptococci associated with infections in animals and human beings. This taxon is now considered to include animal isolates of α-haemolytic group C streptococci, previously called S. dysgalactiae; animal and human isolates of β-haemolytic group C streptococci, previously called ‘S. equisimilis’; β-haemolytic group L strains associated with infections in animals and, rarely, in humans; and β-hiaemolytic group G strains isolated from humans. DNA-DNA reassociation experiments (hydroxyapatite method) and multilocus enzyme electrophoresis (MEE) were performed on reference strains and clinical isolates to determine the genetic relationships among these different phenotypic categories. DNA-DNA hybridization tests showed that they were related at the species level, despite the phenotypic and host heterogeneity. Both genotypic and phenotypic characterization indicated that S. dysgalactiae could be separated into two major sub-groups. The first subgroup contained α-haemolytic strains that showed levels of DNA relatedness with the type strain of S. dysgalactiae ranging from 84 to 90% and from 82 to 88% under optimal (55 °C) and stringent (70 °C) conditions, respectively. The second sub-group contained βhaemolytic strains showing levels of relatedness ranging from 71 to 79% (55 °C) and from 62 to 73% (70 °C). Percentage divergence varied from 0·5 to 1·0% (α-haemolytic group) and from 20 to 3·5% (α-haemolytic group). A dendrogram based on phenotypic similarity between the enzyme bands produced by MEE showed a Jaccard similarity coefficient of 0·45 between the subclusters formed by the two sub-groups. The results of phenotypic and genotypic characterization were consistent with a published proposal to divide S. dysgalactiae into two subspecies, S. dysgalactiae subsp. dysgalactiae and S. dysgalactiae subsp. equisimilis, with a few modifications.

Eleven motile spore-bearing actinomycetes were isolated from various plant samples and were studied to determine their taxonomic positions. The isolates showed colony appearance and morphology similar to those of the ‘sporedome actinomycetes’ previously described by L. G. Willoughby in 1969. Although the isolates showed variety in composition of isomers of 2,6-diaminopimelic acid in the cell walls and in whole-cell sugar patterns, all of the isolates were classified in the genus Kineosporia Pagani and Parenti 1978 emend. Itoh et al. 1989 on the basis of their morphological and other chemotaxonomic characteristics, i.e. menaquinone, phospholipid and cellular fatty acid compositions. This assignment to the genus was also supported by a phylogenetic analysis using the 16S rRNA gene sequences. By DNA-DNA hybridization experiment, five genospecies were recognized among the isolates, and one of them showed high levels with the type strain of the type species, Kineosporia aurantiaca, which is the sole member of the genus. These genospecies can be distinguished from each other by their cultural, physiological and biochemical characteristics, and the other four genospecies should be placed into four new species for which the names Kineosporia succinea (type strain 1-273T= JCM 9957T), Kineosporia rhizophila (type strain l-449T= JCM 9960T). Kineosporia mikuniensis (type strain l-463T= JCM 9961T) and Kineosporia rhamnosa (type strain l-132T= JCM 9954T) are proposed.

Brachyspira pilosicoli (formerly Serpulina pilosicoli) causes swine spirochaetosis and can also be isolated from human faeces, although its role in human disease remains unclear. The genetic and biochemical variations amongst 19 isolates of human spirochaetes from five different countries were evaluated and compared to those found amongst swine isolates of B. pilosicoli. All isolates were negative for β-glucosidase and all but one were positive for hippurate hydrolysis, which are characteristics typical of B. pilosicoli. The isolates showed variation in indole production and α-galactosidase and α-glucosidase activity, other characteristics which can be used to identify B. pilosicoli. The DNA sequences of part of the 16S rRNA gene differed from each other and from that of B. pilosicoli by 0–3 bp out of 283 bp. It is concluded that there is considerable variation amongst human intestinal spirochaetes. Since few of the isolates reported here match the current criteria for B. pilosicoli, it is concluded that this species is more heterogeneous than previously appreciated. However, it cannot be excluded that some isolates may belong to uncharacterized related BrachyspiralSerpulina species.

Thirty-nine selected Frankia strains belonging to different genomic species were clustered on the basis of their in vitro susceptibility to 17 antibiotics, pigment production and ability to nodulate plants of the genus Alnus and/or the family Elaeagnaceae, or the family Casuarinaceae. The majority of the strains studied fell into three cluster groups, A, E and C, corresponding to the three host-specificity groups, Alnus, Elaeagnus and Casuarina. Within the groups, eight composite clusters, consisting of at least two strains, and five single-member clusters were recovered at the 0·74 distance level, in good agreement with levels of genetic relatedness between the strains. In addition, five strains were recovered as single-member clusters not in the cluster groups, four of them representing single-member genospecies and one strain not assigned to any known genospecies. The concordance between the phenotypic clusters and the genospecies described previously shows that the grouping may reflect the taxonomic structure of the genus Frankia. For some clusters, differentiating phenotypic characters were found which may be useful for species definition.

A group of nodule isolates from Neptunia natans, an indigenous stemnodulated tropical legume found in waterlogged areas of Senegal, was studied. Polyphasic taxonomy was performed, including SDS-PAGE of total proteins, auxanography using API galleries, host-plant specificity, PCR-RFLP of the internal transcribed spacer region between the 16S and the 23S rRNA coding genes, 16S rRNA gene sequencing and DNA-DNA hybridization. It was demonstrated that this group is phenotypically and phylogenetically separate from the known species of Rhizobium, Sinorhizobium, Mesorhizobium, Agrobacterium, Bradyrhizobium and Azorhizobium. Its closest phylogenetic neighbour, as deduced by 16S rRNA gene sequencing, is Agrobacterium vitis (96·2% sequence homology). The name Allorhizobium undicola gen. nov., sp. nov., is proposed for this group of bacteria, which are capable of efficient nitrogen-fixing symbiosis with Neptunia natans, and the type strain is ORS 992T (= LMG 11875T).