- Volume 48, Issue 1, 1998

Two strains of the genus Leptospira, belonging to serogroup Tarassovi, were isolated from kidneys of apparently healthy oxen slaughtered at an abattoir in Zimbabwe. Both strains belonged to the same serovar but could not be assigned to previously known serovars using the cross-agglutinin absorption test. The name ngavi is proposed for the new serovar containing these two strains; strain SBF 16 is the reference strain. The Zimbabwe isolates showed some antigenic similarity to serovar gatuni when analyses were carried out using eight monoclonal antibodies, and had restriction patterns similar to those of serovars tarassovi, tunis, moldaviae and guidae when their chromosomal DNAs were analysed using RFLP analysis. The restriction patterns of the two strains could be distinguished from each other and from those of the four serovars when their Southern blots were hybridized with a probe synthesized from a repetitive sequence element cloned from serovar hardjo strain Hardjo-bovis.

A strictly anaerobic bacterium, strain BT, from termite hindgut homogenates, was isolated in pure culture and grew on 3-hydroxybenzoate as sole source of carbon and energy. No other substrate tested was degraded, sulfate, sulfite, thiosulfate, nitrate, ferric iron, oxygen or fumarate were not reduced, and no electron transfer to partner organisms was observed. 3-Hydroxybenzoate was fermented to butyrate, acetate and CO2. Benzoate was detected in the culture supernatant as an intermediate. The isolate was a slightly motile, endospore- forming Gram-positive rod; 16S rDNA sequence analysis revealed a high similarity to members of the genus Desulfotomaculum. The G+C content of the DNA was 48 mol %. Strain BT differs from the members of the genus Desulfotomaculum significantly due to its lack of dissimilatory sulfate reduction, and is therefore described as the type strain of a new genus and species, Sporotomaculum hydroxybenzoicum gen. nov., sp. nov.

Several psychrophilic, gas vacuolate strains of the Cytophage-Flavobacterium-Bacteroides (CFB) phylogenetic group were isolated from sea ice and water from the Arctic and the Antarctic. The closest taxonomically defined species by 16S rRNA sequence analysis is ‘Flectobacillus glomeratus’. However, ‘FIc. glomeratus’ is phylogenetically distant from the Flectobacillus type species, FIc. major. On the basis of phenotypic, genotypic and 16S rRNA sequence analyses we propose a new genus, Polaribacter, with three new species, Polaribacter irgensii strain 23-P (ATCC 700398), Polaribacter franzmannii strain 301 (ATCC 700399) and Polaribacter filamentus strain 215 (ATCC 700397). P. filamentus is the type species of the genus. None of these species exhibits a cosmopolitan or bipolar distribution. This is the first taxonomic description of gas vacuolate bacteria in the CFB group. Additionally, we propose that ‘FIc. glomeratus’ be reclassified to the genus Polaribacter as P. glomeratus, comb. nov.

Seven strains of bacteria were isolated from Pacific oysters, Crassostrea gigas, with a focal or systemic disease. The strains were aerobic, Gram-positive, acid-fast, produced a mycelium which fragmented into irregular rod-like elements, had a peptidoglycan containing meso-diaminopimelic acid, arabinose and galactose as major sugars, mycolic acids with 46-58 carbon atoms and G+C-rich DNA. All of these properties are consistent with the classification of the organisms in the genus Nocardia. A partial sequence of the 165 rRNA gene of isolate NB4H was determined following isolation and cloning of the PCR-amplified gene. The sequence was aligned with those of representative mycolic-acid-containing taxa and a phylogenetic tree was generated using the neighbour-joining method. It was evident from the phylogenetic tree that the three strains tested, RB1, OB3P and NB4H, were identical and belonged to the Nocardia otitidiscaviarum rRNA sub-group. The biochemical, chemical, morphological and physiological properties of the isolates were also essentially identical and served to distinguish them from representative nocardiae. It is, therefore, proposed that the strains isolated from the diseased Pacific oysters be assigned to a new species, Nocardia crassostreae. The type strain is NB4H (= ATCC 700418).

Four strains of marine, aerobic, agar-decomposing bacteria with one polar flagellum and with DNA G+C contents of 38.9-40.2 mol% were isolated from the Far-Eastern mussels Crenomytilus grayanus and Patinopecten yessoensis. These four strains were identified as Pseudoalteromon as; however, they were phenotypically different from species described previously according to carbon compound utilization tests and the BIOLOG identification system. High agar-decomposing activity was found in two strains, in one of which agarase, α-galactosidase, pustulanase and laminarinase had been detected. The level of DNA homology of three of the strains was 70-100%. The fourth isolate was genetically less related to the others (67 % DNA relatedness) and phenotypically was more distant from other members of this group; however, all four strains were assigned to a single species genotypically. DNA from the strains isolated from mussels showed 40-45% genetic relatedness with the DNA of Alteromonas atlantica, 8-36% with DNA of Pseudoalteromonas haloplanktis subsp. haloplanktis, Pseudoalteromon as haloplanktis subsp. tetraodonis, Pseudoalteromon as undina, Pseudoalteromon as nigrifaciens and Pseudoalteromon as carrageenovora, 53% with Pseudoalteromon as elyakovii, 32-48% with marine P. nigrifaciens from mussels and 14-16% with Alteromonas macleodii. The DNA-DNA hybridization data revealed that the levels of relatedness between the strains isolated and the type strains of Pseudoalteromon as citrea and Pseudoalteromon as fuliginea described recently were significant (95-85 %). These results were confirmed by serological data employing polyclonal antibodies to cell surface antigens. The strains isolated from mussels were identified as P. citrea. The hybridization data showed that the name P. fuliginea Romanenko et al. 1994 should be recognized as a junior subjective synonym of P. citrea Gauthier 1977. A notable phenotypic diversity of P. citrea which might be a reflection of their ecological habitats is discussed.

Marginal chlorosis is a new disease of strawberry which was first seen in France in 1988. A phloem-restricted bacterium-like organism was found associated with the disease. Even though the organism could not be cultured, and resembles in this way most other phloem-restricted pathogens, characterization was achieved from the sequence of its PCR-generated 16S rDNA, and comparison with other organisms. From these studies, the strawberry agent was found to be a new bacterium within group 3 of the gamma subclass of Proteobacteria, a group of Gram-negative bacteria including, in particular, insect symbionts or parasites as well as enterobacteria. Its closest relative, Arsenophonus nasoniae, is the causal agent of the son- killer trait in wasps. The two bacteria share 92% 16S rDNA sequence identity. We propose a Candidates taxon for the marginal-chlorosis-associated bacterium, ‘Candidates Phlomobacter fragariae'.

The nucleotide sequences of the 16S rRNA genes from the type strains of four goat mycoplasmas, Mycoplasma adleri, Mycoplasma auris, Mycoplasma cottewii and Mycoplasma yeatsii, were determined by direct solid-phase DNA sequencing. Polymorphisms were found in two of the 16S rRNA gene sequences, showing the existence of two different rRNA operons. Three polymorphisms were found in M. adleri, and one was found in M. yeatsii. The sequence information was used for the construction of phylogenetic trees. M. adleri was included in the Mycoplasma lipophilum cluster within the hominis group. M. auris was comprised in the Mycoplasma hominis cluster of the hominis group. M. cottewii and M. yeatsii were found to be very closely related with only four nucleotide differences, and they grouped with Mycoplasma putrefaciens in the Mycoplasma mycoides cluster within the spiroplasma group. Sequencing of two field isolates of M. cottewii and M. yeatsii, geographically distant from the type strains, showed that the 16S rRNA gene from the field isolate of M. cottewii was identical to the one from the type strain. The field isolate of M. yeatsii had only two nucleotide differences to the type strain and these were present in only one of the two rRNA operons. Sequencing of the 16S rRNA genes from two unidentified mycoplasma isolates from Nepal indicated that they should both be regarded as M. auris strains.

Strawberry plants exhibiting symptoms of stunting and abnormally small leaves were observed in production fields in central Florida, USA. Since the symptoms were suggestive of phytoplasma infection, plants were assayed for presence of phytoplasma by PCR amplification of 16S rDNA and ribosomal protein (rp) gene sequences. Amplification of phytoplasma-specific DNA sequences by PCR indicated infection of the diseased strawberry plants by phytoplasmas. RFLP analyses of amplified 16S rDNA revealed that the plants were infected by two mutually distinct phytoplasmas that differed from strawberry green petal phytoplasma (group 16Srl-C). Both phytoplasmas were members of 16S rRNA gene group I (16Srl). Based on RFLP analysis of amplified 16S rDNA and rp gene sequences, one was classified in group 16Srl subgroup I and new rp subgroup 16Srl-l(rp); its 16S rRNA-rp subgroup was designated 16Srl-K(rr-rp). The second phytoplasma represented a previously undescribed subgroup, designated K, in 16S rRNA group I but belonged to rp subgroup 16Srl-J(rp); this phytoplasma's 16S rRNA-rp subgroup was designated 16Srl- J(rr-rp). Results of RFLP analyses agreed with putative restriction site maps based on nucleotide sequences determined for the amplified 16S rDNAs and rp gene operon DNAs. Further evidence indicated that the 16Srl-K(rr-rp) strawberry phytoplasma, Mexican periwinkle virescence phytoplasma and stolbur phytoplasma shared sequence homologies that enabled amplification of DNA from all three by PCR using primers previously designed as stolbur- specific.

The nucleotide sequence of the 16S rRNA genes of four rare human mycoplasma species. Mycoplasma faucium, M. buccale, M. primatum and M. spermatophilum, were partially sequenced and compared to published rRNA genes of mycoplasmas to determine their position in the Mollicutes phylogenetic tree. Nucleotide sequence motif and overall similarities allowed positioning of these mycoplasmas in the hominis phylogenetic group, as defined by Weisburg et al. [Weisburg, W. G., Tully, J. G., Rose, D. L. & 9 other authors (1989). J Bacteriol 171, 6455-6467]. Furthermore, these mycoplasmas could be clustered into two different subdivisions of the hominis group: (i) M. faucium and M. buccale were found to be included in the M. fermentans subdivision, and (ii) M. primatum and M. spermatophilum were included in the M. hominis one. Variable regions of the 16S rRNA genes were used to determine specific PCR primers to detect and identify M. faucium.

A sulfate-reducing bacterium, strain Aspo-2, was isolated from granitic groundwater sampled at a depth of 600 m. This and other strains of SRB frequently occur in the deep granitic rock aquifers studied. On the basis of its morphological, physiological and genotypical properties, and its unique habitat, we propose strain Aspo-2 as a new species of the genus Desulfovibrio, Desulfovibrio aespoeensis (DSM 10631T).

The presence of a Burkholderia pseudomallei-like species based upon the significant genotypic and phenotypic dissimilarities exhibited between these organisms and true B. pseudomallei strains has been reported previously. In this study, a comprehensive 16S rDNA-based phylogenetic analysis further supports the existence of this newly described Burkholderia species for which the name Burkholderia thailandensis sp. nov. is proposed.

Spiroplasma group XIV strain EC-1T and other isolates from the lampyrid beetle Ellychnia corrusca form a serogroup with tabanid spiroplasma strains (TC-1 and TS-1). It was hypothesized that similarities among these strains reflect a transmission cycle in which lampyrid beetles serve as overwintering hosts and tabanid adults become infected and transmit a homogeneous population of spiroplasma strains during spring, summer and autumn. In the present study, variations in restriction fragment length patterns suggest the presence of multiple genovars. Genotypic analysis may therefore be a companion to serology in elucidating spiroplasma diversity, and may provide clues to strain host range.

Fellomyces ogasawarensis and Fellomyces distylii, new yeast species isolated from a dead leaf of a plant (Distylium lepidotum Nakai, family Hamamelidaceae) collected in the Ogasawara Islands, isolated islands in the Pacific Ocean about 1000 km south of Japan, are described. The phylogenetic relationship of F. ogasawarensis and F. distylii with other members of the genus Fellomyces was estimated from 18S rRNA gene sequence analysis. The type strain of F. ogasawarensis is strain OK-81 (= JCM 9861) and that of F. distylii is strain OK-83 (= JCM 9862).

The phylogenetic relationships between species of yeasts assigned to the Saccharomyces sensu stricto group, which includes Saccharomyces cerevisiae and Saccharomyces bay anus, were studied together with Saccharomyces pastorianus and Saccharomyces paradoxus. The experimental approaches used were RFLP analysis of the PCR-amplified rDNA internal transcribed spacer (ITS) and intergenic spacer, and total ITS sequence analysis. Both RFLP and sequence analyses gave fairly similar results. The gene trees generated with either of the two data sets showed the distribution of the yeasts into two major, well- separated, phylogenetic clusters called 'cerevisiae' and 'bayanus'. The 'cerevisiae' cluster included the S. cerevisiae type strain, together with most of the species (16 out of 23), whereas the 'bayanus' cluster included the remaining seven type strains. Therefore, analysis of rDNA sequences confirmed 5. cerevisiae and S. bayanus as two well-defined taxa. However, 5. pastorianus and 5. paradoxus, the two other usually accepted taxa of the now- defined Saccharomyces sensu stricto complex, could not be clearly separated from 5. bayanus and S. cerevisiae, respectively. However, in both PCR-RFLP and ITS sequence analyses, 5. paradoxus had the outermost position in the 'cerevisiae' cluster. PCR-RFLP analysis of the ribosomal spacer sequences was also carried out on 26 Saccharomyces strains isolated in various wine-growing regions of France in an attempt to clarify their positions in the Saccharomyces phylogenetic tree. Compared to the diversity of the Saccharomyces type strains, less genetic diversity was detected among these yeasts and several of them exhibited identical RFLP patterns. Most of the wine yeast strains (16 out of 26) were closely related to each other and were found within the 'cerevisiae' cluster. The remaining 10 wine yeast strains branched within the 'bayanus' cluster. PCR-RFLP analysis of ribosomal spacer sequences thus appears to be a useful and appropriate method for the correct characterization of Saccharomyces yeast strains used in food processing.