Hiroko Kawasaki

Asaia bogorensis gen. nov., sp. nov., an unusual acetic acid bacterium in the alpha-Proteobacteria.

Eight Gram-negative, aerobic, rod-shaped and peritrichously flagellated strains were isolated from flowers of the orchid tree (Bauhinia purpurea) and of plumbago (Plumbago auriculata), and from fermented glutinous rice, all collected in Indonesia. The enrichment culture approach for acetic acid bacteria was employed, involving use of sorbitol medium at pH 3.5. All isolates grew well at pH 3.0 and 30 degrees C. They did not oxidize ethanol to acetic acid except for one strain that oxidized ethanol weakly, and 0.35% acetic acid inhibited their growth completely. However, they oxidized acetate and lactate to carbon dioxide and water. The isolates grew well on mannitol agar and on glutamate agar, and assimilated ammonium sulfate for growth on vitamin-free glucose medium. The isolates produced acid from D-glucose, D-fructose, L-sorbose, dulcitol and glycerol. The quinone system was Q-10. DNA base composition ranged from 59.3 to 61.0 mol% G + C. Studies of DNA relatedness showed that the isolates constitute a single species. Phylogenetic analysis based on their 16S rRNA gene sequences indicated that the isolates are located in the acetic acid bacteria lineage, but distant from the genera Acetobacter, Gluconobacter, Acidomonas and Gluconacetobacter. On the basis of the above characteristics, the name Asaia bogorensis gen. nov., sp. nov. is proposed for these isolates. The type strain is isolate 71T (= NRIC 0311T = JCM 10569T).

Asaia siamensis sp. nov., an acetic acid bacterium in the alpha-proteobacteria.

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).

Kozakia baliensis gen. nov., sp. nov., a novel acetic acid bacterium in the alpha-proteobacteria.

Four bacterial strains were isolated from palm brown sugar and ragi collected in Bali and Yogyakarta, Indonesia, by an enrichment culture approach for acetic acid bacteria. Phylogenetic analysis based on 16S rRNA gene sequences showed that the four isolates constituted a cluster separate from the genera Acetobacter, Gluconobacter, Acidomonas, Gluconacetobacter and Asaia with a high bootstrap value in a phylogenetic tree. The isolates had high values of DNA-DNA similarity (78-100%) between one another and low values of the similarity (7-25%) to the type strains of Acetobacter aceti, Gluconobacter oxydans, Gluconacetobacter liquefaciens and Asaia bogorensis. The DNA base composition of the isolates ranged from 56.8 to 57.2 mol% G+C with a range of 0-4 mol%. The major quinone was Q-10. The isolates oxidized acetate and lactate to carbon dioxide and water, but the activity was weak, as with strains of Asaia bogorensis. The isolates differed from Asaia bogorensis strains in phenotypic characteristics. The name Kozakia baliensis gen. nov., sp. nov., is proposed for the four isolates. Strain Yo-3T (= NRIC 0488T = JCM 11301T = IFO 16664T = DSM 14400T) was isolated from palm brown sugar collected in Bali, Indonesia, and was designated as the type strain.

Is intracytoplasmic membrane structure a generic criterion? It does not coincide with phylogenetic interrelationships among phototrophic purple nonsulfur bacteria

The 16S rRNA or rRNA gene sequences of the type strains of 5 species of Rhodobacter, Rhodopseudomonas blastica and Paracoccus denitrificans were determined. The sequence analysis revealed that Rhodobacter species, whose intracytoplasmic membrane systems were characteristically vesicular, composed a sole cluster. Rhodopseudomonas blastica, whose intracytoplasmic membrane system was lamellar, was included in the cluster of Rhodobacter. The phylogenetic co-clustering of these bacteria conformed to their possessing of the identical types of carotenoids. Paracoccus denitrificans, which is nonphototrophic, is a right member of the Rhodobacter cluster. Rhodobacter species, Rhodopseudomonas blastica and Paracoccus denitrificans are apart from the other phototrophic bacteria and have the common deletions of 21 bases at the positions 1258 to 1278 (Escherichia coli numbering system). It was demonstrated that the morphological character “intracyto-plasmic membrane structure”, that has been regarded as a generic criterion does not reflect the phylogeny in the phototrophic bacteria. The transfer of Rhodopseudomonas blastica to the genus Rhodobacter is proposed.

Sulfolobus hakonensis sp. nov., a Novel Species of Acidothermophilic Archaeon

We characterized a microbial strain that was isolated from a hot spring at a geothermal area in Hakone, Japan. This isolate, whose lobed-shaped cells were about 1.0 micron in diameter, was a facultative chemolitho-autotroph that required aerobic conditions for growth. The optimum pH was 3.0 (pH range, 1.0 to 4.0), and the optimum temperature was 70 degrees C (temperature range, 50 to 80 degrees C). Lithotrophically, this strain grew on elemental sulfur and reduced sulfur compounds. The G+C content of the genomic DNA was 38.4 mol%. This organism contained calditoglycerocaldarchaeol, which is characteristic of members of the Sulfolobaceae. The levels of 16S rRNA sequence similarity between the new isolate and Sulfolobus acidocaldarius, Sulfolobus solfataricus, and Sulfolobus shibatae were less than 89.8%. Unlike S. acidocaldarius, S. solfataricus, and S. shibatae, the new isolate utilized sugars and amino acids poorly as sole carbon sources, and the levels of DNA-DNA hybridization between the new isolate and these Sulfolobus species were very low. Phenotypically, the new isolate was also distinct from the obligately lithotrophic organism Sulfolobus metallicus. We concluded that the new organism belongs to a new Sulfolobus species, for which we propose the name Sulfolobus hakonensis.

Evolutionary relationships of members of the genera Taphrina, Protomyces, Schizosaccharomyces, and related taxa within the archiascomycetes: Integrated analysis of genotypic and phenotypic characters

To study the phylogeny and evolution of archiascomycetes, we determined the full sequence of the nuclear 18S rRNA gene from 14Taphrina species and 2Protomyces species, and the partial sequence ofSchizosaccharomyces japonicus var.japonicus. The sequences were phylogenetically analyzed by the neighbor-joining, maximum parsimony, and maximum-likelihood methods. We also looked at their principal phenotypic characters and genotypic character. Relationships within the Ascomycota are concordant with the previously published phylogenies inferred from 18S rDNA sequence divergence and divide the archi-, hemi-and euascomycetes into distinct major lineages. All the trees show that, within the archiascomycete lineage, 11 of the 14Taphrina species and the 2Protomyces species are monophyletic. A core groups ofTaphrina andProtomyces is always monophyletic. The evidence from molecular and phenotypic characters such as cell wall sugar composition, ubiquinone, cell wall ultrastructure, and mode of conidium ontogeny, strongly suggests that ‘T’. californica CBS 374.39, ‘T’. maculans CBS 427.69 and ‘T’. farlowii CBS 376.39 should be excluded from the archiascomycete lineage. ‘Taphrina’ farlowii CBS 376.39 groups withCandida albicans in the Saccharomycetales, whereas ‘T’. californica CBS 374.39 and ‘T’. maculans CBS 427.69 have a basidiomycete affinity and group with Tremellalean members in the hymenomycete lineage.Schizosaccharomyces is monophyletic. The strictly anamorphic yeastSaitoella complicata groups with the apothecial ascomyceteNeolecta vitellina rather than theTaphrina/Protomyces branch.

Citeromyces siamensis sp. nov., a novel halotolerant yeast isolated in Thailand

Two halotolerant yeast strains, H130(T) and H149, were isolated from dry salted squid and fermented soybeans, respectively, in Thailand. Both isolates grew by multilateral budding, produced asci that had one roughened spherical ascospore and contained ubiquinone Q-8. These characteristics were shared by Citeromyces matritensis, the only species of the genus Citeromyces. Strains H130(T) and H149 were differentiated from C matritensis by their ability to assimilate L-sorbose and L-lysine and to grow at 37 degrees C. The novel isolates were more tolerant to higher concentrations of cations (3 M NaCl or 0.8 M LiCI) and to higher osmotic pressure (60% glucose) than C. matritensis. A phylogenetic analysis of 18S rRNA gene sequence data indicated that the two novel isolates represented a sister species to C. matritensis. Furthermore, DNA-DNA hybridization data indicated that the isolates were clearly distinct from the type strain of C. matritensis (IFO 0954(T). Based on the above characteristics, strains H130(T) and H149 are proposed to represent a novel species within the genus Citeromyces, Citeromyces siamensis; the type strain is H130(T) (= IFO 11052(T) = JCM 11522(T) = TISTR 5777(T) = CBS 9153(T)).

Farnesyl diphosphate synthase gene of three phototrophic bacteria and its use as a phylogenetic marker

Farnesyl diphosphate (FPP) synthase is essential not only for phototrophic bacteria in carotenoid biosynthesis, but also for non-phototrophic bacteria in the biosynthesis of physiologically important compounds. The gene encoding FPP synthase was assessed as a molecular marker to investigate the intermingled relationship between the phototropic and non-phototropic bacteria in the alpha-Proteobacteria based on 16S rRNA analysis. The FPP synthase amino acid sequences from three phototropic bacteria, Rhodobacter sphaeroides ATCC 11167(T), Rhodobacter capsulatus ATCC 11166(T) and Rhodovulum sulfidophilum W4(T), were determined and used in conjunction with sequences of other representative members of the alpha-, gamma- and epsilon-Proteobacteria and the low-G+C Gram-positive bacteria for phylogenetic analyses by the neighbour-joining and maximum-likelihood methods. The overall topology of the FPP synthase gene tree is consistent with that of the 16S rRNA tree, producing a distinct cluster of the three phototropic bacteria. A minor discordance between the two trees was observed in the cluster of the non-phototrophic Bradyrhizobiumjaponicum USDA 110 and Mesorhizobium loti MAFF 303099; the FPP synthase genes of these two rhizobial species are highly homologous as compared with their respective 16S rRNA. The results suggest that the FPP synthase and 16S rRNA genes have the same evolutionary pattern, evolving vertically from each common ancestral gene; the FPP synthase gene, therefore, could possibly be used for further study on the molecular systematics of photosynthetic bacteria.