Pattaraporn Yukphan

Subdivision of the genus Gluconacetobacter Yamada, Hoshino and Ishikawa 1998: the proposal of Komagatabacter gen. nov., for strains accommodated to the Gluconacetobacter xylinus group in the α-Proteobacteria

The genus Gluconacetobacter is divided into two groups phylogenetically, phenotypically and ecologically: the Gluconacetobacter liquefaciens group and the Gluconacetobacter xylinus group. For the latter group, the genus Komagatabacter is newly introduced, and the type species of the new genus is designated as Komagatabacter xylinus (Brown 1886) comb. nov. Twelve species of the Gluconacetobacter xylinus group are transferred to the new genus as new combinations.

Heterogeneity of Strains Assigned to Gluconobacter frateurii Mason and Claus 1989 Based on Restriction Analysis of 16S-23S rDNA Internal Transcribed Spacer Regions

Twenty-three strains, which were assigned to Gluconobacter frateurii and maintained at Culture Collection NBRC, were re-identified at the species level on the basis of restriction analysis of 16S-23S rDNA ITS regions by digestion with six restriction endonucleases: Bsp1286I, MboII, AvaII, TaqI, BsoBI, and BstNI. The strains examined were divided into six groups, Group III-1, Group III-2, Group III-3, Group III-4, Group III-5, and Group IV. Group III-1 and Group III-4 respectively were divided into two subgroups, Subgroup III-1a, Subgroup III-1b and Subgroup III-4a, Subgroup III-4b. Gluconobacter frateurii NBRC 3264T was included in Group III-2, along with strains NBRC 3265 and NBRC 3270, and G. thailandicus BCC 14116T was included in Group III-3, along with strains NBRC 3254, NBRC 3256, NBRC 3258, NBRC 3255, and NBRC 3257. These groupings were supported by a phylogenetic tree based on 16S-23S rDNA ITS sequences. Strains of group III-2 and Group IV were unequivocally re-identified as G. frateurii, but strains of Group III-3, Group III-4, and Group III-5 were not necessarily re-identified as G. frateurii. The results obtained indicate that the 23 strains have a taxonomically heterogeneous nature, and they are referred to as the G. frateurii complex.

Identification ofGluconobacter strains isolated in Thailand based on 16S–23S rRNA gene ITS restriction and 16S rRNA gene sequence analyses

Ninety-seven acetic acid bacteria, which were isolated from fruits, flowers and other material collected in Thailand by an enrichment culture approach, were assigned to the genusAcetobacter by phenotypic and chemotaxonomic characterisations. On the basis of 16S–23S rRNA gene ITS restriction and 16S rRNA gene sequence analyses, the ninety-seven isolates were grouped into three groups and identified at the specific level: 1) group A including fifty-three isolates, which were identified asAcetobacter pasteurianus, 2) group B including forty-two isolates, which were identified asAcetobacter orientalis and 3) group C including two isolates, which were identified asAcetobacter lovaniensis. There was no isolate to be assigned to other 15 species of the genusAcetobacter.

Identification of Acetobacter strains from Thai fermented rice products based on the 16S rRNA gene sequence and 16S-23S rRNA gene internal transcribed spacer restriction analyses.

BACKGROUNDnFermented rice flour (khao-khab, a non-glutinous rice) and related products are Thai traditional products. The types of acetic acid bacteria (AAB) microflora in khao-khab have not been reported. In this study, Acetobacter strains were isolated and identified based on the phenotypic and chemotaxonomic characteristics and molecular aspects.nnnRESULTSnTwenty-five acetic acid bacteria isolated from fermented rice products and a starter for sweetened rice in Thailand by an enrichment culture approach, were assigned to the genus Acetobacter by phenotypic and chemotaxonomic characterisations. On the basis of the 16S rRNA gene sequence and 16S-23S rRNA gene ITS restriction analyses, 25 isolates were divided into six groups and identified at the specific level: (1) Group 1 included five isolates, which were identified as A. indonesiensis; (2) Group 2 included two isolates, which were identified as A. lovaniensis; (3) Group 3 included one isolate, which was identified as A. orientalis; (4) Group 4 included eleven isolates, which were identified as A. pasteurianus; (5) Group 5 included three isolates, which were identified as A. syzygii and (6) Group 6 included three isolates, which were unidentified and considered to constitute a new species.nnnCONCLUSIONnResults revealed that various Acetobacter species were distributed in Thai fermented rice flour and related products. A novel Acetobacter species was isolated from the product.

Acetobacter thailandicus sp. nov., for a strain isolated in Thailand

A Gram-negative, rod-shaped, and non-motile bacterium, designated as isolate AD25T, was isolated from a flower of the blue trumpet vine (Thunbergia laurifolia) at Tong Pha Phum, Kanchanaburi, Thailand. Phylogenetic analyses of 16S rRNA gene, 16S-23S rRNA gene internal transcribed spacer (ITS) region, and groEL gene sequences showed that the isolate was quite remote and constituted a cluster independent from the type strains of other Acetobacter species. The isolate was closely related to Acetobacter cibinongensis, one of the closest relatives, with 98.3xa0% 16S rRNA gene sequence similarity. The DNA G -+- C content of the isolate was 51.4xa0mol%. The isolate grew intensely on 10xa0% ethanol with 1.5xa0% D-glucose in the presence of 0.3xa0% peptone and 0.3xa0% yeast extract, and grew weakly on 3.0xa0% D-glucose in the presence of 0.1xa0% ammonium sulfate as the sole source of nitrogen. The isolate produced only D-gluconic acid from D-glucose. Based on physiological, biochemical, and genotypic differences between the isolate and the type strains of the validly named species, it is proposed that the isolate be classified as a novel species of Acetobacter, for which the name Acetobacter thailandicus sp. nov. is introduced. The type strain is isolate AD25T (= BCC 15839T = NBRC 103583T).

Kockiozyma gen. nov., for Zygozyma suomiensis: the phylogeny of the Lipomycetaceous yeasts

In phylogenetic trees based on 18S rRNA gene, 26S rRNA gene, mitochondrial small subunit rRNA gene and the EF-1α gene sequences, and the concatenated sequences of the latter four regions derived from the neighbor-joining method, the four species of the genus Zygozyma constituted four clusters, respectively, with low bootstrap values. In phylogenetic trees based on the concatenated sequences of 18S rRNA genes, 26S rRNA genes, mitochondrial small subunit rRNA genes and the EF-1α genes derived from the neighbor-joining method, the maximum parsimony method and the maximum likelihood method, Lipomyces species including Lipomyces starkeyi (the type species of the genus Lipomyces), Lipomyces tetrasporus and so on, Lipomyces lipofer (= Waltomyces lipofer), Lipomyces japonicus (= Smithiozyma japonica), Babjevia anomala (= Lipomyces anomalus) and Dipodascopsis uninucleata constituted their own respective clusters. The four species of the genus Zygozyma, viz., Zygozyma oligophaga (the type species of the genus Zygozyma), Zygozyma suomiensis, Zygozyma arxii (= Kawasakia arxii) and Zygozyma smithiae (= Limtongia smithiae) were separated from one another and phylogenetically independent from other Lipomycetaceous yeast species. From the data obtained, all four species of the genus Zygozyma were distinguished from one another at the generic level. The name of Kockiozyma was newly suggested for Zygozyma suomiensis, and Kockiozyma suomiensis was introduced as a new combination.

Limtongia gen. nov. for Zygozyma smithiae (Lipomycetaceae)

Teleomorphic species of the genus Zygozyma and anamorphic species of the genus Myxozyma were examined phylogenetically. In phylogenetic trees based on 18S rRNA-, 26S rRNA-, mitochondrial small subunit rRNA- and EF-1α-gene sequences and concatenated sequences of the latter four regions, derived from the neighbor-joining method, the four species of the genus Zygozyma constituted four clusters, respectively, with low bootstrap values, indicating that all four species can be distinguished from one another at the generic level. The name of Limtongia was newly suggested for Zygozyma smithiae, and Limtongia smithiae was proposed as new combination.

Tanticharoenia aidae sp. nov., for acetic acid bacteria isolated in Vietnam

Tanticharoenia aidae sp. nov. was introduced for two strains, designated as VTH-Ai06T and VTH-Ai07, which were isolated from the stems of sugar cane collected in Vietnam. In a phylogenetic tree based on 16S rRNA gene sequences derived from the neighbor-joining method, the two strains formed a cluster along with the type strain of Tanticharoenia sakaeratensis with a bootstrap value of 64xa0%. The pair-wise 16S rRNA gene sequence similarity was 98.3xa0% between strain VTH-Ai06T and the type strain of T. sakaeratensis. The range of DNA G+C contents was from 65.2 to 65.4xa0mol%. In DNA–DNA hybridization, the two strains showed low similarities to T. sakaeratensis BCC 15772T. Cellular fatty acids were of C18:1ω7c as a major. A major isoprenoid quinone was Q-10. Phenotypically, the two strains differed from the type strain of T. sakaeratensis in no production of dihydroxyacetone from glycerol. The type strain is VTH-Ai06T (= VTCC 910001Tu2009=u2009BCC 67839Tu2009=u2009NBRC 110637T), whose DNA G+C content was 65.4xa0mol%.

Identification of acetic acid bacteria isolated in Thailand and assigned to the genus Acetobacter by groEL gene sequence analysis

Twenty-three strains of acetic acid bacteria were isolated by an enrichment culture approach from fruits, flowers, mushrooms, and fermented rice products collected in Thailand. All were assigned to the genus Acetobacter and examined taxonomically for 16S rRNA gene and groEL gene sequences. Based on analysis of 16S rRNA gene and groEL gene sequences, the strains isolated were divided into ten groups: Group 1 comprised four isolates identified as A. tropicalis, Group 2 consisted of three isolates identified as A. indonesiensis, Group 3 had two isolates identified as A. persici, Group 4 was two isolates (A. orientalis), Group 5 was one isolate (A. cibinongensis), Group 6 contained three isolates (A. pasteurianus), Group 7 was one isolate of A. papayae, Group 8 was two isolates identified as A. fabarum, Group 9 was one isolate of A. okinawensis, and Group 10 was four isolates identified as A. ghanensis.

A functionally critical single nucleotide polymorphism in the gene encoding the membrane-bound alcohol dehydrogenase found in ethanol oxidation-deficient Gluconobacter thailandicus.

The Gluconobacter thailandicus strains NBRC3254, NBRC3255, NBRC3256, NBRC3257, and NBRC3258 are naturally deficient in the ethanol-oxidizing respiratory chain because they do not produce the cytochrome subunit of the membrane-bound alcohol dehydrogenase (ADH). Draft genomes of G. thailandicus strains NBRC3255 and NBRC3257 indicated that the adhB gene encoding the cytochrome subunit contains four base differences when compared to a closely related gene in the public database One of the nucleotide differences results in an Opal codon at the -19th tryptophan (Trp) in the signal sequence for translocation to the periplasmic space (here, the position of +1st residue is assigned to the N-terminal amino acid residue after signal peptide cleavage), while the other differences result in one missense and two silent amino acid alterations. All five of the G. thailandicus strains were shown to have the Trp(-19)Opal alteration. Ethanol oxidation and ADH activities in NBRC3255 were restored by transformation with a derivative of the endogenous adhB gene, of which the -19th Opal codon was altered to encode Trp. These results indicate that this sequence is a functionally critical single nucleotide polymorphism in the cytochrome subunit. Comparative genomic analyses between the draft genomes of NBRC3255 and NBRC3257 revealed that although the two genomes are closely related, they both have a significant number of unique open reading frames. We suggest that the closely related NBRC3255 and NBRC3257 diverged from a common ancestor having the mutation in the adhB gene, whereas no additional functionally critical mutation occurred in the adhB pseudogene over the course of evolution.