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

Abstract

Forty-five acetic acid bacteria, which were isolated from fruits, flowers and other materials collected in Thailand by an enrichment culture approach, were assigned to the genus Gluconobacter by phenotypic and chemotaxonomic characterisations. On the basis of 16S-23S rRNA gene ITS restriction and 16S rRNA gene sequence analyses, the forty-five isolates were grouped into five groups and identified at the specific level as follows: 1) seventeen isolates were grouped into Group A and identified as G. oxydans; 2) twelve isolates were grouped into Group B and identified as G. cerinus; 3) nine isolates were grouped into Group C and identified as G. frateurii; 4) six isolates were included into Group D and identified as G. thailandicus; 5) one isolate was grouped into Group E, characterised by a restriction pattern comprised of 667 and 48-bp fragments in AvaII digestion, differing from those of strains of Group C or G. frateurii and Group D or G. thailandicus, and unidentified.

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“…Phenotypic and chemotaxonomic characterizations: Phenotypic characterizations were carried out by incubating test strains at 30°C and pH 6.8 for two days on glucose/yeast extract/peptone/glycerol (GYPG) agar 8 . For Gram staining of bacterial cells, the Hucker-Conn modified method 12 was used.…”

“…The amplified 16S rRNA genes were sequenced with an ABI PRISM BigDye Terminator V3.1 8 . For the sequencing of 16S rRNA genes, the following six primers were used: 20F, 1500R, 520F (5 -CAGCAGCCGCGGTA-ATAC-3 ; positions 519-536), 520R (5 -GTATTAC-CGCGGCTGCTG-3 ; positions 536-519), 920F (5 -AAACTCAAATGAATTGACGG-3 ; positions 907-926), and 920R (5 -CCGTCAATTCATTTGAGTTT-3 ; positions 926-907).…”

“…The purified PCR products for Acetobacter isolates were separately digested with restriction endonucleases TaqI, AluI, HpaII, and AvaII (New England BioLabs, Beverly, Massachusetts, USA) according to the manufacturer's instructions. The resulting reaction products were analysed by 2.5% (w/v) agarose gel electrophoresis 8 .…”

“…The forty-seven isolates were isolated from fruits and flowers collected in Thailand by an enrichment culture approach using glucose/ethanol/yeast extract (GEY) medium 8 . A sample source was incubated at pH 4.5 and 30°C for 3-5 days in a liquid medium.…”

“…They are well known as vinegar producers from ethanol by two sequential catalytic reactions of membrane-bound alcohol dehydrogenase (ADH) and aldehyde dehydrogenase 7 . Thailand is located in the tropical area and there are diverse fruits and flowers from which acetic acid bacteria were isolated 8,9 . In Europe, Acetobacter strains, the most popular strains for making acetic acid in vinegar factories, are mesophilic, with optimum temperature for growth at about 30°C 10 .…”