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 3264(T) was included in Group III-2, along with strains NBRC 3265 and NBRC 3270, and G. thailandicus BCC 14116(T) 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.     

Production of 4-keto-D-arabonate by oxidative fermentation with newly isolated Gluconacetobacter liquefaciens

Production of 4-keto-D-arabonate (4KAB) was confirmed in a culture medium of Gluconacetobacter liquefaciens strains, newly isolated from water kefir in Argentina. The strains rapidly oxidized D-glucose, D-gluconate (GA), and 2-keto-D-gluconate (2KGA), and accumulated 2,5-diketo-D-gluconate (25DKA) exclusively before reaching the stationary phase. 25DKA was in turn converted to 4KAB, and 4KAB remained stable in the culture medium. The occurrence of 4KAB was assumed by Ameyama and Kondo about 50 years ago in their study on the carbohydrate metabolism of acetic acid bacteria (Bull. Agr. Chem. Soc. Jpn., 22, 271-272, 380-386 (1958)). This is the first report confirming microbial production of 4KAB.     

Identification of strains assigned to the genus Gluconobacter Asai 1935 based on the sequence and the restriction analyses of the 16S-23S rDNA internal transcribed spacer regions

Thirteen reference strains, including the type strains of the type species of the genus Gluconobacter, Gluconobacter oxydans (NBRC 14819T), Gluconobacter cerinus (NBRC 3267T), and Gluconobacter frateurii (IFO 3264T) were examined for their species identification based on the sequence and the restriction analyses of the 16S-23S rDNA internal transcribed spacer (ITS) regions. A phylogenetic tree constructed by the neighbor-joining method represented three clusters corresponding respectively to the three species, G. oxydans, G. cerinus, and G. frateurii. The type strain of Gluconobacter asaii (NBRC 3276T), which is a junior subjective synonym of G. cerinus, was included completely in the G. cerinus cluster. Several restriction endonucleases discriminating the three species from one another were selected by computer analyses: Bsp1286I, MboII, SapI, Bpu10I, EarI, BsiHKAI, and FatI. On digestion of the PCR products with restriction endonucleases Bsp1286I and MboII, all the restriction patterns coincided with those of the type strains of the three species except for strain NBRC 3251. This strain gave a different pattern from the type strain of G. frateurii, when digested with MboII. However, strain 3251 was included phylogenetically in the G. frateurii cluster. All the reference strains were thus identified at the species level by the sequence and the restriction analyses of the 16S-23S rDNA ITS regions.     

Identification and histamine formation of Tetragenococcus isolated from Thai fermented food products

Forty-one tetrad-forming halophilic lactic acid bacteria were isolated from 7 kinds of fermented foods in Thailand. All the isolates were identified as the genus Tetragenococcus by their phenotypic characteristics. On the basis of 16S rRNA gene restriction analysis using MboI and AluI and 16S rRNA gene sequence analyses, 41 isolates could be divided into two groups (groups A and B). All 22 isolates in Group A were identified as T. halophilus. 16S rRNA gene sequences of the representative isolates, SP37-2 and KS87-1 exhibited 99.4–99.5 % similarity to that of T. halophilus ATCC 33315^T. Nineteen isolates in Group B were identified as T. muriaticus. 16S rRNA gene sequences of the representative isolates, KM1-5 and KS87-14, showed 99.0–99.6 % similarity to that of T. muriaticus JCM 10006^T. Histamine formation was determined by using HPLC and the histidine decarboxylase (hdc) gene of the newly isolated histamine-producing strain was partially sequenced. The strain KS87-14 prolifically formed histamine 10 times higher than the reported T. muriaticus JCM 10006^T. The positive detection of KS87-14 was achieved by using hdcA gene-specific primers JV16HC and JV17HC.     

Neokomagataea gen. nov., with descriptions of Neokomagataea thailandica sp. nov. and Neokomagataea tanensis sp. nov., osmotolerant acetic acid bacteria of the α-Proteobacteria

Isolates AH11(T) and AH13(T) were isolated from flowers of lantana and candle bush respectively collected in Thailand. In phylogenetic trees based on 16S rRNA gene sequences, the two isolates formed an independent cluster, which was then connected to the type strain of Saccharibacter floricola. The calculated pair-wise 16S rRNA gene sequence similarities of isolate AH11(T) were 95.7-92.3% to the type strains of the type species of the 12 genera of acetic acid bacteria. The DNA base composition was from 51.2 to 56.8 mol % G+C, with a range of 5.6 mol %. When isolate AH11(T) was labeled, DNA-DNA similarities were 100, 12, 4, 5, and 4% respectively to isolates AH11(T) and AH13(T) and the type strains of Saccharibacter floricola, Gluconobacter oxydans, and Acetobacter aceti. The two isolates were non-motile and did not oxidize either acetate or lactate. No growth was found in the presence of 0.35% acetic acid w/v. The two isolates were not osmophilic but osmotolerant, produced 2,5-diketo-D-gluconate from D-glucose, and did not oxidize lactate, thus differing from strains of Saccharibacter floricola, which showed weak lactate oxidation. The two isolates contained unsaturated C(18:1)ω7c fatty acid as the major fatty acid, and were unique in the presence of a considerable amount of straight-chain C(18:1)2OH fatty acid. Q-10 was present as the major isoprenoid quinone. Neokomagataea gen. nov. was proposed with the two species, Neokomagataea thailandica sp. nov. for isolate AH11(T) (=BCC 25710(T)=NBRC 106555(T)), which has 56.8 mol % G+C, and Neokomagataea tanensis sp. nov. for isolate AH13(T) (=BCC 25711(T)=NBRC 106556(T)), which has 51.2 mol % G+C.     

Identification of lactic acid bacteria from fermented tea leaves (miang) in Thailand and proposals of Lactobacillus thailandensis sp. nov., Lactobacillus camelliae sp. nov., and Pediococcus siamensis sp. nov

Eighteen rod-shaped homofermentatives, six heterofermentatives, and a coccal homofermentative lactic acid bacteria were isolated from fermented tea leaves (miang) produced in the northern part of Thailand. The isolates were placed in a monophyletic cluster consisting of Lactobacillus and Pediococcus species. They were divided into seven groups by phenotypic and chemotaxonomic characteristics, DNA-DNA similarity, and 16S rRNA gene sequences. Groups I to VI belonged to Lactobacillus and Group VII to Pediococcus. All of the strains tested produced DL-lactic acid but those in Group IV produced L-lactic acid. The strains tested in Groups I, II and V had meso-diaminopimelic acid in the cell wall. Six strains in Group I were identified as Lactobacillus pantheris; five strains in Group II as Lactobacillus pentosus; and four strains in Group V as Lactobacillus suebicus. Two strains in Group VI showed high DNA-DNA similarity for each other and MCH4-2 was closest to Lactobacillus fermentum CECT 562(T) with 99.5% of 16S rRNA gene sequence similarity. Five strains in Group III are proposed as Lactobacillus thailandensis sp. nov., and MCH5-2(T) (BCC 21235(T)=JCM 13996(T)=NRIC 0671(T)=PCU 272(T)) is the type strain which has 49 mol% G+C of DNA. Two strains in Group IV are proposed as Lactobacillus camelliae sp. nov., and the type strain is MCH3-1(T) (BCC 21233(T)=JCM 13995(T)=NRIC 0672(T)=PCU 273(T)) which has 51.9 mol% G+C of DNA. One strain in Group VII is proposed as Pediococcus siamensis sp. nov., and MCH3-2(T) (BCC 21234(T)=JCM 13997(T)=NRIC 0675(T)=PCU 274(T)) is the type strain which has 42 mol% G+C of DNA.