Three strains, RBY-1(T), PHD-1 and PHD-2, were isolated from fruits in Thailand. The strains were Gram-negative, aerobic rods with polar flagella, produced acetic acid from ethanol and did not oxidize acetate or lactate. In phylogenetic trees based on 16S rRNA gene sequences and 16S-23S rRNA gene internal transcribed spacer (ITS) sequences, the strains formed a cluster separate from the type strains of recognized species of the genus Gluconobacter. The calculated 16S rRNA gene sequence and 16S-23S rRNA gene ITS sequence similarities were respectively 97.7-99.7 % and 77.3-98.1 %. DNA G+C contents ranged from 57.2 to 57.6 mol%. The strains showed high DNA-DNA relatedness of 100 % to one another, but low DNA-DNA relatedness of 11-34 % to the tested type strains of recognized Gluconobacter species. Q-10 was the major quinone. On the basis of the genotypic and phenotypic data obtained, the three strains clearly represent a novel species, for which the name Gluconobacter nephelii sp. nov. is proposed. The type strain is RBY-1(T) (= BCC 36733(T) = NBRC 106061(T) = PCU 318(T)), whose DNA G+C content is 57.2 mol%.

The classification and identification of acetic acid bacteria (AAB) underwent much change during the last two decades. However, several closely related AAB species are difficult to differentiate and rapid and correct species level identification remains challenging. In the present study, 281 well-characterized AAB strains representing AAB species of the family Acetobacteraceae were used to construct a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) identification database. The database was evaluated using 270 poorly characterized AAB strains. 16S rRNA and/or multilocus gene sequence analysis and whole-genome sequencing for the calculation of average nucleotide identity values were performed on a selection of strains to verify the accuracy of the obtained results. Virtually all poorly characterized AAB strains were accurately identified to the species level. Most of the remaining strains were considered to represent novel AAB species or belonged to species represented by too few strains in the identification database. The data also revealed that Gluconobacter nephelii and Gluconobacter uchimurae are later heterotypic synonyms of Gluconobacter japonicus and Gluconobacter oxydans, respectively.

Five strains, NBRC 3271(T), NBRC 3272, NBRC 3263, NBRC 3260 and NBRC 3269 were examined genetically, phylogenetically, phenotypically and chemotaxonomically. The DNA G+C contents of the five strains were 55.1-56.4 mol%. The five strains had low levels of DNA-DNA hybridization of 13-51 % to the type strains of Gluconobacter frateurii, Gluconobacter thailandicus, Gluconobacter oxydans, Gluconobacter cerinus, Gluconobacter albidus and Gluconobacter kondonii and formed a cluster that was separate from the type strains of the six Gluconobacter species given above in phylogenetic trees based on 16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer sequences. The five strains weakly produced dihydroxyacetone from glycerol, but not 2,5-diketo-d-gluconate or a water-soluble brown pigment from d-glucose and contained ubiquinone-10. The five strains were assigned as representing a novel species of the genus Gluconobacter, for which the name Gluconobacter japonicus sp. nov. is proposed. The type strain is NBRC 3271(T) (=BCC 14458(T)=strain 7(T), K. Kondo). Cells of the type strain are motile by means of polar flagella and the DNA G+C content is 56.4 mol%.