A few members of the family Acetobacteraceae are cellulose-producers, while only six members fix nitrogen. Bacterial strain RG3T, isolated from Kombucha tea, displays both of these characteristics. A high bootstrap value in the 16S rRNA gene sequence-based phylogenetic analysis supported the position of this strain within the genus Gluconacetobacter, with Gluconacetobacter hansenii LMG 1527T as its nearest neighbour (99.1 % sequence similarity). It could utilize ethanol, fructose, arabinose, glycerol, sorbitol and mannitol, but not galactose or xylose, as sole sources of carbon. Single amino acids such as L-alanine, L-cysteine and L-threonine served as carbon and nitrogen sources for growth of strain RG3T. Strain RG3T produced cellulose in both nitrogen-free broth and enriched medium. The ubiquinone present was Q-10 and the DNA base composition was 55.8 mol% G+C. It exhibited low values of 5.2-27.77 % DNA-DNA relatedness to the type strains of related gluconacetobacters, which placed it within a separate taxon, for which the name Gluconacetobacter kombuchae sp. nov. is proposed, with the type strain RG3T (=LMG 23726T=MTCC 6913T).

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Amplified fragment length polymorphism (AFLP) DNA fingerprinting was investigated as a tool for fast and accurate identification of acetic acid bacteria (AAB) to the species level. One hundred and thirty five reference strains and 15 additional strains, representing 50 recognized species of the family Acetobacteraceae, were subjected to AFLP analysis using the restriction enzyme combination ApaI/TaqI and the primer combination A03/T03. The reference strains had been previously subjected to either DNA-DNA hybridization or 16S-23S rRNA spacer region gene sequence analysis and were regarded as being accurately classified at the species level. The present study revealed that six of these strains should be reclassified, namely Gluconacetobacter europaeus LMG 1518 and Gluconacetobacter xylinus LMG 1510 as Gluconacetobacter xylinus and Gluconacetobacter europaeus, respectively; Gluconacetobacter kombuchae LMG 23726(T) as Gluconacetobacter hansenii; and Acetobacter orleanensis strains LMG 1545, LMG 1592 and LMG 1608 as Acetobacter cerevisiae. Cluster analysis of the AFLP DNA fingerprints of the reference strains revealed one cluster for each species, showing a linkage level below 50 % with other clusters, except for Acetobacter pasteurianus, Acetobacter indonesiensis and Acetobacter cerevisiae. These three species were separated into two, two, and three clusters, respectively. At present, confusion exists regarding the taxonomic status of Gluconacetobacter oboediens and Gluconacetobacter intermedius; the AFLP data from this study supported their classification as separate taxa. The 15 additional strains could all be identified at the species level. AFLP analysis further revealed that some species harboured genetically diverse strains, whereas other species consisted of strains showing similar banding patterns, indicating a more limited genetic diversity. It can be concluded that AFLP DNA fingerprinting is suitable for accurate identification and classification of a broad range of AAB, as well as for the determination of intraspecific genetic diversity.

Ten strains previously assigned to Acetobacter hansenii (=Gluconacetobacter hansenii), Acetobacter pasteurianus LMG 1584 and eight reference strains of the genus Gluconacetobacter were reclassified by 16S rRNA gene sequencing, DNA-DNA similarity, DNA base composition and phenotypic characteristics. The A. hansenii strains and A. pasteurianus LMG 1584 were included in the cluster of acetic acid bacteria (family Acetobacteraceae) by 16S rRNA gene sequences. Further, they were separated into seven distinct groups by DNA-DNA similarity. DNA-DNA similarity group I was identified as G. hansenii. DNA-DNA similarity group II was retained as Gluconacetobacter sp., because DNA-DNA similarity between the strain and Gluconacetobacter entanii LTH 4560(T) could not be determined. This was due to a lack of availability of the type strain from any source. DNA-DNA similarity group III was regarded as a novel species, for which the name Gluconacetobacter saccharivorans sp. nov. (type strain, LMG 1582(T)=NRIC 0614(T)) is proposed. DNA-DNA similarity group IV included the type strains of Gluconacetobacter oboediens and Gluconacetobacter intermedius, and three A. hansenii strains. This group was identified as G. oboediens because high values of DNA-DNA similarity were obtained between the type strains and G. oboediens has priority over G. intermedius. DNA-DNA similarity group V was identified as Gluconacetobacter europaeus. DNA-DNA similarity group VI was regarded as a novel species, for which the name Gluconacetobacter nataicola sp. nov. (type strain, LMG 1536(T)=NRIC 0616(T)) is proposed. DNA-DNA similarity group VII was reclassified as Gluconacetobacter xylinus. The description of G. hansenii is emended.

Thirty-six strains of acetic acid bacteria classified in the genera Acetobacter, Gluconobacter, and Acidomonas were examined for their partial base sequences in positions 1220 through 1375, 156 bases, of 16S rRNA. The strains of the Q10-equipped Gluconobacter species examined were divided into two subgroups, which included the type strains of Gluconobacter oxydans, the type species of the genus Gluconobacter, and of a second species, Gluconobacter cerinus, respectively. The base differences numbered four between the two type strains. The strains of the Q9-equipped species examined classified in the type subgenus Acetobacter of the genus Acetobacter were not very distant phylogenetically from those of the genus Gluconobacter. The calculated number of base differences was 9-6 between the type strains of G. oxydans and G. cerinus and the type strains of Acetobacter aceti and Acetobacter pasteurianus. In contrast, the strains of the Q10-equipped species examined classified in the subgenus Gluconoacetobacter of the genus Acetobacter were very distant phylogenetically from those of the Acetobacter and Gluconobacter species mentioned above. The number of base differences was calculated to be 14-8. Furthermore, the strains of the methanol-assimilating, Q10-equipped species of the genus Acidomonas examined were located in phylogenetically isolated positions. The type strain of Acidomonas methanolica (identical to Acetobacter methanolicus), the type species of the genus Acidomonas, had 16-9 base differences. The data obtained here indicated that the members of the subgenus Gluconoacetobacter of the genus Acetobacter can be distinguished at the generic level. The new genus Gluconoacetobacter was proposed with the type species, Gluconoacetobacter liquefaciens, in recognition of the genus Acidomonas along with the genera Acetobacter and Gluconobacter in the classification of the acetic acid bacteria.