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Differentiation of species of the family Acetobacteraceae by AFLP DNA fingerprinting: Gluconacetobacter kombuchae is a later heterotypic synonym of Gluconacetobacter hansenii

¹ BCCM/LMG Bacteria Collection, Faculty of Sciences, Ghent University, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium
² Research Group of Industrial Microbiology and Food Biotechnology, Department of Applied Biological Sciences and Engineering, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium
³ Laboratory of Microbiology, Faculty of Sciences, Ghent University, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium

Correspondence
Ilse Cleenwerck
ilse.cleenwerck{at}ugent.be

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.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of LMG 23726^T is AM999342.

A supplementary figure showing the (GTG)₅-PCR fingerprints of strains belonging to the species of the Gluconacetobacter xylinus branch and supplementary tables detailing the DNA–DNA relatedness of the strains of the genera Acetobacter and Gluconacetobacter as determined in this and previous studies are available with the online version of this paper.