International Journal of Systematic and Evolutionary Microbiology, Vol 50, 1981-1987, Copyright © 2000 by Society for General Microbiology

Identification of acetic acid bacteria by RFLP of PCR-amplified 16S rDNA and 16S--23S rDNA intergenic spacer

A Ruiz, M Poblet, A Mas and JM Guillamon
Unitat d'Enologia, Centre de Referencia en Tecnologia d'Aliments (CeRTA), Departament de Bioquimica i Biotecnologia, Facultat d'Enologia, Universitat Rovira i Virgili, Ramon y Cajal 70, 43005 Tarragona, Spain

DNA corresponding to 16S rDNA and the 16S--23S rDNA intergenic spacer (ITS) from 22 reference strains of acetic acid bacteria, representing the diversity of the family Acetobacteraceae, and 24 indigenous acetic acid bacteria isolated from wine fermentations were analysed by PCR--RFLP. Frateuria aurantia LMG 1558(T) and Escherichia coli ATCC 11775(T) were included as outgroups. PCR-amplified products of about 1450 bp were obtained from the 16S rDNA of all the strains and products of between 675 and 800 bp were obtained from the 16S--23S rDNA ITS. PCR products were digested with 4-base-cutting restriction enzymes in order to evaluate the degree of polymorphism existing among these strains. Of the enzymes tested, TaqI and RsaI were the most discriminatory and showed no intraspecific variations in the restriction patterns. Restriction analysis of the 16S rDNA with these enzymes is proposed as a rapid and reliable method to identify acetic acid bacteria at the level of genus and species (or related species group) and its applicability to identification of indigenous acetic acid bacteria was demonstrated. The same degree of distinction as that for the 16S rDNA analysis was obtained within reference strains of acetic acid bacteria by PCR--RFLP of the 16S--23S rDNA ITS. However, 16S--23S rDNA ITS restriction patterns of strains isolated from wine did not match those of any of the reference strains. Thus, PCR--RFLP of the 16S--23S rDNA ITS is not a useful method to identify isolates of acetic acid bacteria at the species level, although it may be an adequate method to detect intraspecific differentiation.

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