RESUMO
AIMS: The aim of this study was to develop a reliable system to analyse the expression of the pyrroloquinoline quinone (PQQ)-alcohol dehydrogenase (ADH) and test its ability to predict the growth and oxidative activity of some acetic acid bacteria (AAB). METHODS AND RESULTS: Specific primers were designed for use in RT-PCR to quantify ADH expression and several housekeeping genes in four species of AAB. 16S rRNA gene was selected as an internal control. The relative expression of adhA was measured in Acetobacter aceti, Acetobacter pasteurianus, Gluconacetobacter hansenii and Gluconobacter oxydans grown in two media that had glucose or ethanol as the carbon source. AAB adhA expression was shown to be related to the two Acetobacter species' ability to oxidise and grow on ethanol, whereas G. oxydans were unable to grow on ethanol and the growth of Ga. hansenii was not related to adhA expression. CONCLUSIONS: The differential expression of ADH could be a marker to analyse both growth and oxidation ability in some AAB, especially those of the genus Acetobacter. SIGNIFICANCE AND IMPACT OF THE STUDY: Several housekeeping genes were tested in AAB and after growth in different media and it was evident that only the ribosomal coding genes were adequate as reference genes for RT-PCR.
Assuntos
Acetobacter/genética , Álcool Desidrogenase/genética , Gluconacetobacter/genética , Gluconobacter oxydans/genética , Acetatos/metabolismo , Acetobacter/enzimologia , Acetobacter/crescimento & desenvolvimento , Álcool Desidrogenase/metabolismo , Meios de Cultura , Primers do DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Etanol/metabolismo , Regulação Bacteriana da Expressão Gênica , Gluconacetobacter/enzimologia , Gluconacetobacter/crescimento & desenvolvimento , Gluconobacter oxydans/enzimologia , Gluconobacter oxydans/crescimento & desenvolvimento , Oxirredução , Cofator PQQ/genética , Cofator PQQ/metabolismo , RNA Ribossômico 16S/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
AIMS: To apply rapid and reliable molecular techniques for typing acetic acid bacteria and studying their population dynamics during wine-making processes. METHODS AND RESULTS: We tested the usefulness of the Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) and Repetitive Extragenic Palindromic-PCR (REP-PCR) techniques with reference strains of most of the species of acetic acid bacteria. We obtained exclusive patterns for each strain with the ERIC-PCR technique, proving the utility for characterizing below species level. REP-PCR technique was not as adequate for this purpose because some strains yielded identical fingerprint. One hundred twenty isolates from a commercial red wine fermentation were fingerprinted using both techniques. We detected a high degree of strain diversity in the first stage of fermentation that decreased throughout the process. However, several strains and species were dominant in the alcoholic fermentation phases. The identification of different strains or genotypes at the species level was carried out by restriction analysis of the 16S ribosomal DNA gene. Gluconobacter oxydans dominated the fresh must, while Acetobacter aceti was the only isolated species at the end of the process. Gluconacetobacter hansenii and G. liquefaciens were also isolated in significant numbers at the beginning of fermentation. CONCLUSIONS: ERIC-PCR and REP-PCR techniques proved useful for characterizing strains of acetic acid bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The availability of molecular techniques for a fast and reliable genotypic characterization should increase our knowledge of the ecology of acetic acid bacteria and determine more accurately their growth behaviour during various stages of vinification.
Assuntos
Bactérias/isolamento & purificação , Microbiologia Industrial , Vinho , Ácido Acético/metabolismo , Bactérias/genética , Bactérias/metabolismo , Técnicas Bacteriológicas , Genótipo , Reação em Cadeia da Polimerase/métodosRESUMO
Acetic acid bacteria (AAB) irreversibly spoil wines and represent a serious problem. Limited studies on the ecology of AAB during winemaking have been done due to the lack of rapid and precise techniques for their identification. RFLP analysis of PCR-amplified fragment of 16S rDNA was performed on AAB reference strains. The amplified rDNAs were approximately 870-bp long for all AAB species while no amplicons were detected for lactic acid bacteria and yeasts. Out of the four restriction enzymes tested, TaqI was the most efficient one and divided the studied AAB into six groups. However, complete differentiation among collection strains of Acetobacter pasteurianus and Gluconoacetobacter hansenii was not possible.