Study on the genetics of acetic acid bacteria for the efficient vinegar production

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1. Two acetic acid-sensitive mutants (AS101 and AS102) of Acetobacter acetisbsp. AcetiAS10 were isolated by mutagenesis with N-metyl-N-nitro-N-nitrosoguanidine. By shotgun cloning of Sau3Al-digested chromosome DNA from the parental-type A.aceti AS10 into mutant AS101 and AS102 with a Acetobacter-E.colishattle vector, pMV24, we isolated DNA fragments that complemented these mutations. One of the recombinant plamids contained a 6.6kb fragment showing the presence of five ORFs which is highly homologous with SecD, SecF, MscL, OmpR and EnvZ, respectively. secF and ompR among these genes were sufficient to complement mutation of AS102. Other recombinant plasmid contained a 2.2kb fragnent showing the presence of a ORF which is homologous with OmpR.ompR gene were complemented mutation of AS101, but not complemented these of AS102.2. Exposure of A.aceti 10-8S2 to 3% ethanol or 1% acetate induced the expression of seven proteins and five proteins, respectively. Two of five proeins induced by 1% acetate were revealed to be aconitase and isocitrate dehydrogenase by analysis of N-terminal amino acid sequence of each proteins. We cloned aconitase gene by southern hybridization, and is going to do overexpression of it in A.aceti. In addition, we are going to clone isocitrate dehydrogenase gene. Other three proteins were shown to have no homology with other proteins by analysis of N-terminal amino acid sequence.