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

1.醋酸杆菌（Acetobacter acetisbsp.）AcetiAS 10通过用N-甲基-N-硝基-N-亚硝基胍诱变而分离。通过用Aceto-E.colishattle载体pMV 24将来自亲本型乙酰曲霉AS 10的Sau 3Al消化的染色体DNA鸟枪克隆到突变体AS 101和AS 102中，我们分离了补充这些突变的DNA片段。其中一个重组质粒含有一个6.6kb的片段，该片段含有5个ORF，分别与SecD、SecF、MscL、OmpR和EnvZ高度同源。其中secF和ompR基因足以弥补AS 102的突变。另一个重组质粒含有一个2.2kb的片段，该片段与OmpR基因同源，OmpR基因与AS 101的突变互补，而与AS102.2的突变不互补。将乙酰曲霉10- 8 S2暴露于3%乙醇或1%乙酸盐分别诱导7种蛋白和5种蛋白的表达。经N-末端氨基酸序列分析，在1%乙酸诱导的5种蛋白质中，有2种为乌头酸酶和异柠檬酸脱氢酶。我们利用Southern杂交技术克隆了乌头酸酶基因，并准备在醋酸曲霉中进行过量表达。另外，我们还准备克隆异柠檬酸脱氢酶基因。N-末端氨基酸序列分析表明，其它3个蛋白与其它蛋白没有同源性。

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.