Gene Analysis and Cloning for Cellulose-Producing Bacteria : A, Xylinum.

纤维素产生菌乙酸乙酯亚种。木霉IFO 3288含有7个质粒，命名为pFF1-7。PFF表达载体的相对分子质量为1.4~9.4kb，限制性内切酶分析表明，pFF具有较强的限制性内切酶活性。将pFF6与大肠杆菌pUC18连接，构建穿梭载体pUF106。它具有适合插入外源DNA片段的独特的限制性内切酶切点，并使宿主对氨苄西林产生抗性。该载体转化产生纤维素的木酸醋杆菌ATCC 10245和E.ColiJM109。因此，穿梭载体pUF106可以作为一种实用的产纤维素菌的克隆载体。作为模型实验，利用LeuB突变宿主E.coliC600和质粒载体pUC18，用鸟枪法克隆了产纤维素菌ATCC 10245与亮氨酸合成有关的β-异丙基苹果酸脱氢酶基因。携带6.5kb BamHI片段的质粒载体pUL1赋予野生型大肠杆菌C600β-IPMDH活性。限制性内切酶图谱和缺失分析表明，β-IPMDH基因位于pULH1 1.15kb的HindIII片段上。克隆的β-ipmdh基因与木霉ATCC 10245的原始基因用生物素标记的探针进行Southern杂交。

Cellulose - producing bacterium Acetobacter aceti subsp. xylinum IFO 3288 was found to harbor seven plasmids named pFF1-7. The pFF plasmids had molecular weights of 1.4 - 9.4 kb and they were characterized with restriction endonucleases. Shuttle vector pUF106 was constructed by ligation of pFF6 to Escherichia coli plasmid pUC18. It had unique restriction sites suitable for insertion of a foreign DNA fragment and conferred ampicillin resistance to a host. This vector transformed cellulose-producing Acetobacter xylinum ATCC 10245 as well as E. coli JM109. Thus shuttle vector pUF106 is available as a practical cloning vector for cellulose- producing bacteria.As a model experiment, the beta -isopropylmalate dehydrogenase (beta -IPMDH) gene, which concerned with Leucine synthesis, of cellulose-producing A. xylinum ATCC 10245 were cloned by shotgun cloning method using leuB-mutant host E. coli C600 and plasmid vector pUC18. Plasmid vector pUL1, carrying a 6.5 kilobase pairs (kb) BamHI-fragment, conferred the beta -IPMDH activity to the wild type of E. coli C600. Restriction mapping and deletion analysis indicated that the beta -IPMDH gene was located on a 1.15kb HindIII-fragment of plasmid pULH1. The cloned beta -IPMDH gene was identified with the original genes of A. Xylinum ATCC 10245 by Southern hybridization method using a biotin-labeled probe.