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

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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.