Construction of Plasmid Vectors and a Genetic Transformation System for Acetobacter aceti

Abstract

The proline auxotrophic strain of Acetobacter aceti No. 1023 treated with CaCl2solution was transformed to the Pro+phenotype at a frequency of up to 102/μg DNA using chromosomal DNA prepared from the wild type prototrophic strain. The CaCl2-treated cells of A. aceti No. 1023 could also be rendered competent for uptake of plasmid DNA. In an attempt to produce an appropriate cloning vector for A. aceti, the restriction patterns of the cryptic plasmids, pTA5001 A (23.5 Kb) and pTA5001B (23 Kb), found in A. aceti No. 1023 were determined. A selectable marker (ampicillin resistance) was introduced onto these cryptic plasmids by fusing them to vector pACYC177 from E. coli, using their single restriction site for Xhol. The hybrid plasmids generated could replicate in and confer ampicillin resistance to both A. aceti and E. coli. The maximum transformation frequency for A. aceti No. 1023 with these vectors was 103/μg DNA. © 1985, Japan Society for Bioscience, Biotechnology, and Agrochemistry. All rights reserved.