TRANSFORMATION OF ACETOBACTER-XYLINUM WITH PLASMID DNA BY ELECTROPORATION

Genetic analysis of Acetobacter xylinum, a cellulose-synthesizing bacterium, has been limited by lack of a successful transformation method. Transformation of A. xylinum was attempted using two broad-host-range plasmids (pUCD2 and pRK248) and a variety of transformation methods. Methods using CaCl2, freeze/thaw treatments, and polyethylene glycol were unsuccessful. Transformation of a cellulose-negative strain of A. xylinum with plasmid DNA has been achieved with high-voltage electroporation. Electroporation conditions of 25 μF capacitance, 2.5 kV, 400 ohms resistance, and pulse lengths of 6-8 ms were applied to a cell/DNA mixture in a 0.2-cm cuvette. Plasmid pUCD2 transformed at an efficiency of 106-107 transformants/μg DNA and pRK248 yielded 105 transformants/μg DNA. The frequency of transformation increased linearly with increasing DNA concentration, while transformation efficiency remained constant. pUCD2 was recovered from transformants following chloramphenicol amplification and observed by agarose gel electrophoresis. Both plasmids could be reisolated from Escherichia coli after back-transformation with alkaline lysis DNA preparations from Acetobacter transformants. Electro-transformation of A. xylinum with plasmid DNA suggests its potential use for analysis of the A. xylinum genome. © 1992.