Transformation of Acidiphilium by electroporation and conjugation

Abstract

Two techniques, electroporation and conjugation, have been used to introduce the RK2-based broad-host-range plasmids pRK415 and pLAFR3 into strains of the bacterial genus Acidiphilium. Using electroporation, cells were also transformed with a series of chimeric plasmids constructed by cloning cryptic Acidiphilium plasmids into the Escherichia coli vector pBR328. Various parameters affecting electroporation were investigaed. Transformation efficiency varied widely with different recipient strains. Growth at an elevated temperature (37 °C) prior to electroporation increased transformation efficiency 10-fold compared with growth at 32 °C. For three strains tested, optimum transformation efficiency was obtained with field strengths of 10–15 kV/cm. Transformation efficiency increased linearly with increasing DNA concentration up to 10 μg/mL. Transformation efficiencies in these experiments ranged up to 10 4 transformants/μg DNA. Mobilization of pRK415 and pLAFR3 from E. coli strain S17.1 into several Acidiphilium strains was achieved following incubation for 3 h on nutrient agar medium (pH 7.0). Conjugation frequencies in the range of 10 −5 –10 −9 per recipient cell were obtained. Conjugation frequency was also dependent on recipient strain. Key words: acidophilic bacteria, endogenous plasmids, broad-host-range plasmids.