FACTORS AFFECTING THE ELECTROPORATION OF BACILLUS-SUBTILIS

Bacillus subtilis 168 trp was found to be transformable with the tetra cycline resistance plasmid pAB124 by electroporation of whole cells, i nconsistently and at very low frequencies. Supplementation of the grow th medium with glycine, or particularly DL-threonine, produced cells t hat could be electrotransformed much more efficiently at frequencies u p to 2.5 x 10(3) transformants per mu g plasmid DNA. Transformation wa s optimal with cells grown in medium containing a racemic mixture of t he D- and L-isomers of threonine, and no transformants were obtained w hen pure forms of the D- and L-threonine isomers were used. The cell w alls of B. subtilis grown in the presence or absence of D-, L- and DL- threonine had a similar amino acid composition which did not include t hreonine. A more complex biochemical explanation of the enhancement of electroporation by growth in DL-threonine is likely, and this is disc ussed. Lysozyme treatments to weaken the cell wall and possibly mimic the effect of DL-threonine did not yield any transformants. The effect s of buffer composition and culture incubation time were also determin ed and the electroporation protocol optimized accordingly. The respons e of a range of other B. subtilis strains to electroporation by the me thod produced was found to be variable. In all cases, transformation w as verified by recovery of the plasmid DNA from putative transformants .