Effect of metabolic inhibitors on entry of exogenous deoxyribonucleic acid into Ca2+-treated Escherichia coli cells.

The effect of various metabolic inhibitors (carbonylcyanid-m-chlorophenylhydrazone, nigericin, valinomycin, dicyclocarbodiimide, arsenate, NaF, etc.) and lipid-soluble synthetic ions (tetraphenylphosphonium bromide and tetraphenylboron sodium) on deoxyribonucleic acid (DNA) entry during transformation of Ca2+-treated Escherichia coli cells with plasmid DNA and on cell viability was investigated. In contrast to intact cells, Ca2+-treated E. coli cells were permeable to nigericin, valinomycin, and the other drugs tested. The inhibitors differentially affected [14C]proline active transport, and whereas some drugs inhibited transformation, the effects did not correlate with the effects on transport. The most potent inhibitors of transformation were nigericin, dicyclocarbodiimide, and tetraphenylboron sodium. Carbonylcyanid-m-chlorophenylhydrazone, tetraphenylphosphonium bromide, and valinomycin were relatively inactive. Tetraphenylboron sodium- and nigericin-treated cells bound were plasmid [14C]DNA in the deoxyribonuclease-resistant form than the control and other sample cells. Nevertheless, te penetration of exogenous plasmid DNA into the cell was greatly reduced, at least in case of nigericin. Unlike the other drugs, nigericin and dicyclocarbodiimide drastically affected the cell viability, the former within very short times of interaction. It is concluded that proton motive force does not play any significant role in DNA entry into Ca2+-treated E. coli cells. The results also suggest that adenosine 5'-triphosphate is not required for DNA entry either. The inhibitory effect of certain drugs is discussed in terms of structural perturbations induced by the drugs in cell envelope membranes.

Proton conductor vs. cold in induction of Ca(2+)-dependent competence in Escherichia coli.

In order to elucidate the molecular mechanisms of Ca(2+)-dependent competence in gram-negative bacteria an attempt was made to induce the competence at room temperature in presence of a proton conductor, carbonylcyanide-m-chlorophenylhydrazone (CCCP). Escherichia coli K12 cells treated with Ca2+ at 25 degrees or 37 degrees C in presence of CCCP became permeable for transforming plasmid and transfecting DNAs and DNA-binding antibiotic actinomycin C (AmC) and rubomycin (Rm) at room temperature. The efficiencies of transformation and transfection, however, were by 1-3 orders of magnitude lower compared to cells, treated with Ca2+ at 0 degree C, though both recipients did not differ significantly in their susceptibility to AmC and Rm. Possible mechanisms of Ca2+ action in both recipient systems are discussed in terms of molecular interactions.