High-level expression of the colicin A lysis protein

Abstract

Two plasmids that overproduce the colicin A lysis proteins, Cal, are described. Plasmid AT1 was constructed by a deletion in the colicin A operon, which placed the cal gene near a truncated caa gene in such a way that both gene products wer synthesized at high levels following induction. Plasmid CK4 was constructed by insertion of the cal gene downstream from the tac promoter of an expression vector. Overproduction of Cal was obtained after mitomycin C induction of pAT1 cells and after IPTG induction of pCK4 cells. The kinetics of Cal synthesis were examined with [35S] methionine and [2-3H] glycerol in lpp or lpp+ host strains. Each of the steps of the lipid modification and maturation pathway of Cal was demonstrated. The modified precursor form of overproduced Cal was not chased as efficiently as when it is produced in pColA cells. After treatment with globomycin, a significant amount of this modified precursor form accumulated and was degraded with time into smaller acylated proteins, but without release of the signal peptide. Release of cellular proteins and quasi-lysis were observed after about 1 hour of induction for cells containing either plasmid. In addition, in Cal-overproducing cells, the rate of quasi-lysis was increased but not its extent. In pldA cells, quasi-lysis was reduced but not abolished. Lethality of the Cal induction in the overproducing cells was in th same range as that in wild-type cells.