GUANOSINE TETRAPHOSPHATE (PPGPP)-MEDIATED INHIBITION OF THE ACTIVITY OF THE BACTERIOPHAGE-LAMBDA P(R) PROMOTER IN ESCHERICHIA-COLI

It was previously demonstrated that the activity of bacteriophage lamb da promoter p(R) is decreased in wild-type Escherichia coli cells star ved for amino acids (during the stringent response). Since p(R) activi ty is necessary for the transcriptional activation of ori lambda, this leads to inhibition of the replication of plasmids derived from phage lambda. These results led to the proposal that the p(R) promoter is s usceptible to control by the stringent response. However, subsequent s tudies demonstrated that this promoter is activated by the host dnaA g ene product and since the dnaA promoter was reported to be controlled by the stringent response, it is possible that the inhibition of p(R) activity in amino acid-starved cells is indirect, and results from the impairment of DnaA-mediated transcriptional activation. Here we prese nt evidence that p(R) is negatively regulated by ppGpp, even when DnaA protein is provided in excess as well as in cells devoid of DnaA func tion. We have checked that the level of ppGpp is increased during prol onged (up to 4 h) starvation for isoleucine in relA(+) cells but not i n the relA(-) mutant. At the same time we observed inhibition of lambd a plasmid replication during the stringent, but not relaxed, response, even when DnaA was overproduced. Finally, we found that the activity of a p(R)-lacZ fusion is inhibited after gratuitously induced overprod uction of ppGpp in unstarved cells, irrespective of the status of the dnaA gene product. We conclude that the activity of the p(R) promoter is inhibited directly by ppGpp.