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It was previously demonstrated that the activity of bacteriophage λ promoter pR is decreased in wild-type Escherichia coli cells starved for amino acids (during the stringent response). Since pR activity is necessary for the transcriptional activation of oriλ, this leads to inhibition of the replication of plasmids derived from phage λ. These results led to the proposal that the pR promoter is susceptible to control by the stringent response. However, subsequent studies demonstrated that this promoter is activated by the host dnaA gene product and since the dnaA promoter was reported to be controlled by the stringent response, it is possible that the inhibition of pR activity in amino acid-starved cells is indirect, and results from the impairment of DnaA-mediated transcriptional activation. Here we present evidence that pR is negatively regulated by ppGpp, even when DnaA protein is provided in excess as well as in cells devoid of DnaA function. We have checked that the level of ppGpp is increased during prolonged (up to 4 h) starvation for isoleucine in relA+ cells but not in the relA− mutant. At the same time we observed inhibition of λ plasmid replication during the stringent, but not relaxed, response, even when DnaA was overproduced. Finally, we found that the activity of a pR- lacZ fusion is inhibited after gratuitously induced overproduction of ppGpp in unstarved cells, irrespective of the status of the dnaA gene product. We conclude that the activity of the pR promoter is inhibited directly by ppGpp. [ABSTRACT FROM AUTHOR]