CLONING, CHARACTERIZATION AND DISRUPTION OF A (P)PPGPP SYNTHETASE GENE (RELA) OF STREPTOMYCES-COELICOLOR A3(2)

An internal segment of the (p)ppGpp synthetase gene, relA, of Streptom yces coelicolor A3(2) was amplified from genomic DNA using the polymer ase chain reaction and used as a hybridization probe to isolate the co mplete gene from a cosmid library. relA lies downstream of a gene (apt ) that apparently encodes adenine phosphoribosyltransferase and is tra nscribed from two promoters, relAp1 and relAp2, and by transcriptional readthrough from apt. While the level of relAp2 transcripts remained relatively constant, relAp1 activity apparently peaked during transiti on phase, following a decline in readthrough transcription from apt Di sruption of relA using an att(-) derivative of the temperate phage phi C31 abolished ppGpp synthesis on amino acid depletion. When grown on agar, the disruptants grew more slowly than a control lysogen made wit h an att(+) phage vector and gave smaller colonies that sporulated nor mally. The relA mutation had no consistent or marked effect on actinor hodin production in either liquid- or agar-grown cultures, indicating that elevated levels of (p)ppGpp are not essential for triggering the onset of antibiotic production.