We have previously identified, cloned, sequenced and preliminarily characterized a complex polycistronic regulatory element, agr, that controls the post-exponential phase synthesis of virulence factors and other exoproteins in Staphylococcus aureus. The 3.3 kb chromosomal agr locus consists of two divergent operons transcribed from promoters P2 and P3. The P2 operon contains four genes, agrA, B, C and D. AgrA and AgrC resemble the two components of the bacterial signal transduction systems and respond to a metabolic signal early in exponential phase by activating P2 and P3, making the system autocatalytic. The P3 operon encodes an agr- regulated exoprotein delta-hemolysin, and its 0.5 kb transcript, RNAIII, is the agr-generated regulator of other, unlinked genes. We have shown that in some strains agr is activated early in exponential phase but that the target genes do not respond until some 2 h later and that a second agr-independent post-exponential phase signal (PEPS) is also required. Protein synthesis-inhibitors such as erythromycin and chloramphenicol cause the immediate activation of target gene transcription, bypassing both agr and the PEPS, suggesting that labile regulatory proteins are involved. We have also observed that the 5' end of RNAIII is required for translation of alpha-hemolysin and that delta- hemolysin is not translated until one h after the appearance of RNAIII. On the basis of these and other findings we have developed a working model in which five distinct stages are discerned in the exoprotein regulatory pathway. A broad based continuation of these studies is proposed, focussing on the different stages in the activation pathway and the mechanisms underlying the activation steps at each stage. Particular attention will continue to be paid to the structure and mechanism of action of the remarkable RNA molecule, RNAIII, that is the intracellular effector of the exoprotein response. We have discovered an external activator and an external inhibitor of the response and studies are proposed to test these for possible therapeutic use in staphylococcal disease.
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