The production of most toxins and other exoproteins in Staphylococcus aureus is controlled globally by a complex polycistronic regulatory locus, agr. Secretory proteins are up‐regulated by agr whereas surface proteins are down‐regulated. agr contains two divergent promoters, one of which directs the synthesis of a 514 nucleotide (nt) transcript, RNAIII. In this report, we show that the cloned RNAIII determinant restores both positive and negative regulatory functions of agr to an agr‐null strain and that the RNA itself, rather than any protein, is the effector molecule. RNAIII acts primarily on the initiation of transcription and, secondarily in some cases, at the level of translation. In these cases, translation and transcription are regulated independently. RNAIII probably regulates translation directly by interacting with target gene transcripts and transcription indirectly by means of intermediary protein factors.

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SummaryTst, the gene for toxic shock syndrome toxin-1 (TSST-1), is part of a 15.2 kb genetic element in Staphylococcus aureus that is absent in TSST-1-negative strains. The prototype, in RN4282, is flanked by a 17 nucleotide direct repeat and contains genes for a second possible superantigen toxin, a Dichelobacter nodosus VapE homologue and a putative integrase. It is readily transferred to a recA ¹ recipient, and it always inserts into a unique chromosomal copy of the 17 nucleotide sequence in the same orientation. It is excised and circularized by staphylococcal phages 13 and 80␣ and replicates during the growth of the latter, which transduces it at very high frequency. Because of its site and orientation specificity and because it lacks other identifiable phage-like genes, we consider it to be a pathogenicity island (PI) rather than a transposon or a defective phage. The tst element in RN4282, near tyrB, is designated SaPI1. That in RN3984 in the trp region is only partially homologous to SaPI1 and is excised by phage 80 but not by 80␣. It is designated SaPI2. These PIs are the first in any Gram-positive species and the first for which mobility has been demonstrated. Their mobility may be responsible for the spread of TSST-1 production among S. aureus strains.

SummaryRNAIII is a 514 nt regulatory RNA that is the effector molecule of the staphylococcal agr quorum-sensing system, regulating a large set of virulence and other accessory genes at the level of transcription. RNAIII was discovered nearly 20 years ago and we long ago hypothesized that it would function by regulating the synthesis or activity of one or more intermediary transcription factors. We have finally confirmed this hypothesis, showing that Staphylococcus aureus RNAIII regulates the synthesis of a major pleiotropic transcription factor, Rot, by blocking its translation. RNAIII has a complex secondary structure with several stable hairpins that have highly C-rich end loops, unusual in an AT-rich organism. We noted that these loops are complementary to two G-rich stem loops of the rot mRNA translation initiation region (TIR). Pairing of the complementary RNAs would be predicted to occlude the rot Shine-Dalgarno (SD) site and to block rot translation. Through a combination of transcriptional and translational fusions and Northern and Western blot hybridization analyses, we show that RNAIII does, indeed, block rot translation. Through alterations in the C-rich loops of RNAIII and the G-rich loops of rot, we show that the sequences of these loops are critical for inhibition of rot translation and suggest that this inhibition is affected by pairing between the complementary stem loops, followed by the cleavage of rot mRNA. We propose that the RNAIII-rot mRNA interaction plays a key role in agr regulation of staphylococcal virulence.

Most small multicopy antibiotic-resistance plasmids of Staphylococcus aureus contain a major axis of hyphenated dyad symmetry (paL4) that is required for normal replication and stability, although located outside of the minimal replicon. Rearrangements affecting paL4 cause plasmid instability, a marked reduction in copy number, and the accumulation of large quantities of strand-specific circular single-stranded plasmid DNA. In view ofthe recent observation that pT181 initiates replication by a nick and 3'-extension mechanism (S. Khan, personal communication), it is suggested that these plasmids replicate by an asymmetric rolling-circle mechanism in which the displaced plus strand remains single stranded until paL4 is exposed, forming a hairpin that serves as the lagging strand origin.Inverted duplications of nucleotide sequences (palindromes) have been increasingly recognized to play important roles in a variety ofgenetic activities such as control ofgene function, initiation of replication, genetic transposition, transcription termination, and formation of deletions and inversions. Palindromes, which form hairpins when single-stranded, have been shown to function in the initiation and termination of replication of single-stranded DNA phages. In particular, in replicative form (RF)-to-RF rolling-circle replication, the displaced leading (plus) strand remains single-stranded until a palindromic element is exposed, forming a hairpin that serves as the lagging strand origin (1,2). In duplex DNA,hairpins have yet to be demonstrated in vivo, so that palindromic elements are usually regarded as symmetry elements rather than as physical secondary structures. Palindromic elements also exist in the replication origins of many double-stranded DNA replicons such as the lambdoid phages (3), various plasmids, etc. (4, 5). With plasmid ColEl and its relatives, palindromic elements in the origin region are involved in the formation of the RNA primer for replication (5); with R1, deletions affecting a large palindromic element near the origin cause instability and a decrease in copy number (6). We report here the identification of a major palindromic element, palA, widespread among plasmids from Gram-positive bacteria, outside of the minimal replicon but required for normal plasmid replication and stability. Analysis of this element suggests that these plasmids replicate by an asymmetric rolling-circle mechanism in whichpalA serves as the lagging strand initiation site.