sar Genetic determinants necessary for transcription of RNAII and RNAIII in the agr locus of Staphylococcus aureus

Abstract

The temporal expression of most virulence factors in Staphylococcus aureus is regulated by pleiotropic loci such as agr and sar. We have previously shown that the sar locus affects hemolysin production because it is required for agr transcription. To delineate the sar genetic determinant required for agr transcription, single copies of fragments from the sar locus, encompassing the individual sar transcripts (sarA, sarC, and sarB), were introduced into a sar mutant via the integration vector pCL84. Although a DNA fragment encompassing the sarA transcript plus a 189-bp upstream region was sufficient for agr expression, complementation analysis revealed that the sarB transcript was the most effective in augmenting agr transcription as determined by RNAII and RNAIII transcription and gel retardation assays with the P2 and P3 promoters of agr. As the region upstream of the sarA transcript encodes a 39-amino-acid open reading frame, ORF3, it is possible that posttranslational cooperation between the sarA gene product and ORF3 may be necessary for optimal agr expression. Deletion studies demonstrated that an intact sarA gene is essential for agr transcription. However, mutagenesis and in vitro translation studies revealed that unlike the agr locus, the required element is the SarA protein and not the RNA molecule. Taken together, these results indicate that the sarA-encoded protein, possibly in conjunction with peptides encoded in the upstream region, regulates hemolysin production by controlling agr P2 and P3 transcription.The control of extracellular protein production in bacteria is dependent upon global regulatory systems in which a single regulatory locus controls the expression of several unlinked target genes, often in a temporal manner. Regulation of virulence determinants in Staphylococcus aureus has been shown to be under the control of at least two genetic loci, agr and sar (6, 15). The agr locus encodes two divergent transcripts (RNAII and RNAIII). The promoter P2 initiates the formation of a 3-kb transcript designated RNAII that encodes four genes (agrA through agrD), while the promoter P3 initiates the formation of a 500-bp transcript called RNAIII that codes for the ␦-hemolysin (15). The agr operon mediates control of extracellular and surface proteins via the transcript RNAIII (26). However, in-frame deletions of any of the four genes in the RNAII coding region resulted in diminished transcription of RNAIII, thus indicating that RNAIII transcription is likely dependent on the activation of RNAII (23). We recently reported that the transcription of RNAII and RNAIII was partially dependent on the sar locus, which is encoded within a 1.2-kb DNA fragment that encompasses three overlapping transcripts (1, 13) (Fig. 1). These transcripts, designated sarA (0.58 kb), sarC (0.84 kb), and sarB (1.15 kb), have common 3Ј ends but three distinct promoters. A major 339-bp open reading frame (ORF), sarA, together with widely spaced upstream promoter sequences, is present in this locus (7, 13).Phenotypic analysis reve...

Regulation of Staphylococcus aureus Pathogenesis via Target of RNAIII-activating Protein (TRAP)

“…5D, RNAIII synthesis was greatly reduced in the trap -mutant strain but was not abolished. These results suggest that trap is important for the activation of RNAIII synthesis but that the synthesis of RNAIII can nevertheless be activated at a later stage in the absence of TRAP, possibly by alternate pathways, such as sar (28,29). As also shown in Fig.…”

SarA, a Global Regulator of Virulence Determinants inStaphylococcus aureus, Binds to a Conserved Motif Essential for sar-dependent Gene Regulation

“…We previously showed that the SarA protein binds to the agr promoter region, presumably stimulating transcription, in particular, from the agr P2 promoter (11). Activation of RNAII and, subsequently, RNAIII would lead to alterations in target gene expression (e.g.…”

“…These transcripts, all encoding the major 372-bp sarA open reading frame, have common 3Ј ends but originate from three distinct promoters (10). Transcription and gel shift studies (11,12) revealed that the SarA protein preferentially binds to the P2-, and to a lesser extent, to the P3-agr promoter region, thereby augmenting RNAII and the ensuing RNAIII transcription. RNAIII would then modulate the transcription of sar target genes (e.g.…”

Transmembrane Topology of AgrB, the Protein Involved in the Post-translational Modification of AgrD in Staphylococcus aureus

“…AgrA resembles a response regulator, which is required for the activation of both agr promoters P2 and P3 (2,7,15), although it is not clear whether AgrA binds to these two promoters directly (16,17). It is possible that either phosphorylated AgrA would bind to the agr promoters or AgrA would interact with another global regulator, SarA, to control the agr expression (17)(18)(19).…”