The VraS/VraR two-component system (VraSR) regulates transcriptional induction of penicillin-binding protein 2 (encoded by pbp2) by vancomycin in Staphylococcus aureus. We have now defined the vraSR operon and determined that its induction by ␤-lactams as well as by vancomycin is autoregulated. Induction of the pbp2 and vraSR operons by ␤-lactams and related compounds within 1 hour after exposure to the antimicrobials was dependent on vraS. However, when a disk diffusion assay that can detect induction of genes over an extended time period was used, induction of the pbp2 operon was mediated by some ␤-lactams, including oxacillin; this induction was independent of vraS.

Methicillin/oxacillin (Oxa) resistance in Staphylococcus aureus is primarily mediated by the acquired penicillin-binding protein (PBP2a) encoded by mecA. PBP2a acts together with native PBP2 to mediate oxacillin resistance by contributing complementary transpeptidase and transglycosylase activities, respectively. The VraS/VraR two-component regulatory system is inducible by cell-wall antimicrobials (beta-lactams, glycopeptides) and controls transcriptional induction of many cell-wall genes including pbp2 and itself. We investigated the role of VraS/VraR in the phenotypic expression of oxacillin resistance by inactivating vraS in community-acquired MRSA clinical isolates that lack functional genes encoding the mecA regulatory sequences mecI and mecR1. Inactivation of vraS abrogated oxacillin resistance, and complementation with the vraS operon restored the resistance phenotype. mecA transcription increased in the vraS mutants; however, PBP2a abundance was similar to that of the wild type. Although pbp2 transcription decreased in the vraS mutants, overexpression of the pbp2 operon did not restore resistance. These data demonstrate that although expressions of mecA and pbp2 are required for oxacillin resistance, they are not sufficient. Therefore, the vraS/vraR regulatory system plays a crucial role in allowing MRSA to respond to beta-lactams by regulation of a gene target other than the known effectors of methicillin resistance.

The promoter for a tobacco (Nicofiana tabacum) sesquiterpene cyclase gene, a key regulatory step in sesquiterpene phytoalexin biosynthesis, has been analyzed. The EAS4 promoter was fused to the P-glucuronidase (CUS) reporter gene, and the temporal and spatial expression patterns of C U S activity were examined in stably transformed plants and in transient expression assays using electroporated protoplasts of tobacco. N o C U S activity was observed i n any tissues under normal growth conditions. A low level of C U S activity was detected in wounded leaf, root, and stem tissues, whereas a much higher level was observed when these tissues were challenged with elicitors or microbial pathogens. l h e CUS expression pattern directed by the EAS4 promoter was identical to the induction patterns observed for the endogenous sesquiterpene cyclase genes. Neither exogenous salicylic acid nor methyl iasmonate induced C U S expression; and H,O, induced GUS expression to only a limited extent. Although the EAS4 promoter contains cissequences resembling previously identified transcriptional control motifs, other cis-sequences important for quantitative and qualitative gene expression were identified by deletion and gain-offunction analyses. The EAS4 promoter differs from previously described pathogen-/elicitor-inducible promoters because it only supports inducible gene expression and directs unique spatial expression patterns.

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