Previous studies in our laboratory have shown that the Staphylococcus aureus LytSR two-component regulatory system affects murein hydrolase activity and autolysis. A LytSR-regulated dicistronic operon has also been identified and shown to encode two potential membrane-associated proteins, designated LrgA and LrgB, hypothesized to be involved in the control of murein hydrolase activity. In the present study, a lrgAB mutant strain was generated and analyzed to test this hypothesis. Zymographic and quantitative analysis of murein hydrolase activity revealed that the lrgAB mutant produced increased extracellular murein hydrolase activity compared to that of the wild-type strain. Complementation of the lrgAB defect by providing the lrgAB genes in trans restored the wild-type phenotype, indicating that these genes confer negative control on extracellular murein hydrolase activity. In addition to these effects, the influence of the lrgAB mutation on penicillin-induced lysis and killing was examined. These studies demonstrated that the lrgAB mutation enhanced penicillininduced killing of cells approaching the stationary phase of growth, the time at which the lrgAB operon was shown to be maximally expressed. This effect of the lrgAB mutation on penicillin-induced killing was shown to be independent of cell lysis. In contrast, the lrgAB mutation did not affect penicillin-induced killing of cells growing in early-exponential phase, a time in which lrgAB expression was shown to be minimal. However, expression of the lrgAB operon in early-exponential-phase cells inhibited penicillin-induced killing, again independent of cell lysis. The data generated by this study suggest that penicillin-induced killing of S. aureus involves a novel regulator of murein hydrolase activity.

SummaryStaphylococcus aureus produces a wide array of virulence factors and causes a correspondingly diverse array of infections. Production of these virulence factors is under the control of a complex network of global regulatory elements, one of which is sarA. sarA encodes a DNA binding protein that is considered to function as a transcription factor capable of acting as either a repressor or an activator. Using competitive ELISA assays, we demonstrate that SarA is present at approximately 50 000 copies per cell, which is not characteristic of classical transcription factors. We also demonstrate that SarA is present at all stages of growth in vitro and is capable of binding DNA with high affinity but that its binding affinity and pattern of shifted complexes in electrophoretic mobility shift assays is responsive to the redox state. We also show that SarA binds to the bacteriophage lambda (l) attachment site, attL, producing SarA-DNA complexes similar to intasomes, which consist of bacteriophage lambda integrase, Escherichia coli integration host factor and attL DNA. In addition, SarA stimulates intramolecular excision recombination in the absence of l excisionase, a DNA binding accessory protein.Taken together, these data suggest that SarA may function as an architectural accessory protein.

Penicillin-induced killing and murein hydrolase activity in Staphylococcus aureus are dependent on a variety of regulatory elements, including the LytSR two-component regulatory system and the virulence factor regulators Agr and Sar. The LytSR effects on these processes can be explained, in part, by the recent finding that a LytSR-regulated operon, designated lrgAB, affects murein hydrolase activity and penicillin tolerance. To examine the regulation of lrgAB expression in greater detail, we performed Northern blot and promoter fusion analyses. Both methods revealed that Agr and Sar, like LytSR, positively regulate lrgAB expression. A mutation in the agr locus reduced lrgAB expression approximately sixfold, while the sar mutation reduced lrgAB expression to undetectable levels. cis-acting regulatory elements involved in lrgAB expression were identified by fusing various fragments of the lrgAB promoter region to the xylE reporter gene and integrating these constructs into the chromosome. Catechol 2,3-dioxygenase assays identified DNA sequences, including an inverted repeat and intrinsic bend sites, that contribute to maximal lrgAB expression. Confirmation of the importance of the inverted repeat was achieved by demonstrating that multiple copies of the inverted repeat reduced lrgAB promoter activity, presumably by titrating out a positive regulatory factor. The results of this study demonstrate that lrgAB expression responds to a variety of positive regulatory factors and suggest that specific DNA topology requirements are important for optimal expression.Peptidoglycan or murein hydrolases are a family of enzymes that catalyze the cleavage of specific structural components of the bacterial cell wall. These enzymes are involved in several important physiological processes, including peptidoglycan turnover and recycling, cell wall expansion during bacterial growth, septation, and daughter cell separation (13, 32). Due to the potential of these enzymes to compromise cell wall integrity, leading to cell lysis (autolysis), murein hydrolase activity must be carefully regulated. This regulation includes transcriptional control, blocked access to the specific substrate, inhibition by choline or teichoic acid, and substrate modification (13, 32).The Staphylococcus aureus lytS and lytR genes, whose products are members of the two-component regulator family of proteins, are involved in the control of murein hydrolase activity (3). A lytS mutant phenotypically displays altered murein hydrolase activity and an increased rate of penicillin-and Triton X-100-induced lysis (3, 11). Immediately downstream of lytS and lytR are two genes, lrgA and lrgB, whose transcription is dependent upon lytS and lytR. LrgA and LrgB show no sequence similarity to known murein hydrolases. Instead, LrgA has structural characteristics in common with the bacteriophage-encoded holin proteins involved in murein hydrolase export (4, 11). Recent data generated in our laboratory indicate that the LrgA and LrgB gene products inhibit extracellular murein hy...

The regulation of murein hydrolases is a critical aspect of peptidoglycan growth and metabolism. In the present study, we demonstrate that mutations within the Staphylococcus aureusvirulence factor regulatory genes, agr and sar, affect autolysis, resulting in decreased and increased autolysis rates, respectively. Zymographic analyses of these mutant strains suggest thatagr and sar exert their effects on autolysis, in part, by modulating murein hydrolase expression and/or activity.

SUMMARYTopoisomerases relieve topological tension in DNA by breaking and rejoining DNA phosphodiester bonds. Type IB topoisomerases such as vaccinia topoisomerase (vTopo) and human topoisomerase I are structurally and mechanistically similar to the tyrosine recombinase family of enzymes including bacteriophage lambda Integrase (Int). Previously, our laboratory identified peptide inhibitors of Int from a synthetic peptide combinatorial library. The most potent of these peptides also inhibit vTopo. Here we used the same mixture-based screening procedure to identify peptide inhibitors directly against vTopo using a plasmid relaxation assay. The two most potent new peptides identified, WYCRCK and KCCRCK, inhibit plasmid relaxation, DNA cleavage and Holliday junction (HJ) resolution mediated by vTopo. The peptides tested bind double stranded DNA at high concentration but do not appear to displace the enzyme from its DNA substrate. WYCRCK binds specifically to HJ and perturbs their central base pairing. This peptide also accumulates HJ intermediates when it inhibits Int-mediated recombination, whereas KCCRCK does not. Interestingly, WYCRCK shares four amino acids with a peptide identified against Int, WRWYCR. The octapeptide WRWYCRCK, containing amino acids from both hexapeptides, is more potent than either against vTopo. All peptides are less potent against the type IA E. coli topoisomerase I or against restriction endonucleases. Like the Int-inhibitory peptide WRWYCR, WYCRCK binds to Holliday junctions, and both inhibit junction resolution by vTopo. Our results suggest that the newly identified WYCRCK and peptide WRWYCR interact with a distorted DNA intermediate arising during vTopo-mediated catalysis, or interfere with specific interactions between vTopo and DNA.

No abstract