The Staphylococcus aureus lrgAB Operon Modulates Murein Hydrolase Activity and Penicillin Tolerance

Abstract

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.

Analysis of Genetic Elements ControllingStaphylococcus aureus lrgABExpression: Potential Role of DNA Topology in SarA Regulation

“…In previous studies, it was shown that a lytS mutation resulted in altered murein hydrolase activity, increased Triton X-100-and penicillin-induced lysis, and undetectable lrgAB transcription (3,4,11). Given that the lrgAB operon is located immediately downstream of the lytSR locus (Fig.…”

“…To test this hypothesis, Northern blot analyses were used to compare lrgAB mRNA levels in agr and sar mutants and the parental wild-type strain. Using a lrgAB-specific probe, the level of lrgAB transcription was monitored in late-exponential-phase cells, when the lrgAB operon is maximally expressed (11). As shown in Fig.…”

“…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 hydrolase activity and increase tolerance to penicillin (11).…”

“…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 hydrolase activity and increase tolerance to penicillin (11). Based on these data, it was proposed that the function of LrgA, and possibly LrgB, is analogous to that of an antiholin, i.e., blocking murein hydrolase access to the substrate peptidoglycan (1).…”

“…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.…”