The Staphylococcus aureus cidAB Operon: Evaluation of Its Role in Regulation of Murein Hydrolase Activity and Penicillin Tolerance

Bacterial murein hydrolases, or autolysins, comprise a broad and diverse family of enzymes that specifically cleave structural components of the bacterial cell wall. These enzymes have been shown previously to participate in a number of important biological functions during cell growth and division, including daughter cell separation, cell wall growth, and peptidoglycan recycling and turnover (14, 15, 17, 23, 38, 42, 55). Murein hydrolases also appear to play an important role in several bacterial developmental processes, such as spore formation, swarming motility, and competence (10, 24, 29, 40). Because of their potentially lethal capacity to hydrolyze the cell wall, it is critical that tight control be exercised over the expression and activity of murein hydrolases. For example, compartment-specific and temporal expression of the complement of murein hydrolases involved during Bacillus subtilis sporulation is dependent on several sporulation-specific sigma factors (11, 30, 32, 51), as well as the late-growth regulator Sin (30, 47). At the posttranscriptional level, murein hydrolase activity has been shown elsewhere to be modulated by several mechanisms, such as substrate modification, selective transport, interactions with lipoteichoic acids and cationic peptides, and cleavage by proteolytic enzymes (7, 13, 17, 23, 34, 36, 49, 54). Recent evidence has suggested that the proton motive force (PMF) and its effect on the cell wall pH also regulate murein hydrolase activity in B. subtilis (4, 5). In the case of Staphylococcus aureus, Mani et al. (31) have provided initial evidence for a murein hydrolase regulatory locus. Two Tn917-lacZ mutants were identified that displayed negligible rates of autolysis and exhibited normal cell division (31). Furthermore, our group had previously identified a second regulatory locus, designated lytSR, whose mutation resulted in a significantly increased rate of autolysis (2). The lytSR gene products are members of the bacterial two-component regulatory family of proteins and positively regulate expression of the lrgAB operon that is located immediately downstream of lytSR (3). Recent studies in our laboratory have demonstrated that lrgAB expression inhibits extracellular murein hydrolase activity and promotes penicillin tolerance (19). Interestingly, the lrgA gene product shares many structural similarities to the bacteriophage holin family of proteins (19). These small membrane proteins control the timing of bacteriophage-induced lysis by regulating access of the bacteriophage-encoded murein hydrolase, or endolysin, to the cell wall peptidoglycan (53). The activity of a holin is usually inhibited by the presence of a homologous protein, called the antiholin (53). Since the lrgAB locus inhibits murein hydrolase activity, it was hypothesized that LrgA functions in a manner analogous to an antiholin that, accordingly, functions by inhibiting an unidentified holin (19). In the present study, we have identified an operon homologous to lrgAB, designated cidAB, within the S. aureus genome. Like LrgA, the cidA gene product shared many holin-like characteristics. Analysis of a cidA mutant indicated that this gene enhances extracellular murein hydrolase activity and promotes sensitivity to penicillin. Therefore, we propose that the proteins encoded by the cidAB locus may indeed function in a holin-like manner that is inhibited by the products of the lrgAB operon.