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The Staphylococcus aureus cidABC and lrgAB operons have been shown to regulate murein hydrolase activity and affect antibiotic tolerance. The cid operon enhances murein hydrolase activity and antibiotic sensitivity, whereas the lrg operon inhibits these processes. Based on these findings and the structural similarities of the cidA and lrgA gene products to the bacteriophage holin family of proteins, we have proposed that the cid and lrg operons encode holin- and antiholin-like proteins, respectively, that function to control the murein hydrolase activity produced by the bacteria. Analysis of cid operon transcription revealed the presence of two transcripts, one spanning all three cid genes and whose expression is induced by growth in the presence of acetic acid and the other spanning cidB and cidC only that is produced in a sigma B-dependent manner. The cidABC operon lies immediately downstream from the cidR gene, encoding a potential LysR-type transcriptional regulator. In this study, we demonstrate that cidR is involved in the regulation of cidABC expression. Northern blot analyses revealed that the cidR gene product positively regulates cidABC expression by increasing transcription in the presence of acetic acid produced as a result of the metabolism of glucose. As expected for an operon that encodes a positive effector of murein hydrolase activity, the upregulation of cidABC expression resulted in increased murein hydrolase activity produced by these cells. Furthermore, it was demonstrated that antibiotic tolerance and stationary-phase survival of S. aureus are affected by the cidR gene. Taken together, these results demonstrate that the cidR gene product functions as a transcriptional activator of cidABC transcription in response to acetic acid accumulation in the growth medium.

The Staphylococcus aureus lrg and cid operons encode homologous proteins that regulate extracellular murein hydrolase activity and penicillin tolerance in a diametrically opposing manner. Although their specific regulatory functions remain unknown, it has been postulated that the functions of CidA and LrgA are analogous to those of bacteriophage holins and antiholins, respectively, and that these proteins serve as molecular control elements of bacterial programmed cell death. Although these studies demonstrated that cidBC transcription is abundant in [[sigma].sup.B]-proficient strains, cidABC transcription was only minimally expressed under standard growth conditions. In this study, we demonstrate that cidABC and lrgAB transcription in the clinical isolate UAMS-1 is induced by growth in the presence of 35 mM glucose and that this enhances murein hydrolase activity and decreases tolerance to vancomycin and rifampin. The effect of glucose on murein hydrolase activity was not observed in the cidA mutant, indicating that the induction of this activity was dependent on enhanced cidABC expression. Furthermore, we demonstrate that the effects of glucose on cidABC and lrgAB transcription are mediated by the generation of acetic acid produced by the metabolism of this and other carbon sources. These results shed new light on the control of the S. aureus cidABC and lrgAB genes and demonstrate that these operons, as well as murein hydrolase activity and antibiotic tolerance, are responsive to carbohydrate metabolism.

The Staphylococcus aureus lrg and cid loci are homologous operons that have been shown to regulate murein hydrolase activity and affect sensitivity to penicillin. Although the mode of action of these operons has not been demonstrated, a model based on the similarities of the lrgA and cidA gene products to the bacteriophage holin family of proteins has been proposed. In this study, the transcription organization and regulation of these operons were examined by Northern blot analyses. Unexpectedly, cidB and a gene located immediately downstream, designated cidC, were found to be cotranscribed on a 2.7-kb transcript. Maximal cidBC transcription occurred during early exponential growth, and high-level transcription of cidBC was dependent on the rsbU-mediated activation of the alternative sigma factor B ([[sigma].sup.B]). In contrast, lrgAB transcription in stationary phase was negatively regulated by [[sigma].sup.B]. Although cidABC transcription was not detected by Northern blot analysis, reverse transcriptase PCR revealed that these genes are also cotranscribed as a single RNA message in early exponential growth. Primer extension analysis revealed the presence of two cidBC transcription start sites, but no apparent [[sigma].sup.B]-dependent promoter consensus sequence was identified in these regions. The rsbU gene was also shown to have a positive impact on murein hydrolase activity but a negligible effect on sensitivity to penicillin-induced killing. These results suggest that the lrgAB and cidBC genes may be part of the S. aureus [[sigma].sup.B]-controlled stress regulon.

Results indicate that the virulence factor regulators Agr and Sar in combination with the LytSR two-component regulatory system positively influence lrgAB expression. Data further indicte that DNA topology has a role in the optimal expression of lrgAB.

Results show that lrgAB operon is involved in the penicillin reactivity of Staphylococcus aureus depending on the cell growth status. In the stationary growth phase, induction of penicillin results in the cell death which in turn is mediated by a novel regulator of murein hydrolase activity.

The staphylococcus aureus scdA gene's role in cell division and morphogenesis was investigated by analyzing a new scdA mutant strain, KB323. The mutant scdA strain was obtained through the insertion of the gene's internal fragment into the NCTC 8325-4. Results have shown that the scdA is essential to the peptidoglycan metabolism and cell development even though its transcription is unaffected by LytS and LytR.

The expression of the gene encoding a putative ABC transporter (abcA) and the PBP4 penicillin-binding protein (pbpD) was analyzed in Staphylococcus aureus via Northern blot analysis and promoter-fusion technology. Staphylococcus aureus strains with abcA mutations exhibited increased pbpD and abcA promoter activity that were subjected to positive regulatory control. Furthermore, the increased expression of pbpD was accompanied by higher levels of peptidoglycan cross-linking in cell walls of the abcA mutants.

A genetic strategy based on the polymerase chain reaction (PCR) technique was developed to isolate and identify potential sensor-like genes from Staphylococcus aureus. The DNA surrounding the PCR product origin contained two genes, termed lytS and lytR, which contained amino acid sequences similar to those of the members of two-component regulatory system family of proteins. Cells containing disruptions of lytS had surfaces that were rough and diffused. A large portion of the cells had also undergone lysis, exhibited increased autolysis and an altered level of murein hydrolase activity.

A deletion study of damage-inducible A (dinA) and recA promoter sites was used to examine the assumption that the new consensus sequence (GAAC-N4-GTTC) acts as an operator region necessary for regulation of the SOS system of B. subtilis. The sequence is located in the promoter sites of DNA din genes of Bacillus subtilis. The competence-inducible expression of the recA gene is totally eliminated by mutations inside the comA, srfA and degU loci.

Experiments were performed to examine and characterize the virulence factor regulatory genes agr and sar, and their regulation of autolysis in Staphylococcus aureus. Mutant strains of the agr and sar genes were analyzed under a Triton X-100 and penicillin-induced autolysis conditions. Zymographic analyses of the mutant strains showed that agr and sar influenced autolysis through the modulation of murein hydrolase expression and activity.

Cloning and sequence analysis of Staphylococcus aureus strains have identified a pckA gene encoding phosphoenolpyruvate kinase (PEPCK) which is an essential enzyme for gluconeogenesis. Transcription analysis of a DNA fragment containing a pckA promoter region has shown that pckA promoter activity and enzyme activity are not closely tied to the presence of glucose. Mutants in which pckA has been deleted have shown at least a 22-fold increase in enzyme and promoter activity when grown in the absence of glucose.

The established, simple mechanisms considered to be involved in controlling bacterial death and lysis are discussed, highlighting research findings on the real biological functions of the processes. Bacterial cell death and lysis are shown to be complex, highly regulated processes controlled by the action of molecular components in several enzymes and acids.

Research has been conducted on Staphylococcus aureus lrgAB operon which inhibits murein hydrolase activity and decreases sensitivity to penicillin-induced killing. The authors suggest that cidAB operon encodes a holin-like counterpart of lrgAB and increases murein hydrolase activity and sensitivity to penicillin-induced killing.

The authors review the results of the Wind River Conference on prokaryotic biology- 2002. The topics of interest include physiology of a diverse series of microorganisms.