The role of the Bacillus anthracis LytSR two-component regulatory system in bacterial cell death and lysis

The Staphylococcus aureus cid and lrg operons have been shown to control cell death and lysis in a manner thought to be analogous to that mediated by bacteriophage holins and antiholins, respectively. It has been proposed that the cid and lrg gene products serve as molecular control elements of cell death and lysis during biofilm development. Interestingly, a total of four cid-/lrg -like operons have been identified in the genome of Bacillus anthracis, the causative agent of anthrax. Two of these operons appear to be similar to the S. aureus cid and lrg operons while the other two (designated clhAB-1 and clhAB-2) are unique to B. anthracis. Previous studies have shown that the B. anthracis cidR gene plays an important role in the regulation of cid and lrg operons and cell death in stationary phase. In this study, we have examined the phenotypic and regulatory effects of disrupting the lytSR homologue in B. anthracis. Similar to S. aureus, B. anthracis lrgAB transcription was shown to be induced by dissipation of proton motive force, in a lytSR-dependent manner. Interestingly, lytSR was also shown to regulate clhAB-2 expression in a proton motive force-independent manner suggesting that alternate signals may mediate this control. Zymographic analysis of murein hydrolase activity revealed that the B. anthracis LytSR two-component regulatory system plays a role in regulating murein hydrolase activity, predominantly affecting Sap, an S-layer protein previously shown to possess this activity. Furthermore, the B. anthracis lrgAB mutant displayed decreased stationary phase survival, whereas the clhAB-2 mutant exhibited increased stationary phase survival compared to the parental and complementation strains. A quantitative analysis of sporulation efficiency revealed both mutants formed fewer spores compared to the parental strain. Analyses of sporulation-specific gene transcription demonstrated that the lrgAB mutation affects the expression of spoIID, whose gene product is involved in the engulfment of forespore. Further studies will be necessary to establish the direct role of these operons in sporulation. Taken together, the results of this study suggest that B. anthracis LytSR is a major regulator of lrgAB and clhAB-2 transcription and plays an important role in the control of cell death.