CLONING, EXPRESSION, AND MUTAGENESIS OF PHOSPHATIDYLINOSITOL-SPECIFICPHOSPHOLIPASE-C FROM STAPHYLOCOCCUS-AUREUS - A POTENTIAL STAPHYLOCOCCAL VIRULENCE FACTOR

Staphylococcus aureus secretes a phosphatidylinositol (PI)-specific ph ospholipase C (PI-PLC) which is able to hydrolyze the membrane lipid P I and membrane protein anchors containing glycosyl-PI. The gene for PI -PLC (plc) was cloned from S. aureus into Escherichia coli. Oligonucle otide probes based on partial protein sequence and polyclonal antibodi es raised against the purified protein were used to identify positive clones. E. coli transformed with a plasmid containing the plc gene exp ressed PI-PLC enzyme activity which was abolished by mutagenesis with a tetracycline resistance gene. The plc gene was present in all 15 S. aureus strains examined but not in any of 6 coagulase-negative staphyl ococcal species. The plc gene contained 984 bp and coded for a mature protein with a calculated molecular mass of 34,107 Da. Amino acid sequ ence comparisons indicated that the staphylococcal plc gene was simila r (51 to 56%) to the PI-PLCs from Bacillus cereus, Bacillus thuringien sis, and Listeria monocytogenes. The recombinant PI-PLC expressed in E . coli was purified and exhibited biochemical properties identical to those of the native PI-PLC from S. aureus. PI-PLC production was decre ased in agr mutant strains of S. aureus. However, PI-PLC production by both agr(+) and agr mutant strains exhibited a similar dependence on the type of medium used. These data suggested that PI-PLC production w as regulated by both agr-dependent and agr-independent mechanisms.