title: Cloning, expression, and mutagenesis of phosphatidylinositol-specific phospholipase C from Staphylococcus aureus: a potential staphylococcal virulence factor.
abstract: Staphylococcus aureus secretes a phosphatidylinositol (PI)-specific phospholipase C (PI-PLC) which is able to hydrolyze the membrane lipid PI and membrane protein anchors containing glycosyl-PI. The gene for PI-PLC (plc) was cloned from S. aureus into Escherichia coli. Oligonucleotide probes based on partial protein sequence and polyclonal antibodies raised against the purified protein were used to identify positive clones. E. coli transformed with a plasmid containing the plc gene expressed 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 staphylococcal species. The plc gene contained 984 bp and coded for a mature protein with a calculated molecular mass of 34,107 Da. Amino acid sequence comparisons indicated that the staphylococcal plc gene was similar (51 to 56%) to the PI-PLCs from Bacillus cereus, Bacillus thuringiensis, 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 decreased 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 was regulated by both agr-dependent and agr-independent mechanisms.
authors.affiliation: Department of Physiology and Cellular Biophysics, College of Physicians and Surgeons of Columbia University, New York, New York 10032.
authors.forename: S
authors.initials: S
authors.lastname: Daugherty
authors.forename: M G
authors.initials: MG
authors.lastname: Low
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016296:Mutagenesis; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics* / Q000472:pathogenicity; D013379:Substrate Specificity; D014774:Virulence / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase