Phosphatidylcholine-Specific Phospholipase C and Sphingomyelinase Activities in Bacteria of theBacillus cereusGroup

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Abstract
Bacillus anthracisis nonhemolytic, even though it is closely related to the highly hemolyticBacillus cereus.Hemolysis byB. cereusresults largely from the action of phosphatidylcholine-specific phospholipase C (PC-PLC) and sphingomyelinase (SPH), encoded by theplcandsphgenes, respectively. InB. cereus, these genes are organized in an operon regulated by the global regulator PlcR.B. anthraciscontains a highly similar cereolysin operon, but it is transcriptionally silent because theB. anthracisPlcR is truncated at the C terminus. Here we report the cloning, expression, purification, and enzymatic characterization of PC-PLC and SPH fromB. cereusandB. anthracis.We also investigated the effects of expressing PlcR on the expression ofplcandsph. InB. cereus, PlcR was found to be a positive regulator ofplcbut a negative regulator ofsph. Replacement of theB. cereus plcRgene by its truncated orthologue fromB. anthraciseliminated the activities of both PC-PLC and SPH, whereas introduction intoB. anthracisof theB. cereus plcRgene with its own promoter did not activate cereolysin expression. Hemolytic activity was detected inB. anthracisstrains containing theB. cereus plcRgene on a multicopy plasmid under control of the strongB. anthracisprotective antigen gene promoter or in a strain carrying a multicopy plasmid containing the entireB. cereus plc-sphoperon. Slight hemolysis and PC-PLC activation were found when PlcR-producingB. anthracisstrains were grown under anaerobic-plus-CO2or especially under aerobic-plus-CO2conditions. Unmodified parentalB. anthracisstrains did not demonstrate obvious hemolysis under the same conditions.