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Infection and Immunity, November 2003, p. 6591-6606, Vol. 71, No. 11
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.11.6591-6606.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Phosphatidylcholine-Specific Phospholipase C and Sphingomyelinase Activities in Bacteria of the Bacillus cereus Group

A. P. Pomerantsev, K. V. Kalnin,{dagger} M. Osorio,{ddagger} and S. H. Leppla*

National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-4350

Received 10 April 2003/ Returned for modification 13 June 2003/ Accepted 10 August 2003

Bacillus anthracis is nonhemolytic, even though it is closely related to the highly hemolytic Bacillus cereus. Hemolysis by B. cereus results largely from the action of phosphatidylcholine-specific phospholipase C (PC-PLC) and sphingomyelinase (SPH), encoded by the plc and sph genes, respectively. In B. cereus, these genes are organized in an operon regulated by the global regulator PlcR. B. anthracis contains a highly similar cereolysin operon, but it is transcriptionally silent because the B. anthracis PlcR is truncated at the C terminus. Here we report the cloning, expression, purification, and enzymatic characterization of PC-PLC and SPH from B. cereus and B. anthracis. We also investigated the effects of expressing PlcR on the expression of plc and sph. In B. cereus, PlcR was found to be a positive regulator of plc but a negative regulator of sph. Replacement of the B. cereus plcR gene by its truncated orthologue from B. anthracis eliminated the activities of both PC-PLC and SPH, whereas introduction into B. anthracis of the B. cereus plcR gene with its own promoter did not activate cereolysin expression. Hemolytic activity was detected in B. anthracis strains containing the B. cereus plcR gene on a multicopy plasmid under control of the strong B. anthracis protective antigen gene promoter or in a strain carrying a multicopy plasmid containing the entire B. cereus plc-sph operon. Slight hemolysis and PC-PLC activation were found when PlcR-producing B. anthracis strains were grown under anaerobic-plus-CO2 or especially under aerobic-plus-CO2 conditions. Unmodified parental B. anthracis strains did not demonstrate obvious hemolysis under the same conditions.

* Corresponding author. Mailing address: National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-4350. Phone: (301) 594-2865. Fax: (301) 480-0326. E-mail: sleppla{at}niaid.nih.gov.

Editor: J. T. Barbieri

{dagger} Present address: Acambis, Cambridge, MA 02139.

{ddagger} Present address: Center for Biologics Research and Review, U.S. Food and Drug Administration, Bethesda, MD 20892.

Infection and Immunity, November 2003, p. 6591-6606, Vol. 71, No. 11
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.11.6591-6606.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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