Phosphatidylcholine-specific phospholipase C and sphingomyelinase activities in bacteria of the Bacillus cereus group

Abstract

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-CO(2) or especially under aerobic-plus-CO(2) conditions. Unmodified parental B. anthracis strains did not demonstrate obvious hemolysis under the same conditions.

FIG. 1.

Sequence alignment of the promoter regions for plc and sph genes of B. anthracis strain UM44-1C9 (B.a.) and B. cereus (B.c.) strains SE-1, BKM-B164, GP-4, and 569. Putative PlcR binding sites are located upstream of σ^A −35 and −10 sequences for the plc gene and downstream of the sph σ^A −35 and −10 sequences. Consensus sequences of regulatory elements are indicated in bold type under the corresponding aligned sequences. Gray areas indicate nucleotide sequence differences.

FIG. 2.

Molecular and functional properties of recombinant B. anthracis and B. cereus PC-PLC and SPH. (A) SDS-PAGE of the proteins purified from E. coli, stained with Coomassie blue R-250. M_r, molecular mass marker. (B and C) SPH and PC-PLC activities of the four recombinant phospholipases. The order of the samples is the same as in panel A. (D) Ability of PC-PLC, SPH, and a 1:1 mixture of these proteins to lyse sheep red blood cells. Each well was filled with 20 μl of a 0.5-mg/ml solution of enzyme. Wells with both phospholipases were filled with 20 μl of each enzyme.

FIG. 3.

Inactivation of B. cereus 569 plcR gene. (A) Scheme of plasmid pYJ335-anti-plcR integration into plcR gene of B. cereus 569 chromosome. The truncated antisense-oriented B. anthracis plcR gene (ΔplcR) was recombined with the B. cereus 569 chromosomal plcR gene so that the only intact plcR, at the right end, lacks a promoter. PCR primers used in analysis include P1 and P2 (external primers matching chromosomal DNA), M13f and M13r (internal primers that match only the pYJ335-antisense plasmid), and BAP1 and BAP2 (primers that belong to chromosomal and plasmid DNAs). (B) DNA analysis of recombinant strains. Panel 1, PCR fragments from the left end of the inserted plasmid; panel 2, plasmid content of B. cereus 569 derivatives having integrated (B. cereus 541) and extrachromosomal (B. cereus 540) pYJ335-anti-plcR plasmids. The arrow (panel 2) indicates a band of plasmid pYJ335-anti-plcR. Other bands are endogenous B. cereus 569 plasmids. Panel 3, PCR fragments from the right end of the inserted plasmid. M_r, molecular mass marker. (C) Hemolysis of B. cereus 541 is weaker than that of B. cereus 569 (panel I). B. cereus 541 does not hydrolyze lecithin (panel II).

FIG. 4.

Genetic and restriction map of plasmid pAE5. The plcR gene of B. cereus 569 is under control of the B. anthracis protective antigen gene promoter. The signal peptide of the protective antigen gene was eliminated in order to retain PlcR inside the cell. The location of the inserted IS10 found in pAE5::IS10 is indicated by the arrow. H-T-H, helix-turn-helix motif of PlcR.

FIG. 5.

PlcR antiserum characterization. (A) B. cereus 569 PlcR amino acid sequence. The N-PlcR peptide immunogen corresponds to aa 93 to 107, and the C-PlcR peptide corresponds to aa 246 to 260. The vertical arrow indicates the site at which the B. anthracis PlcR protein is truncated. (B) ELISA reactivity of N-PlcR and C-PlcR antiserum at 1:3,000 dilutions on plates coated with either the N-PlcR peptide (left graph) or the C-PlcR peptide (right graph). (C) Western blot of whole-cell proteins from B. anthracis SdT (lane 1) and B. anthracis SdT2 (lane 2). Membranes were treated with N-PlcR antiserum (serum 1447) or C-PlcR antiserum (serum 1451) at a 1:2,000 dilution. M_r, position of external molecular mass marker.

FIG. 6.

Growth, PlcR production, and enzyme activities for B. cereus and B. anthracis strains. (A) Growth curves for strains incubated at 37°C in BHI broth. OD, optical density. (B) Western blot analyses of whole-cell proteins from B. cereus 569 (left panel) and B. anthracis strain SdT2 (right panel) with C-PlcR antiserum 1451 as described above. No PlcR production was found for B. cereus 541 or B. anthracis SdT (not shown). (C and D) PC-PLC (C) and SPH (D) activities of extracellular proteins. Equal amounts of extracellular protein (2 to 10 μg for PC-PLC and 0.1 to 0.5 μg for SPH) were assayed by the Red Amplex reagents. Maximum root-mean-square deviations did not exceed 10% for the PC-PLC and SPH determinations. Filled square, B. cereus 541; filled diamond, B. cereus 569.

FIG. 7.

Hemolysis and lecithinase production by B. anthracis and B. cereus strains grown for 48 h at 37°C in aerobic, aerobic-plus-CO₂, or anaerobic-plus-CO₂ atmospheres. Lecithin (left) column shows agar plates containing egg yolk suspension (full recipe given in Materials and Methods), and sheep blood (center) and human blood (right) columns show agar plates with 5% sheep and human blood, respectively. On each of the nine plates, B. anthracis strains SdT2, SdT, and Sterne were spotted in the top horizontal row and B. cereus strains 541 and 569 were spotted in the vertical column. The arrangement of strains on all plates is that shown in the upper left panel.

FIG. 8.

Spot tests showing that both B. cereus and B. thuringiensis release activators of B. anthracis SdT2 PC-PLC production. For each test, 5 μl of B. cereus or B. thuringiensis overnight culture was spotted on LB agar containing lecithin between spots of B. anthracis SdT and SdT2. The top illustration demonstrates growth of B. anthracis strains SdT2 and SdT on LB agar with 5% sheep blood. Bacteria were grown on the plates for 24 h at 37°C.

FIG. 9.

PlcR production and PC-PLC and SPH activities of B. anthracis SdT2, B. cereus 569, and B. anthracis SdT4. The strains were grown for 16 h in LB broth at 37°C. Production of PlcR was determined by Western blot analysis of whole-cell proteins with C-PlcR antiserum as described above. The PC-PLC and SPH activities of culture supernatants of the same three strains were determined as described in the legend to Fig. 6.