Figure 3

Inactivation of B. cereus 569 papR gene. (A) Diagram of the genetic organization of the B. cereus 569 papR mutant, B. cereus 535. Arrows indicate the position and orientation of plcR, papR and loxP. Half-head arrows mark the position and orientation of the primers used for PCR analysis. (B) (Upper panel) The PCR fragments amplified with PapRF/PapRR primers from the chromosomal DNA of B. cereus 569, B. cereus 535, and B. cereus 535 with plasmid pUTE29-papR. Locations of the primers correspond to those shown in panel A. The upper fragment (624 bp) presents the plcR-papR allele; the lower fragment (550 bp) presents the plcR-loxP allele. (Lower panel) Ability of B. cereus 569 to hydrolyze lecithin is appreciably depressed in B. cereus 535. Transformation with pUTE29-papR restored ability of B. cereus 535 to hydrolyze lecithin. The locations of the samples correspond to those of the above PCR. The strains were grown at 37°C in LB broth for 8 h. Equal amounts of extracellular protein were assayed by Red Amplex (PC-PLC). (C) Complementation of both mutant B. cereus 535 and 536 with plasmid producing NprB (pB148Lac-) restores PC-PLC activity only in B. cereus 536 while supplementation of growth medium with 10 μM of 27-aa C-terminal PapR peptide restores phosphatidylcholine hydrolyzing activity only in B. cereus 535. Bacteria were grown at 37°C in LB broth for 8 h with or without the PapR peptide. Equal amounts of extracellular protein were assayed by Red Amplex (PC-PLC).