A cell-cell signaling peptide activates the PlcR virulence regulon in bacteria of the Bacillus cereus group

Abstract

PlcR is a pleiotropic regulator that activates the expression of genes encoding various virulence factors, such as phospholipases C, proteases and hemolysins, in Bacillus thuringiensis and Bacillus cereus. Here we show that the activation mechanism is under the control of a small peptide: PapR. The papR gene belongs to the PlcR regulon and is located 70 bp downstream from plcR. It encodes a 48-amino-acid peptide. Disruption of the papR gene abolished expression of the PlcR regulon, resulting in a large decrease in hemolysis and virulence in insect larvae. We demonstrated that the PapR polypeptide was secreted, then reimported via the oligopeptide permease Opp. Once inside the cell, a processed form of PapR, presumably a pentapeptide, activated the PlcR regulon by allowing PlcR to bind to its DNA target. This activating mechanism was found to be strain specific, with this specificity determined by the first residue of the penta peptide.

Fig. 1. β-galactosidase activity of the B.thuringiensis 407 Cry^– [plcA′Z] and 407 Cry^– A′Z Δorf2 strains. The cells were grown at 30°C in LB medium. Time zero indicates the onset of the stationary phase, and tn is the number of hours before (–) or after time zero.

Fig. 2. Intercellular activation of PlcR regulon expression in the B.thuringiensis 407 Cry^– A′Z ΔpapR mutant strain. (A) The cells were grown on LB plates containing X-gal for 24 h at 37°C. The name of the strain is indicated beside each streak. (B) β-galactosidase activity of the B.thuringiensis 407 Cry^– A′Z ΔpapR cells resuspended in filtered B.thuringiensis 407 Cry^– (wild type) culture supernatant (circles) or in their own filtered culture supernatant (diamonds). The cells were grown at 37°C in LB medium.

Fig. 3. Complementation of the B.thuringiensis 407 Cry^– A′Z ΔpapR mutant strain with synthetic peptides. (A) Amino acid sequence of the PapR protein and of the synthetic peptides used for the complementation experiment. The characters in bold correspond to the N-terminal signal peptide sequence. The potential cleavage site was predicted with the SignalP VI.I program (Nielsen et al., 1997). (B) β-galactosidase activity of the B.thuringiensis 407 Cry^– A′Z ΔpapR mutant strain. The cells were grown at 37°C in LB medium and each peptide (OS3, OS4, OS5, OS7 and OS9) was added to a final concentration of 5 µM at t1 (1 h after the onset of the stationary phase).

Fig. 4. Effect of the intracellular production of OS5 in the ΔoppB and ΔpapR mutant strains. (A) The expression of os5i in ΔoppB mutant cells restores hemolytic activity in the mutant. Colonies were grown on sheep blood agar plates in the presence of 20 mM xylose for 24 h at 37°C. Bt 407 Cry^–, B.thuringiensis 407 Cry^– (wild type); Bt 407 Cry^– ΔoppB, B.thuringiensis 407 Cry^–ΔoppB mutant strain; Bt 407 Cry^– ΔoppB (pHT304Ωos5i), B.thuringiensis 407 Cry^–ΔoppB (pHT304Ωos5i). (B) Expression of os5i in ΔpapR mutant cells restores plcA-directed transcription in the mutant. β-galactosidase activity of the B.thuringiensis 407 Cry^– A′Z ΔpapR mutant strain carrying, or not carrying, pHT304Ωos5i, and in the presence or absence of 20 mM xylose. The cells were grown at 30°C in LB medium.

Fig. 5. Expression of the plcA′-lacZ transcriptional fusion in the B.thuringiensis 407 Cry^– A′Z and B.thuringiensis 407 Cry^– A′Z ΔpapR strains carrying pHT304.18Ωxyl′-plcR. The cells were grown at 30°C in HC medium in the presence or absence of 1 mM xylose. As determined using a xyl′-lacZ transcriptional fusion, this concentration of xylose induces a level of transcriptional activity similar to that induced by the native plcR promoter (result not shown).

Fig. 6. DNase I footprinting analysis of PlcR binding to the plcA promoter region. Radiolabeled fragments (50 000 c.p.m.) were incubated with various concentrations of PlcR and OS5. (A) Lane 1, G+A Maxam and Gilbert reaction; lane 2, no protein; lane 3, 0.1 µg of PlcR; lane 4, 10 µg of PlcR; lane 5, 20 µg of OS5; lane 6, 0.1 µg of PlcR and 10 µg of OS5; lane 7, 0.1 µg of PlcR and 20 µg of OS5; lane 8, 10 µg of PlcR and 10 µg of OS5; lane 9, 10 µg of PlcR and 20 µg of OS5. The region protected by PlcR is indicated by a bracket. (B) Lane 1, G+A Maxam and Gilbert reaction; lanes 2–8, 50 pmol of PlcR (1.7 µg) and OS5: lane 2, 1000 pmol (0.65 µg); lane 3, 500 pmol; lane 4, 150 pmol; lane 5, 70 pmol; lane 6, 35 pmol; lane 7, 10 pmol; lane 8, 5 pmol; lane 9, 50 pmol of PlcR; lane 10, neither protein nor peptide. (C) plcA promoter region. The DNase I-protected area is indicated by a bracket and the PlcR box is in bold. Positions are relative to the transcription start point. The –35 and –10 promoter regions, and the transcription start (+ 1) of the plcA gene, are indicated as described previously (Lereclus et al., 1996).

Fig. 7. Comparison of the PapR peptide sequences from various B.thuringiensis, B.cereus (B c), B.anthracis (B a) and B.mycoides (B m) strains. The number beside each sequence refers to the serotype of the B.thuringiensis strain. 1* and 1# designate strain 407 Cry– and strain berliner 1715, respectively. The sequences were aligned using Megalign (DNASTAR).

Fig. 8. (A) β-galactosidase activity of the B.thuringiensis 407 Cry^– A′Z ΔpapR mutant strain. The cells were grown at 37°C in LB medium and each peptide (OS5, OS5-I1, OS5-M1 and OS5-V1; all 0.5 µM) was added at t1 (1 h after the onset of the stationary phase). The amino acid sequences of the peptides are as follows: OS5, LPFEF; OS5-I1, IPFEF; OS5-M1, MPFEF; OS5-V1, VPFEF. (B) DNase I footprinting analysis of PlcR binding to the plcA promoter region in the presence of various pentapeptides. Lane 1, G+A Maxam and Gilbert reaction; lane 2, no protein; lane 3, 1.2 µg of PlcR (35 pmol); lane 4, 1.2 µg of PlcR and of OS5 10 µg; lane 5, 1.2 µg of PlcR and 10 µg of OS5-I1; lane 6, 1.2 µg of PlcR and 10 µg of OS5-M1; lane 7, 1.2 µg of PlcR and 10 µg of OS5-V1. The region protected by PlcR is indicated by a bracket and corresponds to the PlcR box upstream from the plcA gene.