The YvfTU two-component system is involved in plcR expression in Bacillus cereus

Abstract

Background: Most extracellular virulence factors produced by Bacillus cereus are regulated by the pleiotropic transcriptional activator PlcR. Among strains belonging to the B. cereus group, the plcR gene is always located in the vicinity of genes encoding the YvfTU two-component system. The putative role of YvfTU in the expression of the PlcR regulon was therefore investigated.

Results: Expression of the plcR gene was monitored using a transcriptional fusion with a lacZ reporter gene in a yvfTU mutant and in its B. cereus ATCC 14579 parental strain. Two hours after the onset of the stationary phase, a stage at which the PlcR regulon is highly expressed, the plcR expression in the yvfTU mutant was only 50% of that of its parental strain. In addition to the reduced plcR expression in the yvfTU mutant, a few members of the PlcR regulon showed a differential expression, as revealed by transcriptomic and proteomic analyses. The virulence of the yvfTU mutant in a Galleria mellonella insect model was slightly lower than that of the parental strain.

Conclusion: The YvfTU two-component system is not required for the expression of most of the virulence factors belonging to the PlcR regulon. However, YvfTU is involved in expression of plcR, a major regulator of virulence in B. cereus.

Figure 1

The B. cereus yvfTU chromosomal region. (A) Map of the B. cereus ATCC 14579 yvfTU chromosomal region. Grey arrows represent ORFs. The position of the kanamycin resistance gene integrated in the chromosome of the mutant to disrupt yvfTU is indicated. Small arrows represent promoters or putative promoters. Function or putative function of gene products: BC5355, ABC transporter ATP-binding protein; BC5354, ABC transporter permease protein; yvfT, two-component sensor kinase; yvfU, two-component response regulator; nprB, metalloprotease; plcR, transcriptional activator; papR, PlcR activating peptide; Black box: PlcR recognition site. Lines annotated by numbers in brackets correspond to RT-PCR amplicons (see text and Fig. 2). In the yvfT promoter region, putative -10 and -35 sigma A boxes (underlined), initiation of transcription (bold underlined) and RBS (bold italic) are indicated In the yvfU 3'-region, one cytosine (bold underlined) which leads to a stop codon (italic underlined) was indicated in the ATCC 14579 genome sequence, but was lacking in our sequence data which gives a different stop codon (bold). The predicted stem-loop at the end of yvfU is represented. (B) Synteny of the yvfTU chromosomal region among the B. cereus group. Taxonomic group determined as previously described [24]. Studied strains were: Group III, B. cereus ATCC 10987; B. cereus ZK (E33L); B. thuringiensis konkukian; B. anthracis Sterne; B. anthracis Ames; B. anthracis Ames 0581; B. anthracis Ames ancestor; B. cereus W, B. thuringiensis Al Hakam. Group IV, B. cereus ATCC 14579; B. thuringiensis israelensis ATCC 35646. Group VI, B. weihenstephanensis KBAB4. Group VII, B. cereus NVH 391/98, B. cereus NVH 883/00. ^a: truncated in B. anthracis strains.

Figure 2

RT-PCR detection of yvfTU, BC5354 and BC5355 in B. cereus strain ATCC 14579. Lane "+": Positive control (PCR on genomic DNA). Lanes "RT": RT-PCR on 500 ng RNA. Lanes "-": Negative control (RT-PCR on 500 ng RNA with a heat-inactivated reverse-transcriptase). Numbers in brackets refer to the positions of the RT-PCR products on the yvfTU locus, as represented on Fig. 1A. RNA extraction was performed on strains grown at 37°C in LB broth and harvested in exponential phase (E) (OD₆₀₀ 1), or 2 hours after the onset of stationary phase (T2).

Figure 3

yvfT directed lacZ expression in B. cereus WT or yvfTU mutant strains. β-galactosidase activity was measured in either the WT strain (circles) or in the yvfTU mutant (triangles) harbouring pHT-yvfT'Z (649 bp yvfT promoter region cloned upstream from the promoterless lacZ reporter gene in pHT304-18Z). Time 0 indicates the onset of the stationary phase of growth. Each curve is the mean value of triplicate measurements, representative of 3 independent experiments. Bars represent standard deviation.

Figure 4

plcR directed lacZ expression in B. cereus WT or yvfTU mutant strains. β-galactosidase activity was measured in either the WT strain (circles) or in the yvfTU mutant (triangles) harbouring pHT-plcR'Z (plcR promoter region cloned upstream from the promoterless lacZ reporter gene in pHT304-18Z). Time 0 indicates the onset of stationary phase. Each curve is the mean value of triplicate measurements, representative of 3 independent experiments. Bars represent standard deviation.

Figure 5

2D-GE of WT and yvfTU mutant culture supernatants collected 2 hours after the onset of stationary phase. Spots found in lower amount in the extracellular proteome of the yvfTU mutant correspond to NprB and PC-PLC proteins, as confirmed by Mass Spectrometry identification. The different spots correspond to isoforms of the same protein, with distinct charges but identical molecular weights. The gel areas shown are located around 37 kDa with a pI between 5.5 and 6 for NprB, and 25 kDa with a pI between 6.0 and 7.0 for PC-PLC.