Characterization of a small PlcR-regulated gene co-expressed with cereolysin O
© Brillard and Lereclus; licensee BioMed Central Ltd. 2007
Received: 05 January 2007
Accepted: 07 June 2007
Published: 07 June 2007
In the human pathogen Bacillus cereus, the expression of most extracellular virulence factors is controlled by the transcriptional activator PlcR. Among these virulence factors, cereolysin O (Clo) is an haemolysin belonging to the cholesterol-dependant cytolysins, a protein family extensively studied in Gram-positive bacteria.
In the genomes of bacteria belonging to the B. cereus group, including Bacillus anthracis and Bacillus thuringiensis, a small gene encoding a 26-amino acid peptide was present in multicopy. One copy was always found upstream from the gene encoding Clo. In B. cereus ATCC 14579, the small gene and the clo gene are co-transcribed. Transcriptional fusions showed that the three paralogues identified in this strain were expressed in a PlcR-dependent manner. We propose to name these peptides Spp for small PlcR-regulated peptides. We show that a synthetic peptide corresponding to the deduced product of the spp genes displayed antibacterial activity.
The co-expression of spp, a small PlcR-regulated multicopy gene with clo suggests a yet unidentified relationship between Spp and the cholesterol-dependent cytolysin in bacteria belonging to the B.cereus group.
Bacillus cereus is an opportunistic pathogen of humans, causing local and systemic infections, and is a frequent cause of food poisoning. This species belongs to the B. cereus group, which includes the closely related species Bacillus anthracis, Bacillus thuringiensis, Bacillus weihenstephanensis,Bacillus mycoide s and Bacillus pseudomycoide s [1, 2]. B. cereus produces several secreted proteins, including enterotoxins, cytolysins, phospholipases and proteases that may contribute to B. cereus pathogenicity. The expression of most of these virulence factors is controlled by the pleiotropic transcriptional activator PlcR [3, 4]. This global regulator has been shown to contribute to B. cereus virulence in mice and insects  and in rabbit endophthalmitis . Expression of the PlcR regulon is activated at the onset of the stationary phase of growth . This activation results from cell-cell communication under the control of PapR, a small peptide that is exported, processed, and re-imported into bacterial cells in its mature form, presumably a pentapeptide, by the oligopeptide permease [8, 9].
Haemolysins of the cholesterol-dependent cytolysins (CDC) family (also known as thiol-activated cytolysins) have been identified in several genera of Gram-positive bacteria . These pore-forming toxins appear to play a significant role in the pathogenesis of the organisms producing them [11, 12]. Listeriolysin O has been extensively studied, and this CDC has been shown to be an important virulence factor, essential for the cellulosome escape and intracellular multiplication of Listeria monocytogenes . In Streptococcus pyogenes, the spn gene, which encodes an effector protein, is located upstream from the gene encoding Streptolysin (Slo). Cytolysin-mediated translocation involving these two proteins has been described in this bacterium . In this process, Slo acts as a gate when anchored in the target-cell membrane. SPN is thus translocated into the cytoplasm of the target cell, increasing cytotoxicity [14, 15]. The study of genes present in the same operons as CDC-encoding genes may therefore increase our understanding of virulence mechanisms in these bacterial pathogens.
CDC have been identified in bacteria of the B. cereus group. These proteins are named cereolysin O (Clo) in B. cereus, thuringiolysin O (Tlo) in B.thuringiensis and anthrolysin O (Alo) in B. anthracis [16–18]. We show here that three paralogous copies of an unannotated gene encoding a 26-amino acid peptide are present in the B. cereus ATCC 14579 genome . One of these paralogues was co-transcribed with the gene encoding cereolysin O, and all three paralogues were expressed in a PlcR-dependent manner.
Results and discussion
Identification of a small gene, co-transcribed with clo
Primer extension was carried out in order to map the transcription start site of the clo gene, using B. cereus total RNA extracted after various culture times. The 5'-end of the mRNA corresponding to clo was located downstream from the PlcR box, and upstream from the pep1 gene, indicating that a bicistronic transcript consisting of pep1-clo had been produced (Fig. 1b). This result suggests that pep1 and clo were co-transcribed from a single transcription start point in the conditions tested. The -10 and -35 regions of this promoter are highly similar to the -35 region (TTGACA) and -10 region (TATAAT) of vegetative promoters recognised by the σA RNA polymerase of B. subtilis (Fig. 1a). Similar experiments were performed with RNA extracted from the B. cereus Δ plcR strain. No signal was detected at T0, T2 and T4 in such conditions (data not shown), indicating that expression of the pep1-clo operon was PlcR-dependent. This result is consistent with the lack of detection of the Clo protein in the extracellular fraction of the B. cereus ΔplcR strain .
Identification of pep paralogues and orthologues in the B. cereus group
No sequence displaying significant similarity to Pep1 was identified in bacteria outside the B. cereus group, or in other sequences in the databases, indicating that Pep1 orthologues are probably restricted to the B. cereus group. However, in the genome of the atypical B. cereus strain NVH 391–98, no pep1 orthologue could be identified. In this strain, the genome has a reduced size (4 Mb) compared to the other B. cereus group members , and no CDC encoding gene is present. This finding is consistent with the fact that this strain is genetically distant from other B. cereus group members .
PlcR-dependent expression of pep1, pep2 and pep3
Strains and plasmids used in this study
Strain or plasmid
Source or reference
B. cereus ATCC14579
B. cereus Δ plcR
B. subtilis 168
B. cereus F4430/73
B. thuringiensis 407 Cry-
E. coli ET12567
(F- dam-13::Tn9 dcm-6 hsdM hsdR recF143 zjj-202::Tn10 galK2 galT22 ara14 pacY1 xyl-5 leuB6 thi-1)
ApR and EmR cloning vehicle; lacZ reporter gene
433 bp region upstream from clo start codon inserted between Pst I and Bam HI sites of pHT304-18'Z
448 bp region upstream from pep2 start codon inserted between Hin dIII and Bam HI sites of pHT304-18'Z
480 bp region upstream from pep3 start codon inserted between Hin dIII and Bam HI sites of pHT304-18'Z
Putative role of the Spp peptides
Bacillus species are known to produce and export an abundance of small peptides. Several of these peptides are involved in signalling or have antimicrobial activity . Analysis of the deduced amino-acid sequence (26 aa) of spp1 (pep1) with the SignalP 3.0 server showed there to be no predicted signal peptide. However, a double-glycine motif was found at positions 12 to 13 in all the Spp orthologues (Fig. 2). This double-glycine motif is a characteristic of some secreted peptides, such as competence-stimulating peptides in streptococci and bacteriocins in lactic acid bacteria . The leader region of such peptides is cleaved after the double-glycine motif by an ABC transporter . The presence of the double-glycine motif suggested that Spp is exported. By analogy to the described functions of double-glycine peptides in other Gram-positive bacteria, and given that competence has never been described in B. cereus, we hypothesized that Spp has a bacteriocin-like function.
Among the indicator strains tested, B. cereus strains which are Spp producers, were the most affected by the Pep13 antibacterial activity. Thus, other maturation process such as posttranslational modifications, are probably required to protect the bacterial cells against their own peptide.
When B. cereus vegetative cells were incubated 1 hour in a phosphate buffer supplemented with Pep13 (to a final concentration of 0.7 mM), the number of CFU decreased from 1.5 (+/-0.1) × 107/ml to 3.3 (+/- 0.7) × 106/ml (experiments were repeated twice). In the same conditions, the number of B. subtilis CFU decreased from 2.1 × 107/ml to 2.3 × 105/ml. This indicates that Pep13 was bactericidal rather than bacteriostatic against these target cells.
However, although spp is expressed, there is no evidence that Spp is actually synthesized and secreted. Furthermore, given the high concentrations of Pep13 required in our assays, we cannot rule out that the antibacterial activity detected is caused by the high Pep13 hydrophobicity rather than by a specific antibacterial activity.
B. cereus has been isolated from soil, and from the gut of insects and nematodes . Like many other bacteria isolated from such ecological niches in which there is strong competition between numerous species of micro-organisms for colonisation, B.cereus has been shown to produce antimicrobial peptides [29, 30]. Recently, an antibacterial substance with a molecular mass of 3.4 kDa, active only against Gram-positive bacteria, was described in B. cereus ATCC 14579 . This antibacterial activity is probably not caused by Spp, because its antibacterial spectrum is different and the predicted molecular mass of Spp1 is lower: 2.9 kDa (26 aa), and 1.5 kDa for the C-terminal fragment of Spp1 (13 aa). However, we cannot rule out the possibility that Spp1 undergoes post-translational modifications, accounting for differences in molecular mass and antibacterial spectrum.
Two small peptides with double-glycine leader sequences produced by competent cells of S. pneumoniae were recently shown to be involved in the lysis of non-competent S.pneumoniae cells, leading to the release of pneumolysin, a non-secreted CDC. This work revealed the existence of co-operation between bacteriocins and a CDC . In B. cereus, Clo, which is found in the extracellular fraction , is most probably exported by the SEC machinery because it has a signal peptide. Thus, the link between Spp and Clo is probably different from that described in S. pneumoniae.
In S. pyogenes, a co-operative effect between a CDC (Slo) and a protein (Spn) encoded by a gene from the same operon has been observed. This co-operative effect increases toxicity to target cells . We showed that spp1 (pep1) and clo are co-transcribed in B. cereus ATCC 14579. This operon structure was found to be conserved among bacteria belonging to the B. cereus group. These findings suggest that Clo and Spp might co-operate to play a role similar to that of Slo and Spn in S.pyogenes, in specific ecological niches or growth conditions that remain to be determined.
This work has led to the identification of spp genes present in all members of the B. cereus group. We showed that the three spp genes of B. cereus ATCC14579 were expressed in a PlcR-dependent manner. In all the B. cereus group strains, a spp gene is coexpressed with the CDC genes encoding cereolysin, thuringiolysin or anthrolysin. The biological signification of this co-expression and the proposed Spp antibacterial role will have to be clarified.
Strains and growth conditions
The strains used in this study are listed in Table 1. E. coli, and B. cereus cells were routinely grown in Luria broth (LB), at 37°C with vigorous shaking. The antibiotic concentrations used for bacterial selection were: ampicillin, 100 μg.ml-1; erythromycin, 10 μg.ml-1 and kanamycin, 150 μg.ml-1. Bacteria with the Lac+ phenotype were identified on LB agar containing 40 μg.ml-1X-Gal.
Database comparison and sequence analysis
TBLASTN alignments were performed with the deduced amino-acid sequence of the protein encoded by pep1 from B. cereus ATCC 14579 to screen the NR database . The putative signal peptide in the polypeptide sequence was identified with the SignalP 3.0 server .
Plasmid DNA was purified from E. coli using QIAprep spin columns (Qiagen). Chromosomal DNA was extracted from B. cereus cells as previously described . Restriction enzymes and T4 DNA ligase were used as recommended by the manufacturer (New England Biolabs). Oligonucleotide primers were synthesised by Proligo-Genset (Paris, France). PCR was performed in a GeneAmp PCR system 2400 thermocycler (Perkin-Elmer), using the high-fidelity Pfx DNA polymerase (Invitrogen). Amplified DNA fragments were purified with the QIAquick PCR Purification Kit (Qiagen), digested and separated on 0.7% agarose gels. Digested DNA fragments were extracted from agarose gels by centrifugation in a filter device (Ultrafree DA, Millipore). All constructs were verified by DNA sequencing (GenomeExpress, France). Electroporation was used to transform E. coli and B. cereus, as previously described [36, 37].
Construction of pep'-lacZ transcriptional fusions
β-Galactosidase specific activities from cells of B. cereus strains harbouring plasmids with lacZ transcriptional fusions were measured as previously described , and were expressed in units of β-galactosidase per milligram of protein (Miller units). The Bradford method (BioRad protein assay) was used for total protein quantification.
RNA extraction and primer extension
Total RNA was extracted from B. cereus ATCC 14579 wild-type and ΔplcR cells grown in LB at 37°C, at the onset of stationary phase (T0), two hours (T2) and four hours (T4) after T0, as previously described . The clo transcription start site was identified by primer extension with the ExtsnClo oligonucleotide (Table 2), as previously described . DNA sequencing was performed by the dideoxy chain termination method, with the same primer and the corresponding PCR product used as the template, with the T7 sequenase PCR product sequencing kit (USB Corporation).
The entire deduced amino-acid sequence of the pep1 ORF (26 aa: MEIAMAVLKFVGGVIPLIQELLKAFM), and the 13 aa C-terminal region of this peptide were synthesised chemically by Millegen (Toulouse, France). These molecules were called Pep26 and Pep13, respectively. Due to their strong hydrophobicity, these molecules were dissolved in DMSO, as recommended by the manufacturer. The resulting stock solution was then diluted with H2O to 7 mg.ml-1 (2.45 mM) in 65% (v/v) DMSO/H2O for Pep26, and to 11 mg.ml-1 (7.26 mM) in 25% (v/v) DMSO/H2O for Pep13. These solutions were further diluted in H2O and assayed on target bacterial cells. Indicator strains were grown in LB at 37°C with vigorous shaking, until an OD600nmof 0.6 was reached. They were then diluted in fresh LB to give an OD of 0.2 and 5 ml were spread on LB-agar plates. The plates were incubated for 10 min and excess liquid was then removed. Plates were allowed to dry at room temperature for 10 min under laminar air flow. Then, 15 μl of Pep26, Pep13, or DMSO (diluted to a final concentration of 65% as negative control) were applied to the plates inoculated with indicator strains. Plates were incubated overnight at 37°C before checking for a putative zone of growth inhibition.
In order to determine whether Pep13 was bactericidal or bacteriostatic, indicator strains were cultured as described above until an OD of 0.7 was reached. They were diluted 10 fold in a 0.1 M potassium phosphate buffer (pH 7) and 200 μl were incubated with 20 μl of a 7.2 mM Pep13 solution for 1 hour at 37°C. Then, the mixture was serially diluted to determine the number of CFU on LB agar medium.
We would like to thank Sinda Fedhila for assistance with unpublished experiments. We also thank N. Boemare, C. Laroui, C. Nielsen-Leroux, P. Serror for providing some of the strains used in this study and C. Braun-Breton for providing human blood. The initial stage of the work was supported by a grant "AQS: Caractérisation de la virulence de Bacillus cereus" from the Ministère de la Recherche and the Ministère de l'Agriculture et de la Pêche. This work was supported by INRA (Institut National de la Recherche Agronomique).
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