Regulation of expression of type I signal peptidases in Listeria monocytogenes

Catherine Raynaud and Alain Charbit

INSERM-U570, Faculté de Médecine Necker-Enfants Malades, 156 rue de Vaugirard, 75730 Paris Cedex-15, France

Correspondence
Catherine Raynaud
cathraynaud{at}yahoo.fr
Alain Charbit
charbit{at}necker.fr


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The role of type I signal peptidases (SPases I) is to remove the signal peptides of preproteins exported by the general secretory pathway. The genome of Listeria monocytogenes contains a locus encoding three contiguous SPases I (denoted SipX, SipY and SipZ). The authors recently showed that SipX and SipZ perform distinct functions in protein secretion and bacterial pathogenicity. Here, the regulation of sip gene expression in broth and in infected eukaryotic cells was studied. The results show that expression of the three sip genes is (i) controlled by two distinct promoter regions that respond differently to growth phase and temperature variations, and (ii) influenced by PrfA (the transcriptional activator regulating most of the virulence genes of L. monocytogenes) and the stress proteins ClpC and ClpP. It was found that sip gene expression was strongly upregulated upon infection of eukaryotic cells when bacteria were still entrapped in the phagosomal compartment. This upregulation is compatible with the need of L. monocytogenes to optimize its production of virulence factors in the early stage of the intracellular cycle.


Abbreviations: BMM, bone-marrow-derived macrophages; LLO, listeriolysin O; PI-PLC, phosphatidylinositol-specific phospholipase C; SPase, signal peptidase; SPases I, type I signal peptidases


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The Gram-positive bacterium Listeria monocytogenes is a food-borne pathogen that is widespread in the environment, where it survives hostile conditions, presumably due to the adaptation of various regulatory mechanisms. L. monocytogenes is a facultative intracellular bacterial pathogen that causes serious disease in immunocompromised individuals, pregnant women and neonates. Its virulence is due to a capacity to invade and multiply within a wide variety of mammalian cells, including macrophages, hepatocytes, epithelial and endothelial cells (Vazquez-Boland et al., 2001). The molecular mechanisms of this intracellular parasitism have been investigated extensively, and these studies have revealed the crucial role of several secreted proteins, such as listeriolysin O (LLO) and phospholipases, and of surface-exposed proteins, such as internalin A (InlA) and InlB, in the virulence of this pathogen (Cossart, 2004; Dussurget et al., 2004). To date, most of the proteins shown to be directly involved in the virulence of L. monocytogenes are either secreted or cell-wall associated.

Bacterial proteins that are exported from the cytoplasm through the general secretory pathway (Sec machinery) are synthesized as precursors with an amino-terminal signal peptide (Pugsley & Possot, 1993). The signal peptide is required for the targeting of precursor proteins to the cytoplasmic membrane, and for the initiation of their translocation across this membrane. During, or shortly after, the translocation process, most signal peptides are removed by signal peptidases (SPases), which is a prerequisite for the release of secretory proteins from the extracytoplasmic side of the membrane (Paetzel et al., 2000). Thus, SPases play a key role in the transport of protein across membranes in all living organisms (van Wely et al., 2001). In Gram-positive bacteria, the exported proteins are either secreted into the medium, or they remain associated by various means with the bacterial envelope. In silico analysis of the genome of L. monocytogenes EGD-e revealed that more than 5 % of the coding sequences carry a typical signal peptide cleavable by type I SPases (SPases I) (Cabanes et al., 2002; Glaser et al., 2001).

We have recently reported the identification of a locus containing three contiguous genes encoding three SPases I in the genome of L. monocytogenes (Bonnemain et al., 2004). The construction of single and multiple knockout mutants in the sip genes showed that SipX and SipZ perform distinct functions in pathogenicity, and that SipZ is the major SPase I of L. monocytogenes. Most biological membranes contain one or two SPases I for the removal of signal peptides from the secretory precursor proteins. In this respect, the Gram-positive bacteria L. monocytogenes and Bacillus subtilis seem to be exceptional, since they possess three and five chromosomally encoded SPases I, respectively (van Roosmalen et al., 2001). To date, the largest number of SPases I has been found in Bacillus anthracis and Bacillus cereus, which contain six and seven paralogous enzymes, respectively (see van Roosmalen et al., 2004 for a recent review). The unique SPase I of Escherichia coli, also known as leader peptidase (Lep), is essential for cell viability, and SPase I limitation results in accumulation of precursors of exported proteins. Similarly, the SPases I SpsB, from Staphylococcus aureus, and Sec11p, of the yeast endoplasmic reticulum membrane, are enzymes that are essential for cell viability. In contrast, the SPase I SipS of B. subtilis is not essential for cell viability, and mutant B. subtilis strains with a disrupted sipS gene are still able to process secreted preproteins (Bolhuis et al., 1999). This observation seems to indicate that multiple SPases I serve to guarantee a sufficient capacity for protein secretion under various conditions (Tjalsma et al., 1997).

In the present work, we studied the regulation of L. monocytogenes sip gene expression in broth and in infected eukaryotic cells. We show that the three sip genes are under the control of two distinct promoter regions. Our data reveal that the regulation of sip gene expression is temporally controlled, and influenced by complex global regulatory circuits.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and growth conditions.
We used the reference strain L. monocytogenes EGD-e belonging to serovar 1/2a (Glaser et al., 2001), and the strain L. monocytogenes L028, which was isolated from a healthy pregnant woman (provided by P. Cossart, Institut Pasteur, Paris, France). E. coli strain DH5{alpha} was used for all plasmid constructions prior to their introduction into L. monocytogenes. E. coli was grown in Luria–Bertani (LB) medium; L. monocytogenes was grown in BHI medium or RPMI synthetic medium.

Real-time quantitative PCR.
Total RNA isolated from cultures of L. monocytogenes grown in two different conditions was converted into DNA with reverse transcriptase, and quantitative PCR using sets of primers from different genes was performed to evaluate the relative levels of expression. Quantification of expression level was performed on EGD-e grown in BHI at 37 °C. We also compared EGD-e growing inside cells [bone-marrow-derived macrophages (BMM) from BALB/c mice and human enterocyte Caco-2 cells] with EGD-e growing in RPMI medium. For RNA preparations, cells were broken in a solution of 1 ml Trizol (Invitrogen) containing mini glass beads using a Bead Beater (Savant). Total RNA was extracted with 300 µl chloroform/isoamyl alcohol (24 : 1, v/v), then precipitated with 2-propanol, and resuspended in diethyl-pyrocarbonate-treated water. Contaminating DNA was removed by digestion with DNase I.

The absence of contaminating DNA after DNase treatment was confirmed by including a control reaction without reverse transcriptase in the RT-PCR assay (data not shown).

RT-PCR experiments were done with 1 µg RNA, with 2·5 pmol specific primers for the genes gyrA (gyrase, constitutively expressed), and sipX, sipY and sipZ, in a volume of 8 µl. After denaturation at 65 °C for 10 min, 12 µl of the mixture containing 2 µl dNTP (25 mM), 4 µl 4x buffer (provided with the Superscript II), 2 µl DTT, 1 µl RNasin (Promega), and 1·5 µl Superscript II (Invitrogen), was added. Samples were incubated for 60 min at 42 °C, heated at 75 °C for 15 min, and then chilled on ice. PCR conditions were identical for all reactions. The 25 µl reactions, performed in sealed tubes, consisted of 12·5 µl PCR master mix (PE Applied Biosystems) containing Sybr Green, 4 µl template, and 5 pmol each primer. For real-time quantitative PCR, we used the ABI Prism 7700 sequence detection system with TaqMan Universal PCR master mix (PE Applied Biosystems).

For the infection experiments, three different infections were performed, and each point corresponds to three measurements. The human colon carcinoma cell line Caco-2 (ATCC HTB37) was propagated in Dulbecco's modified Eagle medium (DMEM; Gibco) supplemented with 10 % fetal bovine serum at 37 °C with 5 % CO2. Cells were seeded at approximately 2x105 cells ml–1 in tissue culture plates. Monolayers were used 24 h after seeding. Cells were infected with bacteria grown at 37 °C overnight at a multiplicity of 50 bacteria per cell. In experiments involving Caco-2 cells, the induction was measured in infected cells cultivated in RPMI, as compared with L. monocytogenes grown in RPMI.

Data analyses.
Results were normalized to the amount of gyrase mRNA, which was constant under our growth conditions (Réglier-Poupet et al., 2003). For example, to compare sipZ gene expression at pH 7 and 5, for each experiment (carried out three times) we calculated the ratio: (sipZ pH 5/gyrA pH 5)/(sipZ pH 7/gyrA pH 7), and then we calculated the means and standard deviations of the three values obtained.

{beta}-Galactosidase assays.
We used pTCV-lac, a mobilizable shuttle vector that enables transcriptional fusion to {beta}-galactosidase in a wide range of Gram-positive bacteria, in which it replicates at a low copy number per bacterium (Poyart & Trieu-Cuot, 1997). All oligonucleotides were designed to add EcoRI and BamHI sites upstream and downstream of the amplified potential promoters (Table 1). The amplified fragments were digested with EcoRI and BamHI, and cloned into pTCV-lac. Bacteria were grown overnight in BHI broth containing kanamycin. The cultures were then diluted 50-fold in BHI broth supplemented with kanamycin, and cells were collected at different time points during the growth phase. {beta}-Galactosidase was assayed as described by Miller (1972), except that the cells were permeabilized by treatment with 0·5 % toluene and 4·5 % ethanol. The {beta}-galactosidase specific activities, determined in three separate experiments, were expressed as [103x(OD420 of the reaction mixture–1·75 OD550 of the reaction mixture)] divided by [reaction time (min) xOD600 of the quantity of cells used in the assay].


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Table 1. Strains, plasmids and primers

 
The {beta}-galactosidase assays were performed at least seven times (each time with a new culture). The result given for each experiment is the mean of three measurements.

Gel-mobility-shift DNA-binding assays.
EcoRI/BamHI DNA fragments, corresponding to the promoter regions of different genes, were generated by PCR, and labelled radioactively by treatment with the Klenow fragment of DNA polymerase I in the presence of a mixture of dGTP, dATP, dTTP (0·08 mM) and [{alpha}-32P]dCTP (40 µCi; 1·48 MBq). Binding of protein to DNA was carried out in a 20 µl reaction mixture containing 32P-labelled DNA, 1 µg poly(dI-dC) (Pharmacia), 25 mM sodium phosphate (pH 7), 150 mM NaCl, 0·1 mM EDTA, 2 mM MgSO4, 1 mM DTT, 10 % (v/v) glycerol. The DNA-binding reaction was initiated by the addition of total crude protein, and incubated at room temperature for 30 min. Samples were then loaded directly onto an SDS-6 % polyacrylamide gel (50 mM Tris, 400 mM glycine, 1·73 mM EDTA) for electrophoresis (10 V cm–1). After overnight electrophoresis, the gels were dried, and scanned with a Molecular Dynamics PhosphorImager. The autoradiographs shown correspond to a 24 h exposure.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The sip genes are under the control of two distinct promoters
The sip genes belong to a cluster of five genes (Fig. 1a). Two major transcripts were previously identified by Northern blot analysis (Bonnemain et al., 2004): a large one compatible with a transcript covering the entire locus (sipXlmo1273), and a shorter one compatible with a transcription starting upstream of sipZ (sipZlmo1273). Here, we decided to evaluate quantitatively the potential promoter activities of the regions preceding each sip gene. For this, we inserted a 200 bp DNA fragment, corresponding to the region immediately upstream of each sip coding sequence (denoted PsipX, PsipY and PsipZ), upstream of the reporter lacZ gene carried on a Gram-negative/Gram-positive shuttle plasmid (pTCV-lac derivatives, see Methods). The recombinant plasmids were transferred into L. monocytogenes, and Psip-dependent {beta}-galactosidase expression was monitored in BHI at different stages of bacterial growth, and in different growth conditions. We used the promoter of the aph3' gene as a positive control. Two regions, PsipX and PsipZ, promoted {beta}-galactosidase expression, indicating promoter activity (Fig. 1b). In contrast, no promoter activity was recorded in the fragment upstream of sipY, strongly suggesting that sipY is transcribed from PsipX (PsipY was used as a negative control in the remaining experiments). In broth, PsipX activity remained relatively constant throughout the entire period of growth, while that of PsipZ reached a peak after 1 h, when cultures were not yet in the exponential phase of growth, and then decreased sharply after 1 h 30 min, which was the beginning of the exponential phase. The maximal {beta}-galactosidase activity recorded with PsipZ was similar to that recorded with PsipX. Such differences in promoter activities have also been observed in B. subtilis, where sipU and sipV are constitutively transcribed, whereas sipS and sipT are temporally controlled (Tjalsma et al., 1997).



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Fig. 1. (a) Schematic representation of the sip locus of L. monocytogenes. Putative transcription terminators are indicated (black, strong terminator; grey, weak terminator). Grey arrows indicate the potential promoter fragment upstream of each sip gene. (b) Quantification of promoter activity. Expression of {beta}-galactosidase ({beta}-gal) activity for PsipX ({lozenge}), PsipY ({square}), PsipZ ({triangleup}) and aphA-3' ({circ}; positive control) in strain EGD-e growing in LB medium at 37 °C. Bacteria were removed at different time points for measurement of growth (OD600) ({blacktriangleup}) and {beta}-galactosidase activity.

 
sip promoter activities are influenced by environmental conditions
We monitored PsipX and PsipZ activity at acid and neutral pH values, and at different temperatures. After a preculture at pH 7·2, the bacteria (OD600 0·3) were placed in a new medium at pH 7·2 or 5·5 (bacteria from the initial culture were collected by centrifugation, and resuspended in the same volume of the new medium before inoculation). The {beta}-galactosidase activity was measured at t0 and 30 min after inoculation. PsipX and PsipZ activities were similar at pH 7·2 and 5·5, indicating that the two major SPases I of L. monocytogenes (sipX and sipZ encoded) are not regulated by acidification (data not shown). We then compared promoter activities at different temperatures (37, 42 and 4 °C, Fig. 2). The activity of PsipX was identical after 30 min at 37 and 42 °C, but increased almost threefold after 30 min growth at 4 °C (Fig. 2a). In contrast, PsipZ activity decreased twofold at 37 °C, was unchanged at 4 °C, and increased weakly at 42 °C (Fig. 2b). We quantified by real-time PCR the expression of sipX and sipZ at 42 and 4 °C, as compared with 37 °C. The values recorded confirmed the results of the {beta}-galactosidase assays. Transcription of sipX increased at 4 °C, whereas that of sipZ increased at 42 °C (data not shown), revealing that, PsipX and PsipZ respond differently to temperature variations.



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Fig. 2. Effect of temperature on sip expression. (a) PsipX and (b) PsipZ at 4, 37 and 42 °C. Cells were grown in LB medium at 37 °C. At an OD600 of 0·3, they were placed at different temperatures. Then {beta}-galactosidase activity was measured at t0 (black bars) and after 30 min (grey bars). The analysis was carried out three times, and the mean values (±1 SD) are indicated.

 
Pleiotropic regulators modulate the expression of SPase I proteins
Previous studies with B. subtilis suggested regulation of SPase I genes by a ClpXP-dependent activator (Pummi et al., 2002). In L. monocytogenes, the proteases ClpP and ClpC play an essential role in virulence: ClpP is involved in the rapid adaptative response of the bacterium during the infectious process, and a mutant lacking ClpP is avirulent in the mouse model (Gaillot et al., 2000); ClpC is required for stress tolerance, and contributes to early escape from the phagosomal compartment of macrophages, and in vivo survival (Nair et al., 2000).

We tested the role of ClpC and ClpP in the expression of the sip locus by introducing plasmid pTCV-lac-PsipX or pTCV-lac-PsipZ into the two knockout mutants {Delta}clpC and {Delta}clpP of L. monocytogenes. Since the mutations had been constructed in strain LO28, we used wild-type EGD-e and LO28 strains as controls. The activities of PsipX and PsipZ were essentially identical in EGD-e and LO28 (approx. 40 {beta}-galactosidase units). The promoter activities of PsipX and PsipZ were three- to fourfold higher in the clpC and clpP mutants (174±2 and 162±2·6 {beta}-galactosidase units for PsipX in the clpC and clpP mutants, respectively; 158±0·3 and 167±3 {beta}-galactosidase units for PsipZ in the clpC and clpP mutants, respectively) compared with the wild-type strains. These results suggest that, as in B. subtilis, expression of ClpC and ClpP downregulates transcription of the sip genes.

PrfA, the central virulence regulator of L. monocytogenes, has been shown to exert a negative control on ClpC expression at the transcriptional level, reflecting the existence of crosstalk between these two systems (Ripio et al., 1998). Moreover, in B. subtilis, ClpC controls the expression of competence by forming a complex with MecA to negatively regulate ComK (Bockmann et al., 1996). We therefore tested whether MecA and PrfA could also modulate the expression of the sip genes, by monitoring PsipX- and PsipZ-dependent lacZ transcription in {Delta}prfA and {Delta}mecA mutant backgrounds. In these two backgrounds, the activity of both PsipX and PsipZ was two- to threefold lower than in the wild-type strains (approx. 20 and 10 {beta}-galactosidase units for PsipX in the mecA and prfA mutants, respectively; approx. 15 and 10 {beta}-galactosidase units for PsipZ in the mecA and prfA mutants, respectively). Thus, while ClpC and ClpP are involved in the negative regulation of sip gene expression, PrfA and MecA are involved in the positive regulation of their expression.

Proteins bind specifically to the promoter regions PsipX and PsipZ
We performed different gel-mobility-shift DNA-binding assays to the visualize the binding of proteins to the promoter regions PsipX and PsipZ. First, we used total protein extract from L. monocytogenes cells in DNA-binding assays, with 200 bp double-stranded DNA fragments corresponding to PsipX and PsipZ. All the assays were performed in the presence of an excess of non-specific competitor DNA [1 µg poly(dI-dC)]. Radiolabelled probes corresponding to PsipX and PsipZ were incubated with increasing amounts of total protein extract. The displacement of radiolabelled fragment was proportional to the amount of protein extract added, and was complete at a concentration of 15 µg total protein (Fig. 3). The same experiment was performed with a radiolabelled probe corresponding to PsipY (a region showing no promoter activity in the previous experiment), and no displacement of radiolabelled fragment among the amount of protein extract was observed (data not shown). Cold competitor chase experiments (as described by Chastanet et al., 2003) were used to demonstrate the specificity of protein binding. The addition of increasing amounts of unlabelled DNA fragments corresponding to PsipX or PsipZ efficiently prevented binding of the radiolabelled probes (Fig. 3a, b). These results indicate that one or several proteins bind directly to the promoter regions of sipX and sipZ.



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Fig. 3. Gel-mobility-shift DNA-binding assays. (a) Assays were performed with 0·5 pmol 32P-labelled PsipX DNA fragment. Lanes: 1, no protein; 2–4, increasing amounts of total protein extract from strain LO28 (2, 5 and 15 µg in lanes 2, 3 and 4, respectively); 5–8, 15 µg protein and increasing amounts of unlabelled PsipX DNA fragment (0·1, 0·5, 1 and 2 pmol in lanes 5, 6, 7 and 8, respectively). (b) Assays were performed with 0·5 pmol labelled PsipZ fragment. Lanes: 1, no protein; 2 and 3, increasing amounts of total protein extract from LO28 strain, (5 and 15 µg in lanes 2 and 3, respectively); 4–6, 15 µg protein and increasing amounts of unlabelled PsipX DNA fragment (0·1, 0·5 and 2 pmol in lanes 4, 5 and 6, respectively). (c) Assays were performed with 0·5 pmol labelled PsipX fragment. Lanes: 1, no protein; 2–5, 15 µg total protein extracts from mutant strains {Delta}clpC, {Delta}clpP, {Delta}mecA and {Delta}prfA (lanes 2, 3, 4 and 5, respectively).

 
To determine whether ClpC, ClpP, PrfA and MecA would influence sip expression. either by direct binding, or indirectly by modulating the binding of other proteins, to PsipX and PsipZ, the same experiment was performed using total protein extracts from {Delta}prfA, {Delta}mecA, {Delta}clpC and {Delta}clpP isogenic mutant strains. With all the extracts, a similar displacement of the radiolabelled probe was observed (Fig. 3c). Thus, the role of PrfA, MecA, ClpC and ClpP on sip gene expression is indirect, and is most likely to involve complex regulatory circuits.

These preliminary observations will need to be followed up with additional experiments to define the actual regulatory mechanisms involved.

Expression of the sip genes is upregulated in infected cells
We monitored quantitatively the transcription of each sip gene by real-time PCR, upon infection by L. monocytogenes of either mouse bone marrow-derived macrophages (BMM) or the human enterocyte Caco-2 cell line (Fig. 4). In both cell types, an increase in the transcription of the three sip genes was observed shortly after infection. In mouse BMM (Fig. 4a), a sevenfold induction of sipZ was recorded after 30 min of infection; induction levels of sipX and sipY were similar to each other, and slightly lower (approximately fivefold induction). In Caco-2 cells (Fig. 4b), significant induction of sip gene transcription was also observed after 30 min of infection (approximately fivefold induction of sipZ, and three- to fourfold induction of sipX and sipY). Our electron microscopy observations suggest that, at this early stage of the infection, the majority of bacteria are still localized inside phagosomes (data not shown; Réglier-Poupet et al., 2003). In contrast, after 1 h of infection, about 65 % of the bacteria were surrounded by a meshwork of polymerized actin. After 1 h of infection, the induction of each of the three sip genes decreased in the two cell types, but was still about twofold higher than that recorded in broth. These assays suggest that sip gene expression might be preferentially upregulated during the intraphagosomal stage of the infectious cycle of L. monocytogenes.



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Fig. 4. Induction of sip gene expression in mouse BMM and Caco-2 cells. The transcription of sip was monitored by real-time quantitative PCR in (a) mouse BMM and (b) Caco-2 cells after 30 min (black bars) and 1 h (grey bars) of infection. The induction ratio is the number of transcripts detected in bacteria in cells during infection divided by the number of transcripts detected in bacteria grown in RPMI medium. Values are expressed relative to the number of gyrA transcripts. Errors bars show standard deviations.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Regulation of Spase I expression in infected cells
During infection, L. monocytogenes modulates its production of virulence factors according to its needs (Bubert et al., 1999). For example, expression of LLO and phosphatidylinositol-specific phospholipase C (PI-PLC) is upregulated in the phagosomal compartment, where these proteins are essential for phagosomal escape; while ActA expression is strongly induced when bacteria have reached the cytosolic compartment of the infected host cell, where the protein is required for actin polymerization and bacterial motility. The upregulation of sipZ in the phagosomal compartment is compatible with the necessity to process LLO and PI-PLC efficiently and rapidly in this compartment. In this respect, it is worth recalling that inactivation of sipZ impairs the secretion of LLO and the broad-range phospholipase (phosphatidylcholine-specific phospholipase C), restricts intracellular multiplication, and almost abolishes virulence (Bonnemain et al., 2004). The fact that, in broth, acidification of the medium did not affect sip expression, suggests that phagosomal acidification is not the signal responsible for the early induction of sip expression in cells.

It is tempting to suggest that L. monocytogenes has evolved this regulation of SPase I expression as an additional means to control its production of virulence factors. However, it is possible that L. monocytogenes simply adapts the synthesis of SPases according to its needs.

PrfA and other factors are involved in the regulation of sip gene expression
In agreement with our earlier observations (Bonnemain et al., 2004), we found that the three sip genes were regulated by two different promoters, PsipX and PsipZ. Expression of the sip genes appeared to be coordinated by complex regulatory signals, including the pleiotropic transcriptional activator PrfA, and stress proteins involved in L. monocytogenes pathogenicity. Clp ATPases are believed to form part of post-translation regulatory networks that ensure survival in stress conditions, presumably by acting as molecular chaperones that mediate the repair or scavenging of damaged proteins (Wawrzynow et al., 1996). In B. subtilis, Clp ATPases mediate adaptive responses to stress conditions, as well as to many other different processes, including the development of competence, sporulation, exoenzyme synthesis and cell-cycle regulation. Of particular interest, the inactivation of clp genes has been shown to upregulate the transcription of three SPase I genes, sipS, sipT and sipV, and of the lipoprotein signal peptidase gene lsp, suggesting that Clp proteins control a step in the secretion pathway that is common to both non-lipoproteins and lipoproteins (Pummi et al., 2002).

In L. monocytogenes, we found that inactivation of clpC or clpP increased sip gene expression, suggesting that Clp proteins act to downregulate sip expression, and we found that inactivation of MecA downregulated sip expression, suggesting that MecA could act as both a negative and a positive regulator. We also observed that PrfA influenced sip expression, while a large-scale transcriptomic analysis of PrfA-dependent promoters (Milohanic et al., 2003) failed to identify the sip genes. Thus, either sip gene regulation was below the detection threshold determined by the transcriptomic assay, or, under the studied conditions, the sip genes were not regulated.

To date, the interconnection between the PrfA regulon and the regulatory pathways that control stress responses in L. monocytogenes is not clearly understood. ClpC expression is negatively controlled at the transcriptional level by the central virulence regulator PrfA. Gel-mobility DNA-binding assays, performed here with total protein extracts devoid of PrfA, ClpC, ClpP or MecA, confirmed the absence of direct action of these proteins on the sip promoters, suggesting complex control mechanisms of sip gene expression in L. monocytogenes.


   ACKNOWLEDGEMENTS
 
We are greatly indebted to Philip Draper for critical reading of the manuscript. This work was supported by CNRS, INSERM and Université Paris V. C. R. was supported by a fellowship from INSERM.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 24 March 2005; revised 30 June 2005; accepted 6 July 2005.



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