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1 Unité de Biologie des Bactéries Pathogènes à Gram Positif, CNRS URA 2172, Institut Pasteur, 25 Rue du Dr. Roux, 75724 Paris Cedex 15, France
2 Laboratory of Bacterial Biofilms, Instituto De Agrobiotecnologia Y Recursos Naturales, Universidad Pública de Navarra, 31006 Pamplona, Spain
3 Unité des Interactions Bactéries Cellules, INSERM U604, INRA USC2020, Institut Pasteur, 25 Rue du Dr. Roux, 75724 Paris Cedex 15, France
Correspondence
Tarek Msadek
tmsadek{at}pasteur.fr
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These authors contributed equally to this work.
Present address: AbAg, 17 Avenue du Parc, 91380 Chilly Mazarin, France.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). TCSs are involved in a broad range of bacterial responses, including sporulation, virulence, biofilm formation, and synthesis of extracellular enzymes. Many of these systems are known to interact, effectively forming a bacterial sensory transduction network, an aspect that has been particularly well studied in the Gram-positive model bacterium Bacillus subtilis (Msadek et al., 1995; Msadek, 1999). Listeria monocytogenes, a food-borne Gram-positive facultative intracellular pathogen very closely related to B. subtilis, can cause severe diseases in immunocompromised hosts such as pregnant women and neonates. These infections include meningitis, septicaemia and gastroenteritis, with a high degree of mortality. This bacterium has been extensively studied, and because of its ability to escape from the phagosome, to grow in the cytosol and to efficiently invade neighbouring cells, has become an established model for intracellular growth (Cossart & Portnoy, 2000).
Analysis of the L. monocytogenes genome sequence reveals the presence of 17 TCSs (Glaser et al., 2001). Some of these have been studied, including the CheA/CheY system that controls chemotaxis and motility, LisK/LisR, involved in tolerance to stress and virulence (Cotter et al., 2002), VirS/VirR, critical for L. monocytogenes virulence (Mandin et al., 2005), and CesK/CesR, which responds to the presence of cell wall-acting antibiotics and affects β-lactam resistance (Kallipolitis et al., 2003).
The DegS/DegU TCS of B. subtilis was one of the first to be identified in Gram-positive bacteria (Kunst et al., 1988; Msadek et al., 1990), and has been shown to play a central role in the signal transduction network that controls stationary phase adaptive responses (Msadek et al., 1995; Msadek, 1999). Expression of motility and chemotaxis genes requires the secondary sigma factor 
D, whose synthesis and activity are controlled by several different mechanisms (Helmann, 1991). The DegS/DegU TCS is also involved in controlling motility and chemotaxis, since an excess of the phosphorylated form of the DegU response regulator has been shown to abolish expression of the sigD gene encoding D (Dahl et al., 1992; Msadek et al., 1990; Tokunaga et al., 1994).
Carboxy-terminal amino acid sequence similarities place DegU within the FixJ/UhpA response regulator subfamily with a characteristic helix–turn–helix DNA-binding motif (Msadek et al., 1990; Stock et al., 1989). Indeed, DegU has been shown to bind directly in vitro to the upstream regulatory regions of the comK gene (Hamoen et al., 2000) and the fla/che operon (Amati et al., 2004), although a consensus binding site for DegU has yet to be established. Interestingly, although an orthologue of the degU gene is present in L. monocytogenes, the gene encoding the DegS kinase is absent, raising the question of whether the orphan DegU response regulator is functional in L. monocytogenes and if so how signal acquisition occurs. While this work was in progress, DegU of L. monocytogenes was independently reported to play a role in motility and virulence (Knudsen et al., 2004; Williams et al., 2005a).
In L. monocytogenes, cells do not produce flagella and are non-motile when grown at the mammalian host physiological temperature of 37 °C, with flagellar synthesis and motility only occurring when cells are grown at 30 °C and below (Peel et al., 1988). Regulation of flagellar synthesis in L. monocytogenes differs quite markedly from that of B. subtilis model. Indeed, no orthologue of D is present, nor are there any secondary sigma factors dedicated to flagellar synthesis (Glaser et al., 2001). Nevertheless, many regulatory proteins are known to control motility gene expression in L. monocytogenes, such as PrfA, the central virulence gene regulator (Michel et al., 1998); CtsR, which represses class III stress response genes (Karatzas et al., 2003; Nair et al., 2000); FlaR, a histone-like osmoregulated protein which positively regulates flaA expression at 25 °C and acts as a repressor at 37 °C (Sanchez-Campillo et al., 1995); MogR, a transcriptional repressor of flagellar motility genes (Shen & Higgins, 2006); and GmaR, an anti-repressor of MogR (Shen et al., 2006).
Flagellar motility has also been shown to be important for biofilm formation in Listeria (Lemon et al., 2007). Indeed, bacteria in the environment are predominantly sessile, adhering to inert surfaces and developing as multicellular colonies sheathed within an exopolysaccharide matrix, a structure referred to as a biofilm. Listeria biofilm development varies greatly depending on the strain, environmental conditions (pH, growth medium composition, and temperature), and surface properties (Tresse et al., 2006).
In this study, we investigated regulation of degU expression in L. monocytogenes. We purified the L. monocytogenes DegU protein and showed that DegU negatively regulates its own synthesis by binding directly to its promoter region, and that it also binds upstream of the motB motility operon, which contains the gmaR gene. In addition, we demonstrated that DegU is required not only for flagellar synthesis, motility and virulence, but also for biofilm formation, by complementation of a 
degU mutant, restoring the phenotype of the parental EGDe strain. This is, to our knowledge, the first report of L. monocytogenes genes directly regulated by DegU and of the involvement of DegU in biofilm formation in this bacterium.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Escherichia coli K-12 strain DH5
[F– (
80dlacZM15) (lacZYA-argF) U169 recA1 endA1 hsdR17 (
, 
) phoA supE44 
– thi-1 gyrA96 relA1] (Invitrogen) was used for cloning experiments, and E. coli strain BL21 DE3 (Studier & Moffatt, 1986) (Novagen) for protein overproduction and purification. B. subtilis strain 168 trpC2 was used as a Gram-positive cloning host. E. coli strains were grown in Luria–Bertani (LB) medium and transformed by electroporation (Sambrook et al., 1989), with selection on plates supplemented with ampicillin (100 µg ml–1) and kanamycin (25 µg ml–1) when required. B. subtilis cells were grown in LB medium supplemented with 5 µg chloramphenicol ml–1, and transformed as described previously (Msadek et al., 1998). L. monocytogenes strain EGDe and its derivatives were grown in Brain Heart Infusion (BHI) medium (Difco), Modified Welshimer's Broth (MWB) minimal medium (Premaratne et al., 1991) or Roswell Park Memorial Institute (RPMI) 1640 synthetic medium (Sigma-Aldrich) and transformed by electroporation, with selection on BHI plates supplemented with chloramphenicol (10 µg ml–1) or erythromycin (1 µg ml–1) when necessary.
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. Chromosomal DNA from L. monocytogenes strains was isolated using the MasterPure Gram-positive DNA purification kit (Epicentre Biotechnologies). DNA fragments were purified following agarose gel electrophoresis using a Qiaquick gel extraction kit (Qiagen). Plasmid DNA was isolated using a QIAprep Spin Miniprep kit (Qiagen), and PCR fragments were purified using the Qiaquick PCR purification kit (Qiagen). T4 DNA ligase and restriction enzymes (New England Biolabs), PCR reagents and Pwo thermostable DNA polymerase (Roche) were used according to the manufacturer's recommendations. Nucleotide sequencing of plasmid constructs was carried out by Genome Express-Cogenics.
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degU was used to generate a markerless degU mutation in L. monocytogenes strain EGDe. Two DNA fragments, of 761 and 781 bp, were generated by PCR using oligonucleotide pairs OSA14/OSA15 and OSA5/OSA6, respectively (Table 2
), corresponding to the chromosomal DNA regions located directly upstream and downstream from the degU gene. These two DNA fragments were cloned in tandem between the EcoRI and BamHI sites of the pMAD vector (Arnaud et al., 2004) in two consecutive steps, resulting in plasmid pMADdegU. The plasmid was introduced by electroporation into L. monocytogenes strain EGDe, and transformants were selected at 30 °C on BHI plates containing erythromycin and X-Gal (50 µg ml–1). Integration and excision of pMADdegU was performed as described previously (Arnaud et al., 2004) with a non-permissive growth temperature of 42 °C, yielding strain LM1001 (degU), in which the entire degU coding sequence was removed. The gene deletion was confirmed by PCR amplification.
The same strategy was used to remove the coding sequence of the yviA gene in L. monocytogenes strain EGDe. Plasmid pMADyviA was constructed by cloning two PCR-generated fragments, of 244 and 229 bp, using oligonucleotide pairs HD119/HD120 and HD121/HD124 between the EcoRI and BamHI sites of the pMAD vector (Arnaud et al., 2004). Plasmid transformation, integration and excision were then carried out as described above, resulting in strain LM1010 (yviA), and the gene deletion was verified by PCR amplification using oligonucleotides HD118/HD123.
In order to complement the L. monocytogenes degU mutant, the degU gene was cloned into the pMK4 shuttle plasmid (Sullivan et al., 1984). It has been reported previously that the presence of the B. subtilis degU gene is toxic when expressed in E. coli, and that the gene can only be cloned in B. subtilis (Kunst et al., 1988). We found that this was also the case for degU of L. monocytogenes, and therefore used B. subtilis directly as the cloning host. A 1456 bp DNA fragment corresponding to the entire coding sequence of degU with its upstream native promoter was generated by PCR using oligonucleotides OSA14 and HD125. Following restriction with EcoRI and BamHI, the fragment was cloned between the corresponding sites of plasmid pMK4. We used a DNA concentration of 115 µg ml–1 (7 : 1 insert to vector molar ratio) in the ligation mixture, since direct transformation of naturally competent B. subtilis cells requires that ligations be performed at high DNA concentrations in order to form multimeric DNA molecules, favouring DNA uptake and subsequent resolution within the bacterial host (Kunst et al., 1988).
Plasmid pET28/16 (Chastanet et al., 2003), a derivative of plasmid pET28a (Novagen), was used for protein overproduction in E. coli. DegU of L. monocytogenes was overproduced using plasmid pETDegULmo, constructed by cloning a PCR-generated NcoI/XhoI DNA fragment corresponding to the L. monocytogenes degU coding sequence (698 bp; oligonucleotides degUgenF/degUgenR) between the NcoI and XhoI sites of plasmid pET28/16, replacing the stop codon with a XhoI restriction site. This allows the creation of a translational fusion adding six histidine residues to the carboxy-terminus of the protein, while placing expression of the gene under the control of a T7 bacteriophage promoter.
Motility plate assays.
Bacterial swimming was investigated on swim plates as described elsewhere (Kathariou et al., 1995; Knudsen et al., 2004). Single colonies were inoculated in Tryptic Soy Broth (TSB) with 0.25 % agar and incubated at either 25 or 37 °C for 48 h.
Overproduction and purification of DegS and DegU.
Plasmid pETDegULmo was introduced into a BL21 DE3 strain, in which the T7 RNA polymerase gene is under the control of the inducible lacUV5 promoter, which also carries the pREP4 plasmid, allowing coproduction of the GroESL chaperonin in order to optimize recombinant protein solubility (Amrein et al., 1995). The resulting strain was grown in 2 l LB medium at room temperature, expression was induced during the mid-exponential growth phase by addition of 1 mM IPTG, and incubation was continued for 4 h. The DegU protein was then purified using a two-step procedure as follows. Cells were centrifuged at 10 800 g for 30 min and resuspended in one-fiftieth of the culture volume of buffer A (20 mM Tris/HCl, pH 8, 300 mM NaCl, 0.25 % Tween 20). Cells were disrupted by sonication, and cell debris was removed by two consecutive 30 min centrifugation steps at 17 200 g. E. coli crude protein extracts were loaded onto a 0.2 ml Ni-nitrilotriacetic acid (Ni-NTA) agarose column (Qiagen) equilibrated with buffer A. The column was then washed with 10 volumes of buffer B (20 mM Tris/HCl, pH 8, 300 mM NaCl), and the protein was eluted using the same buffer with a linear imidazole gradient (30–500 mM). Fractions were analysed by SDS-PAGE, pooled and dialysed against buffer C (20 mM Tris/HCl, pH 8, 1 mM EDTA) and loaded onto a 0.5 ml DEAE-Sepharose (Pharmacia) column equilibrated with buffer D (20 mM Tris/HCl, pH 8, 1 mM EDTA, 0.5 mM DTT). The protein was then eluted with a 0–1 M linear NaCl gradient. Fractions were pooled and dialysed against buffer E (20 mM Tris/HCl, pH 8, 1 mM EDTA, 200 mM NaCl, 50 %, v/v, glycerol) to remove salt and concentrate the protein solution approximately fourfold. Protein concentrations were determined using the Bio-Rad protein assay (Bradford, 1976).
Biofilm formation assays.
Bacterial attachment and surface growth on polystyrene microtitre plates were studied during growth of L. monocytogenes in freshly prepared MWB minimal medium (Premaratne et al., 1991). Overnight cultures grown in BHI were used to inoculate MWB medium at OD600 0.1, were vortexed briefly, and 200 µl volumes were dispensed into microtitre plate wells, followed by incubation at either 37 or 25 °C for 40 h. The OD600 of each culture was measured to ensure that all cells had reached stationary phase, and the wells were washed five times with PBS and air-dried for 30 min. Biofilms were stained with 0.1 % crystal violet for 30 min (200 µl per well), and the wells were again washed five times with PBS and air-dried. The stained biomass was resuspended for quantification in ethanol/acetone (80 : 20) and A595 was measured. The assay was performed in triplicate.
Extraction of total RNA.
Total RNA was extracted from Listeria cultures grown at either 25 or 37 °C, as previously described (Chastanet et al., 2001; Glatron & Rapoport, 1972), with some minor modifications. Briefly, cells from 40 ml cultures were centrifuged (2 min, 20 800 g) and the cell pellet was resuspended in 1 ml water-saturated phenol. The cell suspension was added to a 2 ml screw-cap microcentrifuge tube containing 0.4 g glass beads (106 µm, Sigma) and 400 µl 2 % Macaloid slurry (Bentone MA, Rheox). Cells were disrupted in a FastPrep cell disintegrator (Bio 101) for 40 s at 4 °C. After centrifugation at 20 800 g for 15 min, the supernatants were extracted twice with 1 volume of phenol/chloroform (1 : 1, v/v), then with 1 volume chloroform. RNA was then precipitated with 2-propanol in the presence of 0.2 M NaCl and resuspended in 50 µl water. RNA concentrations were determined by measuring A260 and samples were stored at –80 °C.
Primer extension reactions.
Total RNA was used as a template for primer extension reactions using a radiolabelled degU-specific oligonucleotide (OSA30, Table 2), as previously described (Chastanet et al., 2001). The corresponding dideoxy chain termination DNA sequencing reactions were carried out by using the same oligonucleotide primer and a PCR-amplified fragment corresponding to the degU (622 bp) upstream region (oligonucleotide pair OSA14/OSA30; Table 2) with the Sequenase PCR product sequencing kit (USB).
RT-PCR reactions.
RT-PCRs were used to show that the degU and yviA genes are cotranscribed, using 22 µg total RNA, oligonucleotides degUmid and yviAmid (100 pmol of each) and the SuperScript One-Step RT-PCR system with RT/Platinum Taq DNA polymerase as recommended by the manufacturer (Invitrogen). The absence of genomic DNA in RNA preparations was verified by omitting the RT/Platinum Taq Mix and substituting 2 U Platinum Taq DNA polymerase in the control reaction. L. monocytogenes genomic DNA was used as a template for the positive control PCR. The cDNA synthesis step was carried out at 50 °C for 30 min, and the subsequent PCR conditions were 5 min at 94 °C for one cycle, followed by 1 min at 94 °C, 30 s at 55 °C, and 1 min at 72 °C for 40 cycles. Agarose gel electrophoresis (1 %) was used to visualize RT-PCR products with a Smartladder DNA molecular mass marker (Eurogentec).
cDNA synthesis and quantitative real-time PCR (qRT-PCR).
RNA samples for qRT-PCR reactions were treated with DNase I using the TURBO DNA-free reagent (Ambion) in order to eliminate residual contaminating genomic DNA. cDNA synthesis and qRT-PCR were then carried out as described previously (Dubrac et al., 2007), using the L. monocytogenes rpoB gene as an internal standard (Schmittgen & Zakrajsek, 2000) and specific oligonucleotide pairs for each gene (see Table 2).
Gel electrophoresis mobility shift assays (EMSAs).
DNA fragments corresponding to the degU (418 bp) and lmo0675 (446 bp) upstream promoter regions and a gmaR intragenic region (463 bp) were generated by PCR with Pwo polymerase (Roche) and oligonucleotide pairs degUupF/degUupR, HD113/HD114 and HD115/HD116, respectively (Table 2). Labelling, DNA binding and gel electrophoresis mobility shift DNA-binding assays were performed as described previously (Derré et al., 1999).
Electron microscopy.
L. monocytogenes strains were grown for 14–16 h at 25 °C in BHI medium with shaking. A drop of bacterial suspension was placed onto a 300-mesh copper carbon-coated grid. The excess was carefully removed and the preparations were negatively stained in 2 % uranyl acetate or in phosphotungstic acid (2 %). Samples were examined at 80 kV with a transmission electron microscope (JEOL 1200EXII), and electron micrographs were recorded using a Mega view charge-coupled device camera (Eloise SARL) (original magnification x20 000).
Virulence assays.
LD50s were determined by intravenously injecting 8-week-old BALB/c mice with 0.3 ml of serial dilutions of L. monocytogenes EGDe and the otherwise isogenic degU mutant (strain LM1001; 103–107 bacteria). Mortality was checked over a 10-day period.
Database comparisons and sequence analysis.
Computations were performed with the SubtiList and ListiList relational databases (http://genolist.pasteur.fr) (Moszer et al., 2002). Sequence comparisons with the GenBank database were accomplished using the National Center for Biotechnology Information BLAST2 (Altschul et al., 1997) web server with the default parameter values provided.
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RESULTS |
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) is also significantly different, despite the presence of an orthologue of the upstream yvyE gene (Fig. 1a). Furthermore, the transcription terminator separating the B. subtilis degU gene from the downstream yviA gene (Kunst et al., 1988) is not present in L. monocytogenes, in which the degU–yviA intergenic region is much shorter (20 bp compared with 97 bp in B. subtilis), strongly suggesting that the two genes are cotranscribed in L. monocytogenes. To verify that this was indeed the case, total RNA was isolated from L. monocytogenes EGDe cells growing exponentially in BHI medium at 37 °C (OD600 0.6) and used in RT-PCR experiments (see Methods) using oligonucleotides degUmid and yviAmid, designed to hybridize within the degU and yviA coding sequences and amplify a 879 bp fragment encompassing the intergenic region (Table 2). As shown in Fig. 1(b), the corresponding DNA fragment was amplified successfully either from total RNA following treatment with reverse transcriptase (lane 2) or from genomic DNA (lane 4), but was not amplified from RNA samples that were not treated with reverse transcriptase (lane 3), indicating that degU and yviA form an operon in L. monocytogenes although they are not cotranscribed in B. subtilis. In order to investigate the function of the degU and yviA genes, we constructed the LM1001 (degU) and LM1010 (yviA) mutant strains of L. monocytogenes EGDe, in which the entire coding sequences of the genes were removed (see Methods). While this work was in progress, two independent reports described the inactivation of degU in L. monocytogenes strain EGD (Knudsen et al., 2004; Williams et al., 2005a). However, there are significant differences between strain EGD and the strain whose genome sequence is available, EGDe. Indeed, strain EGDe is more virulent than strain EGD and also differs significantly in its surface protein profile (O. Dussurget, unpublished observations).
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degU) during mid-exponential growth in BHI medium at 37 °C and used for primer extension experiments. The nucleotide sequence of the region preceding the degU transcription start site revealed appropriately spaced potential –10 and –35 regions sharing strong similarities with the consensus sequences of promoters recognized by the vegetative form of RNA polymerase, EA (Fig. 2a). Interestingly, the degU gene transcript of L. monocytogenes is preceded by a fairly long untranslated region (UTR) of 261 nt, whereas the UTR of the B. subtilis degS–degU operon is only 116 bases. We did not detect any potential ORFs within this UTR.
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degU), in which the degU coding sequence had been removed. Transcription from the degU promoter was found to be increased systematically in the degU mutant, indicating that DegU negatively regulates its own synthesis (Fig. 2b). This was confirmed by qRT-PCR analysis (data not shown).
Purification of DegU
In order to determine whether DegU binds directly to its own promoter region, the L. monocytogenes DegU protein was overproduced by cloning its coding sequence in plasmid pET28/16, placing the gene under the control of an inducible T7 bacteriophage promoter and creating a translational fusion that adds a carboxy-terminal extension containing six histidine residues, and was purified by immobilized metal affinity chromatography (IMAC) using an Ni-NTA agarose column (see Methods). As described for the B. subtilis DegU regulator (Hamoen et al., 2000), DegU of L. monocytogenes was found to be associated with E. coli chromosomal DNA following purification by IMAC, which interfered with its ability to bind DNA in EMSA experiments (data not shown). The IMAC step was therefore followed by a second affinity chromatography purification using DEAE Sepharose (see Methods). As shown in Fig. 3, the Listeria DegU protein was obtained with a purity greater than 95 % and displayed the expected apparent molecular mass of 
26.8 kDa (Fig. 3, lane 4).
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, DegU bound specifically to the radiolabelled fragment, leading to progressive displacement of the probe to a higher-molecular-mass protein–DNA complex (Fig. 4a, lanes 1–4). As DegU concentrations were progressively increased, two additional larger protein–DNA complexes were observed (Fig. 4a, lanes 5 and 6), suggesting the existence of up to three binding sites with different affinities in this promoter region. These results demonstrate that DegU of L. monocytogenes represses its own synthesis by binding directly to the degU promoter region.
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) with respect to the transcription initiation site, in agreement with the different sized protein–DNA complexes observed in the gel mobility shift DNA-binding assay (Fig. 4a). All three binding sites either overlapped or were downstream from the transcription initiation site, in agreement with the role of DegU in repressing expression from this promoter. DegU appears to display low affinity for region three, as it only interacted with this sequence at high DegU concentrations, as seen in the EMSA and DNase I experiments (Fig. 4a, lane 6, b, lanes 5–7). We observed the appearance of DNase I hypersensitive sites within the regions protected by DegU, suggesting that the DNA undergoes bending once the regulator is bound (Fig. 4b).
Close inspection of the three regions protected by DegU failed to reveal any direct or inverted repeat sequences; however, all three binding sites contained a conserved pentanucleotide motif GTAA T/G on either strand, which may be involved in recognition by DegU (Fig. 4b). These results indicate that DegU negatively regulates degU expression by binding directly to its operator sequence in the promoter region. These data constitute the first demonstration, to our knowledge, of direct binding of the DegU regulator to a promoter region in L. monocytogenes.
DegU is required for growth in RPMI 1640 synthetic medium, growth at high temperature, and motility and flagellar synthesis
DegU is known to be a highly pleiotropic regulator in B. subtilis, playing a central role in a signal transduction network that controls stationary phase adaptive responses, including motility, competence and degradative enzyme production (Msadek, 1999). A phenotypic analysis of the degU strain LM1001 was therefore undertaken to verify that DegU is also a pleiotropic regulator in L. monocytogenes, and the mutant was found to be deficient for growth in RPMI 1640 synthetic medium and in BHI when grown at 44 °C (Fig. 5a, b). DegU is known to control motility in B. subtilis and has also been reported to affect motility in L. monocytogenes strain EGD (Knudsen et al., 2004; Msadek et al., 1995; Williams et al., 2005a, b). As shown in Fig. 5(c) using a soft agar swim plate assay, DegU is also required for motility of strain EGDe.
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degU mutant strain was complemented by the introduction of an intact copy of the gene on a multicopy plasmid, resulting in strain LM1003 (degU pMK4-degU). Strains EGDe, LM1001 and LM1003 were observed by transmission electron microscopy, revealing the loss of flagella in the LM1001 degU mutant and restored flagellar synthesis in the complemented strain LM1003 (Fig. 6a).
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, expression of the flaR gene, encoding a repressor of flagellar synthesis, was not lowered markedly in the degU mutant, whereas expression of motB as well as gmaR, known to control flaA expression, was abolished in strain LM1001 (degU). As expected, expression of flaA and cheA was abolished in the degU mutant and fully restored in the complemented strain LM1003 (Fig. 6c).
DegU binds specifically to the gmaR promoter region
GmaR was described recently as an antirepressor of MogR, playing an important role in the control of flagellar synthesis and motility (Shen et al., 2006). GmaR is the 14th gene in the motB operon. In order to test whether DegU controls gmaR expression directly, a 446 bp radiolabelled DNA fragment corresponding to the gmaR promoter region, upstream from the lmo0675 gene, was generated by PCR using oligonucleotides HD113/HD114 (Table 2). The radiolabelled gmaR promoter fragment extends from positions –378 to +68 relative to the translational start site of lmo0675, and was incubated with increasing concentrations of DegU in the presence of an excess of non-specific competitor DNA [1 µg poly-(dI-dC)]. As shown in Fig. 7(a), DegU bound specifically to the radiolabelled fragment, leading to progressive displacement of the probe to a higher-molecular-mass protein–DNA complex (Fig. 7a, lanes 1–4). As DegU concentrations were progressively increased, two additional larger protein–DNA complexes were observed (Fig. 7a, lanes 5 and 6), suggesting the existence of multiple binding sites, as observed for the degU promoter region. DegU did not bind, however, under the same conditions and even at the highest concentrations, to a DNA fragment of approximately the same size (463 bp) corresponding to a region internal to the gmaR gene (Fig. 7b, lanes 1–6), indicating that DegU binding to the gmaR promoter region is specific. This was further demonstrated by cold competitor chase experiments. Indeed, binding of DegU to the gmaR promoter was lost when a 30- to 50-fold excess of unlabelled specific competitor probe DNA was added to the reaction (Fig. 7a, lanes 7 and 8), but not in the presence of a 50-fold excess of unlabelled DNA fragment corresponding to the internal fragment of the gmaR gene (Fig. 7a, lane 9). Thus, DegU binds specifically to its own promoter region as well as to that of the motB gmaR operon.
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, b; Verhamme et al., 2007). In order to examine whether this is also true in L. monocytogenes, strains EGDe, LM1001 (degU) and LM1003 (degU/pMK4degU) were grown on microtitre plates in MWB medium at 25 or 37 °C. Adherent cells were washed with PBS and stained with crystal violet (see Methods), and the mean OD595 values were calculated from replicate experiments. As shown in Fig. 8, the degU mutant formed less biofilm at 25 °C than the parental strain EGDe, and biofilm formation was restored in the complemented strain LM1003.
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degU mutant in a murine model. Virulence of the degU mutant was assayed by intravenous injection as described in Methods, and compared with that of the wild-type EGDe strain. The LD50 of the degU mutant was 4.2x104 c.f.u., whereas that of EGDe was 3.9x103 c.f.u. The degU mutant thus displays a significant decrease in virulence (11-fold). We followed the survival of mice over a 10-day post-inoculation period. Mice infected with the EGDe strain began to die after 2.5 days and were all dead after 4 days, whereas 80 % of the animals infected by strain LM1001 (degU) were still alive after 10 days (Fig. 9). These results clearly show that degU plays a significant role in the pathogenicity of L. monocytogenes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). During a systematic investigation of L. monocytogenes TCSs, our interest was prompted by the fact that although an orthologue of DegU (63 % amino acid sequence identity) is present in this intracellular pathogen, the gene encoding the cognate DegS kinase is missing. Since the B. subtilis system is known to function as a molecular switch, with the phosphorylated and unphosphorylated forms of the response regulator required for expression of distinct sets of genes, the absence of the DegS kinase in L. monocytogenes was intriguing, and suggested that this orphan response regulator was also active in its unphosphorylated form.
An investigation of the role of DegU in Listeria allowed us to show that, in contrast to the situatoin in B. subtilis, DegU negatively regulates its own synthesis by binding to its own promoter. Indeed, in B. subtilis DegU is known to positively regulate its own synthesis (Kobayashi, 2007b). DNase I footprinting experiments allowed us to define three binding sites for DegU which overlap the promoter region. Although no clear consensus sequence could be determined from the binding sites, they each contain a conserved pentanucleotide sequence 5'-GTAA T/G-3' in either orientation, which may be involved in recognition by DegU. Interestingly, a clear-cut consensus sequence for DegU binding has yet to be defined in B. subtilis as well, in which DNase I footprinting was performed on the comK promoter (Hamoen et al., 2000). Since the DNA recognition helix of the DNA-binding helix–turn–helix domain is invariant between DegU of Bacillus and Listeria, it is likely that they bind to the same DNA sequence, as shown for other orthologous TCSs (Dubrac & Msadek, 2004).
DegU is equally pleiotropic in L. monocytogenes. Indeed, the degU mutant displays many phenotypes, including, as shown here, growth deficiency in RPMI 1640 synthetic medium, inability to grow at high temperatures (44 °C), loss of motility and flagellar synthesis, and a deficiency in biofilm formation. Although we have shown that the degU and yviA genes are cotranscribed in Listeria, in contrast to the situation in Bacillus, YviA does not appear to play a role in the DegU regulatory pathway, since the yviA mutation (strain LM1010) did not affect growth in RPMI 1640 or at high temperature, flagellar synthesis and motility, or biofilm formation (data not shown), in agreement with results for B. subtilis in which a yviA mutant has no obvious phenotype (Henner et al., 1988).
Many factors influence biofilm formation, and we noted that biofilms formed in rich media (BHI) were loosely attached and as a result not easily reproducible, whereas when cells were grown in MWB minimal medium adherence was much stronger. It has been reported recently that flagella are essential for biofilm formation in L. monocytogenes (Lemon et al., 2007). Nevertheless, our results suggest that the role of DegU in biofilm formation is not only due to its effect on flagellar synthesis. Indeed, Listeria is capable of forming biofilms at 37 °C, even though flagella are not expressed at the host temperature. Whereas no difference was observed in biofilm formation at 37 °C between the EGDe parental strain and the degU mutant when cells were grown in MWB minimal medium, we observed lowered biofilm formation by the mutant strain when cells were grown at 37 °C in BHI medium, although cells are loosely attached when grown in this medium (data not shown). This observation could suggest a role for DegU in biofilm formation that is at least in part distinct from its role in controlling flagellar synthesis. Indeed, the L. monocytogenes degU mutant is unable to grow in the presence of 5 % ethanol (Knudsen et al., 2004) or in RPMI 1640 minimal medium as shown here, suggesting that DegU is also involved in other cell processes.
Regulation of flagellar motility in Listeria is quite different from that in Bacillus, particularly since there is no D secondary sigma factor dedicated to the expression of motility, chemotaxis, flagellar synthesis and autolysin genes in Listeria. FlaR is a regulator known to control expression of flaA, which encodes flagellin, activating its expression at 25 °C and repressing it at 37 °C (Sanchez-Campillo et al., 1995). However, it appears that DegU does not have a significant effect on flaR expression at 25 or 37 °C, suggesting that it does not act through this regulator. It has been shown recently that DegU controls expression of gmaR, which encodes a bifunctional O-GlcNac transferase that regulates flagellar motility by acting as an anti-repressor of MogR, a repressor of most chemotaxis and motility genes (Grundling et al., 2004; Shen & Higgins, 2006; Shen et al., 2006). We have shown by qRT-PCR that transcription of mogR is not regulated by DegU at either 24 or 37 °C (data not shown). It has been suggested that DegU either directly or indirectly activates gmaR expression, which antagonizes MogR repression activity to restore the expression of flagellar synthesis and motility genes (Shen & Higgins, 2006; Shen et al., 2006).
We show here that DegU binds directly to the promoter region of the motB–gmaR operon, indicating that it likely controls gmaR expression directly. In B. subtilis, DegU has been shown to bind directly to the promoter region of the fla–che operon, which also contains the sigD gene encoding the D secondary sigma factor (Amati et al., 2004). An excess of phosphorylated DegU represses expression of sigD in B. subtilis, and thus leads to loss of flagellar synthesis and motility, yet expression of sigD is only lowered approximately twofold in a degU mutant and cells are fully motile, in contrast to L. monocytogenes (Msadek et al., 1993; Tokunaga et al., 1994). The situation in Listeria is reminiscent of that described in the so-called undomesticated strains of B. subtilis, known to differ significantly in their phenotypes and regulatory pathways from laboratory strains that derive from strain 168, which was originally subjected to repeated rounds of UV and X-ray mutagenesis (Branda et al., 2001; Burkholder & Giles, 1947). Indeed, in contrast to laboratory strains, a degU mutation in the undomesticated strain of B. subtilis ATCC 6051, also known as NCIB 3610, has been reported to lead to loss of flagellar synthesis, much as in Listeria (Kobayashi, 2007a). However, this finding is contradicted by a recent report in which the authors claim that the same mutation in the same strain background does not affect flagellar-based motility (Verhamme et al., 2007).
There are several interesting parallels between the DegU regulatory pathways of Bacillus and Listeria. Indeed, in B. subtilis, DegU acts in the competence regulatory pathway by assisting the ComK regulator to act as an antirepressor of the Rok and CodY repressors to allow expression of its own gene, leading into a positive feedback loop, which allows expression of competence genes and formation of the competence bistability state (Hamoen et al., 2000; Smits et al., 2005, 2007). Interestingly, GmaR acts as an antirepressor of MogR, and DegU both activates GmaR synthesis and acts as an indirect antagonist of MogR (Shen & Higgins, 2006; Shen et al., 2006). It is therefore intriguing to note that the ComK-binding site sequence is AAAA-N5-TTTT (Hamoen et al., 1998, 2000), which is identical to the sequence on the antiparallel strand that is bound by MogR, TTTT-N5-AAAA (Shen & Higgins, 2006; Shen et al., 2006). It is therefore tempting to speculate that DegU may be acting in a similar manner in the two systems, by assisting in an antirepressor mechanism, which will likely be the subject of further investigation. Although many orthologues of Bacillus competence genes are present in Listeria (Borezée et al., 2000), no link could be established between competence gene expression and DegU.
In agreement with previous results obtained with an L. monocytogenes degU mutant in the EGD background and administered orally or intraperitoneally (Knudsen et al., 2004; Williams et al., 2005a, b), we show here that in the EGDe background virulence is attenuated when the mutant is injected intravenously into mice. This virulence defect cannot be attributed solely to the lack of flagellar synthesis, since the flaA and cheA mutants have little or no effect on bacterial virulence in the murine model (Bigot et al., 2005; Dons et al., 2004; Way et al., 2004), although it has been reported that flagella influence Listeria pathogenicity soon after oral ingestion in a murine model (O'Neil & Marquis, 2006). However, since flagellar synthesis genes are not expressed at the host temperature of 37 °C, the specific role of DegU in virulence remains to be established.
It has been shown recently that DegU is required for biofilm and pellicle formation as well as multicellular behaviour in B. subtilis undomesticated strains (Kobayashi, 2007a, b; Verhamme et al., 2007), and that gradual increases in DegU phosphorylation levels are critical in the transition from motile to sessile biofilm-forming cells. DegU is also known to affect biofilm formation in some B. subtilis laboratory strains (Stanley & Lazazzera, 2005). Given our results that link DegU not only with motility but also with biofilm formation in L. monocytogenes, and the fact that the DegS kinase is lacking in this bacterium, we are currently investigating the role of DegU phosphorylation in Listeria and the importance that it may have in the many different phenotypes linked to this pleiotropic regulator.
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ACKNOWLEDGEMENTS |
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Edited by: J. M. van Dijl
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REFERENCES |
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Received 15 February 2008;
revised 4 May 2008;
accepted 7 May 2008.
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); LM1001 (
); LM1003 (
). (b) Growth defect of the 
