The Response Regulator CroR Modulates Expression of the Secreted Stress-Induced SalB Protein in Enterococcus faecalis
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《细菌学杂志》
USC INRA 2017 Microbiologie de l'Environnement, EA 956, IRBA, Universite de Caen, 14032 CAEN cedex, France
ABSTRACT
The Enterococcus faecalis two-component signal transduction system CroRS, also referred as the RR-HK05 pair, is required for intrinsic -lactam resistance (Y. R. Comenge, R. Quintiliani, Jr., L. Li, L. Dubost, J. P. Brouard, J. E. Hugonnet, and M. Arthur, J. Bacteriol. 185:7184-7192, 2003) and is also suspected to be involved in the expression of salB (previously referred to as sagA), a gene important for resistance to environmental stress and cell morphology (Y. Le Breton, G. Bol, A. Benachour, H. Prevost, Y. Auffray, and A. Rince, Environ. Microbiol. 5:329-337, 2003). In this report, we provide genetic and biochemical evidence that salB encodes a secreted protein that is expressed from a monocistronic stress-inducible operon. Consistent with CroR being a direct transcriptional activator of the salB expression, CroR was found to bind to the salB promoter region in electrophoretic mobility shift assays. Interestingly, we provide evidence that SalB does not play a role in the intrinsic -lactam resistance associated with CroRS. We also show that the CroRS system is able to regulate its own expression. The sequence of the CroRS binding site in the salB and croR promoter regions was determined using DNase I footprinting assays.
INTRODUCTION
Enterococci have been traditionally considered commensal inhabitants of the gastrointestinal tract of human and animals and able to colonize a large range of ecological niches. Their presence in food products has been regarded as an indicator of insufficient sanitary quality (14), even though they contribute in some cases to flavor development of European dairy products (9). Enterococci were also regarded as low-grade pathogens, involved in few cases of food poisoning, mainly due to production of biogenic amines (7, 14). Over the last two decades, however, they have emerged as significant opportunistic pathogens in intensive care units, particularly affecting immunosuppressed patients (13, 15). Enterococcus faecalis is the most common species involved in these enterococcal nosocomial infections. The mechanisms by which this bacterium is able to cross the barrier from inoffensive commensal to major hospital-acquired pathogen are still not well understood. Virulence traits carried principally by mobile genetic elements have been described previously (13, 34), but intrinsic physiological properties of E. faecalis, such as its exceptional stress response capacities (29) and inherent antibiotic resistance (21), may also provide an advantage during the infection process as they do for other opportunistic pathogens (39).
The recent availability of the E. faecalis V583 genome sequence (28) provides a tool for the identification of potential E. faecalis regulatory components, such as alternative sigma factors (2), transcriptional regulators (41, 42), and two-component systems (6, 17, 25, 37), that could be involved in the infection process.
Many aspects of bacterial physiology are under the control of two-component systems (for reviews, see references 18, 19, and 36). These regulatory pathways involve an archetypical mode of signal transduction based on a phosphotransfer from a stress-activated sensor histidine-kinase to its cognate response regulator (35). The response regulator then adjusts gene expression in order for the cell to respond to the signal that initiates the process. Inactivation of two-component system genes in different E. faecalis strains (6, 17, 25, 37) failed to uncover a general role for these systems, largely due to heterogeneity within the pool of two-component pathways present in the different E. faecalis strains studied. Nevertheless, some E. faecalis two-component systems have been shown to be involved in virulence (37), biofilm formation (17), intrinsic antibiotic resistance (6, 17), and/or stress responses (6, 17, 25, 37).
Among the systems analyzed, the CroRS two-component system ("ceftriaxone resistance") (6), previously referred as the RR-HK05 pair by Hancock and Perego (16), appears to be common to several E. faecalis strains (16, 25, 37). The CroRS system, composed of the transmembrane CroS sensor histidine-kinase and the OmpR-family CroR response regulator, is essential for intrinsic -lactam antibiotic resistance (6). The signal recognized by CroS remains unknown, but evidence suggests that it could be related to cell wall perturbations (6). The physiological role of the CroRS system also remains unclear. In a previous study, we noted that a croR mutation causes defects in cell morphology and growth (25). These phenotypical features are similar to those we described for a mutation in salB (previously referred as sagA), a gene whose product is likely involved in cell morphology and stress resistance (23). Additionally, salB expression is altered in a croR mutant, suggesting that the CroRS two-component system could be involved in the regulation of salB (25).
In the present study, we found genetic and biochemical evidence that SalB is a stress-activated secreted protein. We then investigated the role of the CroRS two-component system in the regulation of the salB gene in E. faecalis JH2-2 and showed using electrophoretic mobility shift assays (EMSA) that CroR is able to bind specifically to the salB promoter. However, we demonstrated that the intrinsic -lactam resistance associated with the CroRS system cannot be attributed to the lack of SalB. Additional experiments provided evidence that CroRS autoregulates its own expression, and DNA-binding sites of CroR on these first targets were determined.
MATERIALS AND METHODS
Bacterial strains, plasmids, and growth conditions. E. faecalis strain JH2-2 (22, 43), a derivative err05 mutant (affected in croR) (25), and a derivative salB mutant were used in this study. Cultivation was performed using M17 medium (40) supplemented with 0.5% glucose (GM17) or using brain heart infusion medium at 37°C without shaking. When necessary, erythromycin was added at a concentration of 150 μg ml–1. Escherichia coli strains DH5 (32), XL1Blue (Stratagene, La Jolla, CA), and M15(pREP4) (QIAGEN, Valencia, CA) were cultivated under conditions of vigorous agitation at 37°C in LB medium (32) with ampicillin (100 μg ml–1) or kanamycin (25 μg ml–1) when required. MICs of cell wall-active antimicrobials (D-cycloserine, cefotaxime, and ampicillin) were determined with 105 CFU per spot on GM17 agar after 48 h of incubation.
Construction of a salB mutant by homologous recombination. A 444-bp internal fragment of the salB gene was first amplified by PCR using oligonucleotides SalB1 and SalB2 (Fig. 1A; Table 1), digested with SphI and EcoRI, and cloned into these sites in the insertional vector pUCB300. The resulting plasmid, obtained after transformation of E. coli XL1Blue, was introduced into E. faecalis JH2-2. Integrations by single-crossover recombination within salB in erythromycin-resistant colonies were verified by PCR and Southern blot hybridization.
Overexpression and purification of the H6-CroR protein. A QIAexpress system (QIAGEN, Valencia, CA) was used for the expression of a six-His-tagged CroR recombinant protein (H6-CroR) as follows. First, the croR gene was amplified by PCR using primers CroRN and CroRC (Table 1). The PCR product was then digested using SphI and SalI endonucleases and cloned into the SphI and SalI sites of the pQE-30 plasmid (QIAGEN). The resulting plasmid, pQCroR, was then introduced in E. coli M15(pREP4) cells (QIAGEN). Overexpression of the H6-CroR protein was induced by adding 0.5 mM IPTG (isopropyl--D-thiogalactopyranoside) to the culture and performing an additional 2-h incubation. Cells were then harvested, washed twice in buffer I (50 mM Tris-HCl, pH 7.5; 50 mM Na2SO4; 15% glycerol), resuspended in 5 ml of buffer I, and finally lysed using a cell disrupter (One Shot; Constant Systems, Daventry, England). The H6-CroR protein was then purified by immobilized metal affinity chromatography from the cell lysate by use of nickel-nitrilotriacetic acid resin (QIAGEN, Valencia, CA), followed by desalting on a PD-10 column (Amersham Biosciences, Piscataway, NJ). Protein concentrations were determined using a method described by Lowry et al. (26).
EMSA. DNA promoter regions were PCR amplified in the presence of 2 μCi [-32P]dATP. The H6-CroR protein was diluted in protein buffer (20 mM Tris-HCl, pH 7.5; 2 mM dithiothreitol [DTT]; 50 mM acetyl phosphate). A 10-μl volume of protein was then added to 10 μl of 2x dilution buffer [40 mM Tris-HCl, pH 7.5; 2 mM CaCl2; 2 mM DTT; 20 μg ml–1 of poly(dI-dC); 0.2% bovine serum albumin] containing 2.5 ng of DNA. The mixture was incubated for 15 min at room temperature, after which 10 μl of 30% glycerol was added. The products of the reaction were then separated by electrophoresis onto 12.5% polyacrylamide gel and analyzed by autoradiography.
Footprinting experiments. DNase I footprinting assays were performed with a CEQ8000 automated capillary DNA sequencer (Beckman Coulter) using a method based on that previously described by Yindeeyoungyeon and Schell (45). The PsalB (299 bp) and PcroRS (358 bp) DNA fragments amplified with primers PsalB1 and PsalB2 and primers Pcro1 and Pcro2, respectively (Table 1), were cloned into in the pGEM-T easy vector by use of a pGEM-T easy vector system (Promega, Madison, WI) as recommended by the supplier. Then, a labeled DNA fragment was obtained by PCR amplification using vector-designed oligonucleotides PU and D4-PR (D4-PR corresponds to the PR oligonucleotide labeled at the 5' end with Beckman dye D4; Table 1) or oligonucleotides PR and D4-PU and purified using a QIAquick PCR purification kit (QIAGEN). The binding reactions were carried out at room temperature for 5 min in 70-μl reaction volumes containing 33 mM Tris-HCl (pH 7.5), 2 mM CaCl2, 2 mM DTT, 20 μg ml–1 poly(dI-dC), 0.02% of bovine serum albumin, 103 ng H6-CroR, 250 ng of labeled DNA fragment, and 1.5 μl of BeF3– (a component used to mimic effect of phosphorylation) (44) and generated by mixing NaF and BeCl2 at final concentrations of 0.187 M and 2.27 mM, respectively). The DNase treatment was then performed by addition of 70 μl of a solution containing 80 mM Tris-HCl (pH 7.5), 12 mM MgCl2, and 250 U of DNase I (Amersham Biosciences) and incubation for 1 min at room temperature. The reaction was stopped by addition of 35 μl of 25 mM EDTA and incubation for 5 min at 94°C. DNA was then purified using a QIAquick PCR purification kit (QIAGEN), precipitated by addition of 10% (vol/vol) 100 mM EDTA-10% (vol/vol) 3 M sodium acetate (pH 3.2)-5% (vol/vol) glycogen-3 volumes of ethanol (100%) and resuspended in 40 μl of SLS buffer (Beckman Coulter) before capillary electrophoresis was performed using a CEQ8000 sequencing apparatus (Beckman Coulter). The determination of the DNA sequence of the protected region was performed after comigration of the footprinting assay and the corresponding sequence reaction.
Extraction and separation of secreted proteins. Proteins secreted from E. faecalis JH2-2 and mutants croR and salB were prepared as follows. Cultures were grown to mid-log phase (optical density at 600 nm = 0.5) in 50 ml of brain heart infusion medium. Cells were removed by two rounds of centrifugation for 10 min at 3,220 x g. The supernatant was then incubated for 1 h at 37°C with Benzonase (100 U l–1) (Merck) to digest nucleic acids. The supernatant was next mixed with tricholoro-acetic acid to a final concentration of 7% and further incubated at 4°C for 30 min. After a centrifugation at 10,000 x g for 10 min, the precipitated proteins were washed with cold acetone and, after an ultimate centrifugation, resuspended in sodium dodecyl sulfate (SDS) gel loading buffer (32). Proteins were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (10% acrylamide).
Mass spectrometry analysis. The peptides of interest were extracted from electrophoresis gel essentially as described by Sauvageot et al. (33) and analyzed with electrospray ionization tandem mass spectrometry. These analyses were carried out with an electrospray ion trap mass spectrometer (LCQ DECA XP; ThermoFinnigan) coupled on line with a high-pressure liquid chromatography apparatus (Surveyor LC; ThermoFinnigan). The peptides were separated by reverse-phase high-pressure liquid chromatography on a C8 capillary column (HyPurity C8) (150 by 0.5 mm), with the parameters set as previously described (33). TurboSEQUEST software was used to compare the amino acid sequences obtained to those of the proteome of E. faecalis strain V583 available at the Internet site of The Institute of Genomic Research (http://www.tigr.org).
Transcriptional analysis. Total RNA of E. faecalis was isolated using an RNeasy Midi kit (QIAGEN, Valencia, CA). Northern blots of 10 μg RNA normalized by optical density measurement at 260 nm were prepared by using Hybond-N+ membranes (Amersham Biosciences, Piscataway, NJ) and standard procedures (32). Dot blots of 2 μg RNA were prepared using a method previously described by Rince et al. (31) after analysis of RNA with an Experion automated electrophoresis station (Bio-Rad) for normalization and verification of the absence of RNA degradation. Membrane-bound nucleic acids were hybridized to single-strand-labeled probes at 55°C in 1 M sodium phosphate buffer (pH 7.0) containing 5% SDS. After hybridization, membranes were washed twice in 2x SSC (1xSSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% SDS (10 min) and then twice in 0.5x SSC-0.1% SDS (10 min) at 55°C and exposed to a storage phosphor screen (Packard Instrument Company, Canberra, FL) for 5 h.
A single-strand-labeled probe was obtained as follows: first, a DNA fragment was PCR amplified using E. faecalis JH2-2 chromosomal DNA as a template and primer pairs SalB1 and SalB2 (461 bp) (Table 1). The probe was then synthesized by elongating one oligonucleotide (SalB2) with Taq DNA polymerase, 2 μM concentrations of dCTP, dGTP, and dTTP, 2 μCi of [-32P]dATP, and 10 ng of the previously obtained PCR DNA fragment as a template. Thirty cycles of 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C were performed.
The 5' end of salB mRNA was mapped from a 5'-RACE (5'-rapid amplification of cDNA ends) PCR product obtained with a 3'/5'-RACE kit (Roche Molecular Biochemicals) using primer SalBRace for cDNA synthesis from total RNA and PsalB2 for DNA sequencing.
General methods. Restriction endonucleases, alkaline phosphatase, and T4 DNA ligase were obtained from Amersham International, Promega (Promega, Madison, WI), and Roche Applied Science (Roche, Indianapolis, IN) and used according to the manufacturers' instructions. PCR was carried out in a reaction volume of 25 μl using 5 ng of chromosomal DNA of E. faecalis strains and Taq DNA polymerase from Amersham International. The annealing temperature was 5°C below the melting temperature of the primers; 30 cycles were performed, and PCR products were purified using a QIAquick PCR purification kit (QIAGEN). E. coli and E. faecalis were transformed using a Gene Pulser apparatus (Bio-Rad Laboratories, Richmond, CA) as described by Dower et al. (8) and Holo and Nes (20), respectively. Plasmids were purified using a QIAprep Miniprep kit (QIAGEN). DNA and amino acid sequences were analyzed using Mac Vector software (Kodak; Scientific Imaging Systems, New Haven, CT), and databases searches were performed with the BLAST program (1). Other standard techniques were carried out as described by Sambrook et al. (32).
RESULTS
Genetic organization of the salB locus. We previously identified the salB gene during screening for stress response mutations following random mutagenesis of E. faecalis JH2-2 (23). The salB mutant displayed enhanced sensitivity toward different stress challenges (sensitivity to NaCl, heat shock, SDS, ethanol, H2O2, alkaline, or acid pH) as well as altered cell shape and septation anomalies.
The 1,350-nucleotide salB sequence is preceded by two open reading frames (ORFs), EF0392 and EF0393, encoding proteins of unknown function; the EF0395 gene, located downstream of salB, encodes a 42.2-kDa protein homologous to methionine synthetase (Fig. 1A). To determine the size of the salB transcription unit, Northern blot hybridizations were performed using total RNA extracted from exponentially grown E. faecalis strain JH2-2 and a DNA probe complementary to the salB gene (Fig. 1C). A 1.4-kb unique transcript, similar in size to the salB ORF itself, was detected in the assay, suggesting that the salB gene is transcribed independently of the surrounding genes. Consistent with this result, we also identified 24 nucleotides downstream of the salB stop codon (TAA), an invert-repeat sequence that likely corresponds to a Rho-independent terminator (G = –18.6 kcal mol–1) (Fig. 1A). RNA extracted from exponentially growing E. faecalis JH2-2 cells was then used to map the salB transcriptional start site by 5'-RACE PCR. The resulting electropherogram presented in Fig. 1B localizes the transcriptional start point (+1) of the salB gene at a site 49 nucleotides upstream of a potential ribosome-binding site (GGAGGA). Seven base pairs upstream of the transcriptional start site lies a A-like promoter element with a putative –10 box (TAGAAT) separated by 19 nucleotides from a putative –35 box (TTGCTT) (Fig. 1A). These results strongly suggest that salB belongs to a monocistronic operon that is transcribed in mid-exponential phase from a promoter recognized by RNA polymerase carrying the housekeeping sigma factor A.
Previous data showed that a salB mutant was more sensitive to several stress conditions, including heat shock and hyperosmotic stress (23). This prompted us to test whether salB expression could be influenced by stress. Northern blot hybridization of total RNA extracted from E. faecalis cells exposed for 10 min to different culture conditions (the presence of NaCl, 50°C, acid pH) demonstrated that the level of salB mRNA increases when E. faecalis cells are subjected to an osmotic stress or a high temperature challenge (Fig. 1C). The stress inducibility of salB is consistent with the anticipated role of its product as a protein that has a protective function needed when the cell encounters detrimental environmental situations. The effect of the inactivation of croR on the induction of salB was tested by RNA hybridization (Fig. 1D). Dot blot hybridization of total RNA extracted from E. faecalis cells exposed for 5, 10, or 20 min to 0.3 M NaCl or to 50°C showed that CroR is required for salB induction.
salB encodes a secreted protein. The salB sequence encodes a protein of 449 amino acids (aa) (Fig. 2) with a calculated molecular mass of 47.3 kDa and an estimated pI of 4.7. The first 27 amino acids contain the elements found in gram-positive signal peptides. Cleavage after residue 27 by a signal peptidase would result in a secreted SalB protein of 44.5 kDa. Homology searches revealed that the N-terminal portion of SalB (from aa 1 to aa 260) is similar to the N-terminal portions of the proteins SagA (also referred as P60) and P54 from Enterococcus faecium (10, 38) and SagBb from Enterococcus hirae (F. Teng, B. E. Murray, and G. M. Weinstock, unpublished data) and the SalA antigen (putative secreted lipase) from E. faecalis (28). SalB also shares homology with the N-terminal portion of PcsB-like proteins from different species of streptococci such as Streptococcus mutans GbpB (4, 5, 27) (Fig. 2). Interestingly, SagA, P54, GbpB, and a number of "PcsB-like" proteins have been shown to be associated with the cell wall or secreted into the growth medium. At least some of these proteins appear to be involved in cell wall hydrolysis and in the regulation of cell wall biosynthesis (4, 10, 27, 38). The central domain of these proteins, as with that of SalB (aa 261 to aa 341), presents an unusual composition: 46 amino acids out of 81 of this SalB segment are serine, threonine, or proline. In its C-terminal portion, SalB shares significant homology with proteins deduced from the Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 genome (accession no. ZP00063637 and ZP00063635) containing Lys7 domains, which are usually found in proteins that bind to cell wall peptidoglycan (3).
We extracted and separated through SDS-PAGE the secreted proteins from the E. faecalis JH2-2 wild-type strain and from the salB and err05 mutant strains (Fig. 3). Results showed that the salB and err05 mutants were missing a protein that migrated between 50- and 75-kDa molecular mass markers. Mass spectrometry analyses revealed that the missing protein corresponds to SalB (Table 2). The other protein closely migrating with SalB was also identified as the putative secreted lipase SalA (28).
Our results indicate that SalB is a secreted protein. Additionally, the absence of SalB in the media of the strain with the croR mutation is consistent with the notion that the CroRS two-component system is involved in salB expression or secretion.
As previously shown by Comenge et al. (6), a croR mutation led to a decrease in the MICs of cell wall-active antimicrobials (Table 3). In order to investigate whether this phenotype could be attributed to a CroR effect on the SalB protein, we tested the susceptibility of the salB mutant to the cell wall-active antimicrobials. As presented in Table 3, similar resistance levels were observed for the salB mutant and the wild-type strain, showing that the antibiotic sensitivity of the err05 mutant cannot be attributed to the nonexpression of the SalB protein.
CroR binds to the salB promoter region. To test whether the CroR response regulator could bind directly to the salB promoter region, we purified a CroR protein harboring an N-terminal six-histidine tag (H6-CroR) as described in Materials and Methods. The purified protein was visualized on a Coomassie-stained SDS-PAGE gel (Fig. 4A) and was shown to migrate in accordance with its estimated molecular size of 27 kDa.
A 299-bp DNA fragment containing the promoter region of salB (PsalB) (nucleotide –212 to nucleotide +87 relative to the transcriptional start point) was PCR amplified with primers PsalB1 and PsalB2 (Table 1) and used as a DNA target for EMSA. As shown in Fig. 4B, when the PsalB fragment is incubated with different amounts of H6-CroR in the presence of 50 mM acetyl phosphate, three bands with reduced mobility, designated complex 1 (C1), complex 2 (C2), and complex 3 (C3), were observed. Decreases in the amount of H6-CroR in the binding assay mixture progressively led to a reduction in the amounts of C1, C2, and C3 and a concomitant increase in the amount of free unbound target DNA (Fig. 4B). The binding of H6-CroR to the salB promoter region appeared to be specific in that the addition of unlabeled 299-bp PsalB DNA reduced the abundance of the putative complexes whereas addition of a nonspecific competitor (an unlabeled internal fragment of the salB ORF) did not (Fig. 4B). In keeping with the expectation that it is the phosphorylated form of CroR that is the DNA-binding species, no H6-CroR-dependent retardation of PsalB was observed when the EMSA was performed in the absence of the phosphate donor acetyl-phosphate (data not shown).
DNase I footprinting experiments were then performed in order to determine the sequence of the CroRS binding site in the PsalB DNA fragment. As shown in Fig. 5, H6-CroR protects a 45-bp region extending from positions –1 to +44 relative to the transcription initiation site.
The results of these experiments, taken together with those showing the croR dependency of the SalB expression, support the idea of a direct involvement of the CroRS two-component system in the activation of salB.
The CroRS system regulates its own expression. Autoregulation is a common feature for two-component systems. Comenge et al. (6) suggested that this could also be true for CroRS. The croRS locus is expressed as a bicistronic operon from a unique promoter located upstream of the croR gene (6, 25). A 358-bp DNA fragment containing the promoter region of croRS (PcroRS; nucleotide –133 to nucleotide +125 relative to the transcription initiation site determined by Comenge et al.) (6) was obtained by PCR amplification using the primers Pcro1 and Pcro2 (Table 1) and used for EMSA with different amounts of phosphorylated H6-CroR. As was the case with the DNA fragment containing the salB promoter, slower migrating bands (C1 and C2) were observed whose abundance was specifically reduced by the addition of unlabeled PcroRS DNA (Fig. 4C). The apparent binding of CroR-PO4 to the croR promoter is consistent with autoregulation of the croRS operon.
The DNA sequence of the CroRS binding site in the PcroRS DNA fragment was determined using DNase I footprinting experiments and both DNA strands (Fig. 6). Results revealed a 45-bp common protected region corresponding to positions –87 to –43 relative to the transcription initiation site. Comparison of this DNA sequence to that protected in the salB promoter region revealed three conserved 6- to 7-bp motifs (TTCTAAA, AAAGTT, and GTTTATT) which could be candidates for a CroR recognition sequence.
DISCUSSION
Over the last several years, we investigated the E. faecalis environmental stress response by use of two-dimensional electrophoresis (for reviews, see references 12 and 29) and genetic analyses of E. faecalis strain JH2-2 (11, 23-25, 29-31). The genetic approach allowed us to identify two independent mutants presenting similar growth defects and altered cell morphology (23, 25). Genotypic analysis revealed that one mutation (BS9) lies in the salB gene (previously referred as sagA) (23), encoding a putative secreted protein with unknown function (23). The second mutation mapped to a gene encoding an OmpR-like response regulator (25), recently redesignated CroR (6). Northern blot hybridization experiments revealed impaired salB expression in the err05 mutant, suggesting a possible involvement of the CroR regulator in the control of salB expression (25).
In the present report, the salB gene is shown to be transcribed as a 1.4-kb monocistronic mRNA, the expression of which is induced by environmental stresses such as high osmolarity and temperature. Interestingly, dot blot hybridization experiments also revealed that when the err05 mutant strain is subjected to these two stress conditions, the salB expression is not activated. This result strongly suggests that CroR might be required for salB expression when E. faecalis cells are subjected to high osmolarity and temperature.
We then tested the hypothesis of the direct involvement of CroR in salB regulation, and EMSA experiments gave evidence that the phosphorylated CroR response regulator is able to bind specifically to the salB promoter region. This result, in conjunction to the previous demonstration of the reduced salB expression in the err05 mutant strain lacking croR (25), argues that CroR is likely to be a transcriptional regulator involved in salB activation in E. faecalis JH2-2. Given that CroR can be phosphoactivated by its mated histidine-kinase CroS (6), we can conclude that the CroRS two-component system is involved in the salB regulation. To our knowledge, this result corresponds to the first demonstration of the presence in E. faecalis of a signal transduction pathway involved in the direct regulation of a stress protein.
Inactivation of salB or croR genes confers growth defects and alterations of cell morphology (23, 25), so we can suggest that these phenotypes in the err05 mutant strain may be the consequence of the low level of SalB. The first role for the CroRS system to be described was its involvement in E. faecalis intrinsic -lactam resistance (6), and it was tempting to see whether SalB could actually be involved in -lactam resistance. The sensitivity to cell wall-active antimicrobials was tested, and the results showed that the decreases of the MICs of D-cycloserine, cefotaxime, and ampicillin were observed only in the err05 mutant strain but not in the salB mutant strain, demonstrating that SalB is not involved in the CroRS-associated -lactam resistance.
A previous work showed that a mutation in the E. faecalis salB locus led to cell shape anomalies, nonsymmetrical divisions, and sensitivity to different environmental stresses (23). Here, we show that SalB is a secreted protein with homology to PcsB-like proteins involved in cell wall hydrolysis and the regulation of cell wall biosynthesis. SalB shares homologies with the E. faecalis SalA protein. The latter is also a PcsB-like protein, and chromosomal location of salA is similar to that observed for other pcsB-like genes, as it is located downstream of the mreCD locus, encoding proteins known to play a role in cell shape. In our experiments, we demonstrated that SalA is also a secreted protein. However, in contrast to the results seen with salB, salA seems not to be regulated by the CroRS two-component system.
EMSA analyses also showed that CroR binds to the promoter region of the croRS locus. This result demonstrates the validity of the hypothesis, based on analyses of the activity of a PcroRS-lacZ fusion in croRS mutants (6), that the CroRS two-component system is able to regulate its own expression.
DNase I footprinting experiments allowed determination of the sequences of 45-bp DNA segments protected from DNase I by H6-CroR binding in the promoter region of genes croR and salB. Both protected sequences contain low (<18%) G+C content, and this is also the case for the three 6- to 7-bp motifs common in both protected regions. Thus, we can expect a high occurrence of the CroR recognition sequence in the AT-rich E. faecalis genome and consequently a large number of genes controlled by the two-component system CroRS.
The sensitivity of the err05 mutant strain lacking croR to the cell wall-active antimicrobials cannot be explained solely by the low level of SalB in this strain, and additional target(s) might correspond to genes involved in this phenotype. Experiments are now in progress in our laboratory to identify new genes regulated by CroRS.
ACKNOWLEDGMENTS
Y.L.B. was the recipient of a doctoral fellowship from the "Ministère de la Recherche et de l'Enseignement Superieur", France. C.M. was the recipient of a doctoral fellowship from "INRA" and "Region Basse-Normandie." T. Morin was the recipient of a postdoctoral fellowship from "Region Basse-Normandie."
C.M. and Y.L.B. contributed equally to this work.
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ABSTRACT
The Enterococcus faecalis two-component signal transduction system CroRS, also referred as the RR-HK05 pair, is required for intrinsic -lactam resistance (Y. R. Comenge, R. Quintiliani, Jr., L. Li, L. Dubost, J. P. Brouard, J. E. Hugonnet, and M. Arthur, J. Bacteriol. 185:7184-7192, 2003) and is also suspected to be involved in the expression of salB (previously referred to as sagA), a gene important for resistance to environmental stress and cell morphology (Y. Le Breton, G. Bol, A. Benachour, H. Prevost, Y. Auffray, and A. Rince, Environ. Microbiol. 5:329-337, 2003). In this report, we provide genetic and biochemical evidence that salB encodes a secreted protein that is expressed from a monocistronic stress-inducible operon. Consistent with CroR being a direct transcriptional activator of the salB expression, CroR was found to bind to the salB promoter region in electrophoretic mobility shift assays. Interestingly, we provide evidence that SalB does not play a role in the intrinsic -lactam resistance associated with CroRS. We also show that the CroRS system is able to regulate its own expression. The sequence of the CroRS binding site in the salB and croR promoter regions was determined using DNase I footprinting assays.
INTRODUCTION
Enterococci have been traditionally considered commensal inhabitants of the gastrointestinal tract of human and animals and able to colonize a large range of ecological niches. Their presence in food products has been regarded as an indicator of insufficient sanitary quality (14), even though they contribute in some cases to flavor development of European dairy products (9). Enterococci were also regarded as low-grade pathogens, involved in few cases of food poisoning, mainly due to production of biogenic amines (7, 14). Over the last two decades, however, they have emerged as significant opportunistic pathogens in intensive care units, particularly affecting immunosuppressed patients (13, 15). Enterococcus faecalis is the most common species involved in these enterococcal nosocomial infections. The mechanisms by which this bacterium is able to cross the barrier from inoffensive commensal to major hospital-acquired pathogen are still not well understood. Virulence traits carried principally by mobile genetic elements have been described previously (13, 34), but intrinsic physiological properties of E. faecalis, such as its exceptional stress response capacities (29) and inherent antibiotic resistance (21), may also provide an advantage during the infection process as they do for other opportunistic pathogens (39).
The recent availability of the E. faecalis V583 genome sequence (28) provides a tool for the identification of potential E. faecalis regulatory components, such as alternative sigma factors (2), transcriptional regulators (41, 42), and two-component systems (6, 17, 25, 37), that could be involved in the infection process.
Many aspects of bacterial physiology are under the control of two-component systems (for reviews, see references 18, 19, and 36). These regulatory pathways involve an archetypical mode of signal transduction based on a phosphotransfer from a stress-activated sensor histidine-kinase to its cognate response regulator (35). The response regulator then adjusts gene expression in order for the cell to respond to the signal that initiates the process. Inactivation of two-component system genes in different E. faecalis strains (6, 17, 25, 37) failed to uncover a general role for these systems, largely due to heterogeneity within the pool of two-component pathways present in the different E. faecalis strains studied. Nevertheless, some E. faecalis two-component systems have been shown to be involved in virulence (37), biofilm formation (17), intrinsic antibiotic resistance (6, 17), and/or stress responses (6, 17, 25, 37).
Among the systems analyzed, the CroRS two-component system ("ceftriaxone resistance") (6), previously referred as the RR-HK05 pair by Hancock and Perego (16), appears to be common to several E. faecalis strains (16, 25, 37). The CroRS system, composed of the transmembrane CroS sensor histidine-kinase and the OmpR-family CroR response regulator, is essential for intrinsic -lactam antibiotic resistance (6). The signal recognized by CroS remains unknown, but evidence suggests that it could be related to cell wall perturbations (6). The physiological role of the CroRS system also remains unclear. In a previous study, we noted that a croR mutation causes defects in cell morphology and growth (25). These phenotypical features are similar to those we described for a mutation in salB (previously referred as sagA), a gene whose product is likely involved in cell morphology and stress resistance (23). Additionally, salB expression is altered in a croR mutant, suggesting that the CroRS two-component system could be involved in the regulation of salB (25).
In the present study, we found genetic and biochemical evidence that SalB is a stress-activated secreted protein. We then investigated the role of the CroRS two-component system in the regulation of the salB gene in E. faecalis JH2-2 and showed using electrophoretic mobility shift assays (EMSA) that CroR is able to bind specifically to the salB promoter. However, we demonstrated that the intrinsic -lactam resistance associated with the CroRS system cannot be attributed to the lack of SalB. Additional experiments provided evidence that CroRS autoregulates its own expression, and DNA-binding sites of CroR on these first targets were determined.
MATERIALS AND METHODS
Bacterial strains, plasmids, and growth conditions. E. faecalis strain JH2-2 (22, 43), a derivative err05 mutant (affected in croR) (25), and a derivative salB mutant were used in this study. Cultivation was performed using M17 medium (40) supplemented with 0.5% glucose (GM17) or using brain heart infusion medium at 37°C without shaking. When necessary, erythromycin was added at a concentration of 150 μg ml–1. Escherichia coli strains DH5 (32), XL1Blue (Stratagene, La Jolla, CA), and M15(pREP4) (QIAGEN, Valencia, CA) were cultivated under conditions of vigorous agitation at 37°C in LB medium (32) with ampicillin (100 μg ml–1) or kanamycin (25 μg ml–1) when required. MICs of cell wall-active antimicrobials (D-cycloserine, cefotaxime, and ampicillin) were determined with 105 CFU per spot on GM17 agar after 48 h of incubation.
Construction of a salB mutant by homologous recombination. A 444-bp internal fragment of the salB gene was first amplified by PCR using oligonucleotides SalB1 and SalB2 (Fig. 1A; Table 1), digested with SphI and EcoRI, and cloned into these sites in the insertional vector pUCB300. The resulting plasmid, obtained after transformation of E. coli XL1Blue, was introduced into E. faecalis JH2-2. Integrations by single-crossover recombination within salB in erythromycin-resistant colonies were verified by PCR and Southern blot hybridization.
Overexpression and purification of the H6-CroR protein. A QIAexpress system (QIAGEN, Valencia, CA) was used for the expression of a six-His-tagged CroR recombinant protein (H6-CroR) as follows. First, the croR gene was amplified by PCR using primers CroRN and CroRC (Table 1). The PCR product was then digested using SphI and SalI endonucleases and cloned into the SphI and SalI sites of the pQE-30 plasmid (QIAGEN). The resulting plasmid, pQCroR, was then introduced in E. coli M15(pREP4) cells (QIAGEN). Overexpression of the H6-CroR protein was induced by adding 0.5 mM IPTG (isopropyl--D-thiogalactopyranoside) to the culture and performing an additional 2-h incubation. Cells were then harvested, washed twice in buffer I (50 mM Tris-HCl, pH 7.5; 50 mM Na2SO4; 15% glycerol), resuspended in 5 ml of buffer I, and finally lysed using a cell disrupter (One Shot; Constant Systems, Daventry, England). The H6-CroR protein was then purified by immobilized metal affinity chromatography from the cell lysate by use of nickel-nitrilotriacetic acid resin (QIAGEN, Valencia, CA), followed by desalting on a PD-10 column (Amersham Biosciences, Piscataway, NJ). Protein concentrations were determined using a method described by Lowry et al. (26).
EMSA. DNA promoter regions were PCR amplified in the presence of 2 μCi [-32P]dATP. The H6-CroR protein was diluted in protein buffer (20 mM Tris-HCl, pH 7.5; 2 mM dithiothreitol [DTT]; 50 mM acetyl phosphate). A 10-μl volume of protein was then added to 10 μl of 2x dilution buffer [40 mM Tris-HCl, pH 7.5; 2 mM CaCl2; 2 mM DTT; 20 μg ml–1 of poly(dI-dC); 0.2% bovine serum albumin] containing 2.5 ng of DNA. The mixture was incubated for 15 min at room temperature, after which 10 μl of 30% glycerol was added. The products of the reaction were then separated by electrophoresis onto 12.5% polyacrylamide gel and analyzed by autoradiography.
Footprinting experiments. DNase I footprinting assays were performed with a CEQ8000 automated capillary DNA sequencer (Beckman Coulter) using a method based on that previously described by Yindeeyoungyeon and Schell (45). The PsalB (299 bp) and PcroRS (358 bp) DNA fragments amplified with primers PsalB1 and PsalB2 and primers Pcro1 and Pcro2, respectively (Table 1), were cloned into in the pGEM-T easy vector by use of a pGEM-T easy vector system (Promega, Madison, WI) as recommended by the supplier. Then, a labeled DNA fragment was obtained by PCR amplification using vector-designed oligonucleotides PU and D4-PR (D4-PR corresponds to the PR oligonucleotide labeled at the 5' end with Beckman dye D4; Table 1) or oligonucleotides PR and D4-PU and purified using a QIAquick PCR purification kit (QIAGEN). The binding reactions were carried out at room temperature for 5 min in 70-μl reaction volumes containing 33 mM Tris-HCl (pH 7.5), 2 mM CaCl2, 2 mM DTT, 20 μg ml–1 poly(dI-dC), 0.02% of bovine serum albumin, 103 ng H6-CroR, 250 ng of labeled DNA fragment, and 1.5 μl of BeF3– (a component used to mimic effect of phosphorylation) (44) and generated by mixing NaF and BeCl2 at final concentrations of 0.187 M and 2.27 mM, respectively). The DNase treatment was then performed by addition of 70 μl of a solution containing 80 mM Tris-HCl (pH 7.5), 12 mM MgCl2, and 250 U of DNase I (Amersham Biosciences) and incubation for 1 min at room temperature. The reaction was stopped by addition of 35 μl of 25 mM EDTA and incubation for 5 min at 94°C. DNA was then purified using a QIAquick PCR purification kit (QIAGEN), precipitated by addition of 10% (vol/vol) 100 mM EDTA-10% (vol/vol) 3 M sodium acetate (pH 3.2)-5% (vol/vol) glycogen-3 volumes of ethanol (100%) and resuspended in 40 μl of SLS buffer (Beckman Coulter) before capillary electrophoresis was performed using a CEQ8000 sequencing apparatus (Beckman Coulter). The determination of the DNA sequence of the protected region was performed after comigration of the footprinting assay and the corresponding sequence reaction.
Extraction and separation of secreted proteins. Proteins secreted from E. faecalis JH2-2 and mutants croR and salB were prepared as follows. Cultures were grown to mid-log phase (optical density at 600 nm = 0.5) in 50 ml of brain heart infusion medium. Cells were removed by two rounds of centrifugation for 10 min at 3,220 x g. The supernatant was then incubated for 1 h at 37°C with Benzonase (100 U l–1) (Merck) to digest nucleic acids. The supernatant was next mixed with tricholoro-acetic acid to a final concentration of 7% and further incubated at 4°C for 30 min. After a centrifugation at 10,000 x g for 10 min, the precipitated proteins were washed with cold acetone and, after an ultimate centrifugation, resuspended in sodium dodecyl sulfate (SDS) gel loading buffer (32). Proteins were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (10% acrylamide).
Mass spectrometry analysis. The peptides of interest were extracted from electrophoresis gel essentially as described by Sauvageot et al. (33) and analyzed with electrospray ionization tandem mass spectrometry. These analyses were carried out with an electrospray ion trap mass spectrometer (LCQ DECA XP; ThermoFinnigan) coupled on line with a high-pressure liquid chromatography apparatus (Surveyor LC; ThermoFinnigan). The peptides were separated by reverse-phase high-pressure liquid chromatography on a C8 capillary column (HyPurity C8) (150 by 0.5 mm), with the parameters set as previously described (33). TurboSEQUEST software was used to compare the amino acid sequences obtained to those of the proteome of E. faecalis strain V583 available at the Internet site of The Institute of Genomic Research (http://www.tigr.org).
Transcriptional analysis. Total RNA of E. faecalis was isolated using an RNeasy Midi kit (QIAGEN, Valencia, CA). Northern blots of 10 μg RNA normalized by optical density measurement at 260 nm were prepared by using Hybond-N+ membranes (Amersham Biosciences, Piscataway, NJ) and standard procedures (32). Dot blots of 2 μg RNA were prepared using a method previously described by Rince et al. (31) after analysis of RNA with an Experion automated electrophoresis station (Bio-Rad) for normalization and verification of the absence of RNA degradation. Membrane-bound nucleic acids were hybridized to single-strand-labeled probes at 55°C in 1 M sodium phosphate buffer (pH 7.0) containing 5% SDS. After hybridization, membranes were washed twice in 2x SSC (1xSSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% SDS (10 min) and then twice in 0.5x SSC-0.1% SDS (10 min) at 55°C and exposed to a storage phosphor screen (Packard Instrument Company, Canberra, FL) for 5 h.
A single-strand-labeled probe was obtained as follows: first, a DNA fragment was PCR amplified using E. faecalis JH2-2 chromosomal DNA as a template and primer pairs SalB1 and SalB2 (461 bp) (Table 1). The probe was then synthesized by elongating one oligonucleotide (SalB2) with Taq DNA polymerase, 2 μM concentrations of dCTP, dGTP, and dTTP, 2 μCi of [-32P]dATP, and 10 ng of the previously obtained PCR DNA fragment as a template. Thirty cycles of 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C were performed.
The 5' end of salB mRNA was mapped from a 5'-RACE (5'-rapid amplification of cDNA ends) PCR product obtained with a 3'/5'-RACE kit (Roche Molecular Biochemicals) using primer SalBRace for cDNA synthesis from total RNA and PsalB2 for DNA sequencing.
General methods. Restriction endonucleases, alkaline phosphatase, and T4 DNA ligase were obtained from Amersham International, Promega (Promega, Madison, WI), and Roche Applied Science (Roche, Indianapolis, IN) and used according to the manufacturers' instructions. PCR was carried out in a reaction volume of 25 μl using 5 ng of chromosomal DNA of E. faecalis strains and Taq DNA polymerase from Amersham International. The annealing temperature was 5°C below the melting temperature of the primers; 30 cycles were performed, and PCR products were purified using a QIAquick PCR purification kit (QIAGEN). E. coli and E. faecalis were transformed using a Gene Pulser apparatus (Bio-Rad Laboratories, Richmond, CA) as described by Dower et al. (8) and Holo and Nes (20), respectively. Plasmids were purified using a QIAprep Miniprep kit (QIAGEN). DNA and amino acid sequences were analyzed using Mac Vector software (Kodak; Scientific Imaging Systems, New Haven, CT), and databases searches were performed with the BLAST program (1). Other standard techniques were carried out as described by Sambrook et al. (32).
RESULTS
Genetic organization of the salB locus. We previously identified the salB gene during screening for stress response mutations following random mutagenesis of E. faecalis JH2-2 (23). The salB mutant displayed enhanced sensitivity toward different stress challenges (sensitivity to NaCl, heat shock, SDS, ethanol, H2O2, alkaline, or acid pH) as well as altered cell shape and septation anomalies.
The 1,350-nucleotide salB sequence is preceded by two open reading frames (ORFs), EF0392 and EF0393, encoding proteins of unknown function; the EF0395 gene, located downstream of salB, encodes a 42.2-kDa protein homologous to methionine synthetase (Fig. 1A). To determine the size of the salB transcription unit, Northern blot hybridizations were performed using total RNA extracted from exponentially grown E. faecalis strain JH2-2 and a DNA probe complementary to the salB gene (Fig. 1C). A 1.4-kb unique transcript, similar in size to the salB ORF itself, was detected in the assay, suggesting that the salB gene is transcribed independently of the surrounding genes. Consistent with this result, we also identified 24 nucleotides downstream of the salB stop codon (TAA), an invert-repeat sequence that likely corresponds to a Rho-independent terminator (G = –18.6 kcal mol–1) (Fig. 1A). RNA extracted from exponentially growing E. faecalis JH2-2 cells was then used to map the salB transcriptional start site by 5'-RACE PCR. The resulting electropherogram presented in Fig. 1B localizes the transcriptional start point (+1) of the salB gene at a site 49 nucleotides upstream of a potential ribosome-binding site (GGAGGA). Seven base pairs upstream of the transcriptional start site lies a A-like promoter element with a putative –10 box (TAGAAT) separated by 19 nucleotides from a putative –35 box (TTGCTT) (Fig. 1A). These results strongly suggest that salB belongs to a monocistronic operon that is transcribed in mid-exponential phase from a promoter recognized by RNA polymerase carrying the housekeeping sigma factor A.
Previous data showed that a salB mutant was more sensitive to several stress conditions, including heat shock and hyperosmotic stress (23). This prompted us to test whether salB expression could be influenced by stress. Northern blot hybridization of total RNA extracted from E. faecalis cells exposed for 10 min to different culture conditions (the presence of NaCl, 50°C, acid pH) demonstrated that the level of salB mRNA increases when E. faecalis cells are subjected to an osmotic stress or a high temperature challenge (Fig. 1C). The stress inducibility of salB is consistent with the anticipated role of its product as a protein that has a protective function needed when the cell encounters detrimental environmental situations. The effect of the inactivation of croR on the induction of salB was tested by RNA hybridization (Fig. 1D). Dot blot hybridization of total RNA extracted from E. faecalis cells exposed for 5, 10, or 20 min to 0.3 M NaCl or to 50°C showed that CroR is required for salB induction.
salB encodes a secreted protein. The salB sequence encodes a protein of 449 amino acids (aa) (Fig. 2) with a calculated molecular mass of 47.3 kDa and an estimated pI of 4.7. The first 27 amino acids contain the elements found in gram-positive signal peptides. Cleavage after residue 27 by a signal peptidase would result in a secreted SalB protein of 44.5 kDa. Homology searches revealed that the N-terminal portion of SalB (from aa 1 to aa 260) is similar to the N-terminal portions of the proteins SagA (also referred as P60) and P54 from Enterococcus faecium (10, 38) and SagBb from Enterococcus hirae (F. Teng, B. E. Murray, and G. M. Weinstock, unpublished data) and the SalA antigen (putative secreted lipase) from E. faecalis (28). SalB also shares homology with the N-terminal portion of PcsB-like proteins from different species of streptococci such as Streptococcus mutans GbpB (4, 5, 27) (Fig. 2). Interestingly, SagA, P54, GbpB, and a number of "PcsB-like" proteins have been shown to be associated with the cell wall or secreted into the growth medium. At least some of these proteins appear to be involved in cell wall hydrolysis and in the regulation of cell wall biosynthesis (4, 10, 27, 38). The central domain of these proteins, as with that of SalB (aa 261 to aa 341), presents an unusual composition: 46 amino acids out of 81 of this SalB segment are serine, threonine, or proline. In its C-terminal portion, SalB shares significant homology with proteins deduced from the Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 genome (accession no. ZP00063637 and ZP00063635) containing Lys7 domains, which are usually found in proteins that bind to cell wall peptidoglycan (3).
We extracted and separated through SDS-PAGE the secreted proteins from the E. faecalis JH2-2 wild-type strain and from the salB and err05 mutant strains (Fig. 3). Results showed that the salB and err05 mutants were missing a protein that migrated between 50- and 75-kDa molecular mass markers. Mass spectrometry analyses revealed that the missing protein corresponds to SalB (Table 2). The other protein closely migrating with SalB was also identified as the putative secreted lipase SalA (28).
Our results indicate that SalB is a secreted protein. Additionally, the absence of SalB in the media of the strain with the croR mutation is consistent with the notion that the CroRS two-component system is involved in salB expression or secretion.
As previously shown by Comenge et al. (6), a croR mutation led to a decrease in the MICs of cell wall-active antimicrobials (Table 3). In order to investigate whether this phenotype could be attributed to a CroR effect on the SalB protein, we tested the susceptibility of the salB mutant to the cell wall-active antimicrobials. As presented in Table 3, similar resistance levels were observed for the salB mutant and the wild-type strain, showing that the antibiotic sensitivity of the err05 mutant cannot be attributed to the nonexpression of the SalB protein.
CroR binds to the salB promoter region. To test whether the CroR response regulator could bind directly to the salB promoter region, we purified a CroR protein harboring an N-terminal six-histidine tag (H6-CroR) as described in Materials and Methods. The purified protein was visualized on a Coomassie-stained SDS-PAGE gel (Fig. 4A) and was shown to migrate in accordance with its estimated molecular size of 27 kDa.
A 299-bp DNA fragment containing the promoter region of salB (PsalB) (nucleotide –212 to nucleotide +87 relative to the transcriptional start point) was PCR amplified with primers PsalB1 and PsalB2 (Table 1) and used as a DNA target for EMSA. As shown in Fig. 4B, when the PsalB fragment is incubated with different amounts of H6-CroR in the presence of 50 mM acetyl phosphate, three bands with reduced mobility, designated complex 1 (C1), complex 2 (C2), and complex 3 (C3), were observed. Decreases in the amount of H6-CroR in the binding assay mixture progressively led to a reduction in the amounts of C1, C2, and C3 and a concomitant increase in the amount of free unbound target DNA (Fig. 4B). The binding of H6-CroR to the salB promoter region appeared to be specific in that the addition of unlabeled 299-bp PsalB DNA reduced the abundance of the putative complexes whereas addition of a nonspecific competitor (an unlabeled internal fragment of the salB ORF) did not (Fig. 4B). In keeping with the expectation that it is the phosphorylated form of CroR that is the DNA-binding species, no H6-CroR-dependent retardation of PsalB was observed when the EMSA was performed in the absence of the phosphate donor acetyl-phosphate (data not shown).
DNase I footprinting experiments were then performed in order to determine the sequence of the CroRS binding site in the PsalB DNA fragment. As shown in Fig. 5, H6-CroR protects a 45-bp region extending from positions –1 to +44 relative to the transcription initiation site.
The results of these experiments, taken together with those showing the croR dependency of the SalB expression, support the idea of a direct involvement of the CroRS two-component system in the activation of salB.
The CroRS system regulates its own expression. Autoregulation is a common feature for two-component systems. Comenge et al. (6) suggested that this could also be true for CroRS. The croRS locus is expressed as a bicistronic operon from a unique promoter located upstream of the croR gene (6, 25). A 358-bp DNA fragment containing the promoter region of croRS (PcroRS; nucleotide –133 to nucleotide +125 relative to the transcription initiation site determined by Comenge et al.) (6) was obtained by PCR amplification using the primers Pcro1 and Pcro2 (Table 1) and used for EMSA with different amounts of phosphorylated H6-CroR. As was the case with the DNA fragment containing the salB promoter, slower migrating bands (C1 and C2) were observed whose abundance was specifically reduced by the addition of unlabeled PcroRS DNA (Fig. 4C). The apparent binding of CroR-PO4 to the croR promoter is consistent with autoregulation of the croRS operon.
The DNA sequence of the CroRS binding site in the PcroRS DNA fragment was determined using DNase I footprinting experiments and both DNA strands (Fig. 6). Results revealed a 45-bp common protected region corresponding to positions –87 to –43 relative to the transcription initiation site. Comparison of this DNA sequence to that protected in the salB promoter region revealed three conserved 6- to 7-bp motifs (TTCTAAA, AAAGTT, and GTTTATT) which could be candidates for a CroR recognition sequence.
DISCUSSION
Over the last several years, we investigated the E. faecalis environmental stress response by use of two-dimensional electrophoresis (for reviews, see references 12 and 29) and genetic analyses of E. faecalis strain JH2-2 (11, 23-25, 29-31). The genetic approach allowed us to identify two independent mutants presenting similar growth defects and altered cell morphology (23, 25). Genotypic analysis revealed that one mutation (BS9) lies in the salB gene (previously referred as sagA) (23), encoding a putative secreted protein with unknown function (23). The second mutation mapped to a gene encoding an OmpR-like response regulator (25), recently redesignated CroR (6). Northern blot hybridization experiments revealed impaired salB expression in the err05 mutant, suggesting a possible involvement of the CroR regulator in the control of salB expression (25).
In the present report, the salB gene is shown to be transcribed as a 1.4-kb monocistronic mRNA, the expression of which is induced by environmental stresses such as high osmolarity and temperature. Interestingly, dot blot hybridization experiments also revealed that when the err05 mutant strain is subjected to these two stress conditions, the salB expression is not activated. This result strongly suggests that CroR might be required for salB expression when E. faecalis cells are subjected to high osmolarity and temperature.
We then tested the hypothesis of the direct involvement of CroR in salB regulation, and EMSA experiments gave evidence that the phosphorylated CroR response regulator is able to bind specifically to the salB promoter region. This result, in conjunction to the previous demonstration of the reduced salB expression in the err05 mutant strain lacking croR (25), argues that CroR is likely to be a transcriptional regulator involved in salB activation in E. faecalis JH2-2. Given that CroR can be phosphoactivated by its mated histidine-kinase CroS (6), we can conclude that the CroRS two-component system is involved in the salB regulation. To our knowledge, this result corresponds to the first demonstration of the presence in E. faecalis of a signal transduction pathway involved in the direct regulation of a stress protein.
Inactivation of salB or croR genes confers growth defects and alterations of cell morphology (23, 25), so we can suggest that these phenotypes in the err05 mutant strain may be the consequence of the low level of SalB. The first role for the CroRS system to be described was its involvement in E. faecalis intrinsic -lactam resistance (6), and it was tempting to see whether SalB could actually be involved in -lactam resistance. The sensitivity to cell wall-active antimicrobials was tested, and the results showed that the decreases of the MICs of D-cycloserine, cefotaxime, and ampicillin were observed only in the err05 mutant strain but not in the salB mutant strain, demonstrating that SalB is not involved in the CroRS-associated -lactam resistance.
A previous work showed that a mutation in the E. faecalis salB locus led to cell shape anomalies, nonsymmetrical divisions, and sensitivity to different environmental stresses (23). Here, we show that SalB is a secreted protein with homology to PcsB-like proteins involved in cell wall hydrolysis and the regulation of cell wall biosynthesis. SalB shares homologies with the E. faecalis SalA protein. The latter is also a PcsB-like protein, and chromosomal location of salA is similar to that observed for other pcsB-like genes, as it is located downstream of the mreCD locus, encoding proteins known to play a role in cell shape. In our experiments, we demonstrated that SalA is also a secreted protein. However, in contrast to the results seen with salB, salA seems not to be regulated by the CroRS two-component system.
EMSA analyses also showed that CroR binds to the promoter region of the croRS locus. This result demonstrates the validity of the hypothesis, based on analyses of the activity of a PcroRS-lacZ fusion in croRS mutants (6), that the CroRS two-component system is able to regulate its own expression.
DNase I footprinting experiments allowed determination of the sequences of 45-bp DNA segments protected from DNase I by H6-CroR binding in the promoter region of genes croR and salB. Both protected sequences contain low (<18%) G+C content, and this is also the case for the three 6- to 7-bp motifs common in both protected regions. Thus, we can expect a high occurrence of the CroR recognition sequence in the AT-rich E. faecalis genome and consequently a large number of genes controlled by the two-component system CroRS.
The sensitivity of the err05 mutant strain lacking croR to the cell wall-active antimicrobials cannot be explained solely by the low level of SalB in this strain, and additional target(s) might correspond to genes involved in this phenotype. Experiments are now in progress in our laboratory to identify new genes regulated by CroRS.
ACKNOWLEDGMENTS
Y.L.B. was the recipient of a doctoral fellowship from the "Ministère de la Recherche et de l'Enseignement Superieur", France. C.M. was the recipient of a doctoral fellowship from "INRA" and "Region Basse-Normandie." T. Morin was the recipient of a postdoctoral fellowship from "Region Basse-Normandie."
C.M. and Y.L.B. contributed equally to this work.
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