OmpR phosphorylation regulates omps1 expression by differentially controlling the use of promoters

Transcription

1 Microbiology (214), 16, DOI 1.199/mic OmpR phosphorylation regulates omps1 expression by differentially controlling the use of promoters Mario Alberto Flores-Valdez, 1 3 Marcos Fernández-Mora, 2 3 Miguel Ángel Ares, 3 Jorge A. Girón, 4 Edmundo Calva 2 and Miguel Ángel De la Cruz 2,3 Correspondence Miguel Ángel De la Cruz miguel_angel_81@live.com 1 Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A. C., Normalistas 8, Colinas de la Normal, Guadalajara 4427, Jalisco, Mexico 2 Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 21, Cuernavaca, Morelos 6221, Mexico 3 Unidad de InvestigaciónMédica en Enfermedades Infecciosas y Parasitarias, Hospital de Pediatría, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Mexico 4 Department of Molecular Genetics and Microbiology, Emerging Pathogens Institute, University of Florida, Gainesville, FL, USA Received 16 July 213 Accepted 15 January 214 The Salmonella enterica omps1 gene encodes a quiescent porin that belongs to the OmpC/ OmpF family. In the present work we analysed the regulatory effects of OmpR phosphorylation on omps1 expression. We found that in vivo, OmpR in its phosphorylated form (OmpR-P) was important in the regulation of the two omps1 promoters: OmpR-P activated the P1 promoter and repressed the P2 promoter in an EnvZ-dependent manner; expression occurs from the P2 promoter in an ompr mutant. In vitro, OmpR-P had a higher DNA-binding-affinity to the omps1 promoter region than OmpR and OmpRD55A, showing an affinity even higher than that of equivalent DNA regions in the 59-upstream regulatory sequence of the major porin-encoding genes ompc and ompf. By analysing different environmental conditions, we found that glucose and glycerol enhanced omps1 expression in the wild-type strain. Interestingly the stimulation by glycerol was OmpR-dependent while the effect of glucose was still observed in the absence of OmpR. Acetyl phosphate produced by the AckA-Pta pathway did not influence omps1 regulation. These data indicate the important role of the phosphorylation in the activity of OmpR on the differential regulation of both omps1 promoters and its impact on the pathogenesis. INTRODUCTION Salmonella enterica serovar Typhi (S. Typhi) is the aetiological agent of typhoid fever in humans (Pang et al., 1998). S. Typhi synthesizes three major outer-membrane proteins, OmpC, OmpF and OmpA. OmpC and OmpF are under the control of the EnvZ-OmpR two-component system encoded by the ompb locus (Puente et al., 1991) that responds to several environmental stimuli, among which osmolarity and ph have been the most studied (Russo & Silhavy, 1991; Bang et al., 2). EnvZ senses an environmental signal, which modulates OmpR function by a mechanism involving phosphorylation and dephosphorylation (Forst et al., 1989; Forst & Roberts, 1994; Mizuno & Mizushima, 199; Pratt et al., 1996). OmpR is phosphorylated at the aspartate 55 residue to produce OmpR- P, an event that enhances its ability to bind to the upstream 3These authors contributed equally to this work. regulatory sequences of the ompc and ompf promoters (Forst et al., 1989; Delgado et al., 1993; Head et al., 1998). The S. enterica omps1 gene codes for the OmpS1 quiescent porin that belongs to the OmpC/OmpF super family (Fernández-Mora et al., 1995). OmpS1 appears to have a role in swarming motility, biofilm formation and virulence in mice (Toguchi et al., 2; Mireles et al., 21; Rodríguez- Morales et al., 26). Expression of the omps1 gene is mainly dependent on two overlapping promoters, P1 and P2 (Oropeza et al., 1999; Flores-Valdez et al., 23; De la Cruz et al., 27; Fig. 1a). OmpR activates P1, which is located in the omps1 upstream regulatory region and possesses six OmpR-binding sites, and thus in the absence of OmpR there is no P1 activity. The P2 promoter is located in the overlapping OmpR 235 and 21 boxes and does not require OmpR for activation; instead P2 is only active in the absence of OmpR (Fig. 1a). A third promoter with low expression was described by using RNA from plasmid G 214 The Authors Printed in Great Britain 733 On: Sat, 22 Dec 218 1:35:38

2 M. A. Flores-Valdez and others (a) OmpR-binding box III (b) 2 omps1 pro88 Bits 1 OmpR-binding box II OmpR-binding box I P P1 Bits 2 1 ompc (c) 3 β-galactosidase (U mg 1 protein) Vector pfm2 pfvd55a Vector pfm2 pfvd55a (d) a-ompr a-dnak IMSS-1 2. IMSS-4 3. IMSS-4 pfm2 4. IMSS-4 pfvd55a IMSS-1 IMSS-4 IMSS-1 IMSS-4 (wt) (ompr) (wt) (ompr) pscz1 (ompc-lacz) pro88 (omps1-lacz) (e) +1 P2 pro88 C T A G P2 omps1 P1 omps1 1. IMSS-1 2. IMSS-4 (ompr) +1 P1 3. IMSS-4 pfm2 4. IMSS-4 pfvd55a Fig. 1. OmpR-P differentially regulates both omps1 promoters. (a) Nucleotide sequence of the omps1 promoter region. Startpoints and 1 and 35 boxes for both promoters are shown. OmpR-binding boxes (I II) overlap with the P2 promoter. The sequence of the promoter region from the pro88 plasmid is shown. (b) Logo motif analysis (Crooks et al., 24) using the six and three OmpR-binding sites for omps1 and ompc promoter regions, respectively. (c) b-galactosidase activity of ompc-lacz (pscz1) and omps1-lacz (pro88) gene reporter fusions in S. Typhi wild-type (IMSS-1) and a null ompr mutant (IMSS-4) 734 Microbiology 16 On: Sat, 22 Dec 218 1:35:38

3 OmpR phosphorylation controls both omps1 promoters complemented with OmpR wt (pfm2) and OmpR D55A (pfvd55a). The data represent means±sd of three independent experiments. (d) Western blotting experiments showing OmpR levels in different backgrounds: wt, ompr, ompr (pfm2) and ompr (pfvd55a). DnaK is shown as a loading control. (e) Primer extension analysis of the omps1 P1 and P2 promoters from the pro88 in S. Typhi wild-type and in the null ompr mutant complemented with OmpR wt (pfm2) and OmpR D55A (pfvd55a). The ompa transcript was included as a loading control. fusions (Oropeza et al.,1999);however,thispromoter hasnot been detected from chromosomal RNA (Flores-Valdez et al., 23; De la Cruz et al., 27). OmpR dimerizes and recognizes around 18 2 nt (approximately 1 nt recognized by each OmpR molecule; Yoshida et al., 26). In a similar manner to the ompc promoter, the OmpR-binding sites in the omps1 promoter showed conservation of AT-rich sequences (nt 1 7) and a CATNT motif (nt 12 16; Fig. 1b). Sequences upstream of position 288 are required for negative regulation, and sequences downstream are required for positive regulation (Oropeza et al., 1999; Flores-Valdez et al., 23). OmpRbinding boxes I II and part of III were identified in this positive regulation zone (Oropeza et al., 1999; Fig. 1a). Thus, OmpR acts as a modulator of omps1 expression by selectively binding to the P1 or P2 promoters (De la Cruz et al., 27). In contrast to OmpC and OmpF, no osmoregulation was seen for OmpS1 (Oropeza et al., 1999). However, the role of OmpR-P in omps1 regulation remains unclear. In addition to OmpR, H-NS and StpA nucleoid-binding proteins negatively regulated omps1 expression by binding to P1 and P2 (Flores-Valdez et al., 23; De la Cruz et al., 27). Interestingly, we recently reported that DNA static curvature favours the repressive effect of both nucleoid proteins on omps1 expression (De la Cruz et al., 29). LeuO, a LysR-type regulator, antagonizes the repression mediated by H-NS and StpA on the S. Typhi omps1 gene by displacement of these proteins (De la Cruz et al., 27). The aim of this study was to elucidate the role of the P1 and P2 promoters in the regulation of omps1 expression mediated by OmpR-P. METHODS Bacterial strains, plasmids, and recombinant DNA techniques. Bacterial strains and plasmids used in this study are listed in Table 1. DNA manipulations were performed according to standard protocols (Sambrook & Russell, 21). Oligonucleotides used for amplification by PCR were provided by the Oligonucleotide Synthesis Facility at the Instituto de Biotecnología, Universidad Nacional Autónoma de México, and are listed in Table 2. PCRs were performed with Taq DNA polymerase (Invitrogen), according to the manufacturer s instructions. The one-step mutagenesis procedure described by Datsenko & Wanner (2) for bacterial chromosomal genes was used to generate gene deletions and replacements for antibiotic resistance markers. The S. Typhi ompr D55A allele was subcloned from a BlueScript KS vector (Tran et al., 2) by using XbaI and HindIII, and ligated to pmpm-a3 (Mayer, 1995) digested with the same enzymes generating pfvd55a. Bacterial culture and b-galactosidase assays. The culture conditions and microplate protein and b-galactosidase assays were as previously described (Flores-Valdez et al., 23). Acid ph (5.2), high osmolarity (.3M NaCl) or oxidative stress (5 mm H 2 O 2 6 min) was tested in Nutrient Broth (NB) or M9 medium containing glucose or glycerol to a final concentration of.4 %. Western blotting. Whole-cell extracts of S. Typhi strains grown in NB cultures at 37 uc were subjected to SDS-PAGE (12 % polyacrylamide) and blotted onto nitrocellulose membranes (Millipore) as previously described (De la Cruz et al., 27). After blocking with non-fat milk, the membranes were incubated with a primary antibody against OmpR (1 : 2). Goat anti-rabbit (Biomeda) conjugated to horseradish peroxidase (1 : 1 ) was used as a secondary antibody, and the reactions were visualized with chemiluminescence reagents (PerkinElmer). Primer extension analysis. Total RNA was isolated from samples of cultures grown in NB at 37 uc and collected at mid-exponential phase. Five micrograms of total RNA (for ompa) or25mg of total RNA (for omps1), isolated using a commercial kit (RNeasy; Qiagen), were denatured at 9 uc for 3 min and then slowly cooled to 5 uc. The RNA was annealed with [c- 32 P]ATP-labelled ompa-pe 59- TTTGCGCCTCGTTATCATCCAA-39 (annealing from position +3 to 224 with respect to the translational start site) or primer omps1-pe 59-TTGCTGCGCCTGCCACTAATAAC-39 (annealing from +57 to +35 with respect to the translational start site). Primers were extended with Moloney murine leukaemia virus reverse transcriptase at 37 uc for 2 h, and the extended products were collected with a Microcon-3 microconcentrator (Amicon) and analysed by electrophoresis in urea 8 % polyacrylamide gels alongside sequencing ladders. OmpR purification. OmpR was overexpressed and purified from E. coli BL21 (Novagen) carrying pqr254 that encodes a His6x-OmpR fusion protein as previously described (Oropeza et al., 1999). This plasmid was used as a template to generate pqrd55a by inverse PCR as was previously described (Oropeza & Calva, 29). The primers used are listed in Table 2. Phosphorylation of OmpR by acetyl phosphate. OmpR protein was phosphorylated using 25 mm acetyl phosphate (Sigma) as previously described (Kenney et al., 1995). Fluorescence anisotropy. Equilibrium binding measurements were performed using fluorescence anisotropy (Head et al., 1998). OmpR was labelled at the amino terminus using an amine fluorescein labelling kit (Panvera). The labelled protein was purified using a series of three 5 ml desalting columns (Amersham Biosciences). Fluorescent OmpR was excited at 49 nm, and emission was measured at 53 nm in a Beacon fluorometer (Panvera). The samples were incubated in the fluorometer for 3 s, and five measurements were taken at 1 s intervals after each protein addition. A typical binding experiment lasted approximately 3 min. Each point plotted in the figures represents the mean of at least three independent measurements. The change in anisotropy, where (A2A o )/A o represents the difference in anisotropy in the presence of protein minus the anisotropy in the absence of protein divided by the anisotropy in the absence of protein, was plotted as a function of the total protein concentration. The results from the binding curves were fitted by non-linear leastsquares regression as described previously (Head et al., 1998). The On: Sat, 22 Dec 218 1:35:38

4 M. A. Flores-Valdez and others Table 1. Bacterial strains and plasmids Strain or plasmid Genotype and/or relevant markers Reference E. coli MC41 F9 arad139 D (argf-lac)u169 rpsl15 rela1 flb531 deoc1 ptsf25 rbsr Casadaban (1976) BL21(DE3) F 2 ompt gal (dcm) (lon) hsds B (r 2 B m 2 B ) metbl21(de3) Novagen S. enterica IMSS-1 Salmonella enterica serovar Typhi 9.12, d, Vi serotype; Mexican reference clinical strain Puente et al. (1987) IMSS-4 IMSS-1 DompR ::Km r Fernández-Mora et al. (24) IMSS-81 IMSS-1 DompB ::Km r Martínez-Flores et al. (1999) IMSS-45 IMSS-1 D(ackA-pta)::Km r This work IMSS-85 IMSS-81 D(ackA-pta)::Cm r This work Plasmids pro31 pro88 pscz1 pmc1871-derived plasmid, containing a translational fusion of the omps1 59 regulatory region, up to 31 to 27 bp upstream of the transcriptional start point, to the lacz reporter gene pmc1871-derived plasmid, containing a translational fusion of the omps1 59 regulatory region, up to 88 to 27 bp upstream of the transcriptional start point, to the lacz reporter gene pmc1871-derived plasmid, containing a translational fusion of the ompc 59 regulatory region, 145 bp upstream of the transcriptional star point and the first codon, to the lacz reporter gene Oropeza et al. (1999) Oropeza et al. (1999) Martínez-Flores et al. (1999) pfm2 pacyc184 carrying the S. Typhi ompr gene; p15a Fernández-Mora et al. (24) pfvd55a pmpm-a3 carrying the S. Typhi ompr gene with a point mutation in the Asp-55 This work (D55A); p15a pqr254 pqe32 carrying the S. Typhi ompr gene with His 6 in the N-terminal Oropeza et al. (1999) pqrd55a pqr254 carrying the S. Typhi ompr gene with a point mutation in the Asp-55 (D55A) This work apparent dissociation constants reported in the text represent the mean. RESULTS OmpR phosphorylation is key for the differential regulation of the P1 and P2 omps1 promoters In order to test the role of OmpR phosphorylation on omps1 expression, two plasmids coding for OmpR wildtype (pfm2) or for OmpR mutated in the D55 (aspartate 55) phosphorylation site (pfvd55a) were used. D55A OmpR protein is unable to become phosphorylated and thus has a decreased affinity for DNA (Delgado et al., 1993; Head et al., 1998). As a control of OmpR function, the expression of the major OmpR-dependent porin gene ompc was assessed by using the pscz1 fusion, which contains the ompc regulatory region fused to lacz (Martínez-Flores et al., 1999). The expression of ompc was measured in two S. Typhi backgrounds, wild-type (IMSS-1) and an ompr null mutant (IMSS-4). In IMSS-4, ompc expression was abolished, which is in agreement with the reported dependency of ompc expression on OmpR-mediated regulation (Hall & Silhavy, 1981). Introduction of an ompr wild-type allele (pfm2) into the mutant resulted Table 2. Oligonucleotides used in this work Oligonucleotide Sequence Reference acka-h1p1 GTA TCA TAA ATA GGT ACT TCC ATG TCG AGT AAG TTA GTA CTG GTT TGT AGG This work CTG GAG CTG CTT C pta-h2p2 ATC CGG CAT TAG CTT TTA CTG TTA CTG CTG CTG TTG AGA AGC CTG CAT ATG This work AAT ATC CTC CTT AGT TCC acka-5 ATA AAT GTC AGG GCT ATC ATG CGC This work pta-3 AAC GCG TCT TAT CCG GCC TAC AAC GG This work ompr-d55a-59 CAT CTC ATG GTA CTG GCT TTA ATG CTG CCA GGT GAA GAT This work ompr-d55a-39 CAT CTT CAC CTG GCA GCA TTA AAG CCA GTA CCA TGA GAT This work 736 Microbiology 16 On: Sat, 22 Dec 218 1:35:38

5 OmpR phosphorylation controls both omps1 promoters in restoration of ompc expression, as expected. Conversely, the mutated ompr allele coding for a phosphorylationdeficient OmpR (pfvd55a) did not restore ompc expression (Fig. 1c), strongly showing a central role for OmpR phosphorylation on ompc expression. To determine the OmpR levels produced in the plasmids, we performed Western blotting experiments detecting OmpR protein in both the wild-type and the ompr mutant complemented with pfm2 and pfvd55a. OmpR wt and OmpRD55A proteins expressed in trans showed similar levels compared to the wild-type strain (Fig. 1d). The role of OmpR-P in regulating omps1 expression was further examined by using the pro88 fusion, which attains the maximum level of expression and contains the two and half OmpR-binding sites that are required for activation of P1 (Fig. 1a, b; Oropeza et al., 1999; Flores-Valdez et al., 23; De la Cruz et al., 27). As reported previously, in an ompr null mutant, omps1 expression was diminished to half of the value observed in the wild-type strain (Fig. 1c). When this mutant was complemented with pfm2, omps1 expression reached wild-type-like levels as expected (Fig. 1c). Moreover, upon introduction of pfvd55a the level of omps1 expression remained similar to that of the ompr mutant, at half of the wild-type levels (Fig. 1c), consistent with the notion that OmpR (D55A) is nonfunctional and thus neither binds to DNA nor activates P1, allowing for expression of omps1 from P2. In agreement with these results, primer extension experiments showed that in the wild-type strain omps1 expression started from the P1 promoter, while in the null ompr mutant expression was started from P2 (Fig. 1e). In the ompr mutant transformed with pfm2, omps1 activity was restored, indicating it was P1-dependent, but when complemented with pfvd55a, omps1 activity was P2-dependent (Fig. 1e). Therefore, transcriptional activity from P2 was observed only in the absence of OmpR or when OmpR was not phosphorylated at D55. Our data demonstrate that OmpR- P positively regulates the P1 promoter and negatively affects P2. OmpR-P has a higher DNA-binding affinity than OmpR for the omps1 promoter region The differential expression of both omps1 P1 and P2 promoters demonstrated that the phosphorylation status of OmpR determines which promoter is to be used for initiation of transcription of this gene. In order to determine the relevance of OmpR phosphorylation status to omps1 regulation, we compared the DNA-binding affinity of OmpR, OmpR-P and OmpRD55A for the omps1 promoter region. We phosphorylated OmpR in vitro by incubation with acetyl phosphate, and the proportion of OmpR-P obtained was in the range of % as assessed by HPLC (data not shown). Next, the equilibrium binding of OmpR and OmpR-P to the omps1 promoter region was compared by fluorescence anisotropy experiments. The apparent dissociation constant (K d ) for OmpR binding to OmpR-boxes I-II and the half of III on omps1 was nm (Fig. 2a, b). Strikingly, OmpR-P showed a 72-fold increase in DNA-binding affinity, with a K d of 2.2 nm (Fig. 2a). To corroborate the relevance of the phosphorylation site, we purified the OmpRD55A protein. The K d for OmpRD55A on omps1 was nm, very similar to non-phosphorylated OmpR (Fig. 2a). These compelling data indicate that OmpR-P displays a high affinity for the omps1 promoter region activating P1 promoter and repressing P2 (Figs 1 and 2). EnvZ is required for the differential regulation of both P1 and P2 promoters of omps1 Under certain conditions, acetyl phosphate promotes EnvZ-independent OmpR phosphorylation (Hsing & Silhavy, 1997; Matsubara & Mizuno, 1999; Bang et al., 2; Heyde et al., 2). In order to define whether phosphorylation of OmpR mediated by EnvZ was the main source of transcriptionally active OmpR-P for regulating omps1 expression, we monitored gene expression in several genetic backgrounds, including an ompb (ompr-envz) background (IMSS-81; Martínez-Flores et al., 1999). In an ompb mutant, omps1 expression reached levels similar to those observed in the single ompr mutant (Fig. 3a). Interestingly, when pfm2 (OmpR wt) was introduced into the ompb mutant, omps1 expression remained as if no OmpR-P was produced (Fig. 3a). We further demonstrated by primer extension experiments the participation of EnvZ in producing OmpR-P for omps1 differential promoter use. In an ompb mutant, omps1 transcription initiated from P2 and when this mutant was transformed with pfm2 or pfvd55a, P2 remained as an active promoter (Fig. 3b). Production of acetyl phosphate in the cell is mediated by the acetate kinase (AckA) and phosphotrascetylase (Pta) enzymes (Wanner & Wilmes-Riesenberg, 1992; McCleary & Stock, 1994; Wolfe, 25). To discard the effect of the acetyl phosphate produced in the cell on OmpR phosphorylation, we analysed omps1 transcription in the absence of the AckA-Pta pathway. No differences were observed in both acka-pta and ompb acka-pta compared to the wild-type and ompb backgrounds (Fig. 4b). These results strongly indicate that EnvZ is the main kinase involved in the production of phosphorylated OmpR for omps1 expression, since in the absence of EnvZ such expression was P2-dependent, while the OmpR- P dependent P1-promoter was unable to become activated at least to detectable levels. Glucose and glycerol stimulate omps1 expression To analyse the omps1 expression under different environmental conditions, we monitored pro88 fusion both in the wild-type strain and in the ompr mutant. The conditions used were ph, osmolarity, oxidative stress and carbon source. No differences were found in acid ph (5.2), high osmolarity (.3 M NaCl) or oxidative stress (5 mm H 2 O 2 for 6 min, Fig. 4a) in agreement with previous reports On: Sat, 22 Dec 218 1:35:38

6 M. A. Flores-Valdez and others (a) (A-Ao)/Ao.15.1 K d =157.3 nm (A-Ao)/Ao.15.1 K d =2.2 nm [OmpR] mm [OmpR-P] mm.25.2 (A-Ao)/Ao.15.1 K d =121.2 nm [OmpR-D55A] mm (b) 5 3 Fig. 2. Binding of OmpR and OmpR-P to the omps1 promoter region. (a) DNA-binding curves of OmpR, OmpR-P and OmpRD55A to omps1 OmpR-binding boxes I II, and half of III, contained in a 54 bp oligonucleotide. The fractional change in anisotropy is plotted against OmpR or OmpR-P concentration. A o is the anisotropy value of the oligonucleotide in the absence of the protein and A is the measured anisotropy value at each protein concentration. The apparent dissociation constants calculated from these curves were 2.2 nm for OmpR-P, nm for OmpR and for OmpRD55A. (b) Sequence of the oligonucleotide used in this study. (Oropeza et al., 1999). However, when S. Typhi harbouring the pro88 plasmid was grown in M9 medium with glucose or glycerol, omps1 transcription was enhanced (Fig. 4a). Interestingly, this stimulation was differential: induction by glycerol and glucose was dependent and independent of OmpR, respectively. By evaluating the role of the AckA-Pta pathway in stimulation by glucose and glycerol in omps1 expression, we measured pro88 levels in wild-type, ompr, ompb, acka-pta and ompb acka-pta backgrounds. pro88 levels were similar between the wild-type strain and the acka-pta single mutant (Fig. 4b). Similarly, DompB D(ackApta) and DompB had a similar phenotype (Fig. 4b). Acetyl phosphate produced in the cell did not influence the omps1 expression stimulated by glycerol and glucose. DISCUSSION The expression of omps1 is dependent on two promoters, P1 and P2. OmpR differentially regulates both promoters in its phosphorylated state, acting as a modulator of omps1 expression and selectively choosing one or the other promoter (Fig. 1). OmpR-P regulates P1 and P2 positively and negatively, respectively (Fig. 1b). In contrast to the regulation of both ompc and ompf porin genes, whose expression is strictly OmpR-P-dependent, omps1 expression can be independent of OmpR-P; even though OmpR- P had a much higher affinity to the omps1 promoter region than OmpR and OmpRD55A (72-fold; Fig. 2). Similar fluorescence anisotropy experiments performed by other workers showed that OmpR-P had a 32-fold higher affinity for ompf than OmpR, in contrast to a threefold affinity displayed for ompc (Head et al., 1998). Moreover, acetyl phosphate is a phospho-donor in the cell, which can phosphorylate response regulators including OmpR (Hsing & Silhavy, 1997; Matsubara & Mizuno, 1999; Bang et al., 2; Heyde et al., 2). Nevertheless, we observed that omps1 expression from the P1 promoter, under our culture conditions (NB), was completely 738 Microbiology 16 On: Sat, 22 Dec 218 1:35:38

7 OmpR phosphorylation controls both omps1 promoters (b) +1 P2 pro88 C T A G (a) β-galactosidase (U mg 1 protein) P2 omps1 IMSS-1 (wt) Vector pfm2 pfvd55a IMSS-4 (ompr) Vector pro88 (omps1-lacz) pfm2 pfvd55a IMSS-81 (ompr-envz) +1 P1 1. IMSS-1 2. IMSS-81 (ompb) 3. IMSS-81 pfm2 4. IMSS-81 pfvd55a P1 omps1 Fig. 3. EnvZ is necessary for OmpR-P-mediated omps1 regulation. (a) b-galactosidase activity of the omps1-lacz (pro88) gene reporter fusion in S. Typhi wild-type (IMSS-1), a null ompr mutant (IMSS-4) and in an ompb (ompr-envz) mutant (IMSS-81) complemented with OmpR wt (pfm2) or the OmpR D55A variant mutated in the phosphorylation site (pfvd55a). The data represent means±sd of three independent experiments. (b) Primer extension analysis of the omps1 P1 and P2 promoters from pro88 in S. Typhi wild-type, the null ompr mutant and in the ompb mutant complemented with OmpR wt (pfm2) or OmpR D55A (pfvd55a). The ompa transcript was included as a loading control. (a) β-galactosidase (U mg 1 protein) NB NBS NBH NBGlu NBGly NBA (b) 6 β-galactosidase (U mg 1 protein) IMSS-1 IMSS-4 IMSS-1 IMSS-4 IMSS-81 IMSS-45 IMSS-85 (wt) (ompr) (wt) (ompr) (ompb) (acka-pta) (ompb acka-pta) NB NBS NBGlu NBGly Fig. 4. Environmental conditions and omps1 regulation. (a) b-galactosidase activity of the omps1-lacz (pro88) gene reporter fusion in S. Typhi wild-type (IMSS-1) and a null ompr in low osmolarity (NB), high osmolarity (NBS), oxidative stress (NBH), acid ph (NBA) and the presence of glucose (NBGlu) or glycerol (NBGly). (b) b-galactosidase activity of the omps1-lacz (pro88) gene reporter fusion in S. Typhi wild-type (IMSS-1), ompr, ompb (ompr-envz), acka-pta and ompb acka-pta in low osmolarity (NB), high osmolarity (NBS) and the presence of glucose (NBGlu) or glycerol (NBGly). The data represent means±sd of three independent experiments On: Sat, 22 Dec 218 1:35:38

8 M. A. Flores-Valdez and others dependent on EnvZ indicating a lack of cross-talk or the involvement of acetyl phosphate (AckA-Pta pathway) (Figs 3 and 4). Therefore, in the absence of EnvZ or in the presence of OmpR D55A, which results in the absence of OmpR-P, omps1 expression was P2-dependent (Fig. 3b). While for the P1 promoter OmpR-P is a transcriptional activator, it acts as a repressor for the P2 promoter (Fig. 1b). A repressor effect of OmpR-P has been reported for flagella and invasin in Escherichia coli and Yersinia enterocolitica, respectively (Shin & Park, 1995; Brzostek et al., 27). The work presented here indicates that the P2 promoter can act as a transcriptional support for P1 where, in the absence of OmpR-P, S. Typhi uses the second promoter, in contrast with ompc and ompf that are strictly OmpR-P-dependent. Presumably, discriminating between both omps1 promoters will depend on the response of Salmonella to the conditions in different ecological niches. In this manner, glycerol and glucose differentially regulated omps1 expression: induction by glycerol and glucose was dependent and independent of OmpR, respectively (Fig. 4). These results suggest specific functions for both omps1 promoters in responding to different environmental signals. Mechanistic details about how both glucose and glycerol stimulated omps1 expression are being studied. OmpR-P, in addition to regulating the porin repertoire (De la Cruz & Calva, 21), activates the expression of Salmonella pathogenicity island 2 (SPI-2) via ssrab, conferring Salmonella enterica with the ability to replicate and survive within the macrophage (Lee et al., 2). Hence, omps1 would have a selective advantage to be expressed under various environments found in the host. In this respect, omps1 has been previously implicated in virulence in the mouse (Lawley et al., 26; Rodríguez-Morales et al., 26). ACKNOWLEDGEMENTS We thank Dr Linda Kenney for assistance with the fluorescence anisotropy assays; Dr Ann Stock for anti-ompr antibody; Dr Víctor H. Bustamante and Dr José Luis Puente for helpful discussions; Dr Verónica I. Martinez and Dr Alejandra Vázquez for excellent technical assistance. E. C. was supported by a grant from the CONACYT, Mexico (No ). REFERENCES Bang, I. S., Kim, B. H., Foster, J. W. & Park, Y. K. (2). OmpR regulates the stationary-phase acid tolerance response of Salmonella enterica serovar typhimurium. J Bacteriol 182, Brzostek, K., Brzóstkowska, M., Bukowska, I., Karwicka, E. & Raczkowska, A. (27). OmpR negatively regulates expression of invasin in Yersinia enterocolitica. Microbiology 153, Casadaban, M. J. (1976). Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol 14, Crooks, G. E., Hon, G., Chandonia, J. M. & Brenner, S. E. (24). WebLogo: a sequence logo generator. Genome Res 14, Datsenko, K. A. & Wanner, B. L. (2). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97, De la Cruz, M. A. & Calva, E. (21). The complexities of porin genetic regulation. J Mol Microbiol Biotechnol 18, De la Cruz, M. A., Fernández-Mora, M., Guadarrama, C., Flores- Valdez, M. A., Bustamante, V. H., Vázquez, A. & Calva, E. (27). LeuO antagonizes H-NS and StpA-dependent repression in Salmonella enterica omps1. Mol Microbiol 66, De la Cruz, M. A., Merino, E., Oropeza, R., Téllez, J. & Calva, E. (29). The DNA static curvature has a role in the regulation of the omps1 porin gene in Salmonella enterica serovar Typhi. Microbiology 155, Delgado, J., Forst, S., Harlocker, S. & Inouye, M. (1993). Identification of a phosphorylation site and functional analysis of conserved aspartic acid residues of OmpR, a transcriptional activator for ompf and ompc in Escherichia coli. Mol Microbiol 1, Fernández-Mora, M., Oropeza, R., Puente, J. L. & Calva, E. (1995). Isolation and characterization of omps1, a novel Salmonella typhi outer membrane protein-encoding gene. Gene 158, Fernández-Mora, M., Puente, J. L. & Calva, E. (24). OmpR and LeuO positively regulate the Salmonella enterica serovar Typhi omps2 porin gene. J Bacteriol 186, Flores-Valdez, M. A., Puente, J. L. & Calva, E. (23). Negative osmoregulation of the Salmonella omps1 porin gene independently of OmpR in an hns background. J Bacteriol 185, Forst, S. A. & Roberts, D. L. (1994). Signal transduction by the EnvZ- OmpR phosphotransfer system in bacteria. Res Microbiol 145, Forst, S., Delgado, J. & Inouye, M. (1989). Phosphorylation of OmpR by the osmosensor EnvZ modulates expression of the ompf and ompc genes in Escherichia coli. Proc Natl Acad Sci U S A 86, Hall, M. N. & Silhavy, T. J. (1981). The ompb locus and the regulation of the major outer membrane porin proteins of Escherichia coli K12. J Mol Biol 146, Head, C. G., Tardy, A. & Kenney, L. J. (1998). Relative binding affinities of OmpR and OmpR-phosphate at the ompf and ompc regulatory sites. J Mol Biol 281, Heyde, M., Laloi, P. & Portalier, R. (2). Involvement of carbon source and acetyl phosphate in the external-ph-dependent expression of porin genes in Escherichia coli. J Bacteriol 182, Hsing, W. & Silhavy, T. J. (1997). Function of conserved histidine-243 in phosphatase activity of EnvZ, the sensor for porin osmoregulation in Escherichia coli. J Bacteriol 179, Kenney, L. J., Bauer, M. D. & Silhavy, T. J. (1995). Phosphorylationdependent conformational changes in OmpR, an osmoregulatory DNA-binding protein of Escherichia coli. Proc Natl Acad Sci U S A 92, Lawley, T. D., Chan, K., Thompson, L. J., Kim, C. C., Govoni, G. R. & Monack, D. M. (26). Genome-wide screen for Salmonella genes required for long-term systemic infection of the mouse. PLoS Pathog 2, e11. Lee, A. K., Detweiler, C. S. & Falkow, S. (2). OmpR regulates the two-component system SsrA-ssrB in Salmonella pathogenicity island 2. J Bacteriol 182, Martínez-Flores, I., Cano, R., Bustamante, V. H., Calva, E. & Puente, J. L. (1999). The ompb operon partially determines differential expression of OmpC in Salmonella typhi and Escherichia coli. J Bacteriol 181, Matsubara, M. & Mizuno, T. (1999). EnvZ-independent phosphotransfer signaling pathway of the OmpR-mediated osmoregulatory expression of OmpC and OmpF in Escherichia coli. Biosci Biotechnol Biochem 63, Microbiology 16 On: Sat, 22 Dec 218 1:35:38

9 OmpR phosphorylation controls both omps1 promoters Mayer, M. P. (1995). A new set of useful cloning and expression vectors derived from pbluescript. Gene 163, McCleary, W. R. & Stock, J. B. (1994). Acetyl phosphate and the activation of two-component response regulators. J Biol Chem 269, Mireles, J. R., II, Toguchi, A. & Harshey, R. M. (21). Salmonella enterica serovar typhimurium swarming mutants with altered biofilm-forming abilities: surfactin inhibits biofilm formation. J Bacteriol 183, Mizuno, T. & Mizushima, S. (199). Signal transduction and gene regulation through the phosphorylation of two regulatory components: the molecular basis for the osmotic regulation of the porin genes. Mol Microbiol 4, Oropeza, R. & Calva, E. (29). The cysteine 354 and 277 residues of Salmonella enterica serovar Typhi EnvZ are determinants of autophosphorylation and OmpR phosphorylation. FEMS Microbiol Lett 292, Oropeza, R., Sampieri, C. L., Puente, J. L. & Calva, E. (1999). Negative and positive regulation of the non-osmoregulated omps1 porin gene in Salmonella typhi: a novel regulatory mechanism that involves OmpR. Mol Microbiol 32, Pang, T., Levine, M. M., Ivanoff, B., Wain, J. & Finlay, B. B. (1998). Typhoid fever important issues still remain. Trends Microbiol 6, Pratt, L. A., Hsing, W., Gibson, K. E. & Silhavy, T. J. (1996). From acids to osmz: multiple factors influence synthesis of the OmpF and OmpC porins in Escherichia coli. Mol Microbiol 2, Puente, J. L., Flores, V., Fernández, M., Fuchs, Y. & Calva, E. (1987). Isolation of an ompc-like outer membrane protein gene from Salmonella typhi. Gene 61, Puente, J. L., Verdugo-Rodríguez, A. & Calva, E. (1991). Expression of Salmonella typhi and Escherichia coli OmpC is influenced differently by medium osmolarity; dependence on Escherichia coli OmpR. Mol Microbiol 5, Rodríguez-Morales, O., Fernández-Mora, M., Hernández-Lucas, I., Vázquez, A., Puente, J. L. & Calva, E. (26). Salmonella enterica serovar Typhimurium omps1 and omps2 mutants are attenuated for virulence in mice. Infect Immun 74, Russo, F. D. & Silhavy, T. J. (1991). EnvZ controls the concentration of phosphorylated OmpR to mediate osmoregulation of the porin genes. J Mol Biol 222, Sambrook, J. & Russell, D. W. (21). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. Shin, S. & Park, C. (1995). Modulation of flagellar expression in Escherichia coli by acetyl phosphate and the osmoregulator OmpR. J Bacteriol 177, Toguchi, A., Siano, M., Burkart, M. & Harshey, R. M. (2). Genetics of swarming motility in Salmonella enterica serovar typhimurium: critical role for lipopolysaccharide. J Bacteriol 182, Tran, V. K., Oropeza, R. & Kenney, L. J. (2). A single amino acid substitution in the C terminus of OmpR alters DNA recognition and phosphorylation. J Mol Biol 299, Wanner, B. L. & Wilmes-Riesenberg, M. R. (1992). Involvement of phosphotransacetylase, acetate kinase, and acetyl phosphate synthesis in control of the phosphate regulon in Escherichia coli. J Bacteriol 174, Wolfe, A. J. (25). The acetate switch. Microbiol Mol Biol Rev 69, Yoshida, T., Qin, L., Egger, L. A. & Inouye, M. (26). Transcription regulation of ompf and ompc by a single transcription factor, OmpR. J Biol Chem 281, Edited by: J. Stülke On: Sat, 22 Dec 218 1:35:38