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J Biol Chem, Vol. 274, Issue 38, 27185-27190, September 17, 1999

Molecular Characterization of the PmrA Regulon^*

Marc M. S. M. Wösten and Eduardo A. Groisman^§

From the Department of Molecular Microbiology, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri 63110

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The two-component system PmrA/PmrB of Salmonella enterica controls expression of several loci including those mediating modifications in the lipopolysaccharide that result in polymyxin resistance. To gain insight in the regulation of polymyxin resistance, we mapped the transcription start sites of the PmrA-regulated genes pmrC, pmrG, pbgPE, and ugd and identified a conserved sequence in the promoter region of the first three genes. His-tagged PmrA protein could gel shift DNA fragments containing the promoters of the pmrC, pmrG, and pbgPE genes but not the udg promoter. DNase I footprinting analysis of the pmrC, pmrG, and pbgPE promoters indicate that phosphorylated as well as unphosphorylated PmrA bind to a 16-base pair imperfect inverted repeat sequence (5'-TTAAKTTCTTAAKGTT-3'), which is found 40, 80, and 38 nucleotides upstream from the transcription start sites of the pmrC, pmrG, and pbgPE genes, respectively. Our data suggest that a PmrA dimer activates transcription of the divergent pmrG and pbgPE promoters by binding to a single site in the pmrG-pbgPE intergenic region and that the ugd gene is regulated by the PmrA/PmrB system only indirectly.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

To survive in different environments, bacteria modulate expression of their genes often using two-component signal transduction systems (1, 2). These systems typically consist of a sensor histidine kinase and a response regulator. In general, sensor kinases contain conserved carboxyl-terminal histidine residues that are autophosphorylated in the presence of ATP (3). Most sensors are membrane-bound and transfer the phosphate group to an aspartic acid residue in the amino terminus of the response regulator. The phosphorylated response regulator usually mediates control at the transcriptional level by binding to specific DNA sequences.

The pmrA and pmrB genes of Salmonella enterica serovar Typhimurium encode products with sequence similarity to DNA binding response regulators and autophosphorylatable histidine kinases, respectively (4). The pmrA locus is required for resistance to polymyxin B and to other antimicrobial compounds in Salmonella (5-7) and controls the production of proteins that mediate the modification of the lipopolysaccharide core and lipid A with ethanolamine and 4-aminoarabinose (8).

Transcription of PmrA-activated genes is induced in response to mild acidic conditions (9) or during growth in a low extracellular magnesium media in a process that requires the PhoP/PhoQ two-component system (10, 11) (Fig. 1). Four PmrA-regulated loci have been identified to date as follows: ugd, encoding UDP-glucose dehydrogenase; the seven-gene operon pbgPE (designated as the pmrF locus by Gunn et al. (12)); pmrG; and the pmrCAB operon, indicating that the pmrA is autoregulated (10, 11). The ugd and pbgPE loci are required for polymyxin resistance A (12, 13), growth in low magnesium solid media (13), and the incorporation of 4-aminoarabinose into lipid A (12, 14). The PmrG protein is homologous to Ais, an aluminum induced protein of Escherichia coli K-12 (12), but its biochemical function as well as that of the putative membrane protein PmrC remain unknown. The pmrG gene is located upstream and transcribed in the opposite orientation of the pbgPE operon. Downstream of the pbgPE operon is the pmrD gene, which may also be PmrA-regulated because when present in a multicopy number plasmid, it confers polymyxin resistance in a PmrA-dependent manner (15).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   The PmrA regulon of S. enterica. Transcription of PmrA-activated genes can be induced by growth in low magnesium in a PhoP/PhoQ-dependent fashion and by mild acidic conditions in a PhoP/PhoQ-independent manner. Inducing conditions are believed to promote autophosphorylation of the PmrB protein that can serve as a phosphate donor for the PmrA protein. Phosphorylated PmrA activates its own operon (pmrCAB), the seven-gene operon pbgPE (designated as the pmrF locus by Gunn et al. (12)) and pmrG by binding to their promoter regions. The ugd gene is probably activated indirectly through a yet unidentified factor.

In this paper, we characterize the PmrA-regulated promoters in Salmonella by determining the transcription start sites of four PmrA-regulated operons and identifying the DNA sequence to which the PmrA protein binds. Our experiments define two classes of PmrA-regulated genes as follows: those that are directly regulated by the PmrA protein (pmrCAB, pmrG, and pbgPE), and those that are regulated indirectly by the PmrA protein (ugd).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Strains and Plasmids-- E. coli JM109 was used as the host for the preparation of plasmid DNA. E. coli BL21(DE3) served as the host for overexpression of the PmrA protein and the cytoplasmic domain of the PmrB (PmrBc) protein. To overproduce the PmrA protein, the chromosomal pmrA gene was PCR¹-amplified with primer 351 (5'-AAG GAT CCA GGA GAC TAA GCG-3') and six histidine tag (H6) containing primer 438 (5'-TCC AAG CTT AGT GGT GGT GGT GGT GGT GGC TTT CCT CAG TGG CAA CC-5'). The resulting 715-bp PCR product was digested with BamHI and HindIII and cloned between the BamHI and HindIII sites of pUHE21-2lacI^q to form pUHE-PmrA-H6. The pmrA gene containing the six histidine codons was cut out of pUHE-PmrA-H6 with NdeI and HindIII and ligated into pT7-7. The cytoplasmic domain of pmrB was amplified with primer 988 (5'-AGA TAT ACA TAT GCA CCA CCA CCA CCA CCA CCG GCG TAT TAC CCG TCC GCT-3') containing an initiation codon followed by a six-histidine tag and primer 353 (5'-ACG AAG CTT ATG CCT TTT TCA-3'). The resulting 845-bp PCR product was digested with NdeI and HindIII and ligated into plasmid pT7-7. The nucleotide sequence of the cloned PCR products was verified by sequencing both strands. S. enterica serovar Typhimurium 14028s (16) was used for chromosomal DNA and RNA isolation.

RNA Isolation and Primer Extension-- Overnight cultures of S. enterica serovar Typhimurium grown in N-minimal medium (17), pH 7.7, with 10 mM MgCl₂ were washed 3 times in N-minimal medium, pH 7.7, without MgCl₂ and diluted 1:50 into 50 ml of N-minimal medium, pH 7.7, containing either 10 µM or 10 mM MgCl₂ or N-minimal medium, pH 5.8, containing 10 mM MgCl₂. Total RNA was extracted from mid-exponential phase cultures (A₆₀₀, 0.4-0.6) with Trizol (Life Technologies, Inc.) according to the manufacturer's specifications. Primers used for cDNA synthesis were 935 (5'-AGA CGG CAA TAT AAA AGG AA-3'), located 56 bp downstream of the pmrC start codon; 955 (5'-CAT TAA CCT CTC AGG CAG AC-3'), situated 14 bp downstream of the start codon of the first gene in the pbgPE operon; 1069 (5'-ATA AAG CGT AGG GTA AAT GC-3'), located 5 bp downstream of the pmrG start codon; and 1007 (5'-GCA ATA AGC AAA CCA TTA GA-3'), located 36 bp downstream of the ugd initiation codon. The cDNA synthesis was performed with 30 µg of total RNA, 2 pmol of primer labeled with T4 polynucleotide kinase, and [-³²P]ATP and 50 units of SuperScript^TMII RnaseH reverse transcriptase (Life Technologies, Inc.). The extension products were analyzed by electrophoresis on a 6% polyacrylamide, 7.5 M urea gel and compared with sequence ladders initiated with primers 935, 955, 1069, or 1007.

Purification of the PmrA and PmrBc Proteins-- Histidine-tagged PmrA and PmrBc proteins were expressed in E. coli BL21(DE3) containing either plasmid pT7-PmrA-H6 (EG10067) or pT7-PmrBc-H6 (EG11751). Cells were grown in 500 ml of LB broth at 30 °C to an A₆₀₀ of 0.6. Then, isopropyl-1-thio--D-galactopyranoside was added to a final concentration of 1 mM, and incubation was continued for 4 h. Cells were harvested and suspended in 10 ml of buffer A (50 mM NaH₂PO₄, pH 8.0; 300 mM NaCl, and 10 mM imidazole) and disrupted by sonication. Cell debris was pelleted by centrifugation at 3,000 × g for 15 min. Three ml of 50% nickel-nitrilotriacetic acid was added to the supernatant and incubated for 1 h at 4 °C on a rocking platform. The PmrA-H6 and PmrBc-H6 proteins were washed 4 times with 10 ml of buffer A containing 50 mM imidazole. The proteins were eluted with 4 ml of buffer A containing 250 mM imidazole.

Gel Mobility Shift Assays-- DNA fragments used for gel mobility shift assay were amplified by the PCR using S. enterica serovar Typhimurium chromosomal DNA as a template. Prior to the PCR, primers 935, 955, and 1007, which anneal to the coding strand of pmrC, pbgPE, and ugd, respectively, were labeled with T4 polynucleotide kinase and [-³²P]ATP. The pmrC promoter region was amplified using primer 454 (5'-TCG AAT TCG ATC ACC GCG CTG-3') and labeled primer 935. The pbgPE/pmrG promoter region was generated by PCR with primer 767 (5'-TCG CCG GAC GGG AGA AAG GC-3') and labeled primer 955. The ugd promoter region was amplified with primer 977 (5'-CGG GAT CCG GGC TTT TTT TTA TCT C-3') and labeled primer 1007. Approximately 25 amol of labeled DNA and 0, 10, 20, 30, 50, 100, or 250 pmol of PmrA-H6 protein in a 100-µl volume were incubated at room temperature for 15 min. The binding buffer used for protein-DNA incubations was 20 mM Tris, pH 7.4, 5 mM MgCl₂, 50 mM KCl, 50 µg/ml bovine serum albumin, 2.5 µg/ml salmon sperm DNA, and 10% glycerol. Samples (20 µl) were run on a 4% non-denaturing Tris glycine polyacrylamide gel at 2 °C. After electrophoresis the gel was dried and autoradiographed.

Phosphorylation of PmrA by PmrBc-- The PmrBc-H6 or PmrA-H6 proteins were incubated at room temperature (RT) for 10 or 40 min, respectively, with 50 µCi of [-³²P]ATP in phosphorylation buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 2 mM MgCl₂, and 1 mM dithiothreitol). Phosphorylation of PmrA-H6 was accomplished by adding 0.5 pmol of autophosphorylated PmrBc-H6 to 1 pmol of PmrA in phosphorylation buffer. The reaction was stopped after 0.1, 0.5, 1, 2, 5, 10, 20, or 40 min with SDS loading buffer. The samples were run on a 15% SDS-polyacrylamide gel.

DNase I Footprinting-- DNase I protection assays were done for both DNA strands using the appropriate labeled primer. PmrA-H6 protein was incubated with phosphorylated or unphosphorylated PmrBc-H6 for 20 min at RT as described above, except that instead of [-³²P]ATP, ATP was used. Binding reactions with 0, 4, 8, 16, 32, 64, or 128 pmol of PmrA-H6 protein and 25 amol of labeled DNA were performed as described for the gel mobility shift assay. DNase I (Life Technologies, Inc.) (0.05 units) was added and incubated for 3 min at room temperature. The reaction was stopped by adding 10 µl of 25 mM EDTA. DNA fragments were purified with Wizard DNA cleanup system (Promega, Madison, WI) and resuspended in 30 µl of H₂O. Samples (6 µl) were analyzed by denaturing polyacrylamide (6%) gel electrophoresis by comparison with a DNA sequence ladder generated with the appropriate primer.

Dimerization of PmrA Protein-- His-tagged PmrA protein (2 µg) was resuspended in SDS sample buffer with or without 2-mercaptoethanol and electrophoresed on a 15% SDS-polyacrylamide gel.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Mapping the Transcription Start Sites of PmrA-regulated Genes-- To characterize the PmrA-regulated promoters, we determined the transcription start sites of the PmrA-activated genes pmrC, pmrG, pbgPE, and ugd. Expression of PmrA-activated genes is promoted during growth in low magnesium media in a process that requires the PhoP/PhoQ two-component system. But transcription of PmrA-activated genes can also be induced by growth in mild acidic conditions in a PhoP/PhoQ-independent manner mechanism (9-11). To distinguish between PhoP/PhoQ-dependent and -independent transcription of PmrA-activated genes, total RNA was isolated from mid-exponential phase cultures of wild-type Salmonella grown in N-minimal media with 10 µM Mg²⁺, pH 7.7, or 10 mM Mg²⁺, pH 5.8, respectively (9). RNA was also isolated from bacteria grown in N-minimal media, 10 mM Mg²⁺, pH 7.7, a condition that does not promote expression of PmrA-activated genes.

A single primer extension product was obtained for the pmrC promoter, corresponding to an A residue located 23 nucleotides upstream of the pmrC start codon, with RNA isolated from bacteria grown under either inducing condition (Fig. 2). Single transcription start sites were also detected for the pmrG and ugd promoters, corresponding to a G residue 104 bp upstream of the pmrG start codon and to an A residue 122 bp upstream of the ugd start codon (Fig. 2). In contrast, two major bands located 5 bp apart were observed for the pbgPE promoter (Fig. 2). S1 mapping experiments revealed that the top band, corresponding to an A residue 73 nucleotides upstream of the start codon of the pbgP1 gene, was the transcription start site (data not shown). The S1 mapping experiments revealed the same transcription start sites (data now shown) identified by primer extension for the pmrC, pmrG, and ugd genes (Fig. 2). On the other hand, no primer extension products were observed with RNA isolated from bacteria grown at 10 mM Mg²⁺, pH 7.7 (Fig. 2), consistent with previous results using lac gene fusions (9). That identical transcription start sites were detected when expression of PmrA-activated genes was induced by growth in low magnesium or by mild acidification suggests that these signals act by modifying the activity of the PmrA protein and argues against a model in which different promoters are used in response to different signals.

View larger version (96K): [in this window] [in a new window]   Fig. 2.   Mapping the transcription start sites of the pmrC, pmrG, pbgPE, and ugd operons. Primer extension products were generated after reverse transcriptase of total RNA isolated from mid-exponential phase cells grown in N-minimal medium, pH 7.7, containing 10 mM Mg2+ (lane 1), 10 µM Mg2+ (lane 2), or N-minimal medium, pH 5.8, containing 10 mM Mg2+ (lane 3). The primer extension products were run on a 6% sequencing gel against dideoxy sequencing reactions primed with the same primer. The sequence spanning the transcription start site is shown, and the transcription start site is marked with an arrow.

We analyzed the DNA sequences 5' to the transcription start sites of the pmrC, pmrG, pbgPE, and ugd genes by comparing them to known promoter consensus sequences. The promoters of theses PmrA-activated genes displayed similarity to E. coli ⁷⁰ promoters: the 10 regions of these promoters contained three out of the six conserved nucleotides, and the 35 regions contained three or four of the six conserved nucleotides (Fig. 6). The pmrG and pbgPE promoters also contain an 18-bp A + T-rich sequence upstream of the 35 region known as UP element (18).

The PmrA Protein Binds to the Promoter Regions of the pmrC, pmrG, and pbgPE Genes-- To examine the ability of the PmrA protein to bind the promoter regions of PmrA-activated genes, we first constructed a derivative of the pmrA gene encoding a protein with six histidine residues at its amino terminus, and we showed that a plasmid encoding this pmrA derivative (pUHE-PmrA-H6) could restore transcription of the pmrC, pbgPE, pmrG, and ugd genes in a pmrA null mutant (data not shown). The PmrA-H6 protein could gel shift a 300-bp DNA fragment that included 215 bp upstream of the initiation codon of pmrC, and a 341-bp DNA fragment that included the initiation codons of the divergent pbgPE and pmrG genes (Fig. 3). The amount of PmrA-H6 protein needed for the gel shift of the pmrC and pbgPE/pmrG DNA fragments was similar: gel shifts were seen with 10 pmol of PmrA-H6 protein, and all of the input pmrC and pbgPE/pmrG DNA was in the complexed state in the presence of 250 pmol of protein. Band retardation was observed despite the presence of a large excess (1,000-fold) of competing salmon sperm DNA, suggesting that PmrA-H6 binds directly and specifically to the pbgPE/pmrG and pmrC promoter regions. In contrast, a 261-bp DNA fragment that includes 202 bp upstream of the ugd start codon could not be gel-shifted by 250 pmol of PmrA-H6 protein (Fig. 3), even when this protein was phosphorylated (data not shown).

View larger version (50K): [in this window] [in a new window]   Fig. 3.   The PmrA protein binds to the promoter region of the pmrC and pbgPE/pmrG genes but not to the ugd promoter region. The 300-bp pmrC PCR fragment includes 215 bp of the region upstream of the initiation codon of pmrC. The 341-bp pbgPE/pmrG PCR fragment contains the complete non-coding region between pbgPE and pmrG genes, and the 261-bp ugd PCR fragment contains 202 bp of the region upstream of the ugd gene. The concentration of PmrA-H6 added to each reaction is indicated at the top of each lane.

Autophosphorylation of the PmrB Protein and Phosphotransfer to the PmrA Protein-- We purified the cytoplasmic domain of the PmrB protein (PmrBc-H6) and investigated its ability to autophosphorylate in the presence of ATP. The PmrBc-H6 protein was capable of efficient autophosphorylation (Fig. 4). Moreover, it could serve as a phosphate donor for the PmrA-H6 protein. Rapid phosphotransfer from the PmrBc-H6 to the PmrA-H6 protein was observed (Fig. 4). The half-life for phosphorylated PmrBc-H6 was 10 min when incubated with the PmrA-H6 protein in a 1:2 PmrBc-H6/PmrA-H6 stoichiometry. As expected, the PmrA-H6 protein alone showed no evidence of phosphorylation when incubated with ATP (Fig. 4, lane 10).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Phosphorylation of the PmrA protein by the cytoplasmic domain of the PmrB protein in vitro. Autophosphorylation of PmrBc-H6 (0.5 pmol) was accomplished by incubation of PmrBc-H6 with [-32P]ATP for 10 min at RT. The PmrA protein was incubated for 40 min with [-32P]ATP. Time course of phosphotransfer from 32P-PmrBc (0.5 pmol) to PmrA (1 pmol) is indicated at the bottom of the figure. The samples were run on a 15% SDS-polyacrylamide gel, and autophosphorylation and phosphotransfer were visualized by autoradiography.

Determination of the PmrA-binding Site-- To define experimentally the DNA sequence that the PmrA protein recognizes, DNase I footprinting analysis was performed on both the coding and non-coding strands of the pmrC and pbgPE/pmrG promoter fragments using both phosphorylated and unphosphorylated PmrA-H6 protein. In the pmrC promoter, the PmrA-H6 protein protected from nt 49 to 28 relative to the transcription start site and from nt 57 to 31 in the non-coding strand of pmrC (Fig. 5). Thus, there was an overlap of 19 bp between the two strands protected by the PmrA protein. In the pbgPE/pmrG promoter region, the PmrA-H6 protein also protected a single region of the coding (nt 46 to 26) and non-coding strand (nt 54 to 27), overlapping a region of 20 bp (Fig. 5). In addition, areas of hypersensitivity were observed flanking portions of the protected sites. The location of the hypersensitive site in the pbgPE coding strand was dependent on the phosphorylated state of the PmrA-H6 protein. At high protein concentrations, phosphorylated and unphosphorylated PmrA-H6 protected the same region in the different promoters. On the other hand, at low protein concentrations phosphorylated PmrA-H6 protected this region better than unphosphorylated PmrA-H6. The protected sequences of the pmrC, pmrG, and pbgPE promoters were highly similar to one another (Fig. 6) and allowed us to define the following PmrA binding sequence: 5'-TTAA(G/T)TTCTTAA(G/T)GTT-3', which includes an imperfect inverted repeat (Fig. 6).

View larger version (119K): [in this window] [in a new window]   Fig. 5.   DNase I footprinting analysis of the pmrC and pbgPE/pmrG promoters. Footprinting analysis of the pmrC and pbgPE/pmrG promoter regions was performed on both end-labeled coding and non-coding strands. The amount of phosphorylated [P-PmrA] or unphosphorylated [PmrA] PmrA-H6 protein added to the DNA fragments is indicated at the top of the figures. Solid lines represent the PmrA-binding region. The position of the binding was determined by comparison with sequence ladders, obtained using the same labeled primer as was used for the probe. The hypersensitive DNase I sites are indicated with arrows.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   The promoter regions of the ugd, pmrC, and pbgPE/pmrG genes. A hooked arrow indicates the transcription start site. Brackets mark the DNase I-protected nucleotides by PmrA-H6 of the coding and non-coding strands. Hypersensitive DNase I sites are indicated by vertical arrows. Imperfect inverted repeats are indicated with horizontal arrows. The proposed 10 and 35 regions are marked in bold, and the UP element is marked with a line. Ambiguous codes used are: K, G or T; M, A or C.

The PmrA Protein Can Form Dimers-- The PmrA-H6 protein was analyzed by SDS-polyacrylamide gel with or without 2-mercaptoethanol in the sample buffer. In the absence of 2-mercaptoethanol the PmrA-H6 protein runs as a 50-kDa species, consistent with the notion that this protein can dimerize (Fig. 7). PmrA harbors a single cysteine at position 27, which may be responsible for disulfide bridge formation between two PmrA molecules. The dimer form of PmrA may bind the inverted repeat in the promoter regions of PmrA-activated genes to promote gene transcription.

View larger version (69K): [in this window] [in a new window]   Fig. 7.   The PmrA protein is a dimer. Purified His-tagged PmrA protein (2 µg) was analyzed on a 15% SDS-polyacrylamide gel with or without 2-mercaptoethanol and stained with Coomassie Brilliant Blue. Disulfide bridge formation may be mediated by the single cysteine residue at position 27 of two PmrA monomers.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The two-component system PmrA/PmrB controls transcription of the pmrC, pmrG, pbgPE, and ugd genes (9, 12). Some of these genes encode products that mediate the modification of the lipid A by the addition of 4-aminoarabinose, which makes the microorganism 1,000-fold less susceptible to polymyxin B (12, 13). We have defined the promoter region of the PmrA-regulated genes pmrC, pmrG, pbgPE, and ugd genes. When compared with known promoter consensus sequences (19), they showed weak identity to the E. coli ⁷⁰ promoter consensus sequence, which is typical for activator-dependent promoters (20, 21). PmrA belongs to the OmpR family of response regulators, which are generally transcribed by ⁷⁰ RNA polymerase (22). Activator proteins bind at various distances from weak promoters to enhance binding or open complex formation by RNA polymerase (23). In the absence of activator protein, RNA polymerase forms open complexes poorly due to unfavorable interactions with the 35 and 10 elements of the promoter. The pmrG and pbgPE promoters both contain a possible 18-bp UP element located at positions 52 and 66, respectively, relative to the transcription start site, which could be a target for the carboxyl-terminal domain of the RNA polymerase  subunit. Binding of this domain to target promoters can increase promoter activity 2-20-fold (18).

Transcription of PmrA-activated genes is induced in response to mild acidic conditions (9) or by a low extracellular magnesium concentration through interaction with the PhoP/PhoQ two-component system (10, 11). Transcription products for the pmrC, pmrG, pbgPE, and ugd genes were detected only under inducing conditions for the PmrA/PmrB two-component system. Moreover, the same transcription start site was detected whether bacteria were grown in low pH or in low extracellular magnesium. This indicates that the same promoter is used under different activating conditions (as supposed to different promoters being activated in response to different signals).

A single retarded DNA-protein complex was detected for the pmrC and the pbgPE/pmrG promoter fragments indicating that these promoters contain only one PmrA-binding site. This notion is further supported by our DNase I footprinting analysis which showed that the PmrA-binding site is located around nt 40, 38, or 80 of the transcription start sites of pmrC, pbgPE, and pmrG genes, respectively. This location is in agreement with the DNA-binding sites of other activators, which are usually found between nt 30 and 80 with respect to the transcription start site (24). The PmrA protein protected a 16-bp sequence (5'-TTAAKTTCTTAAKGTT-3') from DNase I, partially covering the potential 35 promoter region of the pmrC and pbgPE promoters (Fig. 6). This sequence includes an imperfect 9-bp inverted repeat, similar to the DNA-binding sites of many prokaryotic regulators that consist of 5-10-bp inverted repeat sequences (25). The dyad symmetry in the PmrA-binding sequence suggests that the PmrA protein binds to these sites as a dimer, and consistent with this notion we found that the PmrA protein can dimerize (Fig. 7).

There is single PmrA-binding site in the pbgPE-pmrG intergenic region, and this site is closer to the pbgPE promoter than to the pmrG promoter. A similar phenomenon has been described in the agr locus of Staphylococcus aureus where two divergent promoters, P2 and P3, contain a single binding site for the regulatory protein SarA (26). The SarA location is closer to the P2 promoter, which is the most activated promoter. Further work will be required to examine whether the pbgPE promoter is preferentially activated relative to the pmrG promoter.

Phosphorylation of response regulators is believed to prevent the activity of an inhibitory domain, allowing the amino terminus to dimerize or oligomerize and the carboxyl terminus to bind to the target DNA (27). However, like the PhoP protein of Bacillus subtilis, the PmrA protein dimerizes and binds to DNA regardless of its phosphorylation state (28). Several response regulators also bind to their target promoters efficiently in their unphosphorylated form (29, 30), but others, such as NarL and ComA, bind to their target genes only in phosphorylated form (31, 32). Yet, in all cases, phosphorylation of response regulators affects binding to the target promoters, often by increasing binding affinity such as in the case of the OmpR and PhoB proteins (33, 34). A similar phenomenon is seen for PmrA, where 2-4-fold less protein is needed for full DNase I protection if phosphorylated PmrA protein is used instead of unphosphorylated PmrA. The high binding affinity of the unphosphorylated PmrA for its target DNA places PmrA in the same class of response regulators as the PhoP protein of B. subtilis, the UhpA protein of E. coli, and the BvgA protein of Bordetella pertussis (1). Phosphorylation of this class of response regulators appears to only change the conformation of these proteins, resulting in transcription activation.

The PmrA protein exhibited specific binding to the pmrC and pbgPE/pmrG promoter fragments. On the other hand, neither phosphorylated nor unphosphorylated PmrA protein could bind to the ugd promoter. This was surprising because genetic analysis of strains harboring ugd::lac fusions demonstrated that ugd transcription requires a functional pmrA gene (9, 12). The ugd gene codes for UDP glucose dehydrogenase which converts UDP-glucose into UDP-glucuronic acid, a precursor in the biosynthesis of the exopolysaccharide colanic acid (35). When grown at low temperature in defined media, the ugd mutant is not mucoid presumably because it is unable to synthesize colanic acid.² On the other hand, pmrA mutants are mucoid,² implying that the ugd gene can be expressed independently of the PmrA protein. This suggests that the PmrA protein regulates transcription of the ugd gene only indirectly and that a different regulator is likely to bind and activate transcription from the ugd promoter. Thus, the ugd gene defines a new class of PhoP/PhoQ-activated genes, a class that can be transcriptionally induced by at least three signals as follows: growth in low magnesium (in a process that requires both the PhoP/PhoQ and PmrA/PmrB two-component systems), growth in mild acid media (in a process that requires PmrA/PmrB but that is independent of PhoP/PhoQ), and a yet undefined condition (in a process that is independent of both PmrA/PmrB and PhoP/PhoQ).

Finally, we analyzed the genome of E. coli K-12 for the presence of PmrA-binding sites and found such sequences in front of the pmrC homologue yjdB, where it covers the inner part of a 14-bp inverted repeat, and in the intergenic region of the homologues of the pbgPE and pmrG genes. The conservation of the binding sites in E. coli and Salmonella is consistent with the high degree of sequence identity that exists between the Salmonella PmrA protein and its homologue in E. coli K-12 BasR (90% at the amino acid level) (36) and suggests that transcription activation of PmrA-regulated genes will be highly similar in these enteric species.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant AI42236 (to E. A. G).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Research associate of the Howard Hughes Medical Institute.

^§ Associate Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Institute, Washington University School of Medicine, Dept. of Molecular Microbiology, 660 South Euclid Ave., Campus Box 8230, St. Louis, MO 63110. Tel.: 314-362-3692; Fax: 314-747-8228; E-mail: groisman@borcim.wustl.edu.

² M. M. S. M. Wösten and E. A. Groisman, unpublished results.

	ABBREVIATIONS

The abbreviations used are: PCR, polymerase chain reaction; H6, six-histidine tag; bp, base pair(s); RT, room temperature; nt, nucleotide(s).

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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