JOURNAL OF BACTERIOLOGY, Aug. 1995, p. 4696–4702 0021-9193/95/$04.0010 Copyright q 1995, American Society for Microbiology
Vol. 177, No. 16
Modulation of Flagellar Expression in Escherichia coli by Acetyl Phosphate and the Osmoregulator OmpR SOOAN SHIN
AND
CHANKYU PARK*
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Yusong-Ku, Taejon, Republic of Korea Received 19 December 1994/Accepted 3 June 1995
During the search for unknown factors involved in motility, we have found that expression of the flagellar master operon flhDC is affected by mutations of the pta and ackA genes, encoding phosphotransacetylase and acetate kinase, respectively (S. Shin, J. Sheen, and C. Park, Korean J. Microbiol. 31:504–511, 1993). Here we describe results showing that this effect is modulated by externally added acetate, except when both pta and ackA are mutated, suggesting the role of acetyl phosphate, an intermediate of acetate metabolism, as a regulatory effector. Furthermore, the following evidence indicates that the phosphorylation of OmpR, a trans factor for osmoregulation, regulates flagellar expression. First, in a strain lacking ompR, the expression of flhDC is no longer responsive to a change in the level of acetyl phosphate. Second, an increase in medium osmolarity does not decrease flhDC expression in an ompR mutant. It is known that such an increase normally enhances OmpR phosphorylation. Third, OmpR protein binds to the DNA fragment containing the flhDC promoter, and its affinity is increased with phosphorylation by acetyl phosphate. DNase I footprinting revealed the regions of the flhDC promoter protected by OmpR in the presence or absence of phosphorylation. Therefore, we propose that the phosphorylated OmpR, generated by either osmolarity change or the internal level of acetyl phosphate, negatively regulates the expression of flagella. tations (37, 52), on the nutritive state (28), or on the growth phase (37). Acetyl-P is also known to phosphorylate various response regulators, including CheY, NRI, PhoB, and OmpR (11, 28, 52), and is thus suggested to influence either the sensitivity or the magnitude of an adaptive response (29, 51). Recent evidence indicates that the ability to synthesize acetyl-P is required in order to survive glucose starvation (35). Osmoregulation of the major porin proteins, OmpC and OmpF, is known to be mediated by a two-component regulatory system, consisting of OmpR and EnvZ. The sensor protein, EnvZ, controls the phosphorylation of OmpR, a regulatory protein that has a DNA binding property (39). In contrast to the expression of OmpC synthesis, which increases proportionally with increasing osmolarity, OmpF is synthesized at low osmolarity and repressed at high osmolarity (39). In OmpF regulation, phosphorylated OmpR (phospho-OmpR) plays a role as an activator when its level is low and as a repressor when its level is high. This dual role of phospho-OmpR in OmpF regulation was explained by the hierarchical binding of phospho-OmpR to the promoter region of ompF (38). Besides the outer membrane porins, other genes (49)—those encoding the tripeptide permease TppB of Salmonella typhimurium, the outer membrane protease Opr, the PhoA and PhoE proteins of the Pho regulon, and the positive regulator MalT of the maltose operon—are known to be regulated by EnvZ and OmpR. Recently, it was demonstrated that microcin C51 production depends on OmpR (24) and that the promoter of the fatty-acid receptor gene, fadL, is negatively regulated at high osmolarity by interacting with phospho-OmpR (15). The pleiotropic effect of regulation by EnvZ and OmpR suggests the role of OmpR as a global regulator. In this study, we present evidence indicating that flagellar expression at the transcription of the flhDC master operon is modulated by a mutation altering the internal level of acetyl-P. Furthermore, we demonstrate that this transcriptional modulation is mediated by a direct interaction of the osmoregulator OmpR with the promoter region of flhDC, suggesting that the
The flagellar and chemotaxis regulon of Escherichia coli contains more than 40 genes, organized into at least 13 operons, that specify functions for motility and chemosensory signal processing (for a review, see reference 27). These genes are transcriptionally regulated in a cascade fashion in order to be coordinated with flagellar assembly (16, 22, 25). The flagellar synthesis is inhibited when the bacteria are grown under a catabolite-repressive condition (1, 45) or stressful conditions (1, 42) such as high concentrations of salts, sugars, or alcohols, high temperatures, or conditions of blocked DNA replication. The expression of flhDC, the master operon, is required for the expression of the whole flagellar regulon. It is reported that the heterotetramer consisting of two FlhD and two FlhC proteins binds directly to the upstream region of three class II operons (26). The mutation called cfs (constitutive flagellar synthesis), which is insensitive to catabolite repression, was mapped to the flhDC operon (45). In addition, mutations circumventing the high-temperature inhibition of flagellation were mapped in both the flhDC and fliA operons (46). The heat shock proteins, DnaK, DnaJ, and GroEL, were shown to be required for the expression of the flhDC operon (43), while mutations which result in defective in phospholipid biosynthesis, such as those in pss, psd (41), and pgsA (34) genes, and the mutations in galU (23) and mdoA (12) genes inhibit motility. E. coli not only produces acetate as part of the mixed-acid fermentation process but also utilizes it as a carbon source (20). The fermentative production of acetate from acetyl coenzyme A occurs through a constitutive pathway, consisting of phosphotransacetylase (the pta gene product) and acetate kinase (the ackA gene product), via acetyl phosphate (acetyl-P) (6). It was shown that intracellular levels of acetyl-P vary over a wide range, depending on the presence of pta or ackA mu-
* Corresponding author. Phone: 82-42-869-4019. Fax: 82-42-8694010. Electronic mail address (INTERNET):
[email protected]. ac.kr. 4696
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TABLE 1. Bacterial strains, phages, and plasmids used Strain, bacteriophage, or plasmid
E. coli strains BW13635 BW15015 BW16464 BW16777 MC4100 MH1461 OW1 TK827 XA21 CP807 CP992 CP993 CP994 CP996 CP997 CP998 CP999 CP1000 CP1001 CP1003 Bacteriophages lNK1045 lRZ5 lSS10 P1vir Plasmids pAT428 pMC1396 pMY150 pPM61 pT7/T3a19 pSS9 pSS10 pSS11
Relevant genotype
Source or reference
proC::Tn5-132 ackA::TnphoA9-9 (ackA-pta-hisPQJ-dhuA) zej-223::Tn10 pta::TnphoA9-3 F2 D(argF-lac)U169 rpsL relA flhD deoC ptsF rbsR MC4100 envZ11 thiA thr leu his lacY rpsL MC4100 F(ompF9-lacZ)16-23 ompR331::Tn10 lacI D(lac)58 xyl mtl thi rpsL OW1 D(lac)58 CP807 lSS10 CP992 F(pta-1::Tn10-lacZ) CP992 pta::TnphoA9-3 CP992 ackA::TnphoA9-9 CP992 D(ackA)2 zej-223::Tn10 CP992 D(ackA-pta-hisPQJ-dhuA) zej-223::Tn10 CP992 ompR331::Tn10 CP994 ompR331::Tn10 CP996 ompR331::Tn10 CP992 envZ11 zhl::Tn10
B. Wanner 52 53 B. Wanner 14 14 36 5 B. Bachmann This work This work This work This work This work This work This work This work This work This work This work
l Pam cIts857 b221 Tn10-lacZ l 9bla 9lacZ lacY1 lRZ5 flhDC9-lac
53 18 This work Laboratory stock
pBR322 ompB pBR322 9lacZ lacY pBR322 ompC pMK2204 flhDC
8 7 17 4 Bethesda Research Laboratories This work This work This work
pT7/T3a19 ompR pMC1396 flhDC9-lac pT7/T3a19 flhDC promoter
repression of flhDC may occur through the phosphorylation, by either EnvZ or acetyl-P, of OmpR. MATERIALS AND METHODS Bacterial strains. All strains used are derivatives of E. coli K-12 and are listed in Table 1. The fusion pta-1::Tn10-lacZ was obtained by transposon mutagenesis with lNK1045 (53) containing Tn10-lacZ (44). pta-101 was isolated by the method with monofluoroacetate resistance as described (13). To confirm pta and ackA mutations, phosphotransacetylase and acetate kinase assays (6) were performed. The deletion D(ackA)2 lacks the segment of DNA including the 59 end of the gene and several kilobases upstream (36a). The deletion D(pta-ackAdhuA-hisJQP), which eliminates both pta and ackA activities, was derived from BW16464 (Table 1). The transposon insertion zej-223::Tn10, obtained from B. Wanner (52), was used for transduction of the markers pta-101, D(ackA)2, and D(pta-ackA-dhuA-hisJQP). pta::TnphoA9-3 and ackA::TnphoA9-9 (52) exhibit the Lac2 phenotype, presumably because the insertional orientation of lacZ is opposed to the transcriptional direction of pta or ackA. ompR331::Tn10 and envZ11 were derived from the strains TK827 (5) and MH1461 (14). To eliminate the background level of LacZ activity, the lacZ in-frame deletion D(lac)58 (from the strain XA21) was introduced by using the transposon insertion in proC::Tn5-132. Construction of flhDC*-lac fusion. The flhDC9-lac fusion was constructed by subcloning the 1.8-kb PvuII fragment, containing an intact flhD and the Nterminal 14 codons of flhC, from pPM61 (4) into the SmaI site of pMC1396 (7), yielding pSS10. In this construct, the lacZ gene was fused translationally to the flhC gene. The recombinant l phage (lSS10) containing the lac fusion was obtained by in vivo recombination after cells containing pSS10 were infected with lRZ5 (18). The lSS10 was lysogenized into CP807 (Table 1). Flagellar expression was also monitored with transcriptional lacZ fusions to flagellar genes, including flhD, flhC, fliA, and fliC, constructed by Komeda (21), which gave rise to basically similar results. Since Mudlac fusions are unstable at high temperatures, they were not suitable for some of our experiments. The expression of lac
fusions was monitored by measuring b-galactosidase activity as described previously (31). Growth conditions. Luria-Bertani medium, tryptone broth (TB; 1% tryptone and 0.25% NaCl), and M9 minimal salts medium were prepared as described by Miller (31). Sugars (0.2%), organic acid (40 mM), thiamine (0.01%), and amino acids (1 mM) were added to the minimal medium as required. To examine its physiological effect, acetate was added at a final concentration of 40 mM to the liquid medium. For selection and phenotypic confirmation of mutants carrying changes in pta and ackA, 25 mM monofluoroacetate was included in M9 minimal medium containing 40 mM pyruvate as the carbon source. A semisolid swarm plate, containing 0.25% agar, was made with 1% tryptone and 0.25% NaCl (36). Antibiotics were added at the following concentrations: ampicillin, 100 mg/ml; tetracycline, 12.5 mg/ml; kanamycin, 50 mg/ml. Enzymes and chemicals. All restriction enzymes were purchased from KOSCO, Seoul, Republic of Korea, avian myeloblastosis virus (AMV) reverse transcriptase was purchased from Promega, and other modification enzymes for DNA were purchased from Boehringer Mannheim. The column resins were purchased from Bio-Rad and Pharmacia, radioisotopes were purchased from Amersham, and other reagents were purchased from Sigma Chemical Co. and Boehringer Mannheim. Purification of OmpR protein. To overproduce the protein OmpR, the ompR gene was subcloned into plasmid pT7/T3a19 (Bethesda Research Laboratories). The BclI-EcoRI fragment (1.2 kb) from pAT428, harboring the ompB region (8), was ligated with the vector fragment digested with BamHI and EcoRI, yielding pSS9. To increase the lac promoter-dependent expression, MC4100 was used as the lacI host. Cells were grown for 8 h at 358C in Luria-Bertani medium supplemented with ampicillin (100 mg/ml). The purification of OmpR followed the method described in reference 17, with some modification. The cells harvested were sonicated and subjected to ultracentrifugation to remove the membrane fraction. The supernatant, dialyzed with 20 mM Tris-Cl (pH 7.5) buffer containing 10 mM b-mercaptoethanol and 5% (vol/vol) glycerol, was loaded onto a DEAE-Sepharose column, and proteins were eluted with a 0- to 120-mM NaCl gradient. Fractions containing OmpR were then pooled and precipitated with
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ammonium sulfate at a 60% final concentration. The pellet from the ammonium sulfate fractionation was resuspended in 20 mM bis(2-hydroxyethyl)-iminotris(hydroxymethyl)methane (Bis-Tris) (pH 6.5) buffer containing 1 mM dithiothreitol and 100 mM KCl and was passed through a Sephacryl S-200 (Pharmacia) column. The purity of OmpR in the pooled fraction was more than 95%. The identity of the protein was confirmed by rabbit anti-OmpR antiserum. Primer extension analysis. MC4100 harboring pSS11 was grown in TB medium to an optical density at 560 nm (OD560) of 0.4, the same condition for the measurement of flagellar expression. The purification of RNA was performed by buffered phenol extraction and CsCl gradient ultracentrifugation (3). The amount of total RNA was determined by measuring the OD260. The primer extension experiment was carried out as described previously (30). The oligonucleotide primer 59-CCCACCCAGAATAACC-39, complementary to the region from 1161 to 1176 that includes the translation start site of flhDC mRNA (see Fig. 4), was used. 32P-labelled (.5000 cpm) primer (20 pg) was hybridized to 90 mg of purified RNA at 308C for 12 h. The polymerization reaction was performed with 35 U of AMV reverse transcriptase (Promega) at 428C for 1 h. The results were reproducible regardless of modification of experimental conditions for hybridization (378C for 4 h) and polymerization (for 30 min). DNA sequencing. For the determination of the upstream DNA sequence of the flhDC promoter, the EcoRI-HindIII fragment of pSS11 was subcloned into M13mp18 phage. The sequencing was performed by the dideoxy-chain termination method (40). DNA mobility shift assay. A DNA probe containing the promoter region of flhDC was prepared as follows. The DraI-NsiI fragment (490 bp; see Fig. 4) from plasmid pPM61 (4) was treated with T4 DNA polymerase in the presence of deoxyribonucleotides to remove the 39 protruding terminal. This fragment was inserted into the SmaI site of pT7/T3a19 so that the DraI site was located close to the HindIII site in the multiple cloning site. The resulting plasmid, pSS11, could now be digested with BamHI, adjacent to the DraI end, or with EcoRI, adjacent to the NsiI end. The 511-bp BamHI-EcoRI fragment was phosphorylated by T4 polynucleotide kinase with [g-32P]ATP. In order to eliminate the possibility of nonspecific binding of OmpR, the remaining portion of pSS11 DNA was digested with BglI and ScaI, yielding 178-, 364-, 904-, and 1,421-bp fragments of DNA, which were then used as nonspecific DNA probes. The 511-bp flhDC promoter DNA fragment was further cleaved to obtain the 220-bp BamHI-Sau3AI fragment, containing the upstream flhDC promoter and the downstream 291-bp Sau3AI-EcoRI fragment (see Fig. 4). The reaction mixture (10 ml) for the mobility shift assay contained 10 mM Tris-Cl (pH 7.4), 50 mM KCl, 1 mM dithiothreitol, 10 mM MgCl2, 10% glycerol, 0.5 mg of poly(dI-dC), 32P-labelled (about 10,000 cpm) probes, and 0 to 400 ng of OmpR protein. For the phosphorylation of OmpR, 10 mM acetyl-P (Sigma Co.) was added to the reaction mixture. OmpR phosphorylation and binding to probes were performed at 308C for 1 h. After 30 min, the sample was loaded onto 5% polyacrylamide gel (ratio of acrylamide to bisacrylamide, 80:1) containing 2.5% glycerol. The gel was electrophoresed in a low-ionic-strength buffer containing 6.7 mM Tris-Cl (pH 7.4), 3.3 mM sodium acetate, and 1 mM EDTA (3). DNase I footprinting. Various amounts of OmpR protein were added to the binding reaction mixture, which is the same as the one used in the DNA mobility shift assay but contains more labelled (about 30,000 cpm) probe without glycerol. Then, 0.33 U (U as defined by the manufacturer) of DNase I (Boehringer Mannheim) diluted in 10 mM MgCl2 was added, and then the probe was subjected to digestion for 1 min at room temperature. The reaction was stopped by the addition of an equal volume of stop solution (200 mM sodium acetate, 30 mM EDTA, 0.2% sodium dodecyl sulfate, and 65 mg of yeast tRNA per ml). Samples were extracted with phenol-chloroform, precipitated with ethanol, washed with 70% ethanol, dissolved in loading buffer for sequencing, and then loaded onto 6% polyacrylamide–8 M urea gel.
RESULTS Effect of acetyl-P on flagellar expression. In a previous study (44), we obtained a randomly generated insertion in the phosphotransacetylase gene, termed pta-1::Tn10-lacZ, that partially restores the motility inhibited at 398C. The insertion improved swarming on semisolid agar plates because of an increased expression of flagellin (data not shown). Genetic mapping revealed that the insertion lies in the pta gene for phosphotransacetylase (44), suggesting an involvement of the Pta and AckA pathway. We investigated the effect of an intermediate of this pathway, acetyl-P, on flagellar expression by using various acetate mutations that were reported to alter the level of acetyl-P in the cell (37) (the level is also changed by adding acetate externally [28, 52]). The results are summarized in Fig. 1. Expression of flhDC, as monitored with the flhDC9-lac fusion, is generally increased in pta and pta ack double mutants, whereas it is decreased in the ackA mutant (Fig. 1). However,
J. BACTERIOL.
FIG. 1. Effect of acetate mutations on expression of flagellar genes. b-Galactosidase activity expressed from the flhDC9-lac fusion strain was measured in the cells grown to an exponential stage (OD560, 0.3 to 0.5) in TB medium at 35 or 398C, M9-glucose medium at 358C, or M9-glucose medium supplemented with 40 mM acetate. Strains used were CP992 (wild type), CP993 (pta), CP997 (ackA), and CP998 [D(pta-ackA)]. Since pta-1::Tn10-lacZ itself expresses b-galactosidase, the activity of flhDC9-lac in the pta-1::Tn10-lacZ strain was taken as the activity measured for an isogenic background strain of CP975, except that the activity of flhDC9-lac was subtracted. The expression of flhDC9-lac in pta alleles, including pta-1::Tn10-lacZ, pta::TnphoA9-3, and pta-101 (see Materials and Methods for isolation), varied from 81 to 116%.
the effects were no longer observed in mutants other than the pta ackA double mutant when acetate was added externally. Under conditions of catabolite repression, the effects of acetate mutations were still observed, indicating that these effects are independent of catabolite repression. Furthermore, temperature increase reduces the overall expression of flhDC without altering the pattern found in different acetate mutants. The expression of flhDC in TB at 358C was consistent with that of flagellin, a downstream component (data not shown). The results imply that the regulation of flagellar expression by acetyl-P occurs negatively at the level of the flhDC master operon. Involvement of the osmoregulator OmpR. We speculated that the transcriptional effects of acetyl-P on flagellar genes may involve one of the transcriptional regulators of the twocomponent systems (11, 28, 52). However, no such regulators that interact with the promoter of the flhDC operon were identified. On the other hand, there has been evidence indicating that flagellar synthesis is repressed when cells are exposed to high salt concentrations (42). When we examined the expression of the flhDC gene in a strain lacking ompR, we observed that the expression was no longer responsive to a change in acetyl-P level, as demonstrated with pta and ackA mutants (Fig. 2). A higher level of flhDC expression is maintained in an ompR mutational background, which is comparable to that of the strain containing only the pta mutation. This observation implies a negative role of OmpR protein in regulating the flhDC promoter. The chemotactic swarm phenotype on tryptone (data not shown) confirms that the ompR mutation masks the effects of acetate mutations, which is consistent with the observation for flhDC expression, shown in Fig. 2. Repression of flhDC by increasing osmolarity. In order to assess the role of OmpR phosphorylation in motility, we measured the expression of flhDC9-lac under different osmolarity conditions (Fig. 3). Cells were grown at 358C in tryptone medium with various concentrations of NaCl to a density of 0.4 at OD560. The flhDC expression in wild-type cells decreased dras-
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FIG. 2. Involvement of OmpR in acetyl-P effect on flhDC expression. The expression of flhDC in the strains with acetate mutations plus ompR mutations was examined. Because the ompR mutation had a Tcr marker, the pta::TnphoA9-3 and ackA::TnphoA9-9 insertions with Kmr markers were used. Strains used were CP992 (ompR1 [wild type]), CP994 (ompR1 pta), CP996 (ompR1 ackA), CP999 (ompR2 [wild type]), CP1000 (ompR2 pta), and CP1001 (ompR2 ackA).
tically with increasing osmolarity in the range between 0.043 and 0.15 M NaCl. In contrast, minor effects were observed in the ompR mutant, although some reductions with unknown causes were noticed at very high osmolarity. EnvZ protein is known to phosphorylate OmpR by modulating its kinase and phosphatase activities in response to the osmolarity of the medium. The hypersignaling mutation envZ11, which causes increased phosphorylation of OmpR due to a defect in dephosphorylation (14), exhibits a nonmotile phenotype (data not shown), which indeed results in severe reduction in flhDC expression below the level of that of the ackA mutant. This implies that the repression of flagellar synthesis at high osmolarity is due to the phosphorylation of OmpR. Interaction of OmpR with the flhDC promoter. To study the characteristics of the flhDC promoter, we have determined the DNA sequence of the previously unsequenced (4) upstream promoter between the DraI and Sau3AI sites, shown in Fig. 4.
FIG. 3. Dependence of flhDC expression on OmpR. The b-galactosidase activity from the flhDC9-lac fusion was measured for cells grown in media with 1% tryptone and various NaCl concentrations. Overnight cultures in TB were transferred to fresh 1% tryptone media with various NaCl concentrations and incubated at 358C until exponential phase (OD560 5 0.4). The regular TB contains 0.043 M NaCl (0.25%). Strains used were CP992 (wild type) and CP999 (ompR2).
FIG. 4. DNA sequence of the flhDC promoter region. The sequence between the DraI and Sau3AI sites was determined during this study. In comparison with the previously determined sequence, our sequence was missing a T between 286 and 287. The underlined letters in boldface type are OmpR binding sites. 11 represents the initiation site of transcription. The start codon (GTG) for the FlhD translation is shown in boldface capital letters. The novel cAMP receptor protein binding site, based on the consensus sequence aanTGTGAnntnnn NCANAtt (10), is shown in boldface lowercase letters. The previously determined cAMP receptor protein site predicted is located between 1139 and 1159. The capital letters with dots below represent the putative integration host factor binding sites, which are based on the consensus sequence (50) C/TAAnnnnTT GATA/T.
The transcription initiation site was also determined by primer extension analysis with a primer containing the GTG start codon (4) of the flhDC operon (Fig. 4). In addition to a single intense band for the major transcription initiation, two relatively minor bands, indicating the initiations at 1128 and 1129, were consistently found under all conditions for hybridization and polymerization in this study (data not shown). The direct interaction of OmpR with the flhDC promoter was demonstrated by a DNA mobility shift assay. The mobilities of nonspecific DNAs within the pT7/T3a19 vector were unaltered in the presence of different amounts of purified OmpR (Fig. 5). However, the 511-bp BamHI-EcoRI fragment containing the flhDC promoter clearly shows a shift. Adding a competitor DNA, the XbaI-EcoRI fragment (0.9 kb) containing the ompC promoter region of pMY150 (17), to the assay mixture reduced the amount of the band corresponding to the shifted flhDC DNA (data not shown). The BamHI-EcoRI fragment, labelled with [g-32P]ATP at each terminus, was cut with Sau3AI (Fig. 4). A 220-bp BamHI-Sau3AI (upstream sequence) fragment labelled at the BamHI end and a 291-bp Sau3AI-EcoRI (downstream sequence) fragment labelled at the EcoRI end were further investigated by DNase I footprinting. The regions protected from DNase I digestion because of
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J. BACTERIOL.
DISCUSSION
FIG. 5. Mobility shift of the flhDC promoter DNA due to interaction with OmpR protein. The plasmid pSS11, harboring the DraI-NsiI fragment of the flhDC promoter region was digested with BamHI, EcoRI, BglI, and ScaI (see Materials and Methods for details) and labelled with T4 polynucleotide kinase. The 511-bp DNA contains the region of the flhDC promoter. The numbers to the right indicate the sizes (base pair) of the DNA fragments. The solid triangle indicates the position of the shifted DNA interacting with OmpR protein. Other DNA bands were not affected by OmpR. The amounts of OmpR were as follows: lane 1, none; lane 2, 50 ng; lane 3, 100 ng; lane 4, 200 ng; lane 5, 400 ng.
nonphosphorylated OmpR were found at 2135 to 2179 and 14 to 149 when an excess amount of protein, i.e., more than 1.4 mg (50 pmol) of OmpR, was added (Fig. 6A and B). Furthermore, when phospho-OmpR was used, the protected regions were extended from 2135 to 2116 and from 14 to 217, with an increase in affinity of about 10-fold. The windows are clearly visible in the lanes with 100 or 200 ng of OmpR. The region between 14 to 149 is more sensitive to phospho-OmpR than is the upstream sequence (Fig. 6C and D).
We demonstrated that the level of acetyl-P modulates flagellar expression in the flhDC master operon. Evidence, both in vivo and in vitro, indicates that this effect is mediated by the osmoregulator OmpR, involving phosphorylation. Such a regulatory interaction appears to be particularly effective when cells are under osmotic stress or when they encounter a change in nutritional supply. The level of acetyl-P may fluctuate, depending on the nutritional state of the cell (28), thereby serving as a global regulator. Cells unable to synthesize acetyl-P were inefficient in tolerating glucose starvation (35). The effect of acetyl-P on flagellar expression seems independent of catabolite repression. Changes in osmolarity as well as envZ11 mutations affect flagellar synthesis (Fig. 3), suggesting that the effects of high concentrations of salts on the synthesis of flagella (42) involve the phosphorylation of OmpR. The change in porin synthesis is gradually affected by the level of phospho-OmpR generated by acetyl-P (28). In contrast, flagellar synthesis appears to change drastically with the level of acetyl-P (Fig. 1). When acetyl-P is not produced, as in the pta mutant, the level of expression of flhDC is high. Furthermore, since the experimental conditions for growth and the swarm assay are kept at low osmolarity, the level of phospho-OmpR in pta mutants would be almost negligible. Under the same conditions, the phenotypic similarities observed between the pta and ompR null mutants indicate that OmpR and phospho-OmpR are not required for the expression of flagella. When cells accumulate acetyl-P, and thus have increased levels of phospho-OmpR, the flagellar expression is substantially reduced. The same repression, due to the envZ11 mutation, which is known to activate OmpR constitutively, was observed. We therefore propose that the regulatory role of phospho-OmpR in flagellar synthesis is negative, whereas it is both positive and negative in porin expression (39).
FIG. 6. DNase I footprinting of OmpR binding sites in the flhDC operon promoter region. The upstream region in the BamHI-Sau3AI fragment was labelled at the BamHI end (A and C), and the Sau3AI-EcoRI fragment containing the downstream region was labelled at the EcoRI end (B and D). The labelled fragments were then incubated with nonphosphorylated OmpR (A and B) at the following amounts: lane 1, none; lane 2, 44 ng; lane 3, 87.5 ng; lane 4, 175 ng; lane 5, 350 ng; lane 6, 700 ng; lane 7, 1400 ng; lane 8, 2800 ng. OmpR protein phosphorylated with acetyl-P (C and D) was added to the reaction mixture as follows: lane 1, none; lane 2, 25 ng; lane 3, 50 ng; lane 4, 100 ng; lane 5, 200 ng; lane 6, 300 ng; lane 7, 350 ng; lane 8, 400 ng. The numbers to the right of each panel represent the distances (nucleotide base) from the transcription initiation site (shown in Fig. 4).
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In previous studies of Pho, Ntr, and Che systems (9, 11, 52), the effects of acetyl-P were seen exclusively when there were no cognate kinases. However, a recent experiment (28) demonstrated that acetyl-P is able to phosphorylate OmpR in the presence of EnvZ. The change in pattern of outer membrane porins due to acetyl-P was observed by adding acetate to the pta mutant. We have seen a similar difference in porin composition when we introduced acetate mutations (data not shown). The level of OmpC increased more significantly in the ackA mutant than in the wild types or pta or the pta ackA double mutants at low osmolarity. Moreover, even OmpF was detected in pta mutants or the pta ackA double mutant at high osmolarity. This indicates that acetyl-P is able to phosphorylate OmpR, despite the presence of phosphatase and kinase. We believe that the difference in responsiveness to acetyl-P between the two-component system with EnvZ and OmpR and other two-component systems lies in the presence of excess OmpR relative to EnvZ. In the Ntr system, both NRI and NRII are present at low levels (a few molecules per cell [29]). Although the situation is a little more complicated because of the presence of the dephosphorylation facilitator CheZ (24,000 molecules of monomer), the ratio of the molecules of monomer of the kinase CheA (2,500) to those of the substrate CheY (12,000) is not greatly off balance (48). In contrast, EnvZ and OmpR exist at approximately 10 and 1,000 copies per cell, respectively (33, 54). Thus, it is likely that some fraction of OmpR beyond the reach of the phosphatase and kinase activities of EnvZ would be available for phosphorylation by acetyl-P. The large amount of OmpR under the noninduced condition also implies the role of OmpR as a global regulator that might control some physiological events other than the expression of outer membrane porin. Actually, some proportions of OmpR seem to be phosphorylated by acetyl-P in wild types, because the flagellar expression of the wild type is more significantly reduced than in the pta mutant (Fig. 2). The flagellar genes are subjected to regulation by a number of other genes, including galU and mdoA. Membrane-derived oligosaccharide (MDO) was discovered as an acceptor molecule for sn-1-phosphoglycerol during phospholipid turnover (19). The synthesis of MDO in periplasmic space is suppressed by high osmolarity, suggesting that MDO plays a role in adaptation to an osmolarity change. It has been shown that flagellar synthesis is reduced in mdoA mutants, whereas OmpC expression is enhanced. In addition, a reversion from mdoA mutation for better flagellation was mapped in the ompB region where envZ and ompR are located (12). The galU mutation that causes a defect in the synthesis of UDP-glucose, a precursor of MDO, was also reported to inhibit flagellation (23). Our finding on the involvement of OmpR in flagellar regulation appears to shed light on earlier queries raised by independent observations. We propose that the effects of mdoA and galU mutations are mediated by OmpR, although the mechanism by which MDO molecules interact with EnvZ is still unclear. Furthermore, considering that the turnover of phospholipids is affected by changes in osmolarity (19) involving MDOs, it is likely that the mutations in pss, psd (41), and pgsA (34) may also influence flagellation, perhaps through OmpR. The binding of purified OmpR, presumably in a nonphosphorylated form, to the ompC and ompF promoters has been studied extensively. In those studies, it was shown that the function of OmpR phosphorylation is to enhance (approximately 10-fold [2]) the affinity of this protein to target DNA, without altering its specificity. In our study, a nearly 10-fold difference in the binding of OmpR, due to phosphorylation, was observed in addition to the extension of protected sites. As in the case of the OmpF promoter (38), it is likely that OmpR
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binds hierarchically to the flhDC promoter. The region between 14 and 149 first binds the OmpR protein, and then the upstream region from 2135 to 2179 is occupied with increasing OmpR concentrations. The downstream binding region from 14 to 149 is further extended to 217, including the transcription initiation site, when OmpR is phosphorylated. Unlike the regulation of the mode of OmpR binding to ompF, OmpR does not seem to act as an activator in the flagellar promoter, because the flagellar regulon is expressed in ompRnull or pta mutants, in which phospho-OmpR does not exist. Thus, we propose that the binding itself and an extended occupancy upon phosphorylation negatively regulate flhDC expression. Although we do not have evidence for the interaction between OmpR proteins bound to the upstream and downstream regions, it is possible that they associate to form a loop, as in ompF regulation (38, 47). The sequencing of the flhDC upstream region revealed the presence of two integration host factor binding sequences (50), each with one mismatch, between the two OmpR binding sites (Fig. 4). An examination of the newly determined sequence also revealed a candidate for the cyclic AMP (cAMP) receptor protein binding sequence (10), for which the homology is greater than that of the sequence predicted previously (4) (Fig. 4). Although an interaction between the integration host factor and the flagellar promoter has yet to be demonstrated, such an interaction would suggest that the integration host factor mediates the looping involved. We also found many A-T tracks, which might be responsible for the formation of a bent DNA in the flhDC promoter analyzed by two-dimensional gel electrophoresis (20a, 32). ACKNOWLEDGMENTS We thank J. Adler, B. Bachmann, M. S. Cho, G. Hazelbauer, N. Kleckner, Y. Kohara, P. Matsumura, T. Mizuno, W. Shi, B. Wanner, and A. Wolfe for kindly providing bacterial strains and phages. We also thank B. Wanner and A. Wolfe for numerous discussions. This work was supported by grants from the Korea Science and Engineering Foundation and the Ministry of Education of Korea. REFERENCES 1. Adler, J., and B. Templeton. 1967. The effects of environmental conditions on the motility of Escherichia coli. J. Gen. Microbiol. 46:175–184. 2. Aiba, H., F. Nakasai, S. Mizushima, and T. Mizuno. 1989. Phosphorylation of a bacterial activator protein, OmpR, by a protein kinase, EnvZ, results in stimulation of its DNA-binding ability. J. Biochem. 106:5–7. 3. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1992. Short protocols in molecular biology: a compendium of methods from current protocols in molecular biology. Green Publishing Associates and John Wiley and Sons, New York. 4. Bartlett, D. S., B. B. Frantz, and P. Matsumura. 1988. Flagellar transcriptional activators FlbB and FlaI: gene sequences and 59 consensus sequences of operons under FlbB and FlaI control. J. Bacteriol. 170: 1575–1581. 5. Brissette, R. E., K. Tsung, and M. Inouye. 1991. Suppression of a mutation in OmpR at the putative phosphorylation center by a mutant EnvZ protein in Escherichia coli. J. Bacteriol. 173:601–608. 6. Brown, T. D., M. C. Jones-Mortimer, and H. L. Kornberg. 1977. The enzymic interconversion of acetate and acetyl-CoA in Escherichia coli. J. Gen. Microbiol. 102:327–336. 7. Casadaban, M. J., J. Chou, and S. N. Cohen. 1980. In vitro gene fusions that join an enzymatically active b-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J. Bacteriol. 143:971–980. 8. Coleman, J., P. J. Green, and M. Inouye. 1984. The use of RNAs complementary to specific mRNA to regulate the expression of individual bacterial genes. Cell 37:429–436. 9. Dailey, F., and H. C. Berg. 1993. Change in direction of flagellar rotation in Escherichia coli mediated by acetate kinase. J. Bacteriol. 175:3226–3229. 10. de Crombrugghe, B., S. Busby, and H. Buc. 1984. Cyclic AMP receptor protein: role in transcription activation. Science 224:831–838. 11. Feng, J., M. R. Atkinson, W. McCleary, J. B. Stock, B. L. Wanner, and A. J.
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