To test whether OmpR is involved in regulation of the bolA1p, we investigated possible effects of ompR mutation on transcription from bolA1p. In vivo, bolA1p was found to be repressed by OmpR. Furthermore in vitro, the phospho-OmpR was found to bind to the OmpR binding region of bolA1p and repress the transcription by EσS or EσD. These results suggest that the phosphorylated form of OmpR is a negative regulator for the transcription of the bolA1p promoter.
A group of Escherichia coli promoters, called ‘gear box’ promoters, share some unique properties such as: (i) their activity shows an inverse growth rate dependency yielding constant levels of gene expression per cell and per cell cycle at any growth rate; (ii) they are transcribed by EσS, the stationary phase-specific form of RNA polymerase; and (iii) they contain specific sequences at the −10 region and AT-rich sequences upstream from the −35 region . At present, only the ftsQ1p and bolA1p promoters fulfil these requirements, and both of them are involved in control of the expression of genes that are involved in functions associated with cell growth and division.
The intracellular concentration of σD, the major σ for transcription of growth-related genes, is maintained at a constant level throughout growth cycle , but the cellular concentration of σS increases when the cell growth enters the stationary phase [3,4], leading to increased transcription of the bolA and ftsQ genes. However, these promoters are transcribed in vitro by the RNA polymerase containing σD (EσD) as well as σS (EσS) . It remains unclear why these promoters are not expressed in exponential phase cells. An as yet unidentified trans-acting factor(s) or condition(s) may prevent such transcription in vivo.
The mcb promoter (mcbp) also shows a striking homology with bolA1p and ftsQ1p. It contains a gear box-like sequence at the −10 region and an AT-rich sequence upstream from −35 region . However, the stationary phase induction of mcbp transcription does not require σS. Likewise, the rsdp2 promoter for the putative anti-σD factor also contains a gear box-like sequence and is inactive at the exponential phase . The rsdp2 promoter is, however, transcribed by the σD holoenzyme (rsdp1 is transcribed by σS). The activity of mcbp is known to be affected positively by OmpR . In this study, we then analyzed possible effects of OmpR on one gear box promoter, bolA1p.
2Materials and methods
2.1E. coli K-12 strains, bacteriophages and plasmids
E. coli strains, bacteriophages and plasmids were listed on Table 1. To construct pBolA1P, BamHI–EcoRI fragment (239 bp) containing bolA1p of pMAV103  was cloned into the corresponding sites of pBluescriptII SK+ (Stratagene). The rpoS gene between StuI sites on pKTF109 was replaced by HincII fragment containing the chloramphenicol-resistance (Cmr) cassette of pKRP10 to obtain pKTF109Cm . The BamHI-digested pKTF109Cm was transformed into JC7623 . The selection was made for Cmr (25 μg ml−1) to isolate a rpoS mutant (JCΔRS).
BamHI–EcoRI fragment (239 bp) containing bolA1pof pMAV103 was cloned into the corresponding sites of pBluescriptII SK+
rpoS::Cmr, HincII fragment (0.8 kbp) containing Cmr cassette of pKRP10 was cloned into StuI sites of pKTF109
2.2Purification and phosphorylation of OmpR
As described previously , OmpR was expressed and purified by using QIAexpress Ni-NTA Protein Purification System (Qiagen). According to Kenney et al. , 1 μg of OmpR was incubated in 10 μl of 50 mM Tris–HCl (pH 7.4), 50 mM KCl, 20 mM MgCl2, 10 mM acetylphosphate at 37°C for 30 min.
A primer A (5′-TCTATCCGCTCACGTATCAT-3′, 10 pmol, +61 to +42 of bolA1p, Fig. 2A) was labeled with 10 μCi [γ-32P]ATP (5000 Ci mmol−1) by T4 polynucleotide kinase (Toyobo), extracted with phenol:CHCl3, precipitated by ethanol and dissolved in H2O. The labeled primer (100 000 cpm) was incubated with 25 μg of total RNA in hybridization buffer (50% formamide, 0.4 M NaCl, 20 mM PIPES (pH 6.4), 1 mM EDTA) at 80°C for 15 min, followed by further incubation at 37°C overnight. After the hybridized DNA was precipitated with ethanol, it was elongated by Moloney murine leukemia virus reverse transcriptase RNaseH minus (Toyobo) at 37°C for 1 h. The cDNA was precipitated with ethanol, dissolved in formamide dye (95% formamide, 0.05% bromophenol blue, 0.05% xylene cyanol) and analyzed by electrophoresis on a 6% polyacrylamide gel containing 8 M urea. Dideoxy sequencing reaction was carried out with pBolA1P, primer A, and Sequencing PRO DNA sequencing kit (Toyobo). The gel was dried and exposed to an imaging plate that was analyzed in BAS1000 Mac (Fuji film).
A primer C (5′-CGATAGCTTTCATCCACTAC-3′, 10 pmol, +121 to +102) was labeled with 10 μCi [γ-32P]ATP (5000 Ci mmol−1) by T4 polynucleotide kinase (Toyobo). The labeled DNA fragment was amplified by PCR with genome DNA (MC4100, 100 ng) as template, the labeled primer C, the primer B (5′-TGGGTAAGGCATGTAAATTC-3′, 10 pmol, −189 to −170) and KOD DNA polymerase (Toyobo), and then recovered from polyacrylamide gel. The labeled fragment (10 000 cpm) was incubated with 50 μg of total RNA in hybridization buffer (80% formamide, 0.4 M NaCl, 20 mM HEPES (pH 6.4)) at 75°C for 10 min, followed by further incubation at 37°C overnight, and then digested by S1 nuclease. The undigested DNA was precipitated by ethanol and dissolved in formamide dye solution and analyzed by electrophoresis on a 6% polyacrylamide gel containing 8 M urea.
2.5Gel shift assay
The fragments A, B and C (Fig. 3a) were labeled with 10 μCi [γ-32P]ATP (5000 Ci mmol−1; Amersham) and T4 polynucleotide kinase (Toyobo), extracted with phenol:CHCl3, precipitated with ethanol, dissolved with H2O and used for gel shift assay. The labeled fragment D encompassing the bolA1p promoter region (−189 to +61) was amplified by PCR with genome DNA (MC4100, 100 ng) as template, the labeled primer A (see Section 2.3, 10 pmol), the primer B (see Section 2.4, 10 pmol) and KOD DNA polymerase (Toyobo). Each labeled fragment (10 000 cpm, 50 fmol) was incubated with purified OmpR or OmpR phosphorylated by acetylphosphate at 37°C in 10 μl of 50 mM Tris–HCl (pH 7.8 at 37°C), 50 mM NaCl, 3 mM magnesium acetate, 5 mM CaCl2, 0.1 mM EDTA, 0.1 mM dithiothreitol and 25 μg ml−1 bovine serum albumin. After an incubation of 10 min and addition of the dye solution (40% glycerol, 0.025% bromophenol blue, 0.025% xylene cyanol), it was applied to a 6% acrylamide/bisacrylamide (29:1) gel (0.5×TBE, 100 V).
2.6In vitro transcription assay
A BamHI–EcoRI fragment from pBolA1P was used as template DNA of bolA. Purification of RNA polymerase core enzyme and σS subunit has been described previously . Single-round transcription reactions using the RNA polymerase holoenzyme were carried out under standard conditions .
3Results and discussion
3.1Effect of OmpR on expression in vivo of the bolA1p
The effect of OmpR on bolA1p expression was analyzed by primer extension (Fig. 1). Total RNAs were isolated from wild-type MC4100 and ompR disruptant RN101 growing at different cell densities. Equal amounts of these RNA samples were used in the primer extension assay. The mRNA start point from bolA1p was the same as that determined by Nguyen et al. . The level of bolA1p mRNA was higher in RN101 than in MC4100. To see quantification, the bolA1p mRNA in MC4100 (wild-type), RN102 (rpoS mutant), RN101 (ompR mutant), RN103 (ompR and rpoS mutant) and RN403 (envZ mutant) was analyzed by S1 nuclease assay (Fig. 2). At the stationary phase, the level of bolA1p mRNA was two times higher in the envZ (RN403) and ompR (RN101) mutants than in MC4100. However, such an expression was not observed in the rpoS mutants (RN102 and RN103). These results indicate that OmpR phosphorylated by EnvZ negatively regulates the expression of bolA1p dependent on RpoS.
3.2Specific binding of phospho-OmpR to the bolA1p promoter region
We tested whether phospho-OmpR directly interacts with the bolA1p promoter. For this purpose, we employed the gel shift assay using phosphorylated and unphosphorylated forms of OmpR and 32P-labeled bolA1p promoter DNA probes. As shown in Fig. 3, the fragments A, B and D containing the putative OmpR binding region formed complexes only when incubated with 3.6 pmol of OmpR phosphorylated by acetylphosphate (Fig. 3b,c). However, the phospho-OmpR did not bind the fragment C containing −5 to +137 of boA1p (Fig. 3c). These data provide the evidence that the phospho-OmpR binding region is present within the −51 to −5 region of bolA1p promoter, which contains the putative OmpR binding region.
Transcription factor OmpR is a receiver regulator of the two component system for transcription regulation of a set of the osmolarity response genes in bacteria. The activity of OmpR is regulated through a phosphorylation and dephosphorylation cascade by the sensor kinase EnvZ . The consensus sequence of OmpR binding is TTTACTTTTTG-AACAT-TT . For transcription activation, the OmpR protein binds to the OmpR site generally located upstream from the promoter −35, and interacts directly with a subunit of RNA polymerase [19,20]. Thus, OmpR was proposed to be a class-I transcription factor .
bolA1p examined in this study contains OmpR binding site-like sequences with a high level of identify (72%) in the upstream region between −41 and −51 (Fig. 3). The gel shift assay indicates that phospho-OmpR protein indeed binds to the respective fragments containing the putative OmpR sites (Fig. 3). Since the OmpR binding site overlaps with the RNA polymerase binding site, the OmpR binding might interfere with the binding of RNA polymerase to the bolA1p.
Ballesteros et al.  reported that bolA1p promoter retains an AT-rich sequence located upstream of the promoter −35 sequence, which is similar in sequence and position to the UP element with transcription enhancing activity, originally identified in the rrn genes . An alternative explanation for the observed repression of transcription by OmpR is that the enhancing effect of UP element is interfered with OmpR which is bound between the promoter and the UP element.
3.3Repression of transcription in vitro of bolA1p by phospho-OmpR
To prove negative regulation of bolA gene transcription by the phospho-OmpR, we set up a mixed template in vitro transcription assay with one template bearing bolA1p promoter and another tac promoter. The bolA1p promoter was transcribed by EσD as well as EσS (Fig. 4A,B), but was repressed in the presence of phospho-OmpR while transcription of the tac promoter was unaffected by the OmpR protein. These results indicate that phospho-OmpR directly represses the bolA1p promoter.
Recently, Santos et al.  demonstrated that bolA1p is also induced during early exponential growth in response to several forms of stress and that induction can be partially σS independent, where σD should play the important role. However, σD is a constitutive sigma subunit that is not induced by stress. Their results suggest the existence of another transcriptional factor specific for stress-mediated induction of the gene bolA. In this paper, we have shown that bolA1p transcription can be controlled negatively by phospho-OmpR. Although it is not clarified how much OmpR is phosphorylated by stress, bolA1p expression may be induced with a decreased phospho-OmpR concentration in the cells under the stress.
We thank M. Vicente and K. Tanaka for providing plasmids and A. Nishimura for the ompR mutant, JB100. This work was financially supported by the Mishima Kaiun Memorial Foundation and a grant from the Ministry of Education, Science and Culture of Japan.