This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ogasawara, H.
Right arrow Articles by Utsumi, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ogasawara, H.
Right arrow Articles by Utsumi, R.

 Previous Article  |  Next Article 

Journal of Bacteriology, December 2004, p. 8317-8325, Vol. 186, No. 24
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.24.8317-8325.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Negative Regulation of DNA Repair Gene (ung) Expression by the CpxR/CpxA Two-Component System in Escherichia coli K-12 and Induction of Mutations by Increased Expression of CpxR

Hiroshi Ogasawara,1 Jun Teramoto,1 Kiyo Hirao,1 Kaneyoshi Yamamoto,1 Akira Ishihama,2 and Ryutaro Utsumi1*

Department of Bioscience and Biotechnology, Graduate School of Agriculture of Kinki University, Nakamachi, Nara,1 Division of Molecular Biology, Nippon Institute for Biological Science, Ome, Tokyo, Japan2

Received 21 July 2004/ Accepted 10 September 2004


arrow
ABSTRACT
 
In Escherichia coli K-12 overexpressing CpxR, transcription of the ung gene for uracil-DNA glycosylase was repressed, ultimately leading to the induction of mutation. Gel shift, DNase I footprinting, and in vitro transcription assays all indicated negative regulation of ung transcription by phosphorylated CpxR. Based on the accumulated results, we conclude that ung gene expression is negatively regulated by the two-component system of CpxR/CpxA signal transduction.


arrow
INTRODUCTION
 
The cpxRA two-component system of Escherichia coli, consisting of CpxA sensor kinase or phosphatase and CpxR cognate response regulator, regulates biofilm formation, motility, chemotaxis, host cell invasion, and virulence (5, 8, 13, 21). By using the generally accepted DNA sequence for binding CpxR (two tandem GTAAA motifs separated by a 5-bp linker, the CpxR box), DeWulf et al. (6) performed 15-bp weighted matrix analysis for CpxR recognition sites on the entire E. coli genome and identified new target genes (ung, ompC, psd, mviA, aroK, rpoE, secA, and aer) that might be regulated positively or negatively by CpxR/CpxA. The expression of cpxRA increases sharply at the onset of the stationary growth phase (5), suggesting the involvement of the Cpx system in stationary phase survival.

Uracil-DNA glycosylase (Ung), the most abundant type among all of the glycosylases in bacterial cells (16, 20), excises uracil residues from DNA, which arise as a result of either misincorporation of dUTP by DNA polymerase or deamination of DNA cytosine (9, 10, 11, 16, 27). The ung mutants show the specific mutator effect of a G · C-to-A · T transition as Duncan and Weiss reported previously (9). The ung gene expression remains constant up to the early stationary phase of E. coli but declines in the late stationary phase (26). However, the mechanisms underlying these phenomena are not understood well.

In order to clarify the possible involvement of the Cpx system in repression of ung expression, the effects of CpxR overexpression on ung expression are investigated in this study.


arrow
MATERIALS AND METHODS
 
Bacterial strains and growth conditions. The E. coli strains used are listed in Table 1. They were grown at 37°C in a Luria-Bertani (LB) medium (pH 7.5) containing 1% Bacto Tryptone (Difco), 0.5% Bacto Yeast Extract (Difco), and 1% NaCl. The following antibiotics were added to the medium: ampicillin, 100 µg/ml; chloramphenicol, 25 µg/ml; tetracycline, 12.5 µg/ml; and kanamycin, 25 µg/ml.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Bacterial strains and plasmids

Construction of plasmids. The plasmids used are listed in Table 1. A DNA fragment (538 bp) containing the ung promoter region was prepared by PCR using E. coli BW25113 genome DNA as template and a pair of primers, ung-EcoRI-F (5'-TGGAACTTCACGGAATTCAATGTCA-3') and ung-BamHI-R (5'-AAAATAGGGATCCTGCTTCTCTTCA-3'). After digestion with EcoRI and BamHI, the PCR-amplified fragment was inserted at the corresponding site of pRS551 to generate the plasmid pRSung. A DNA fragment (751 bp) containing the nlpE coding region was prepared by PCR using E. coli BW25113 genome DNA as a template along with a pair of primers, NLPEAF (5'-ATGCGCGGCAGAATTCGCAGCGGTCGGGAA-3') and NLPEAR (5'-TGCGTTTGTTTCTAGATCAAGACGGGTTAC-3'). After digestion with EcoRI and BamHI, the PCR-amplified fragment was inserted at the corresponding site of pBAD18 to generate the plasmid pBADnlpE.

Preparation of the labeled probe for S1 nuclease, gel shift, and DNase I footprinting assays. Probe A was generated by PCR amplification of the ung promoter region with the primers ung-S1F (5'-TGATGCCTCCCCGGCAAAAT-3') and 32P-labeled ung-S1R (5'-TAGGGTTGCTGCTTCTCTTC-3'), and E. coli BW25113 genome DNA (100 ng) was used as the template for the Ex Taq DNA polymerase. The PCR product with 32P at its terminus was recovered from a polyacrylamide gel and then used for S1 nuclease, gel shift, and DNase I footprinting (noncoding strand) assays. The ung-S1R and labeled ung-S1F primers were used to prepare probe B for an S1 nuclease assay (transcription of yfiD). Probe C was generated by PCR amplification of the cpxR promoter region with the primers cpxRA-S1F (5'-GTTATCGCCTGAACCGACTT-3') and 32P-labeled cpxRA-S1R (5'-GAAGCCTTCCATCTCGAGCA-3'), and E. coli BW25113 genome DNA (100 ng) was used as the template for the Ex Taq DNA polymerase. The PCR product with 32P at its terminus was recovered from a polyacrylamide gel and then used for S1 nuclease. The cpxRA-S1R and labeled cpxRA-S1F primers were used to prepare probe D for an S1 nuclease assay (transcription of cpxP). The labeled primers were prepared with 10 µCi of [{gamma}-32P]ATP (5,000 Ci/mmol) by T4 polynucleotide kinase (Toyobo).

RNA isolation and S1 nuclease assay. To prepare total RNA for the S1 nuclease assay, overnight cultures were diluted 100-fold in 100 ml of LB medium grown to an optical density at 600 nm (OD600) of 0.6 or 1.2 at 37°C. Subsequent purification steps were carried out as described previously (19). Ten thousand counts per minute of probe A (2 fmol) was incubated with 100 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; it was then digested with 50 U of S1 nuclease. The undigested DNA was precipitated by ethanol, dissolved in formamide dye solution (95% formamide, 0.05% bromophenol blue, 0.05% xylenecyanol), and analyzed by electrophoresis on a 6% polyacrylamide gel containing 8 M urea.

Purification of CpxR. His-tagged CpxR for gel shift, DNase I footprinting, and in vitro transcription assays was purified from pKH50-3/BL21(DE3) as described previously (28).

Gel shift assay. Ten thousand counts per minute of probe A (2 fmol) was incubated at 37°C for 10 min with CpxR in 12.5 ml of 50 mM Tris-HCl (pH 7.8), 50 mM NaCl, 3 mM Mg acetate, 0.1 mM EDTA, and 0.1 mM dithiothreitol. After addition of the DNA dye solution (40% glycerol, 0.025% bromophenol blue, 0.025% xylenecyanol), the mixture was directly subjected to a 6% polyacrylamide gel electrophoresis.

DNase I footprinting assay. Forty thousand counts per minute of probe A or B (8 fmol) was incubated at 37°C for 10 min with CpxR in 25 µl of 50 mM Tris-HCl (pH 7.8), 50 mM NaCl, 3 mM magnesium acetate, 5 mM CaCl2, 0.1 mM EDTA, 0.1 mM dithiothreitol, and 25 mg of bovine serum albumin/ml. After incubation for 10 min, DNA digestion was initiated by the addition of 5 ng of DNase I (Takara). After digestion for 30 s at 25°C, the reaction was terminated by the addition of 45 µl of DNase I stop solution (20 mM EDTA, 200 mM NaCl, 1% sodium dodecyl sulfate, 250 µg of yeast tRNA/ml). Digested products were precipitated by ethanol, dissolved in formamide dye solution, and analyzed by electrophoresis on a 6% polyacrylamide gel containing 8 M urea.

In vitro transcription assay. A 538-bp EcoRI-BamHI fragment from pRSung, including the ung promoter region, or a 205-bp EcoRI fragment from pKB252 (1), including the lacUV5 promoter region, was used as the template DNA. Single-round transcription by the reconstituted holoenzymes was carried out as described previously (14). In brief, 0.1 pmol of template was incubated with 0, 1.25, 2.5, 5, and 10 pmol of CpxR for 10 min at 37°C in the presence or absence of 10 mM acetylphosphate in a total volume of 33 µl. Into this reaction mixture, 0.5 pmol of RNA polymerase was added and incubated for 20 min at 37°C to form an open complex. Then a substrate-heparin mixture containing [{alpha}-32P]UTP was added and further incubated for 10 min at 37°C. The transcripts were subjected to a 6% polyacrylamide gel containing 8 M urea.

Assay of Ung activity. Cell extract for examining Ung activity was prepared as described previously (16). Here, 5 ml of an overnight culture of E. coli cells was inoculated into 200 ml of LB medium (pH 7.5) containing 1 mM of IPTG (isopropyl-ß-D-thiogalactopyranoside) and grown at 37°C until reaching an OD600 of 1.2. After harvesting by centrifugation, the obtained pellet was washed with 50 ml of H2O. The cells were again harvested by centrifugation, and the pellet was resuspended in 3 ml of a lysis buffer (50 mM Tris-HCl, pH 7.6, 100 mM NaCl, 10% glycerol, 1 mM dithiothreitol). After the cells were sonicated, the cell debris was removed by centrifugation at 15,000 rpm for 20 min at 4°C, and the supernatant was used for assaying Ung activity. The DNA oligonucleotide containing uracil (oligo Lac-461Ruracil, 5'-AGCGCCATGGCCTGACUCATTCCCCAGCGA-3') was labeled with 32P at the 5' end by T4 polynucleotide kinase (Toyobo) and hybridized to the unlabeled oligomer, LAC-461F (5'-TCGCTGGGGAATGGGTCAGGCCATGGCGCT-3'), at a molar ratio of 1:10 to form a duplex with a single U · G mismatch. The duplex was separated with a 20% polyacrylamide gel electrophoresis and recovered. It was reacted with cell extract for 30 min at 37°C in a treatment buffer (20 mM Tris-HCl, 10 mM EDTA) and then treated with 0.1 M NaOH to cleave at the apyrimidine site for another 30 min at 37°C. After that, the products were separated in a 20% polyacrylamide containing 8% urea and autoradiographed.

Lac+ mutation assay. To measure the mutation frequency from G · C to A · T, a spontaneous Lac+ mutation assay using E. coli CC102 (2) was performed as described previously (17). A single colony was inoculated into 5 ml of LB medium containing 1 mM IPTG and grown with aeration for 16 h. The overnight culture was diluted 106-fold with minimal A salt (18). Then, 0.1 ml of the diluted culture was added to 10 ml of minimal A medium containing up to 0.2% glucose, 1 mM MgSO4, 0.0005% thiamine hydrochloride, and tetracycline and/or chloramphenicol. After incubation for 16 h, the culture was harvested by centrifugation. The cell pellet was resuspended in 1 ml of minimal A salt, plated on a minimal A medium (0.2% lactose or 0.2% glucose), and incubated at 37°C. The mutation frequency was then calculated as the ratio of lac+ cells to 1 x 109 cells.


arrow
RESULTS AND DISCUSSION
 
Effect of overexpression of CpxR on Ung activity. To determine whether the Cpx system is involved in the regulation of ung expression, we examined the Ung activity in crude cell extracts from E. coli BW25113(p41-5{Delta}GFP) overexpressing CpxR. When E. coli BW25113(p41-5{Delta}GFP) was incubated in the presence of IPTG, it was confirmed that the cpxR transcript from the lac promoter on the plasmid was overexpressed (Fig. 1A, lane 4). To measure Ung activity, we used a model substrate consisting of a synthetic polynucleotide of 30 nucleotides in length containing a single uracil residue at position 17 from the 5' end. When cell extracts from E. coli BW25113(pCA24{Delta}GFP) without CpxR expression were used, the labeled polynucleotide (30-mer) containing uracil was cleaved after treatment with 0.1 M NaOH to form a 16-mer oligonucleotide and two other bands as cleaved DNA products (Fig. 2A, lanes 2 and 3). These bands were not detected in the absence or presence of the cell extracts from BWung (Fig. 2A, lanes 1 and 6). When CpxR was induced by addition of IPTG in BW25113(p41-5{Delta}GFP) (Fig. 2A, lanes 4 and 5), Ung activity decreased to about fivefold lower than that of BW25113(pCA24{Delta}GFP) (Fig. 2A, lanes 2 and 3), implying that the Cpx system repressed ung expression.



View larger version (54K):
[in this window]
[in a new window]
  		FIG. 1. Overexpression of cpxR. The arrow indicates cpxR transcript from the lac promoter on the plasmid p41-5{Delta}GFP. BW25113(pCA24{Delta}GFP) (lanes 1 and 2) and BW25113(p41-5{Delta}GFP) (lanes 3 and 4) were grown to mid-log phase (OD600, 0.6) in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 1 mM IPTG. The S1 nuclease assay for the cpxR transcript was performed as described in Materials and Methods.



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 2. Assay of uracil-DNA glycosylase (Ung) activity. (A) Ung activity was assayed as described in Materials and Methods. For this assay, cell extracts (lanes 2 and 4, 25 ng; lanes 3 and 5, 50 ng) from BW25113(pCA24{Delta}GFP) (lanes 2 and 3), BW25113(p41-5{Delta}GFP) (lanes 4 and 5), and BWung (lane 6, 50 ng) were used. The 30-mer oligonucleotide was cleaved by Ung and NaOH to form a 16-mer product. In lane 1, no cell extract was used. (B) Cleaved DNA products (from panel A) were quantified with BAS 1000 Mac (Fuji film). Percent cleavage was determined by dividing the intensity of the cleaved product (16-mer) by the total intensity, which was defined as the sum of the intensities of the intact substrate and the cleaved products.

As described previously (9), a G · C-to-A · T transition was stimulated in E. coli ung mutants (CC102ung) (Table 2). We investigated the possible effects of CpxR overexpression on the mutator activity of the G · C-to-A · T transition by using a spontaneous Lac+ mutation assay (17). The results in Table 2 indicate that the G · C-to-A · T transition frequency for strain CC102(p41-5{Delta}GFP), overexpressing CpxR, was stimulated to about sixfold more than that for CC102(pCA24{Delta}GFP).


View this table:
[in this window]
[in a new window]
  		TABLE 2. G · C->A · T mutation frequency

Negative regulation of ung gene expression by overexpression of CpxR. To investigate the effect of CpxR overexpression on the transcription of the ung gene, we performed an S1 nuclease assay using RNAs prepared from CpxR-expressing BW25113(p41-5{Delta}GFP) as well as BW25113(pCA24{Delta}GFP). When CpxR was overexpressed by addition of IPTG (Fig. 1A, lane 4; Fig. 3A, lane 4; Fig. 3B, lane 8), the transcription of ung decreased by fivefold (Fig. 3A, lane 4) compared to that of BW25113(pCA24{Delta}GFP) (Fig. 3A, lane 2) while cpxP increased by 10-fold (Fig. 3B, lanes 6 and 8). In addition, we examined whether overproduction of the outer membrane lipoprotein NlpE can repress the ung expression (Fig. 3C, lane 4), since overproduction of NlpE activates the Cpx signal transduction pathway (3, 7, 25). When nlpE expression was induced by arabinose, the ung transcription was repressed by fivefold (Fig. 3C, lane 4) compared to that of BW25113(pBAD18) (Fig. 3C, lane 2), while the transcriptional level of cpxP increased by 10-fold (Fig. 3D, lanes 6 and 8). Neither IPTG nor arabinose repressed ung and cpxP transcriptions in BW25113 (data not shown); therefore, these results suggest that the ung expression was negatively regulated by the Cpx pathway.



View larger version (37K):
[in this window]
[in a new window]
  		FIG. 3. Effects of overexpressed CpxR or NlpE on ung and cpxP transcription. BW25113(pCA24{Delta}GFP) (A and B, lanes 1, 2, 5, and 6) and BW25113(p41-5{Delta}GFP) (A and B, lanes 3, 4, 7, and 8) were grown to mid-log phase (OD600, 0.6) in the absence (A and B, lanes 1, 3, 5, and 7) or presence (A and B, lanes 2, 4, 6, and 8) of 1 mM IPTG. S1 nuclease assays for ung (A) and cpxP (B) transcripts were performed as described in Materials and Methods. BW25113(pBAD18) (C and D, lanes 1, 2, 5, and 6) and BW25113(pBADnlpE) (C and D, lanes 3, 4, 7, and 8) were grown to mid-log phase (OD600, 0.6) in the absence (C and D, lanes 1, 3, 5, and 7) or presence (C and D, lanes 2, 4, 6, and 8) of 0.2% arabinose. After that, S1 nuclease assays for ung (C) and cpxP (D) transcripts were performed. Lane AG represents the Maxam-Gilbert sequence ladder. The transcription start site is marked with an asterisk.

To confirm this result, we next examined the influence of cpxRA deletion on the expression of the ung gene. In wild-type BW25113 cells, the level of ung mRNA decreased in the stationary phase (Fig. 4C, lane 1), but the level of yfiD transcription did not change independently of the growth phase (Fig. 4B and D). The growth phase-coupled reduction in ung transcription was not observed in strain BW27559 (Fig. 4A and C, lane 2). These results indicate that CpxR negatively regulates ung transcription in the stationary phase and that this repression does not take place in the absence of the Cpx system.



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 4. ung and yfiD gene expression dependent on growth phase. E. coli BW25113 (A and C, lane 1; B and D, lane 3) and BW27559 ({Delta}cpxR) (A and C, lane 2; B and D, lane 4) were grown to mid-log phase (OD600 = 0.6) (A, B) or early stationary phase (OD600 = 1.2) (C, D). After that, S1 nuclease assays for ung (A, C) and yfiD (B, D) transcripts were performed. Lane AG represents the Maxam-Gilbert sequence ladder. The transcription start site is marked with an asterisk.

To further confirm the role of CpxR in the transcription of the ung gene, we performed in vitro transcription using reconstituted RNA polymerase holoenzyme E{sigma}70 and truncated DNA fragments (538 bp) containing the promoter region of ung in the presence or absence of CpxR. Addition of CpxR caused repression of ung transcription, while it did not affect the expression from the lacUV5 promoter (Fig. 5A). Such a repression was stimulated in the presence of 10 mM acetylphosphate (Fig. 5B, lanes 5 to 8).



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 5. In vitro transcription assay. Single-round transcription in vitro of 0.1 pmol ung DNA template (–452 to +86 of promoter region) (A, lanes 5 to 8; B, lanes 5 to 8) or lacUV5 DNA template (A, lanes 1 to 4; B, lanes 1 to 4) was performed in the presence (B) or absence (A) of 10 mM acetylphosphate. The amounts of CpxR were as follows: lanes 1 and 5, 0 pmol; lanes 2 and 6, 1.25 pmol; lanes 3 and 7, 2.5 pmol; lanes 4 and 8, 5 pmol. Electrophoresis was performed with a 6% polyacrylamide sequencing gel. Bold arrows indicate the ung and lacUV5 transcripts. (C) ung (circles) and lacUV5 (square) transcripts in the absence (open symbols) and presence (closed symbols) of CpxR were quantified with BAS 1000 Mac (Fuji film). The relative value in shown as a ratio between each transcript and that in the absence of CpxR.

Identification of the CpxR binding site. To define the mechanism underlying the repression of ung transcription by CpxR, we tested whether CpxR directly interacts with the ung promoter region. For this purpose, we carried out gel shift assays using the 32P-labeled DNA fragment containing the ung promoter region. In the absence of acetylphosphate, a marked shift was observed only at higher concentrations of CpxR (Fig. 6A, lanes 4 to 6). In contrast, a significant gel shift was observed even at the lowest concentration of CpxR when 25 mM acetylphosphate was added (Fig. 6A, lanes 8 to 12). CpxR is a member of the OmpR family of winged helix-turn-helix transcription factors. We investigated the binding ability of another OmpR family protein, PhoP, and that of the bovine serum albumin to the ung promoter. No interaction of protein-ung promoter DNA was detected (Fig. 6B). These results indicate that the affinity of CpxR to the CpxR box of the ung promoter region is increased by phosphorylation.



View larger version (38K):
[in this window]
[in a new window]
  		FIG. 6. Gel shift assay. (A) Probe A was incubated at 37°C for 10 min with CpxR (lanes 1 to 6) or CpxR phosphorylated by acetylphosphate at 37°C (lanes 7 to 12). The amounts of CpxR were as follows: lanes 1 and 7, 0 pmol; lanes 2 and 8, 1.25 pmol; lanes 3 and 9, 2.5 pmol; lanes 4 and 10, 5 pmol; lanes 5 and 11, 7.5 pmol; lanes 6 and 12, 10 pmol. (B) Probe A was incubated at 37°C for 10 min with PhoP (lanes 1 to 5) or BSA (lanes 6 to 10) at 37°C. The amounts of PhoP or BSA were as follows: lanes 1 and 6, 0 pmol; lanes 2 and 7, 1.25 pmol; lanes 3 and 8, 2.5 pmol; lanes 4 and 9, 5 pmol; lanes 5 and 10, 10 pmol).

Next, we tried to identify the specific CpxR binding site on the ung promoter region by using a DNase I footprinting assay. CpxR was found to bind to the CpxR box containing the direct repeat between –61 and –86 upstream from the transcriptional start position (Fig. 7). The CpxR box in the ung promoter is located 61 bp upstream of the transcription start site (Fig. 8) and corresponds with the site predicted by DeWulf et al. (6). It is well known that the C-terminal domain of the {alpha} subunit of RNA polymerase ({alpha}-CTD) interacts with an AT-rich sequence (UP element) located upstream of the –35 hexamer (24). The CpxR box in the ung promoter also contains an AT-rich sequence corresponding to the UP element. CpxR binding to the ung promoter may repress its transcription by interfering with the ability of the {alpha}-CTD to bind to the UP element. In Salmonella enterica, the transcriptional factor PmrA binding to the 59 bp upstream site in the pmrD promoter represses pmrD transcription by interfering with the ability of the {alpha}-CTD to interact with a putative UP element that overlaps the AT-rich PmrA binding site (15). CpxR seems to repress the ung expression in a similar manner as shown for the pmrD promoter (15). Taken together, these results indicate that the ung gene transcription is directly controlled by phosphorylated CpxR.



View larger version (70K):
[in this window]
[in a new window]
  		FIG. 7. DNase I footprinting assay. Probe A was incubated with various amounts of the purified CpxR (lane 1, 0 pmol; lane 2, 10 pmol; lane 3, 20 pmol; lane 4, 30 pmol; lane 5, 40 pmol; lane 6, 50 pmol; lane 7, 60 pmol; lane 8, 70 pmol; lane 9, 80 pmol) and subjected to DNase I footprinting assays. Lanes AG represent the Maxam-Gilbert sequence ladder. The black boxes and bold arrows indicate the CpxR binding region and the direct repeat, respectively.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 8. ung promoter and CpxR box. The transcription start site is marked with an asterisk. The nucleotide number represents the distance from the transcription initiation site of the ung promoter. Putative recognition sequences for {sigma}70 (–10 and –35) are boxed. The arrows indicate the direct repeat in the CpxR box shown previously (6).

In the present study, we demonstrated negative regulation of ung expression by overexpression of CpxR (see Fig. 2 and 3). Transcription of the ung gene at the stationary phase is about threefold higher in the cpxRA deletion strain than in the parental strain (Fig. 4C). DeWulf et al. (6) predicted positive control of the ung, ompC, psd, mviM, aroK, and secA genes by phosphorylated CpxR and negative control of rpoErseABC and aer. In this paper, we suggest that ung is expressed in the exponential growth phase but decreases in the stationary phase concomitantly with the increase in phosphorylated CpxR. This finding indicates that ung is a target of negative control by CpxR.

Furthermore, we also found an increase in the mutation level of E. coli overexpressing CpxR. The increased mutation might be attributable, at least in part, to the repression of ung expression by the increased CpxR. This is because it is not possible to repair DNA uracil residues, which arise as a result of either misincorporation of dUMP by DNA polymerase or deamination of DNA cytosine residues. This finding further implies that the CpxR/CpxA two-component system plays a role in controlling the mutation rate.


arrow
ACKNOWLEDGMENTS
 
We thank R. W. Simons, J. H. Miller, B. K. Duncan, H. Mori, and Hirofumi Aiba for providing plasmids and E. coli strains.

This work was supported by the Program for Promoting Advancement of Academic Research at Private Universities and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Department of Bioscience and Biotechnology, Graduate School of Agriculture of Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan. Phone: 81-742-43-7306. Fax: 81-742-43-1445. E-mail: utsumi{at}nara.kindai.ac.jp. Back


arrow
REFERENCES
 
    1
  1. Backman, K., M. Ptashne, and W. Gilbert. 1976. Construction of plasmids carrying the cI gene of bacteriophage {lambda}. Proc. Natl. Acad. Sci. USA 73:4174-4178.[Abstract/Free Full Text]
    2. 2
  2. Cupples, C. G., and J. H. Miller. 1989. A set of lacZ mutations in Escherichia coli that allow rapid detection of each of the six base substitutions. Proc. Natl. Acad. Sci. USA 86:5345-5349.[Abstract/Free Full Text]
    3. 3
  3. Danese, P. N., and T. J. Silhavy. 1997. The {sigma}E and the Cpx signal transduction systems control the synthesis of periplasmic protein-folding enzymes in Escherichia coli. Genes Dev. 11:1183-1193.[Abstract/Free Full Text]
    4. 4
  4. Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97:6640-6645.[Abstract/Free Full Text]
    5. 5
  5. DeWulf, P., O. Kwon, and E. C. C. Lin. 1999. The CpxRA signal transduction system of Escherichia coli: growth-related autoactivation and control of unanticipated target operons. J. Bacteriol. 181:6772-6778.[Abstract/Free Full Text]
    6. 6
  6. DeWulf, P., A. M. McGuire, X. Liu, and E. C. C. Lin. 2002. Genome-wide profiling of promoter recognition by the two-component response regulator CpxR-P in Escherichia coli. J. Biol. Chem. 277:26652-26661.[Abstract/Free Full Text]
    7. 7
  7. DiGiuseppe, P. A., and T. J. Silhavy. 2003. Signal detection and target gene induction by the CpxRA two-component system. J. Bacteriol. 185:2432-2440.[Abstract/Free Full Text]
    8. 8
  8. Dorel, C., O. Vidal, C. Prigent-Combaret, I. Vallet, and P. Lejeune. 1999. Involvement of the Cpx signal transduction pathway of E. coli in biofilm formation. FEMS Microbiol. Lett. 178:169-175.[CrossRef][Medline]
    9. 9
  9. Duncan, B. K., and B. Weiss. 1982. Specific mutator effects of ung (uracil-DNA glycosylase) mutations in Escherichia coli. J. Bacteriol. 151:750-755.[Abstract/Free Full Text]
    10. 10
  10. Duncan, B. K. 1985. Isolation of insertion, deletion, and nonsense mutations of the uracil-DNA glycosylase (ung) gene of Escherichia coli K-12. J. Bacteriol. 164:689-695.[Abstract/Free Full Text]
    11. 11
  11. Gallinali, P., and J. Jiricny. 1996. A new class of uracil-DNA glycosylases related to human thymine-DNA glycosylase. Nature (London) 383:735-738.[CrossRef][Medline]
    12. 12
  12. Guzman, L.-M., D. Belin, M. J. Carson, and J. Beckwith. 1995. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177:4121-4130.[Abstract/Free Full Text]
    13. 13
  13. Hung, D. L., T. L. Raivio, C. H. Jones, T. J. Silhavy, and S. J. Hultgren. 2001. Cpx signaling pathway monitors biogenesis and affects assembly and expression of P pili. EMBO J. 20:1508-1518.[CrossRef][Medline]
    14. 14
  14. Kajitani, M., and A. Ishihama. 1983. Determination of the promoter strength in the mixed transcription system: promoters of lactose, tryptophan and ribosomal protein L10 operons from Escherichia coli. Nucleic Acids Res. 11:671-686.[Abstract/Free Full Text]
    15. 15
  15. Kato, A., T. Latifi, and E. A. Groisman. 2003. Closing the loop: the PmrA/PmrB two-component system negatively controls expression of its posttranscriptional activator PmrD. Proc. Natl. Acad. Sci. USA 100:4706-4711.[Abstract/Free Full Text]
    16. 16
  16. Lutsenko, E., and A. S. Bhagwat. 1999. The role of the Escherichia coli Mug protein in the removal of uracil and 3,N4-ethenocytosine from DNA. J. Biol. Chem. 274:31034-31038.[Abstract/Free Full Text]
    17. 17
  17. Mackay, W. J., S. Han, and L. D. Samson. 1994. DNA alkylation repair limits spontaneous base substitution mutation in Escherichia coli. J. Bacteriol. 176:3224-3230.[Abstract/Free Full Text]
    18. 18
  18. Miller, J. H. (ed.) 1992. A short course in bacterial genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
    19. 19
  19. Minagawa, S., H. Ogasawara, A. Kato, K. Yamamoto, Y. Eguchi, T. Oshima, H. Mori, A. Ishihama, and R. Utsumi. 2003. Identification and molecular characterization of the Mg2+ stimulon of Escherichia coli. J. Bacteriol. 185:3696-3702.[Abstract/Free Full Text]
    20. 20
  20. Mokkapati, S. K., A. R. Fernandez de Henestrosa, and A. S. Bhagwat. 2001. Escherichia coli DNA glycosylase Mug: a growth-regulated enzyme required for mutation avoidance in stationary-phase cells. Mol. Microbiol. 41:1101-1111.[CrossRef][Medline]
    21. 21
  21. Otto, K., and T. J. Silhavy. 2002. Surface sensing and adhesion of Escherichia coli controlled by the Cpx-signaling pathway. Proc. Natl. Acad. Sci. USA 99:2287-2292.[Abstract/Free Full Text]
    22. 22
  22. Mori, H., K. Isono, T. Horiuchi, and T. Miki. 2000. Functional genomics of Escherichia coli in Japan. Res. Microbiol. 151:121-128.[Medline]
    23. 23
  23. Oshima, T., H. Aiba, Y. Masuda, S. Kanaya, M. Sugiura, B. L. Wanner, H. Mori, and T. Mizuno. 2002. Transcriptome analysis of all two-component regulatory system mutants of Escherichia coli K-12. Mol. Microbiol. 46:281-291.[CrossRef][Medline]
    24. 24
  24. Ross, W., K. K. Gosink, J. Salomon, K. Igarashi, C. Zou, A. Ishihama, K. Severinov, and R. L. Gourse. 1993. A third recognition element in bacterial promoter: DNA binding by the {alpha} subunit of RNA polymerase. Science 262:1407-1413.[Abstract/Free Full Text]
    25. 25
  25. Snyder, W. B., L. J. B. Davis, P. N. Danese, C. L. Cosma, and T. J. Silhavy. 1995. Overproduction of NlpE, a new outer membrane lipoprotein, suppresses the toxicity of the Cpx signal transduction pathway. J. Bacteriol. 177:4216-4223.[Abstract/Free Full Text]
    26. 26
  26. Varshney, U., T. Hutcheon, and J. H. van de Sande. 1988. Sequence analysis, expression, and conservation of Escherichia coli uracil DNA glycosylase and its gene (ung). J. Biol. Chem. 263:7776-7784.[Abstract/Free Full Text]
    27. 27
  27. Wyszynski, M., S. Gabbara, and A. S. Bhagwat. 1994. Cytosine deaminations catalyzed by DNA cytosine methyltransferases are unlikely to be the major cause of mutational hot spots at sites of cytosine methylation in Escherichia coli. Proc. Natl. Acad. Sci. USA 91:1574-1578.[Abstract/Free Full Text]
    28. 28
  28. Yamamoto, K., H. Ogasawara, N. Fujita, R. Utsumi, and A. Ishihama. 2002. Novel mode of transcription regulation of divergently overlapping promoters by PhoP, the regulator of two-component system sensing external magnesium availability. Mol. Microbiol. 45:423-438.[CrossRef][Medline]

Journal of Bacteriology, December 2004, p. 8317-8325, Vol. 186, No. 24
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.24.8317-8325.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Price, N. L., Raivio, T. L. (2009). Characterization of the Cpx Regulon in Escherichia coli Strain MC4100. J. Bacteriol. 191: 1798-1815 [Abstract] [Full Text]  
  • Altman, E., Segal, G. (2008). The Response Regulator CpxR Directly Regulates Expression of Several Legionella pneumophila icm/dot Components as Well as New Translocated Substrates. J. Bacteriol. 190: 1985-1996 [Abstract] [Full Text]  
  • Pruss, B. M., Besemann, C., Denton, A., Wolfe, A. J. (2006). A Complex Transcription Network Controls the Early Stages of Biofilm Development by Escherichia coli. J. Bacteriol. 188: 3731-3739 [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ogasawara, H.
Right arrow Articles by Utsumi, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ogasawara, H.
Right arrow Articles by Utsumi, R.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»