FREE
This Article
FREE
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 Related articles in JB
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
Google Scholar
Right arrow Articles by Kimsey, H. H.
Right arrow Articles by Waldor, M. K.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kimsey, H. H.
Right arrow Articles by Waldor, M. K.

 Previous Article  |  Next Article 

Journal of Bacteriology, November 2009, p. 6788-6795, Vol. 191, No. 22
0021-9193/09/$08.00+0     doi:10.1128/JB.00682-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Vibrio cholerae LexA Coordinates CTX Prophage Gene Expression{triangledown}

Harvey H. Kimsey and Matthew K. Waldor*

Channing Laboratory, Brigham and Women's Hospital, and Howard Hughes Medical Institute, Boston, Massachusetts 02115

Received 26 May 2009/ Accepted 1 August 2009


arrow
ABSTRACT
 
The filamentous bacteriophage CTX{Phi} transmits the cholera toxin genes by infecting and lysogenizing its host, Vibrio cholerae. CTX{Phi} genes required for virion production initiate transcription from the strong PA promoter, which is dually repressed in lysogens by the phage-encoded repressor RstR and the host-encoded SOS repressor LexA. Here we identify the neighboring divergent rstR promoter, PR, and show that RstR both positively and negatively autoregulates its own expression from this promoter. LexA is absolutely required for RstR-mediated activation of PR transcription. RstR autoactivation occurs when RstR is bound to an operator site centered 60 bp upstream of the start of transcription, and the coactivator LexA is bound to a 16-bp SOS box centered at position –23.5, within the PR spacer region. Our results indicate that LexA, when bound to its single site in the CTX{Phi} prophage, both represses transcription from PA and coactivates transcription from the divergent PR. We propose that LexA coordinates PA and PR prophage transcription in a gene regulatory circuit. This circuit is predicted to display transient switch behavior upon induction of CTX{Phi} lysogens.


arrow
INTRODUCTION
 
The horizontal transfer of CTX{Phi}, the lysogenic filamentous phage that carries the genes encoding cholera toxin, contributes to the emergence of new toxigenic strains of Vibrio cholerae, the cause of epidemic cholera (6, 31). For stable CTX{Phi} transfer to occur, the infecting single-stranded DNA CTX{Phi} genome must integrate site-specifically into the V. cholerae genome and establish a lysogenic program that involves the transcriptional repression of CTX{Phi} genes involved in bacteriophage replication and morphogenesis (17). The CTX{Phi} prophage expresses a 14-kDa repressor, RstR, that represses the expression of CTX{Phi} structural genes and provides immunity to secondary CTX{Phi} infection (12, 32). The CTX{Phi} prophage can be induced with DNA-damaging agents such as mitomycin C and UV radiation, a process that is mediated by a second repressor, the host SOS repressor LexA (25). Prophage induction does not kill the host bacterium and results in only a modest increase in CTX{Phi} titers, unlike the massive phage growth and host lysis that occurs upon induction of phage lambda.

The CTX{Phi} replication and morphogenesis genes initiate transcription from the strong PA promoter located in ig-2, an intergenic region separating the rstA replication gene from the divergently transcribed rstR gene encoding the CTX{Phi} repressor (12) (Fig. 1B). RstR strongly represses PA transcription in CTX{Phi} lysogens by binding to a set of three operator sites that surround PA (13). The host SOS repressor LexA also represses PA by binding to a single SOS box centered –48.5 bp from the start of PA transcription (25) (Fig. 1B). LexA, together with RecA, regulates a network of more than 30 host genes that monitors and responds to DNA damage (7). Upon DNA damage or replication fork arrest, RecA, together with single-stranded DNA and ATP, stimulates the autoproteolysis of LexA (14), leading to the transient derepression of numerous genes encoding DNA repair functions. We previously showed that LexA repression of PA occurs when DNA-bound LexA occludes a promoter UP element that overlaps the LexA binding site, thereby preventing the C-terminal domain of the {alpha} subunit of RNA polymerase (RNAP) from gaining access to this UP element (24).


Figure 1
View larger version (13K):
[in this window]
[in a new window]

  		FIG. 1. Primer extension analysis of PR activity in V. cholerae. (A) Total V. cholerae RNA was hybridized to P32-labeled primer A or B and extended with Moloney murine leukemia virus reverse transcriptase. Primers A and B anneal to sites on rstR mRNA that are exactly 10 nt apart. Lanes 1 to 4, DNA sequencing ladder using primer A. Lanes 5 and 6, RNA extracted from the CTX{Phi}– V. cholerae strain HK386. Lanes 7 and 8, RNA extracted from HK138, an isogenic derivative containing two integrated copies of CTX{Phi}. (B) The transcription regulatory region of CTX{Phi}. Shown below are the PA and PR promoter elements and their positions relative to the RstR operators O1, O2, and O3, which are depicted as shaded boxes. Promoter –10-bp and –35-bp sequences are represented by black rectangles, and the SOS box is shown as a striped rectangle. The beginning of the RstR protein-coding region is represented by a black arrow. Shown above is the DNA sequence of the PR and PA promoter region. The promoter –10-bp and –35-bp sequences are shown underlined. Transcription start points and the direction of transcription are depicted with angled arrows. The 16-bp SOS box, the binding sequence for LexA, is shown boxed within the PR promoter (25). The SOS box overlaps with a promoter UP element located upstream of the PA promoter (24).

How CTX{Phi} lysogens regulate RstR levels is not understood but seems to be critical to the functioning of the regulatory circuit governing CTX{Phi} lysogeny. For example, the O2 operator and the single SOS box in CTX{Phi} overlap, such that RstR and LexA cannot both bind to their respective sites in O2 (25). However, our finding that CTX{Phi} lysogens can be induced by DNA damage indicates that the O2 region is predominantly occupied by LexA. One hypothesis that could account for these observations is that the lysogenic levels of RstR are tightly regulated, such that the concentration of RstR is maintained at a level high enough to bind O1, thereby repressing CTX{Phi} prophage transcription, but not sufficiently high to allow binding to O2 or O3. To investigate this hypothesis, we have identified the rstR promoter PR and determined that RstR both positively and negatively autoregulates its own expression from PR. Interestingly, RstR autoactivation requires LexA binding to the SOS box that overlaps with the –35 sequence of PR. Thus, RstR and LexA, which were known to function as repressors of CTX{Phi} transcription from PA, also function as coactivators of rstR transcription from PR. These results delineate a CTX{Phi} regulatory circuit with similarities to the classic phage lambda switch but also with unique features that are better suited to a temperate filamentous bacteriophage.


arrow
MATERIALS AND METHODS
 
Strains. Strain HK386 is a {Delta}lacZ derivative of the environmental V. cholerae strain 2740-80 (attRS+ CTX–) (19). The lysogen strain HK138 was constructed by transformation of HK386 with CTXKn plasmid DNA (31). Escherichia coli BW25113 [{Delta}(araD-araB)567 {Delta}lacZ4787(::rrnB-3) lacIp-4000(lacIq) {lambda}– rph-1 {Delta}(rhaD-rhaB)568 hsdR514] (4) and BW27784 (BW25113 {Delta}araFGH {Delta}araEp::PCP18-araE) (11) were obtained from the E. coli stock center. Strain TP608 (AB1157 {Delta}(recC-ptr-recB-recD)::Ptac-gam-red-pae-cI822 sulA6209::tet lexA71::Tn5) was kindly provided by A. R. Poteete (20). sulA6209::tet, followed by lexA71::Tn5, was transduced into strain BW27784 by serial P1 transduction using a P1vir lysate grown with strain TP608, as previously described (18).

Primer extension. Oligonucleotide primers were high-pressure liquid chromatography purified by the manufacturer (Operon Technologies) and end labeled with P32-labeled {gamma}-ATP (10 Ci/mmol) and T4 polynucleotide kinase. Total RNA was isolated from V. cholerae and E. coli using Trizol reagent, according to the manufacturer's instructions (Invitrogen). Ten micrograms of total RNA was annealed to 0.8 pmol of labeled primer and extended using SuperScript III, according to the manufacturer's directions for first-strand cDNA synthesis (Invitrogen). Reactions were terminated by addition of 0.1 M EDTA and extracted with phenol-chloroform (dilution of 24:1). Nucleic acids were ethanol precipitated, resuspended in denaturing sample buffer (99% formamide, 0.1% 1N NaOH, and 0.01% each of bromophenol blue and xylene cyanol FF), and heated briefly to 92°C. The reaction products, together with a set of dideoxy DNA sequencing reactions generated with the same radiolabeled primer, were separated on 8% DNA sequencing gels.

Plasmids. pBAD-RstR has been described previously (12). The lacZ transcription reporter plasmids pCB182 and pCB192 (27) were modified by cloning a 300-bp fragment carrying the rrnBT1 transcription terminator (30) into the unique SmaI site, yielding pCB182N and pCB192N. This modification prevented unregulated PA transcription from disrupting plasmid replication. pHK301 contains CTX{Phi} DNA from positions +5 to –115 relative to the start of rstR transcription, while pHK312 contains sequences from +96 to –160, both cloned into pCRII (Invitrogen) (13). The CTX{Phi} fragments were excised by digestion with SalI and HindIII and ligated to SalI- and HindIII-digested pCB192N DNA, yielding the PR-lacZ reporters pHK301N and pHK312N. The PA-lacZ reporter pHK393 was constructed by cloning the XbaI-HindIII fragment from pHK301 into reporter pCB182N. The O1-18 mutant derivatives were generated by PCR-mediated mutagenesis using a QuikChange site-directed mutagenesis kit (Stratagene). Similarly, a derivative of pHK301N carrying the m1 substitution, pHK883, was generated by site-directed mutagenesis. The mutations were confirmed by DNA sequencing. The m1 SOS box mutation was originally carried on the plasmid pMQm1. For in vitro transcription, the CTX{Phi} region from +5 to –115 (relative to the start of rstR transcription) carrying the m1 SOS box allele was amplified from pMQm1 (M. Quinones, unpublished data) with primers containing XhoI and HindIII restriction sites at their 5' ends. The resulting DNA fragment was cloned into pCRII (Invitrogen), yielding pHK725.

LacZ assays. LacZ assays were performed as described previously (18). Fresh colonies grown on LB agar plus antibiotics were used to inoculate 2 ml LB broth plus antibiotics and L-arabinose as an inducer. Cultures were incubated on a roller wheel at 37°C for 12 to 14 h and diluted 1:10 in phosphate-buffered saline prior to being assayed.

RstR DNA binding assay. RstR activity was measured in cell extracts using a gel-shift assay similar to that used to measure CI concentrations in E. coli (5). A 250-ml sample of cultures of BW27784 carrying pBAD-RstR was grown in LB plus antibiotics and the indicated concentration of L-arabinose to late log phase (optical density at 600 nm of ~0.8). Cells were harvested, washed, concentrated to a volume of 2 to 3 ml in GS buffer (20 mM Tris-HCl [pH 7.5], 150 mM KCl, 10% glycerol), and lysed in a prechilled French pressure cell. Cell debris was removed by centrifugation, and small aliquots were stored frozen at –70°C. Samples were thawed on ice and serial diluted with a similar cell extract made from cultures of BW27784 lacking pBAD-RstR plasmid. A 120-bp DNA probe containing the high-affinity O1 operator site was generated by PCR with one primer fluorescently labeled with 5-FAM (Operon Technologies) and purified using a QIAquick PCR purification kit (Qiagen). Binding reactions were carried out in GS buffer containing 10 mM dithiothreitol and 10 µg/ml sheared salmon sperm DNA as the nonspecific binding competitor. Samples were incubated at 4°C for 30 min with gentle mixing. One microliter of dye solution (0.1% bromophenol blue) was added, and samples were loaded and run on 6% polyacrylamide and 0.5x Tris-borate-EDTA DNA retardation gels (Invitrogen). Gels were scanned using the blue laser of a Typhoon scanner (GE Biosciences), and the band intensities corresponding to the bound and unbound DNA fractions were determined using ImageQuant software. The volume of extract resulting in 50% binding of probe DNA was interpolated from the binding curves, and RstR binding activity (RU) was calculated as [(1/V50)/total protein concentration (µg/ml)] x 100, where V50 is the volume of cell extract (µl) resulting in 50% shifting of probe DNA. Protein concentrations were determined with the Coomassie Plus kit (Pierce).

Western blotting. Anti-His6-tagged RstR (RstR6H) sera were prepared in BALB/c mice as previously described (8) and used in Western blots at a dilution of 1:5,000. Western blots were developed with anti-mouse antibodies conjugated to horse radish peroxidase (Pierce Biotechnology).

Repressor purifications. RstR6H was purified as previously described (13). E. coli LexA was expressed from pJWL228 in E. coli T7 Express lysY/lacIq (New England Biolabs) and purified as previously described (16). The chromatography media were substituted in order to make use of a programmable fast-performance liquid chromatography system. Fraction II material (3 ml, from 500 ml of starting culture) was diluted to 0.1 M NaCl and loaded onto a HiTrap SP fast-flow column (1 ml) connected to an ÄKTApurifier fast-performance liquid chromatography system (GE Biosciences). Buffer conditions used were identical to those described for the original phosphocellulose chromatography step (16). LexA was eluted in a 20-ml gradient from 0.1 M to 1.0 M NaCl. LexA-containing fractions (2.5 ml) were pooled and dialyzed versus 1 liter buffer Q (0.02 M Tris-HCl [pH 8.0], 0.1 mM Na2+-EDTA, 10% glycerol) containing 0.2 M NaCl. Dialyzed material was diluted to 0.08 M NaCl with buffer Q and loaded onto a MonoQ 5/50 column (1 ml) preequilibrated in buffer Q plus 0.08 M NaCl. LexA was eluted using a 40-ml gradient from 0.08 M to 0.6 M NaCl. LexA eluted as a single peak early in the gradient (data not shown). LexA peak fractions were pooled, and small aliquots were stored at –70°C. This material was estimated to be 95% pure, as judged by Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis. LexA concentrations were determined by UV absorption at 280 nm, using a molar extinction coefficient of 6,900 M–1 cm–1.

In vitro transcription. Single-round runoff transcription reactions were carried out using heparin sulfate, as previously described (33). Template DNAs were generated by PCR from pHK301 or pHK725 (m1) plasmid DNA using pairs of primers that anneal to flanking vector sequences. Primer sequences are available upon request. PCR products were purified using a QIAquick PCR purification kit and quantitated by UV absorption at 260 nm. Template DNA and E. coli RNAP ({sigma}70) holoenzyme (Epicentre) were present at 2 nM and 20 nM, respectively. Five units of RNase inhibitor (Agilent Technologies) was added to all reactions. Template DNA and repressors were preincubated at 37°C for 15 min. RNAP was added, and the incubation continued for 15 min. A mix containing 2 µCi of P32-labeled {alpha}-UTP (10 mCi/mmol; Perkin Elmer), nucleoside triphosphates (10 µM each of CTP, GTP, and ATP, and 1 µM UT, final concentration) and heparin sulfate (100 µg/ml, final concentration) was added, and the incubation was continued for a further 30 min. A set of 32P-labeled RNA standards were generated using T7 RNAP, according to the manufacturer's instructions (Agilent Technologies). All reactions were terminated by ethanol precipitation. Pellets were resuspended in denaturing sample buffer and briefly heated to 95°C, and RNA products were fractionated on 6% denaturing polyacrylamide gels.


arrow
RESULTS
 
Primer extension analysis located the 5' end of rstR mRNA at a position 23 bp upstream of the RstR start codon (Fig. 1A). Similar results were obtained by S1 nuclease mapping (data not shown). The putative rstR promoter PR contains the –10 sequence TACAAT, which closely matches the consensus {sigma}70 –10 sequence. However, PR lacks a recognizable DNA sequence that closely matches the {sigma}70 consensus –35 sequence (28) (Fig. 1B). The single LexA binding site in CTX{Phi} (25) is centered –23.5 bp from the start of rstR transcription, within the spacer region of PR and overlapping the presumed –35 sequence (Fig. 1B). The PR promoter is closely opposed to the divergent PA promoter, which directs the transcription of CTX{Phi} genes required for replication and viral assembly. A significant fraction of rstR primer extension products contained 5' extensions as long as 10 residues in length (Fig. 1A). Similar 5' extensions have been observed at promoters whose transcription, like that of rstR, initiates at a string of three A residues (34). During transcription initiation, repeated slippage by RNAP at the string of three T residues on the DNA template strand leads to the incorporation of 5' A tracts in the nascent mRNA. A similar slippage process could produce the 5' extensions we observe in rstR mRNA.

To investigate factors that regulate rstR transcription, we first analyzed LacZ expression from two plasmid-borne PR-lacZ fusions in V. cholerae. pHK301N contains the O1 and O2 operators but lacks O3, while pHK312N includes all three RstR operators (Fig. 1B). The activity of these reporters varied markedly, depending on whether the host strain carried the integrated CTX{Phi} prophage. Both pHK301N and pHK312N expressed high LacZ activity in HK138, which carries tandem integrated copies of CTX{Phi} (1,000 and 1,250 Miller units, respectively), but markedly lower activities (50 and 200 Miller units, respectively) in HK386, an isogenic strain lacking CTX{Phi}. Since CTX{Phi} lysogens are known to express RstR (12, 31), which represses the expression of most other CTX{Phi} genes, we hypothesized that RstR activates its own transcription from PR. The O1 operator site, located at position –40 to –80 from the start of rstR transcription (Fig. 1B), was the likely site of RstR-mediated activation of PR. We therefore constructed derivatives of pHK301N and pHK312N that contain a mutated O1 site (O1-18), such that RstR no longer bound to O1. As shown in Fig. 2, the O1-18 mutation is a deletion/substitution mutation that lies near the center of O1 and results in a complete loss of RstR binding to O1 (Fig. 2B). LacZ activities were determined for the mutant reporter plasmids in the same lysogen/nonlysogen strain pair. In both cases, LacZ activities determined in the CTX{Phi} lysogen strain were reduced to the basal levels observed in the nonlysogen strain (data not shown). These data are consistent with the hypothesis that RstR binding to the upstream O1 site is required for PR transcription.


Figure 2
View larger version (12K):
[in this window]
[in a new window]

  		FIG. 2. RstR does not bind to the mutated O1-18 site. (A) Map of the PR promoter region showing the location of the rstR O1-18 mutation. Only the rstR nontemplate strand is shown. The O1-18 mutation is a deletion/substitution mutation involving 5 bp near the center of O1. The –10 and –35 promoter sequences (in bp) are shown in boldface type. The DNase I footprint of RstR at the O1 operator site is represented by a bracket. (B) A 32P-labeled DNA probe carrying O1-18 was not bound by RstR6H in gel-shift assays. From left to right, RstR6H concentrations were 0, 6, 12, 25, and 50 nM in both panels. The 301 DNA probe also contains the O2 operator site, but RstR binding to O2 is not sufficiently strong to result in additional shifted species. Gel-shift assays were carried out as previously described (13).

Using the same set of reporter plasmids, we investigated whether PR autoactivation could be detected in E. coli by expressing RstR from the arabinose-inducible plasmid pBAD-RstR (12). With pHK301N (O1+ O2+), a strong peak of LacZ activity was observed at inducer concentrations of approximately 2 x 10–5 percent arabinose (Fig. 3A). The peak of PR activation ranged between 15- and 20-fold over basal expression levels (no arabinose). At higher inducer concentrations, a strong decrease in lacZ expression was observed. This RstR-mediated repression cannot be explained by nonspecific binding of RstR to the PR promoter region, since a lacZ reporter to a control promoter that is not regulated by RstR was not detectably repressed by RstR in analogous experiments (data not shown). Primer extension analysis of RNA extracted from the same arabinose-induced cultures showed that lacZ transcription in E. coli originated from the true PR promoter (data not shown). LacZ expression from the mutated reporter pHK301N(O1-18) could not be activated by RstR expressed from pBAD-RstR (Fig. 3A), indicating that PR transcription requires RstR binding to O1 positioned –40 to –80 from the start of rstR transcription.


Figure 3
View larger version (63K):
[in this window]
[in a new window]

  		FIG. 3. RstR activates and represses transcription from PR. (A) Activity of the PR promoter in E. coli in response to RstR. The following PR-lacZ reporters were analyzed in strain BW27784 (BW25113 {Delta}araFGH {Delta}araEp::PCP18-araE) (11): pHK312N (O1+ O2+ O3+), pHK301N (O1+ O2+ O3–), and pHK301N(O1-18) (O1– O2+ O3–). RstR was provided from pBAD-RstR. Cultures were grown in LB with the indicated arabinose (ara) concentrations, and LacZ activity was determined as described in Materials and Methods. LacZ activities are the mean results from two to four experiments, and error bars depict the standard errors of the means. (B) Direct measurement of RstR DNA binding activity in cell extracts of BW27784 expressing RstR from pBAD-RstR. Cell extracts were prepared from cultures grown in the indicated arabinose concentrations, and RstR activity was determined by gel-shift assay, using a DNA probe carrying the O1 operator. RU was defined as: [(1/V50)/total protein concentration (µg/ml)] x 100, where V50 is the volume of cell extract (µl) resulting in shifting of 50% of probe DNA (see Materials and Methods). Between two and four independent measurements were performed on each extract. Inset, Western blot analysis of RstR expression in the same arabinose-induced cultures. Inducer concentrations (in percentages) are as follows: lane 1, no inducer; lane 2, 2.0 x 10–6; lane 3, 1.0 x 10–5; lane 4, 2.0 x 10–5; lane 5, 2.0 x 10–4; lane 6, 2.0 x 10–3; lane 7, 6.0 x 10–2. (C) DNase I footprint analysis of RstR6H bound to O1 and O2. A 120-bp fragment extending from +5 to –115 relative to the start of rstR transcription was amplified by PCR, in which one primer was 32P labeled at the 5'-end position. The first lane in each panel shows the DNase I reaction with no added RstR6H. RstR concentrations in the following lanes were approximately 2, 4, 8, 16, 32, and 64 nM RstR6H. The regions of O1 and O2 protected by RstR are depicted by black and white rectangles, respectively. Filled circles mark the locations of DNase I hypersensitive sites, a characteristic of specific RstR binding. Only the DNase I footprint of the rstR template strand is shown. A small portion of the O2 footprint that includes the +1 start of transcription is not visible at the bottom of the gel.

We were able to relate the changes in PR activity to changes in the cellular concentration of RstR by directly measuring RstR activity in extracts of arabinose-induced cultures of BW27784 (pBAD-RstR) using a gel-shift assay and by Western blotting (Fig. 3B). The peak of PR transcription was observed at approximately 2 x 10–5 percent arabinose, while strong repression was observed at approximately 100-fold-higher inducer concentrations (Fig. 3A). Over that range, RstR activity increased approximately 14-fold (10.7 to 142 RU). In a parallel DNase I footprinting experiment using a DNA probe carrying O1 and O2, we estimated the relative concentrations of RstR required to bind O1 rather than to bind both O1 and O2. As shown in Fig. 3C, an approximately eightfold-higher concentration of RstR was required to observe both O1 and O2 occupancy rather than occupancy of O1 alone. Taken together, these data support the idea that RstR, when present at low cellular concentrations, binds to the high-affinity O1 site upstream of PR and activates PR transcription, while PR repression occurs when high concentrations of RstR lead to additional RstR binding to the lower-affinity O2 site.

When we analyzed LacZ expression in E. coli from pHK312N, a reporter containing all three RstR operators, we observed a 2.5-fold increase in LacZ activity over basal levels, followed again by a decrease in LacZ activity at high inducer concentrations (Fig. 3A). Our previous analysis of RstR-operator DNA interactions indicated that RstR binds cooperatively to O2 and O3 (13). Cooperativity was supported by DNase I footprint studies showing that the difference in the apparent affinity of RstR for O1 and O2 is significantly narrowed by the presence of O3 on the same DNA fragment (22). A likely explanation for the poor activation observed with pHK312N is that pBAD-directed expression of RstR is not sufficiently fine-tuned to allow for full activation of PR, which requires RstR binding to O1, without autorepression, which we propose involves RstR occupancy of O2 and O3. These data are consistent with the hypothesis that RstR autoactivation occurs when RstR is bound only to O1, and autorepression occurs when additional RstR binds to O2 and O3.

We next analyzed the role of LexA in rstR autoregulation. This global SOS regulator represses CTX{Phi} gene expression from PA by binding to a single SOS box centered –48.5 bp from the start of PA transcription (25) (Fig. 1B). We used E. coli for these studies, as we have been unable to delete the lexA gene from V. cholerae (22). This approach is supported by previous studies showing that E. coli LexA fully substitutes for V. cholerae LexA in PA repression (25). To confirm that the test strain constructed for these experiments, BW27784 sulA6209 lexA71, lacks LexA activity, we introduced plasmid pMQLZ, which carries the known V. cholerae LexA-regulated promoter PlexA fused to the lacZ gene of E. coli (25). The resulting strain exhibited an expected sixfold increase in LacZ activity compared to that of an isogenic lexA+ strain. As shown in Fig. 4A, the RstR-mediated activation of PR transcription from pHK301N (O1+ O2+) was abolished by lexA71. lexA71 did not significantly alter the expression of RstR from pBAD-RstR, since a PA-lacZ reporter was efficiently repressed by RstR in the same mutant strain (Fig. 4B; see below). These results suggest that RstR and LexA are both required to activate transcription from PR. To explore whether LexA mediates activation by directly binding to the single SOS box within PR, the left half-site of the LexA binding site in pHK301N was changed from CTGT to CTAT (nucleotide change underlined) (Fig. 1B and 5B). This mutation, m1, was previously shown to abolish LexA binding to the SOS box in CTX{Phi} (25). This mutated PR:lacZ reporter plasmid (pHK883) could not be activated by RstR when assayed in BW27784 sulA6209 carrying a wild-type allele of lexA (data not shown), indicating that a direct interaction between LexA and the SOS box is required for RstR-mediated transcription from PR.


Figure 4
View larger version (12K):
[in this window]
[in a new window]

  		FIG. 4. LexA is required for PR autoactivation and partially represses the divergent PA promoter. RstR was provided from pBAD-RstR. LacZ activity was measured in E. coli strains BW27784 sulA6209 and BW27784 sulA6209 lexA71. The legend shows only the relevant lexA genotype. Cultures were grown in LB plus antibiotics, and the indicated arabinose (ara) concentrations and LacZ activity were determined from three independent cultures. The means ± standard errors of the means are plotted. Shown above each panel is a map of the promoter fusion used for each experiment. The promoters and binding sites for RstR and LexA are as described in the legend to Fig. 1B. (A) The PR-lacZ reporter plasmid is pHK301N(O1+ O2+). (B) The PA-lacZ reporter plasmid pHK393 contains the same CTX{Phi} sequences present in pHK301N inserted into the lacZ reporter plasmid in the opposite orientation. In the absence of RstR (no arabinose), LexA can be seen to repress PA transcription by approximately threefold. In the absence of LexA (open triangles), RstR retains the ability to strongly repress PA transcription.


Figure 5
View larger version (15K):
[in this window]
[in a new window]

  		FIG. 5. In vitro analysis of PR and PA transcription regulation using purified factors. Single-round transcription assays were carried out with E. coli RNAP ({sigma}70) holoenzyme, purified RstR6H and E. coli LexA. DNA template and RNAP were present at 2 nM and 20 nM, respectively. LexA was present at 3.5 µM, where indicated. Amounts of RstR6H added are given in micrograms. The migrations of radiolabeled RNA standards are shown at the left. (A) Maps of the linear DNA template and the predicted sizes of PA and PR runoff transcripts are shown above each panel. The DNA template was made by PCR and contains the same O1+ O2+ DNA sequences present in pHK301N. (B) RstR-mediated activation of PR transcription is blocked by the m1 SOS box mutation. The left panel shows control reactions programmed with the same wild-type DNA template as is shown in panel A. The m1 DNA template was made by PCR and contains the same CTX{Phi} sequences as those of the wild-type template described in the legend to panel A. The sizes of the PA and PR runoff transcripts from the m1 template differ from those in the control reactions due to the use of different sets of PCR primers for template synthesis. The DNA sequences of the E. coli consensus, CTX{Phi}, and m1 SOS boxes are shown below.

Using the same set of mutant E. coli strains, we also analyzed the roles of LexA and RstR in regulating the divergent PA promoter, the primary CTX{Phi} promoter. In the absence of RstR, LexA repressed PA transcription by approximately threefold (no arabinose) (Fig. 4B), consistent with our previous studies of PA regulation (24). Regardless of the presence of LexA, RstR strongly repressed PA transcription (Fig. 4B), confirming our earlier findings showing that LexA and RstR independently repress PA transcription.

Since removal of LexA from the cell could have indirect consequences on PR activity, we investigated the roles of RstR and LexA in CTX{Phi} gene transcription using in vitro transcription assays. Purified RstR (RstR6H) and/or E. coli LexA was incubated with DNA templates containing O1 and O2 but lacking O3. E. coli RNAP({sigma}70) was added, and transcription was allowed to proceed for one round using heparin sulfate. As shown in Fig. 5A, a transcript corresponding to the predicted 211-nucleotide (nt) PR transcript was observed only in the presence of both RstR6H and LexA. PR transcription increased with increasing RstR concentrations, then decreased at the highest RstR levels tested, similar to the profile of PR activity observed in PR-lacZ reporter assays (Fig. 3A). At its peak, PR activation resulted in a 21-fold increase in transcript levels. A transcript corresponding to the predicted 94-nt PA transcript showed the expected pattern of dual repression by LexA and RstR (Fig. 5A). To confirm that these transcripts originated from the PA and PR promoters, a second overlapping DNA template was generated, in which the predicted sizes of the PA and PR runoff transcripts were 175 nt and 115 nt, respectively. Using this DNA template, a transcript corresponding in size to the predicted PR transcript was again observed only in the presence of LexA and RstR, and a 175-nt RNA product corresponding to the PA transcript showed the expected pattern of dual repression by LexA and RstR (data not shown). These results demonstrate that RstR and LexA stimulate transcription from PR in a codependent manner while also repressing transcription from PA. Our results also confirm that RstR represses PR activity at high repressor concentrations.

We investigated the requirement for LexA binding to the SOS box by analyzing transcription from a DNA template containing the G-to-A substitution mutation, m1, in the left half-site of the LexA binding site within PR. m1 was previously shown to abolish LexA binding to this site without affecting RstR binding to the O2 region (25). Using m1 DNA (O1+ O2+) as a transcription template, we did not observe stimulation of the predicted 145-nt PR transcript in reactions containing RstR and LexA (Fig. 5B), indicating that LexA binding to the known SOS box within PR is required for activation. As expected, PA transcription from the m1 template could still be repressed by the addition of RstR6H (Fig. 5B) but was no longer repressed by the addition of LexA, confirming that the presence of m1 results in a nonfunctional SOS box. These data demonstrate that PR activation requires LexA binding to the single SOS box within PR and confirm our in vivo PR-lacZ studies, showing that LexA, as well as LexA binding to the SOS box, is required for PR activation.


arrow
DISCUSSION
 
We previously demonstrated a direct role for both RstR and LexA in repressing CTX{Phi} transcription from the PA promoter (24, 25). Here we show that RstR and LexA perform the additional role of coactivating rstR transcription from the divergent PR promoter. LexA has a relatively minor role in repressing transcription from PA, whereas LexA is essential for rstR expression from PR. Therefore, LexA and RstR function as both repressors and activators of CTX{Phi} gene expression. Additionally, we found that high concentrations of RstR repress PR activity, indicating that CTX{Phi} lysogens use both positive and negative autoregulatory mechanisms to tightly regulate repressor levels. Our findings suggest that maintenance of CTX{Phi} lysogeny uses a regulatory circuit requiring the interplay of host- and phage-encoded transcription factors.

RstR autoactivates its own expression from the PR promoter when bound to O1 and autorepresses its own expression when bound to O2 and O3. Several lines of evidence support this hypothesis. PR-lacZ fusions expressed high levels of LacZ in CTX{Phi} lysogens, and a mutation (O1-18) that abolished RstR binding to the upstream O1 operator eliminated the 20-fold increase in LacZ activity observed in CTX{Phi} lysogens. Also, a shortened PR-lacZ fusion (O1+ O2+) was strongly activated in E. coli when RstR was provided from the arabinose-inducible plasmid pBAD-RstR (Fig. 3A). Since an O1+ O2+ reporter could be easily activated using a heterologous system, we exploited this feature to define the roles of RstR and LexA in PR transcription regulation. By varying the RstR levels using the PBAD promoter system (Fig. 3B), we showed that RstR activated PR transcription at low concentrations and strongly repressed PR transcription at high concentrations. LexA was absolutely required for PR autoactivation in vivo (Fig. 4A). In vitro transcription studies carried out with purified components confirmed that RstR and LexA function directly as coactivators of rstR transcription and also confirmed that RstR negatively autoregulates PR transcription (Fig. 5).

Using a heterologous transcription reporter system in E. coli, we observed only strong PR activation, with constructs lacking the O3 operator positioned downstream of PR (Fig. 1B). The presence of O3 improves RstR binding to O2, probably due to cooperative interactions between neighboring DNA-bound RstR tetramers (13, 22). We propose that the weak activation of a PR-lacZ fusion carrying all three operators (Fig. 3A) is due to the difficulty in modulating RstR concentrations that allow for O1 occupancy but not that of O2 and O3. However, the same full-length lacZ fusion expressed high LacZ activity in V. cholerae CTX{Phi} lysogen strains, where RstR levels are presumably maintained by the positive and negative autoregulatory mechanisms outlined above. Alternatively, there could be physiological differences between V. cholerae and E. coli that limit PR transcription through an unknown mechanism.

Although there have been many studies describing LexA-mediated transcription repression, there is only one previous report of LexA functioning directly as a transcription activator (29). Together with this previous report, our discovery that LexA functions as a coactivator of RstR transcription suggests that the host SOS regulon may contain a subset of genes whose transcription is also activated by LexA. In support of this hypothesis, genome-wide studies of gene expression following DNA damage identified a number of genes in E. coli whose mRNA levels rapidly decrease upon UV exposure (3).

LexA's role as a coactivator of PR transcription is unusual, as it binds to a site centered –23.5 bp from the start of rstR transcription, within the promoter spacer region and overlapping the –35 sequence (Fig. 1B). In contrast, most activators bind upstream of the –35 promoter region, where they make contacts with the C-terminal domain of the {alpha} subunit, the {alpha}-subunit N-terminal domain, or the {sigma} region 4 of RNAP and/or other transcription activators (1). Nevertheless, a small number of transcription activators bind to sites within or overlapping the target promoter spacer region. These include the phage T4-encoded transcription activator MotA (9) and the lambda CII protein (10), which make direct contacts with RNAP, whereas activators such as the E. coli MerR protein act by distorting the DNA to bring the –10 and –35 promoter elements into alignment (2). Future studies will determine whether RstR- and LexA-mediated activation of rstR transcription involves direct interactions with RNAP.

Although we favor a direct model to explain RstR and LexA activation at PR, alternative models could be envisioned in which LexA and RstR stimulate PR transcription indirectly. One such model is that LexA and RstR act by preventing promoter interference between PA and PR. In this model, RNAP, when engaged in transcription at the strong PA promoter, directly represses PR transcription due to the close proximity of the two promoters. LexA and RstR would thereby activate PR transcription indirectly by interfering with PA transcription. To investigate the possibility of promoter interference between PA and PR, we altered the –10 sequence of PA from TATTTT to TATAAC (change underlined) and analyzed the effect of this change on PA and PR activity levels. The mutation reduced PA activity by approximately 50% in the absence of LexA and RstR (2,500 to 1,200 Miller units). However, this alteration to PA did not result in an increase in basal transcription from that of PR (45 to 50 Miller units), indicating that there is no strong promoter interference in this system. Also, if promoter interference was responsible for repressing PR, one would predict that RstR bound to O1, which strongly represses PA transcription (Fig. 4B and 5A), would suffice to activate PR transcription. However, our in vivo and in vitro results demonstrate clearly that RstR is not sufficient to activate PR. Taken together, these observations suggest that PA and PR do not intrinsically interfere with one another.

The gene network governing CTX{Phi} lysogeny shares certain features with the lysogenic switch of {lambda} (21). The promoters are divergently arranged and overlap with multiple repressor operator sites. During lysogeny, the primary phage promoters are repressed by a phage-encoded repressor, and the repressors both activate and repress their own expression. However, in contrast to lambda induction, which trips a highly stable switch that commits lysogens to phage development and host lysis, our findings suggest that CTX{Phi} induction follows transient and reversible switch kinetics. When CTX{Phi} lysogens are exposed to DNA damage-inducing agents, LexA levels would fall. The loss of LexA, which leads to the partial derepression of CTX{Phi} transcription from PA, would also result in a rapid cessation of RstR expression from PR, as LexA is absolutely required for PR transcription activation. CTX{Phi} induction should cease and rstR expression should resume, once the host DNA damage is repaired and LexA recovers to normal levels. The recovery kinetics of the CTX{Phi} switch might vary, as the recovery of LexA levels would likely depend upon the extent of DNA damage and the rate of DNA repair (15, 26). The recruitment by CTX{Phi} of the host LexA repressor appears to have resulted in a transient switch circuit, one that may be particularly suited to a filamentous bacteriophage that reproduces without killing its host.

The negative autoregulation of RstR levels is a critical feature of the proposed CTX{Phi} switch. We previously showed that RstR and LexA compete for binding to their respective overlapping sites at O2 (25). Therefore, RstR concentrations must be maintained at levels that allow for O1 occupancy but leave O2 vacant and accessible to LexA. In cells experiencing high levels of RstR, O2 and O3 would be occupied by RstR, displacing LexA, and SOS treatments would not induce CTX{Phi} gene expression. Negative autoregulation ensures that RstR levels do not drift upwards unchecked. Negative autoregulation is also critically important to the proper functioning of the {lambda} switch, as overproduction of CI due to mutations that disrupt negative autoregulation results in lysogens that cannot efficiently switch to lytic development (5).


arrow
ACKNOWLEDGMENTS
 
We thank B. Davis and A. L. Sonenshein for valuable discussions and A. Poteete and J. W. Little for kindly providing strains and plasmids. We are especially indebted to A. Hochschild and lab members for advice and discussions.

This work was supported by the Howard Hughes Medical Institute and NIH grant AI-42347.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Channing Laboratory, 181 Longwood Ave., Boston, MA 02115. Phone: (617) 525-4646. Fax: (617) 525-4660. E-mail: mwaldor{at}rics.bwh.harvard.edu Back

{triangledown} Published ahead of print on 7 August 2009. Back


arrow
REFERENCES
 
    1
  1. Barnard, A., A. Wolfe, and S. Busby. 2004. Regulation at complex bacterial promoters: how bacteria use different promoter organizations to produce different regulatory outcomes. Curr. Opin. Microbiol. 7:102-108.[CrossRef][Medline]
    2. 2
  2. Brown, N. L., J. V. Stoyanov, S. P. Kidd, and J. L. Hobman. 2003. The MerR family of transcriptional regulators. FEMS Microbiol. Rev. 27:145-163.[CrossRef][Medline]
    3. 3
  3. Courcelle, J., A. Khodursky, B. Peter, P. O. Brown, and P. C. Hanawalt. 2001. Comparative gene expression profiles following UV exposure in wild-type and SOS-deficient Escherichia coli. Genetics 158:41-64.[Medline]
    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. Dodd, I. B., A. J. Perkins, D. Tsemitsidis, and J. B. Egan. 2001. Octamerization of lambda CI repressor is needed for effective repression of PRM and efficient switching from lysogeny. Genes Dev. 15:3013-3022.[Abstract/Free Full Text]
    6. 6
  6. Faruque, S. M., and J. J. Mekalanos. 2003. Pathogenicity islands and phages in Vibrio cholerae evolution. Trends Microbiol. 11:505-510.[CrossRef][Medline]
    7. 7
  7. Friedberg, E. C., G. C. Walker, W. Siede, R. D. Wood, R. A. Schultz, and T. Ellenberger (ed.). 2005. The SOS response of prokaryotes to DNA damage, p. 463-508. In DNA repair and mutagenesis, 2nd ed. ASM Press, Washington, DC.
    8. 8
  8. Harlow, E., and D. Lane. 1999. Using antibodies: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
    9. 9
  9. Hinton, D. M., S. Pande, N. Wais, X. B. Johnson, M. Vuthoori, A. Makela, and I. Hook-Barnard. 2005. Transcriptional takeover by sigma appropriation: remodelling of the sigma70 subunit of Escherichia coli RNA polymerase by the bacteriophage T4 activator MotA and co-activator AsiA. Microbiology 151:1729-1740.[Abstract/Free Full Text]
    10. 10
  10. Jain, D., Y. Kim, K. L. Maxwell, S. Beasley, R. Zhang, G. N. Gussin, A. M. Edwards, and S. A. Darst. 2005. Crystal structure of bacteriophage lambda cII and its DNA complex. Mol. Cell 19:259-269.[CrossRef][Medline]
    11. 11
  11. Khlebnikov, A., K. A. Datsenko, T. Skaug, B. L. Wanner, and J. D. Keasling. 2001. Homogeneous expression of the PBAD promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter. Microbiology 147:3241-3247.[Abstract/Free Full Text]
    12. 12
  12. Kimsey, H. H., and M. K. Waldor. 1998. CTX{Phi} immunity: application in the development of cholera vaccines. Proc. Natl. Acad. Sci. USA 95:7035-7039.[Abstract/Free Full Text]
    13. 13
  13. Kimsey, H. H., and M. K. Waldor. 2004. The CTX{Phi} repressor RstR binds DNA cooperatively to form tetrameric repressor-operator complexes. J. Biol. Chem. 279:2640-2647.[Abstract/Free Full Text]
    14. 14
  14. Little, J. W. 1984. Autodigestion of lexA and phage lambda repressors. Proc. Natl. Acad. Sci. USA 81:1375-1379.[Abstract/Free Full Text]
    15. 15
  15. Little, J. W. 1983. The SOS regulatory system: control of its state by the level of RecA protease. J. Mol. Biol. 167:791-808.[CrossRef][Medline]
    16. 16
  16. Little, J. W., B. Kim, K. L. Roland, M. H. Smith, L. L. Lin, and S. N. Slilaty. 1994. Cleavage of LexA repressor. Methods Enzymol. 244:266-284.[Medline]
    17. 17
  17. McLeod, S. M., H. H. Kimsey, B. M. Davis, and M. K. Waldor. 2005. CTX{Phi} and Vibrio cholerae: exploring a newly recognized type of phage-host cell relationship. Mol. Microbiol. 57:347-356.[CrossRef][Medline]
    18. 18
  18. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
    19. 19
  19. Pearson, G. D. N., A. Woods, S. L. Chiang, and J. J. Mekalanos. 1993. CTX genetic element encodes a site-specific recombination system and an intestinal colonization factor. Proc. Natl. Acad. Sci. USA 90:3750-3754.[Abstract/Free Full Text]
    20. 20
  20. Poteete, A. R., and A. C. Fenton. 2000. Genetic requirements of phage lambda red-mediated gene replacement in Escherichia coli K-12. J. Bacteriol. 182:2336-2340.[Abstract/Free Full Text]
    21. 21
  21. Ptashne, M. 2004. A genetic switch: phage lambda revisited, 3rd ed. Cold Spring Harbor Press, Cold Spring Harbor, NY.
    22. 22
  22. Quinones, M. 2006. Regulation of CTX prophage induction. Tufts University School of Medicine, Medford, MA.
    23. 23
  23. Reference deleted.
    24. 24
  24. Quinones, M., H. H. Kimsey, W. Ross, R. L. Gourse, and M. K. Waldor. 2006. LexA represses CTX{Phi} transcription by blocking access of the alpha C-terminal domain of RNA polymerase to promoter DNA. J. Biol. Chem. 281:39407-39412.[Abstract/Free Full Text]
    25. 25
  25. Quinones, M., H. H. Kimsey, and M. K. Waldor. 2005. LexA cleavage is required for CTX prophage induction. Mol. Cell 17:291-300.[CrossRef][Medline]
    26. 26
  26. Sassanfar, M., and J. W. Roberts. 1990. Nature of the SOS-inducing signal in Escherichia coli. The involvement of DNA replication. J. Mol. Biol. 212:79-96.[CrossRef][Medline]
    27. 27
  27. Schneider, K., and C. F. Beck. 1986. Promoter-probe vectors for the analysis of divergently arranged promoters. Gene 42:37-48.[CrossRef][Medline]
    28. 28
  28. Shultzaberger, R. K., Z. Chen, K. A. Lewis, and T. D. Schneider. 2007. Anatomy of Escherichia coli {sigma}70 promoters. Nucleic Acids Res. 35:771-788.[Abstract/Free Full Text]
    29. 29
  29. Tapias, A., S. Fernandez, J. C. Alonso, and J. Barbe. 2002. Rhodobacter sphaeroides LexA has dual activity: optimising and repressing recA gene transcription. Nucleic Acids Res. 30:1539-1546.[Abstract/Free Full Text]
    30. 30
  30. Uptain, S. M., and M. J. Chamberlin. 1997. Escherichia coli RNA polymerase terminates transcription efficiently at rho-independent terminators on single-stranded DNA templates. Proc. Natl. Acad. Sci. USA 94:13548-13553.[Abstract/Free Full Text]
    31. 31
  31. Waldor, M. K., and J. J. Mekalanos. 1996. Lysogenic conversion by a filamentous phage encoding cholera toxin. Science 272:1910-1914.[Abstract]
    32. 32
  32. Waldor, M. K., E. J. Rubin, G. D. Pearson, H. Kimsey, and J. J. Mekalanos. 1997. Regulation, replication, and integration functions of the Vibrio cholerae CTX{Phi} are encoded by region RS2. Mol. Microbiol. 24:917-926.[CrossRef][Medline]
    33. 33
  33. Whipple, F. W., M. Ptashne, and A. Hochschild. 1997. The activation defect of a lambda cI positive control mutant. J. Mol. Biol. 265:261-265.[Medline]
    34. 34
  34. Xiong, X. F., and W. S. Reznikoff. 1993. Transcriptional slippage during the transcription initiation process at a mutant lac promoter in vivo. J. Mol. Biol. 231:569-580.[CrossRef][Medline]

Journal of Bacteriology, November 2009, p. 6788-6795, Vol. 191, No. 22
0021-9193/09/$08.00+0     doi:10.1128/JB.00682-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Related articles in JB:

A New Twist on a Classic Paradigm: Illumination of a Genetic Switch in Vibrio cholerae Phage CTX{Phi}
Bryce E. Nickels
JB 2009 191: 6779-6781. [Full Text]  



This article has been cited by other articles:

  • Nickels, B. E. (2009). A New Twist on a Classic Paradigm: Illumination of a Genetic Switch in Vibrio cholerae Phage CTX{Phi}. J. Bacteriol. 191: 6779-6781 [Full Text]  

This Article
FREE
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 Related articles in JB
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
Google Scholar
Right arrow Articles by Kimsey, H. H.
Right arrow Articles by Waldor, M. K.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kimsey, H. H.
Right arrow Articles by Waldor, M. K.

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