Requirements for Vibrio cholerae HapR Binding and Transcriptional Repression at the hapR Promoter Are Distinct from Those at the aphA Promoter

Bacterial strains and expression plasmids.The V. cholerae strains and expression plasmids used in this study are listed in Table 1. Strains were maintained at −70°C in Luria-Bertani (LB) medium (18) containing 30% (vol/vol) glycerol. Antibiotics were used at the following concentrations in LB medium: ampicillin, 100 μg/ml; kanamycin, 45 μg/ml; polymyxin B, 50 units/ml; streptomycin, 1 mg/ml. 5-Bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) was used in LB agar at 40 μg/ml.

Construction of hapR-lacZ chromosomal fusions.A ΔhapR plasmid, pKAS187, was previously constructed which contains two 500-bp fragments of DNA that flank the hapR gene joined at a NotI restriction site (13). The V. cholerae ΔhapR-lacZ fusion was constructed by digesting this plasmid with NotI and inserting a promoterless lacZ gene from pVC200 (21) that has its own ribosome binding site and ATG codon, generating pGKK273. The resulting fusion was then introduced into the chromosome of KSK262 by allelic exchange, simultaneously creating a deletion of the hapR gene and a fusion of the hapR promoter to lacZ in the resulting strain, GK972. The merodiploid hapR⁺hapR-lacZ fusion was constructed as follows. A fragment from pGKK273 containing the hapR-lacZ fusion was obtained using an EcoRI site 500 bp upstream of the hapR gene and a BglII site which lies immediately downstream of lacZ and it was ligated into pKAS180 which contains two 500-bp fragments flanking the V. cholerae lacZ gene (14). The resulting fusion in pKAS264 was then introduced in place of the lacZ gene in KSK262, generating strain KSK2226. The ΔhapR mutation was then introduced with pKAS187 (13). The hapR overexpression plasmid pKAS189 (13) contains the hapR coding sequence expressed from a heterologous promoter in pMMB66EH.

Construction of hapR promoter mutations.The base pair changes in the hapR promoter were constructed by overlapping PCR using primers which contain the site for the type IIS restriction enzyme EarI. For each change, two 500-bp products were amplified from C6706 and then ligated into pKAS154 (13). The mutant products were amplified using primers HA24 (+18) (5′-GATCGCTCTTCGATTCTGTTATTTGCTACTTAAAGCCC), HA23 (+21) (5′-GATCGCTCTTCGATTTTGCTATTTGCTACTTAAAGCCCTATG), or HA25 (+18 and +21) (5′-GATCGCTCTTCGATTCTGCTATTTGCTACTTAAAGCCCTATG)together with primer HA16 (5′-GATCGGAATTCGTTTAAGGTATTCCCTGTATCG). The wild-type product was generated using primers HA22 (5′-GATCGCTCTTCGAATAATCATTAGAGCAAAATGC) and HA17 (5′-GATCGTCTAGAGCAGTTGGTTAGTTCGGTTG). After ligation, the resulting plasmids were sequenced. One of these, pWEL85, was used to introduce the +18 mutation into C6706 by allelic exchange to generate KSK2339. To construct the ΔhapR-lacZ chromosomal fusion containing the +18 change, an EcoRI NotI fragment containing the mutation was removed from pWEL85 and used to replace the wild-type fragment in pKAS187 described above. The promoterless lacZ gene described above was then inserted into the plasmid, generating pKAS275. After sequencing, the fusion was introduced into KSK262 by allelic exchange, generating KSK2316. To construct the merodiploid hapR⁺hapR-lacZ chromosomal fusion containing the +18 change, the EcoRI BglII hapR promoter-lacZ fragment analogous to that isolated from pGKK273 above was digested out of pKAS275 and ligated into pKAS180 (14). The resulting fusion in pKAS282 was then introduced in place of the lacZ gene in KSK262, generating KSK2337.

Identification of the hapR transcriptional start site.Total RNA was isolated from C6706 after growth for 7.5 h in LB medium at 37°C with TRIZOL reagent (Invitrogen). The RNA was subjected to 5′ rapid amplification of cDNA ends (Invitrogen) as described previously (14), except that first-strand cDNA synthesis was carried out using the hapR-specific primer HA11 (5′-CTTCTTGTTCACCTAAACGG) and the first and second nested primers were HA12 (5′-GTTGGGTGTGCGGTTGGTTG) and HA13 (5′-CAAACCACTCACCTAAAGCG).

Purification of HapR.HapR was purified using the IMPACT-CN protein fusion and purification system (New England Biolabs). The HapR gene was amplified from C6706 using HA-Sap (5′-GATCGGCTCTTCAGCACGCGTTCTTATAGATACACAGCA) and HA-Nde (5′-GATCGCATATGGACGCATCAATCGAAAAACG). The resulting fragment was digested with NdeI and SapI and ligated into pTXB-1 to generate pWEL20. The nucleotide sequence was confirmed by DNA sequencing. Escherichia coli strain ER2566 containing pWEL20 was grown in LB Amp at 30°C for 3 h, induced with 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside) and shifted to 12°C, and growth was continued overnight. The cells were collected by centrifugation and resuspended in column buffer (20 mM Tris [pH 8.0], 500 mM NaCl, 1 mM EDTA). The extract was sonicated and clarified by centrifugation at 14,000 rpm for 30 min in an SS34 rotor. The supernatant was then loaded onto a chitin column equilibrated with column buffer and washed with 10 volumes of column buffer and high-salt column buffer (containing 1 M NaCl). The column was then quickly washed with 3 volumes of cleavage buffer (column buffer containing 100 mM dithiothreitol [DTT]) and was left overnight at 16°C. HapR was then eluted off the column by using column buffer without DTT. The resulting protein was dialyzed overnight in a solution of 20 mM Tris (pH 7.5), 1 mM EDTA, 10 mM NaCl, and 0.1 mM DTT, glycerol was added to 10%, and it was frozen at −70°C. The final purity was estimated to be greater than 98%.

Gel mobility shift experiments.A 150-bp hapR promoter fragment was amplified by PCR from C6706 or the E. coli strains containing the mutant plasmids pWEL84 (+21G), pWEL85 (+18G), or pWEL86 (+18G, +21G) using primers HA4 (−61) (5′-GATCGGAATTCGTTGCACATTTTTCACCCAAC) and HAN1 (+96) (5′-GATCGGCGGCCGCTTCGATTGATGCGTCCATAG). A 120-bp hapR promoter fragment was amplified from C6706 using primers HA5 (+41) (5′-TGCTCAATCAACAACTCAATTG) and HA6 (+158) (5′-GATCGGGATCCAACGCGATTTCCATCAGTTG). The fragments were gel purified, 3′ end labeled with digoxigenin and visualized using chemiluminescence (12). Binding reactions for HapR were carried out as previously described (13).

DNaseI footprinting.A 350-bp fragment was PCR amplified from C6706 with HA3E (−198) (5′-GATCGGAATTCGCGATGTCAGTATCGCTGAC) and HA6 (+158) and ligated into pBluescript (Stratagene), generating pWEL81. For upper-strand labeling, the inserts were excised with EcoRI and XbaI. For lower-strand labeling, the inserts were excised with BamHI and HindIII. The fragments were gel purified, treated with shrimp alkaline phosphatase, and kinased with [γ-³³P]ATP (NEN; 3,000 Ci/mmol). Singly end-labeled fragments were obtained by digestion with BamHI (for the upper strand) and EcoRI (for the lower strand). Proteins were bound to the singly end-labeled DNA fragment as previously described (13).

Real-time quantitative reverse transcription (RT)-PCR.Overnight cultures of C6706 and KSK2339 were incubated in LB medium at 30°C with shaking. The cultures were diluted 1:00 into fresh LB medium and incubated at 37°C until the optical density at 600 nm (OD₆₀₀) reached 0.5. They were then diluted 1:5 and growth was continued until the OD₆₀₀ reached 2.0. The cells were collected by centrifugation and RNA was isolated using TRIZOL (Invitrogen). The RNA was subjected to DNaseI and purified using RNeasy columns (QIAGEN). Equal amounts of RNA (5 μg) were used to generate cDNA (Invitrogen). To ensure that the RNA preparations were free from genomic DNA, reverse transcription was performed with or without reverse transcriptase. Quantitative PCR was carried out by performing two independent experiments each in triplicate with 20 ng of cDNA using QuantiTect SYBR Green master mix (QIAGEN). PCR was monitored using the ABI Prism 7700 detection system. The primers for hapR were HapRF (5′-AACTGATGGAAATCGCGTTG) and HapRR (5′-AACACTGTTGCAACGGAGAC), which generated a 100-bp product. Primers for the gene encoding ribosomal protein L27 (rpmA) which served as the endogenous control were RpmF (5′-AGCTGGTGGTTCTACTCGTAACG) and RpmR (5′-CGAACGATGATGTTACCTGC), which generated a 101-bp product. Relative hapR expression was determined using the following calculation: 2^{−(ΔC_T target − ΔC_T control)}, where C_T is the threshold cycle. The levels of rpmA were essentially identical in the strains examined.

β-Galactosidase assays.Assays with V. cholerae lacZ fusions were carried out as described previously (18).

HapR influences the transcription of the hapR promoter in V. cholerae.To determine whether the V. cholerae HapR protein functions similarly to LuxR as an autorepressor, a ΔhapR-lacZ transcriptional fusion strain, GK972, was constructed. In this strain, the hapR coding sequence was replaced with an E. coli lacZ gene containing its own ribosome binding site and ATG codon, thus rendering its expression independent of translational regulation through LuxO. A plasmid overexpressing HapR from a heterologous promoter, pKAS189, was then introduced into GK972. Although at low cell density (OD₆₀₀ = 0.2) no significant difference in expression was observed compared to a vector control (Fig. 1), at high cell density (OD₆₀₀ = 4.0) the expression of the fusion increased approximately 15-fold over that at low cell density, and under this condition the presence of HapR from pKAS189 resulted in a fourfold reduction in β-galactosidase activity (Fig. 1). To determine if a similar effect also occurs when hapR is in single copy, a hapR⁺ merodiploid strain was constructed by introducing the hapR-lacZ reporter into the lacZ locus by homologous recombination with regions of the flanking chrA and galR genes (14). The resulting strain, KSK2226, harbors the same hapR promoter-lacZ fusion as in GK972, but it is situated at the lacZ locus, leaving the hapR gene intact at its normal chromosomal location. KSK2226 was then compared with an isogenic strain containing a ΔhapR mutation. Again, at low cell density no significant difference between the strains was observed (Fig. 1), but at high cell density a twofold increase in expression was observed in the ΔhapR strain, consistent with a role for HapR in its negative autoregulation (Fig. 1).

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FIG. 1.

Influence of HapR on hapR-lacZ fusions in V. cholerae. Strains were grown in LB medium at 37°C for 2 h (low cell density, OD₆₀₀ = 0.2) or 8 h (high cell density, OD₆₀₀ = 4.0). From left to right: GK972 (ΔhapR-lacZ) pMMB66EH (vector control), GK972 pKAS189 (pMMB66EH/hapR⁺), KSK2226 (hapR⁺/hapR-lacZ), KSK2234 (KSK2226 ΔhapR). Cultures with pMMB66EH and pKAS189 contained 1 mM IPTG. wt, wild type.

Identification of the hapR transcriptional start site.The transcriptional start site for the hapR promoter was determined using 5′ rapid amplification of cDNA ends (6) with RNA isolated from wild-type C6706 grown in LB medium at 37°C to high cell density. A product corresponding to the size expected for the transcriptional start was identified, and sequencing of it revealed that the +1 of the hapR promoter is a G 77 bp upstream of the ATG (Fig. 2). The −10 sequence, TACACT, shows two mismatches from the consensus TATAAT, and the −35 sequence, TTGACC, has only one mismatch from the consensus TTGACA.

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FIG. 2.

Nucleotide sequence of the proximal region of the hapR promoter. The transcriptional start site (+1), ATG codon, and −10 and −35 regions are shown. Black lines show the region of the promoter protected by HapR, and arrows indicate putative dyad symmetry. The boxed sequence below the nucleotide sequence shows the data used to determine the position of the transcriptional start.

HapR binds to a site in the hapR promoter downstream of the transcriptional start.Gel mobility shift assays were used to localize the HapR binding site in the hapR promoter. As shown in Fig. 3, a single shift was observed in the presence of increasing amounts of purified HapR protein on a DNA fragment encompassing the region from −61 to +96. However, no shift was observed on a fragment from +41 to +158, indicating that the HapR binding site is located between −61 and +41 in the promoter. No additional complexes with slower mobility were observed on a larger fragment extending upstream in the promoter to −116 (data not shown), indicating that HapR does not recognize any other sites in the promoter with a similar affinity.

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FIG. 3.

Binding of purified HapR to specific hapR promoter fragments. Lanes 1-4, a 150-bp fragment from −61 to +96; lanes 5-8, a 120-bp fragment from +41 to +158. The first lane in each set has no protein added, the second lane has 36 nM (18 ng) HapR, the third lane has 70 nM (35 ng) HapR, and the fourth lane has 140 nM (70 ng) HapR.

To further localize the binding site between −61 and +41, DNaseI footprinting was utilized. On a 350-bp fragment extending from −198 to +158, a single site of strong protection was observed (Fig. 4). On the top strand, this site extended from +12 to +36, and on the bottom strand, it extended from +8 to +33 (Fig. 2). No other strong regions of protection were observed on this fragment, suggesting that a single site is necessary for HapR autorepression. Comparison of a 22-bp sequence of partial dyad symmetry within the footprint (Fig. 5A) to the site previously identified at the aphA promoter (13) reveals the lack of a strong HapR binding motif. Of the 10 bp which are conserved between the sites, six of these are symmetrical in both, indicating that there is a degree of functional similarity between them.

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FIG. 4.

DNaseI footprint for HapR at the hapR promoter. Top and bottom strands of a 350-bp fragment from −198 to +158. Lane 1, no protein; lane 2, 1.4 μM (176 ng) HapR.

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FIG. 5.

A. Comparison of the HapR binding sites at the hapR and aphA promoters. Arrows show the extent of dyad symmetry and bold characters show positions that are symmetrical in each site. Asterisks denote positions where single base pair changes prevent HapR binding. B. Binding of purified HapR to the 150-bp fragment from −61 to +96. The first three lanes are wild type (wt), the next three lanes contain the +18 A-to-G mutation, the next three lanes contain the +21 A-to-G mutation, and the last three lanes contain both mutations. The first lane in each set has no protein added, the second lane has 36 nM (18 ng) HapR, and the third lane has 140 nM (70 ng) HapR.

Identification of a point mutation in the HapR binding site that prevents binding.We have previously shown that a naturally occurring base pair change in classical biotype strains at position −77 from G to T in the HapR binding site at the aphA promoter completely prevents HapR binding, indicating it is critical for its recognition (13). Since the analogous position in the HapR binding site at the hapR promoter (+17) is not conserved (Fig. 5A), nor does it appear to be important for symmetry, identification of a base pair change that prevents the protein from binding to the promoter was not straightforward. Therefore, two base pairs were changed to G that appeared to be important for maintaining the dyad symmetry of the site (A at position +18 and A at position +21). When these base pair changes were introduced into the −61 to +96 fragment which is capable of binding HapR (Fig. 3), the +18 change prevented binding whereas the +21 change did not (Fig. 5B). As expected from this, a fragment containing both mutations was also defective for binding (Fig. 5B). These results indicate that HapR binding at its own promoter can be disrupted by a single base pair change at position +18.

The G +18 mutation in the HapR binding site prevents autorepression.To determine if the +18 A-to-G change that interferes with HapR binding to its own promoter also prevents autorepression, its influence on hapR-lacZ fusions in ΔhapR and hapR⁺ strains was determined. As shown in Fig. 6, the expression of the ΔhapR-lacZ fusion containing the G +18 mutation (KSK2316) was only slightly higher than that of the wild-type fusion GK972 at both low and high cell density, indicating that the point mutation does not significantly influence the expression of the hapR promoter in the absence of HapR. However, in the presence of HapR, the expression of the fusion containing the G +18 mutation (KSK2337) showed approximately twofold derepression at high cell density such that it appeared similar to the wild-type fusion lacking HapR. These results indicate that HapR autorepresses by binding to the site identified in the hapR promoter that extends from +8 to +36. Consistent with this, the level of hapR expression as determined by real-time RT-PCR in C6706 containing the +18 A-to-G change at high cell density was approximately twofold higher than in the wild type (Fig. 7).

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FIG. 6.

Influence of the +18 A-to-G change on ΔhapR and hapR⁺lacZ fusions in V. cholerae. Strains were grown in LB medium at 37°C for 2 h (low cell density, OD₆₀₀ = 0.2) or 8 h (high cell density, OD₆₀₀ = 4.0). From left to right: GK972 (ΔhapR-lacZ), KSK2316 (ΔhapR-lacZ +18G), KSK2226 (hapR⁺/hapR-lacZ), KSK2337 (hapR⁺/hapR-lacZ +18G). wt, wild type.

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FIG. 7.

Relative levels of hapR expression determined by real-time PCR. Cultures were grown in LB medium at 37°C to an OD₆₀₀ of 2.0. RNA was isolated, converted to cDNA, and analyzed by real-time PCR. C6706 (wild type [wt]), KSK2339 (+18G).