Mechanism underlying the DNA-binding preferences of the Vibrio cholerae and vibriophage VP882 VqmA quorum-sensing receptors

Olivia P. Duddy; Xiuliang Huang; Justin E. Silpe; Bonnie L. Bassler

doi:10.1371/journal.pgen.1009550

Abstract

Quorum sensing is a chemical communication process that bacteria use to coordinate group behaviors. In the global pathogen Vibrio cholerae, one quorum-sensing receptor and transcription factor, called VqmA (VqmA_Vc), activates expression of the vqmR gene encoding the small regulatory RNA VqmR, which represses genes involved in virulence and biofilm formation. Vibriophage VP882 encodes a VqmA homolog called VqmA_Phage that activates transcription of the phage gene qtip, and Qtip launches the phage lytic program. Curiously, VqmA_Phage can activate vqmR expression but VqmA_Vc cannot activate expression of qtip. Here, we investigate the mechanism underlying this asymmetry. We find that promoter selectivity is driven by each VqmA DNA-binding domain and key DNA sequences in the vqmR and qtip promoters are required to maintain specificity. A protein sequence-guided mutagenesis approach revealed that the residue E194 of VqmA_Phage and A192, the equivalent residue in VqmA_Vc, in the helix-turn-helix motifs contribute to promoter-binding specificity. A genetic screen to identify VqmA_Phage mutants that are incapable of binding the qtip promoter but maintain binding to the vqmR promoter delivered additional VqmA_Phage residues located immediately C-terminal to the helix-turn-helix motif as required for binding the qtip promoter. Surprisingly, these residues are conserved between VqmA_Phage and VqmA_Vc. A second, targeted genetic screen revealed a region located in the VqmA_Vc DNA-binding domain that is necessary to prevent VqmA_Vc from binding the qtip promoter, thus restricting DNA binding to the vqmR promoter. We propose that the VqmA_Vc helix-turn-helix motif and the C-terminal flanking residues function together to prohibit VqmA_Vc from binding the qtip promoter.

Author summary

Bacteria use a chemical communication process called quorum sensing (QS) to orchestrate collective behaviors. Recent studies demonstrate that bacteria-infecting viruses, called phages, also employ chemical communication to regulate collective activities. Phages can encode virus-specific QS-like systems, or they can harbor genes encoding QS components resembling those of bacteria. The latter arrangement suggests the potential for chemical communication across domains, i.e., between bacteria and phages. Ramifications stemming from such cross-domain communication are not understood. Phage VP882 infects the global pathogen Vibrio cholerae, and “eavesdrops” on V. cholerae QS to optimize the timing of its transition from existing as a parasite to killing the host, and moreover, to manipulate V. cholerae biology. To accomplish these feats, phage VP882 relies on VqmA_Phage, the phage-encoded homolog of the V. cholerae VqmA_Vc QS receptor and transcription factor. VqmA_Vc, by contrast, is constrained to the control of only V. cholerae genes and is incapable of regulating phage biology. Here, we discover the molecular mechanism underpinning the asymmetric transcriptional preferences of the phage-encoded and bacteria-encoded VqmA proteins. We demonstrate how VqmA transcriptional regulation is crucial to the survival and persistence of both the pathogen V. cholerae, and the phage that preys on it.

Citation: Duddy OP, Huang X, Silpe JE, Bassler BL (2021) Mechanism underlying the DNA-binding preferences of the Vibrio cholerae and vibriophage VP882 VqmA quorum-sensing receptors. PLoS Genet 17(7): e1009550. https://doi.org/10.1371/journal.pgen.1009550

Editor: Sean Crosson, Michigan State University, UNITED STATES

Received: April 10, 2021; Accepted: June 16, 2021; Published: July 6, 2021

Copyright: © 2021 Duddy et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: This work was supported by the Howard Hughes Medical Institute, National Institutes of Health Grant R37GM065859, and National Science Foundation Grant MCB-1713731 (BLB), NIGMS T32GM007388 (OPD), a Charlotte Elizabeth Procter Fellowship provided by Princeton University, and a National Defense Science and Engineering Graduate Fellowship supported by the Department of Defense (JES). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Quorum sensing (QS) is a cell-cell communication process that allows bacteria to coordinate collective behaviors [1]. QS relies on the production, release, and group-wide detection of extracellular signaling molecules called autoinducers (AIs). In the global pathogen Vibrio cholerae, the AI, 3,5-dimethyl-pyrazin-2-ol (DPO), together with its partner cytoplasmic QS receptor and transcription factor, VqmA (VqmA_Vc), comprises one of the QS circuits that controls group behaviors [2–4]. VqmA_Vc, following binding to DPO, activates transcription of the vqmR gene encoding the small RNA, VqmR, which, in turn, represses the expression of genes required for biofilm formation and virulence factor production [2–4].

Recently, bacteria-specific viruses, called phages, have been shown to engage in density-dependent regulation of their lysis-lysogeny decisions via chemical dialogs [5,6]. Germane to our studies are phages that encode proteins resembling bacterial QS components [5,7]. Vibriophage VP882 is one such phage: It encodes the QS receptor VqmA (VqmA_Phage), a homolog of the V. cholerae QS receptor VqmA_Vc [5]. VqmA_Phage, like VqmA_Vc, binds host-produced DPO. DPO-bound VqmA_Phage activates transcription of the phage gene qtip. Qtip is an antirepressor that sequesters the phage VP882 repressor of lysis, leading to derepression of the phage lytic program and killing of the Vibrio host at high cell density [5,8]. Thus, the DPO AI mediates both bacterial and phage lifestyle decisions. Curiously, VqmA_Phage can substitute for VqmA_Vc to activate the V. cholerae vqmR promoter (PvqmR) [5]. In contrast, VqmA_Vc cannot substitute for VqmA_Phage and recognize the phage VP882 qtip promoter (Pqtip). Presumably, the ability of VqmA_Phage to bind both PvqmR and Pqtip provides phage VP882 the capacity to influence host QS and simultaneously enact its own lysis-lysogeny decision.

VqmA_Phage shares ~43% amino acid sequence identity with VqmA_Vc, and most of the key residues required for ligand and DNA binding are conserved [5,9]. Thus, how VqmA_Phage can recognize two different promoters, while VqmA_Vc cannot, is not understood. Here, we define the mechanism underlying this asymmetry. We show that VqmA selectivity for target promoters is driven by the DNA-binding domain (DBD) of the respective protein. We identify 6 key nucleotides within PvqmR and Pqtip that contribute to VqmA promoter-binding selectivity, as exchanging these critical DNA sequences inverts the DNA-binding preferences of the two VqmA proteins. The 192^nd and 194^th residues in VqmA_Vc and VqmA_Phage, respectively, within the helix-turn-helix (HTH) motifs, contribute to promoter-binding specificity. Isolation of VqmA_Phage mutants capable of activating vqmR expression but incapable of activating qtip expression revealed conserved or functionally conserved residues in VqmA_Phage and VqmA_Vc, indicating that VqmA_Vc likely possesses an additional feature that prevents it from binding Pqtip DNA. A mosaic VqmA_Vc protein containing the VqmA_Phage HTH motif along with the C-terminal 25 flanking VqmA_Phage residues was capable of binding Pqtip. Thus, the two corresponding regions in VqmA_Vc must function in concert to prevent VqmA_Vc from binding to Pqtip. Together, our analyses demonstrate how VqmA_Phage, via its promiscuous DNA-binding activity, can control phage VP882 functions and drive host V. cholerae QS. Moreover, we discover why V. cholerae VqmA_Vc cannot do the reverse, as its DNA binding is strictly constrained to the host V. cholerae genome.

Results

VqmA promoter-binding selectivity is conferred by the DNA-binding domain

VqmA proteins are composed of N-terminal Per-Arnt-Sim (PAS) domains responsible for binding the DPO AI and C-terminal DBDs containing HTH motifs [10]. Both VqmA_Vc and VqmA_Phage bind DPO. By contrast, with respect to DNA binding, VqmA_Phage binds to Pqtip and PvqmR, whereas VqmA_Vc only binds to PvqmR [5]. We reasoned that this asymmetric DNA-binding pattern arises from differences in the DBDs (S1 Fig). To test this idea, we constructed chimeras in which we exchanged the VqmA_Vc and VqmA_Phage C-terminal domains to produce _VcN-C_Phage and _PhageN-C_Vc proteins. We chose to make the junction at a residue near the C-terminal end of the PAS domain immediately following an amino acid stretch (GTIF) that is identical in both VqmA_Vc and VqmA_Phage (S1 Fig). We cloned vqmA_Vc, vqmA_Phage, _VcN-C_Phage, and _PhageN-C_Vc under an arabinose-inducible promoter and transformed each construct into recombinant Δtdh E. coli harboring a PvqmR-lux or a Pqtip-lux reporter. The Tdh enzyme is required for DPO biosynthesis, therefore a Δtdh E. coli strain makes no DPO [3]. Apo-VqmA displays basal transcriptional activity in vivo [9]. Thus, while DPO enhances VqmA DNA-binding activity, it is not an absolute requirement for binding. Using Δtdh E. coli for these studies ensured that any transcriptional activity that occurred was exclusively a consequence of the DNA-binding capabilities of the chimeras and not ligand-binding-driven transcriptional activation of the chimeras. Consistent with our hypothesis, promoter activation by each chimera was determined by the protein from which the DBD originated: All four versions of VqmA activated PvqmR-lux, whereas only VqmA_Phage and _VcN-C_Phage activated Pqtip-lux (Fig 1A and 1B, respectively). Next, we conjugated the four versions of VqmA into Δtdh ΔvqmA_Vc V. cholerae lysogenized by a phage VP882 mutant in which the endogenous vqmA_Phage was inactive (VP882 vqmA_Phage::Tn5). Thus, the only source of VqmA protein was that made from the plasmid. As expected, following arabinose-induction, only VqmA_Phage and _VcN-C_Phage activated qtip expression and induced host-cell lysis (Fig 1C).

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Fig 1. Promoter DNA-binding selectivity is conferred by the VqmA DBD.

(A and B) Normalized reporter activity from Δtdh E. coli harboring (A) PvqmR-lux or (B) Pqtip-lux and arabinose-inducible VqmA_Vc, VqmA_Phage, _VcN-C_Phage, or _PhageN-C_Vc. Black, no arabinose; white, 0.2% arabinose. Data are represented as mean ± SD (error bars) with n = 3 biological replicates. (C) Growth curves of the Δtdh ΔvqmA_Vc V. cholerae harboring phage VP882 vqmA_Phage::Tn5 and arabinose-inducible VqmA_Vc, VqmA_Phage, _VcN-C_Phage, or _PhageN-C_Vc in medium lacking (Control) or containing 0.2% arabinose (Induced). (D) EMSAs showing binding of VqmA proteins to PvqmR and Pqtip DNA. From left to right are, VqmA_Vc, VqmA_Phage, _PhageN-C_Vc, and _VcN-C_Phage. 25 nM PvqmR or Pqtip DNA was used in all EMSAs with no protein (designated -) or 2-fold serial dilutions of proteins. The lowest and highest protein (dimer) concentrations are 18.75 nM and 600 nM, respectively. (E and F) Normalized reporter activity from WT E. coli as in panels A and B harboring arabinose-inducible VqmA_Vc, GST-DBD_Vc, VqmA_Phage, and GST-DBD_Phage. (G) EMSAs showing binding of GST-DBD_Vc and GST-DBD_Phage to PvqmR and Pqtip DNA. Probe and protein concentrations as in panel D.

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We verified the above findings in vitro using electrophoretic mobility shift assays (EMSAs). Consistent with the cell-based assays, the purified VqmA_Vc, VqmA_Phage, _VcN-C_Phage, and _PhageN-C_Vc proteins shifted PvqmR DNA, whereas only the VqmA_Phage and _VcN-C_Phage proteins shifted Pqtip DNA (Fig 1D). Assessing the ratios of bound to total DNA across varying protein concentrations allowed us to calculate the relative binding affinities (EC₅₀) of the VqmA proteins for PvqmR and Pqtip DNA (S2A Fig). Our EMSA analyses show that _PhageN-C_Vc, like VqmA_Vc, only bound PvqmR, but with an estimated ~7-fold lower affinity. Consistent with our previous findings, VqmA_Phage bound Pqtip about 3-fold more strongly than it bound PvqmR [5]. By contrast, _VcN-C_Phage showed a modest increase in its preference for Pqtip relative to that for PvqmR, with binding to both promoters at a level similar to that with which VqmA_Phage bound Pqtip. Indeed, in agreement with our EC₅₀ measurements, when Pqtip and PvqmR DNA were supplied at equimolar concentrations in a competitive DNA-binding assay, lower amounts of VqmA_Phage and _VcN-C_Phage were required to shift Pqtip DNA than to shift PvqmR DNA (S2B Fig). In conclusion and in agreement with our in vivo results, the respective DBD of each purified VqmA protein drives promoter selectively.

We next assayed the VqmA_Vc and VqmA_Phage DBDs lacking their PAS domains (DBD_Vc and DBD_Phage, respectively) for activation of PvqmR-lux and Pqtip-lux. Deletion of the PAS domains resulted in inactive proteins as neither DBD activated transcription (S3A and S3B Fig, respectively), and likewise, EMSA analyses showed that neither DBD bound either promoter (S3C Fig). Gel filtration analyses indicated that the DBD proteins purified as monomers (S3D Fig), suggesting that the DBDs were unable to dimerize in the absence of their partner PAS domains. This result is consistent with previous findings that, in addition to sensing DPO, the VqmA_Vc PAS domain is responsible for dimerization [9,11].

Transcriptional activity driven by HTH-containing proteins typically depends on dimer formation. Soluble glutathione S-transferase (GST) spontaneously forms a homodimer [12], and so GST can be employed as a substitute for native dimerization domains of proteins [13]. Thus, to examine the VqmA requirement for dimerization, we fused GST to the N-terminus of each VqmA DBD to yield recombinant GST-DBD_Vc and GST-DBD_Phage and we tested whether DNA-binding function was restored. Indeed, the GST-DBD proteins purified as dimers (S3D Fig). PvqmR-lux and Pqtip-lux expression analyses revealed that the DBDs, when fused to GST, regained function, with the caveat that the GST-DBD_Vc exhibited 10-fold reduced activity compared to wild-type (WT) VqmA_Vc (Fig 1E). Importantly, the DNA-binding preferences mimicked those of the full-length proteins: GST-DBD_Phage activated both PvqmR-lux and Pqtip-lux, whereas GST-DBD_Vc only activated PvqmR-lux (Fig 1E and 1F). Companion EMSA analyses showed that GST-DBD_Phage bound Pqtip ~5-fold more strongly than it bound PvqmR, whereas GST-DBD_Vc showed almost no binding to PvqmR and, unexpectedly, some weak binding could be detected to the Pqtip DNA (Fig 1G). We confirmed that purified GST alone did not bind either PvqmR or Pqtip (S3E Fig). Given that the GST-DBD_Vc driven activation of Pqtip-lux was undetectable in vivo (Fig 1F), we presume that the observed in vitro GST-DBD_Vc binding to Pqtip DNA is a consequence of the simplified context in which the EMSA is performed. Likely, the DNA:VqmA ratio in the EMSA is far higher than in cells, which, in the case of GST-DBD_Vc, fosters modest non-specific DNA binding. Taken together, our results show that VqmA promoter-binding selectivity is conferred by the DBD, and that dimerization is necessary.

VqmA DNA-binding preferences can be inverted by exchanging key DNA sequences in PvqmR and Pqtip

To study the VqmA promoter-binding asymmetry from the aspect of the DNA, our next goal was to identify the critical DNA sequence within Pqtip that prevents VqmA_Vc from binding. In the phage VP882 genome, Pqtip resides between vqmA_Phage and qtip and VqmA_Phage activates its own and qtip expression, suggesting that VqmA_Phage binding may involve both DNA strands. Similarly, VqmA_Vc has been shown to interact with both strands of PvqmR [11]. Thus, in each case, both DNA strands need to be considered (Fig 2A). Previous work revealed that the critical region in PvqmR required for VqmA_Vc binding is -AGGGGGGATTTCCCCCCT- [2,11]. The corresponding fragment from Pqtip, but on the opposite DNA strand, -TAGGGGGAAAAATACCCT-, possesses ~56% sequence identity to this region suggesting it could be the key stretch of DNA that drives VqmA_Phage promoter selection. The highest divergence in the two promoters is in the central 6 nucleotides: “-AAAATA-” in Pqtip and “-TTTCCC-” in PvqmR. We synthesized DNA probes in which we exchanged the “-AAAATA-” in Pqtip with “-TTTCCC-” from PvqmR and tested VqmA_Vc and VqmA_Phage binding by EMSA analysis. We call these probes PvqmR* and Pqtip*, respectively. Indeed, promoter DNA-binding specificity was exchanged: VqmA_Vc shifted Pqtip*, whereas it only weakly shifted PvqmR* (Fig 2B). VqmA_Phage bound to PvqmR* twice as strongly as it bound to Pqtip*, showing the opposite preference for the two synthetic promoters compared to the native promoters (Fig 2B). PvqmR*-lux and Pqtip*-lux transcriptional fusions mimicked the EMSA results: VqmA_Vc only activated expression of Pqtip*-lux, whereas VqmA_Phage activated expression of PvqmR*-lux and Pqtip*-lux (Fig 2C and 2D). Thus, this 6-nucleotide stretch is the key sequence that determines the DNA-binding specificity for the two VqmA proteins. Moreover, the presence of the -AAAATA- nucleotide sequence in Pqtip is sufficient to prevent VqmA_Vc from activating transcription of Pqtip.

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Fig 2. Promoter selectivity is reversed by exchanging key nucleotide fragments.

(A) DNA sequence alignment (ClustalW) of PvqmR and Pqtip. The reverse strand of Pqtip is shown. Numbering indicates positions relative to the transcription start sites. Identical nucleotides are designated with black shading. The reported 18-bp DNA stretch in PvqmR required for VqmA_Vc to bind (2,11) and the corresponding region in Pqtip are highlighted in the red box. The GG-N₆-CC palindrome in PvqmR (2,11) and the recently identified GG-N₉-CC palindrome in Pqtip (15) are indicated above and below the red box, respectively. (B) EMSAs showing binding of the designated VqmA proteins to PvqmR* and Pqtip* DNA. The cartoon at the left illustrates the key sequences exchanged in the probes. Probe and protein concentrations as in Fig 1D. (C) Normalized reporter activity from Δtdh E. coli harboring PvqmR-lux or PvqmR*-lux and arabinose-inducible VqmA_Vc or VqmA_Phage. Black, no arabinose; white, 0.2% arabinose. Data are represented as mean ± SD (error bars) with n = 3 biological replicates. (D) As in C for Pqtip-lux or Pqtip*-lux.

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Protein sequence-guided mutagenesis reveals that residue E194 in phage VP882 VqmA_Phage and the equivalent A192 residue in V. cholerae VqmA_Vc contribute to specificity for Pqtip

We considered two possible mechanisms that could underpin the asymmetric VqmA DNA-binding patterns: phage VP882 VqmA_Phage could possess a feature that relaxes its DNA-binding specificity, and/or V. cholerae VqmA_Vc could possess a feature that restricts its DNA-binding ability. To distinguish between these possibilities, we first probed which residues drive VqmA_Phage interactions with Pqtip but do not contribute to interactions with PvqmR. To do this, we performed site-directed mutagenesis of VqmA_Phage with the goal of identifying mutants that fail to bind Pqtip but retain binding to PvqmR. Charged residues in HTH motifs typically mediate interactions between VqmA-type transcription factors and DNA, and indeed, both VqmA HTHs are enriched in positively-charged amino acids [9,11,14]. Sequence alignment of the HTHs in VqmA_Phage and VqmA_Vc revealed four obvious differences in charged residues that could underlie the DNA-binding asymmetry between the two proteins (S1 Fig). We mutated those residues in VqmA_Phage to the corresponding VqmA_Vc residues. The changes are: VqmA_Phage^K176Q, VqmA_Phage^R184I, VqmA_Phage^I193E, and VqmA_Phage^E194A. To test the combined effect of these mutations on VqmA_Phage DNA-binding function, we also constructed the quadruple VqmA_Phage^{K176Q, R184I, I193E, E194A} mutant. VqmA_Phage^K176Q, VqmA_Phage^R184I, VqmA_Phage^I193E retained the ability to induce phage lysis showing that in vivo binding to Pqtip was not eliminated (Fig 3A). VqmA_Phage^E194A induced only low-level cell lysis suggesting that, while binding to Pqtip is not eliminated, it is compromised (Fig 3A). Analysis of PvqmR-lux and Pqtip-lux expression revealed that all four VqmA_Phage single point mutants possessed levels of activity within 2-fold of that of WT PvqmR-lux. By contrast, they displayed ~2-7-fold reductions in Pqtip-lux activity, with VqmA_Phage^E194A being the least active (Fig 3B and 3C, respectively). The quadruple mutant was unable to induce phage lysis in a V. cholerae lysogen and it did not activate PvqmR-lux or Pqtip-lux expression showing it is defective in binding to both promoters (Fig 3A–3C). Western blot analysis demonstrated that all of the VqmA_Phage variants were produced at levels similar to WT in both V. cholerae and E. coli (S4A Fig). Thus, our results indicate that, among these charged residues, only the VqmA_Phage residue E194 in the HTH motif plays a role in VqmA_Phage selection of Pqtip.

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Fig 3. VqmA_Phage residue E194 and the corresponding VqmA_Vc residue A192 contribute to specificity for binding to Pqtip.

(A) Growth curves of Δtdh ΔvqmA_Vc V. cholerae harboring phage VP882 vqmA_Phage::Tn5 and the indicated 3xFLAG-VqmA_Phage alleles in medium lacking (Control) or containing 0.2% arabinose (Induced). (B and C) Normalized reporter activity from Δtdh E. coli harboring (B) PvqmR-lux or (C) Pqtip-lux and the indicated arabinose-inducible 3xFLAG-VqmA_Phage alleles. Black, no arabinose; white, 0.2% arabinose. Data are represented as mean ± SD (error bars) with n = 3 biological replicates. (D) Growth curves of Δtdh ΔvqmA_Vc V. cholerae harboring phage VP882 vqmA_Phage::Tn5 and the indicated 3xFLAG-VqmA_Vc alleles in medium lacking (Control) or containing 0.2% arabinose (Induced). (E and F) Normalized reporter activity from Δtdh E. coli harboring (E) PvqmR-lux or (F) Pqtip-lux and the indicated arabinose-inducible 3xFLAG-VqmA_Vc alleles. Black, no arabinose; white, 0.2% arabinose. Data are represented as mean ± SD (error bars) with n = 3 biological replicates. ns = not significant, ****P <0.0001, ***P <0.0005, **P <0.005, *P <0.05 in one-way ANOVA compared to WT VqmA proteins.

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While the residues we mutated in the phage VP882 VqmA_Phage HTH motif do not dramatically perturb site-specific recognition of Pqtip, the corresponding residues in the V. cholerae VqmA_Vc HTH motif could nonetheless restrict its capacity to bind Pqtip. Therefore, we also mutated the analogous VqmA_Vc residues to the corresponding VqmA_Phage residues. We made: VqmA_Vc^Q174K, VqmA_Vc^I182R, VqmA_Vc^E191I, VqmA_Vc^A192E_, and VqmA_Vc^{Q174K, I182R, E191I, A192E}. Here, our goal was to test whether the variants gained the ability to bind Pqtip. Only VqmA_Vc^A192E induced a modest level of lysis in the V. cholerae lysogen, whereas all other VqmA_Vc variants failed to do so (Fig 3D). All of the VqmA_Vc variants drove the WT level of PvqmR-lux activity (Fig 3E). VqmA_Vc^A192E generated low but detectable Pqtip-lux expression, while the other VqmA_Vc variants did not (Fig 3F). The VqmA_Vc variants were produced at similar levels to WT VqmA_Vc in V. cholerae and E. coli (S4B Fig). We conclude that, among the tested residues, only A192 plays a role in preventing VqmA_Vc from binding Pqtip.

Our mutagenesis analyses for VqmA_Vc are consistent with our analyses for VqmA_Phage: The residue at the 192^nd position in V. cholerae VqmA_Vc and the analogous residue at the 194^th position in phage VP882 VqmA_Phage contribute to selection of Pqtip. However, given that the A192E substitution in VqmA_Vc results in only partial activation of Pqtip expression, and the E194A substitution in VqmA_Phage results in only partial loss of activation of Pqtip, the E194 residue in VqmA_Phage cannot be the sole amino acid responsible for the preference VqmA_Phage shows for Pqtip. Rather, additional residues in VqmA_Phage must participate in conferring specificity.

Random mutagenesis of the VqmA_Phage DBD reveals that residues G201, A202, E207, and M211 are required for VqmA_Phage to bind Pqtip but are dispensable for binding PvqmR

Our protein sequence-guided approach did not reveal the primary mechanism underlying promoter-binding specificity for either of the VqmA proteins. We therefore performed a genetic screen to forward our goal of identifying phage VP882 VqmA_Phage mutants that fail to bind Pqtip but retain the ability to bind PvqmR. We constructed a library of random mutations in the region of vqmA_Phage encoding the DBD in the context of the full-length gene, cloned them into a plasmid under an arabinose-inducible promoter, and introduced them into Δtdh ΔvqmA_Vc V. cholerae harboring PvqmR-lux on the chromosome and lysogenized by phage VP882 harboring inactive vqmA_Phage (vqmA_Phage::Tn5). The logic of the screen is as follows: When propagated on agar plates supplemented with arabinose, V. cholerae exconjugants harboring vqmA_Phage alleles possessing reasonable Pqtip-binding activity will lyse because those VqmA_Phage proteins will bind Pqtip on the phage VP882 genome and launch the phage lytic cascade (S5 Fig). Such exconjugants will die and thus be eliminated from the screen. Exconjugants that survive but carry vqmA_Phage null alleles will produce no light because those VqmA_Phage proteins will fail to bind PvqmR-lux, so they also can be eliminated from the screen. The vqmA_Phage alleles of interest to us are those that are maintained in surviving exconjugants (because they encode proteins that cannot bind Pqtip) and produce light (because they encode proteins that can bind PvqmR-lux).

Our screen yielded the following mutants: VqmA_Phage^G201D, VqmA_Phage^G201R, VqmA_Phage^A202V, VqmA_Phage^E207K, VqmA_Phage^E207V, and VqmA_Phage^M211K (Fig 4A). To verify that these VqmA_Phage mutants were indeed defective in binding Pqtip, we individually transformed them into Δtdh E. coli carrying the Pqtip-lux reporter or the PvqmR-lux reporter and measured light production. All variants retained WT capability to activate PvqmR-lux, but they did not harbor WT capability to activate Pqtip-lux expression (>10-fold reductions in activity) (Fig 4B and 4C, respectively). Thus, any residual Pqtip binding by these mutant VqmA_Phage proteins is insufficient to induce host-cell lysis in the phage VP882 lysogen (Fig 4A). We verified that the VqmA_Phage variants are produced at the same level as WT VqmA_Phage in V. cholerae and E. coli (S4C Fig). According to the protein sequence alignment, VqmA_Phage residues (175–200) corresponding to positions 173–198 in VqmA_Vc comprise the VqmA_Phage HTH motif (S1 Fig). Thus, the residues identified in the mutagenesis (G201, A202, E207, and M211) are located C-terminal to the VqmA_Phage HTH motif. Mapping the analogous V. cholerae VqmA_Vc residues (G199, A200, Q205, and L209) to the DPO-VqmA_Vc-PvqmR structure (there is no DPO-VqmA_Phage-Pqtip structure) also shows that all of these residues cluster in a flexible loop region and helix adjacent to, but distinct from the HTH motif that directly contacts DNA (Figs 4D and S1). Surprisingly, the residues identified in the VqmA_Phage mutagenesis are either identical (VqmA_Phage G201 and A202 versus VqmA_Vc G199 and A200) or similar (VqmA_Phage E207 and M211 versus VqmA_Vc Q205 and L209) between VqmA_Phage and VqmA_Vc. To test whether possession of the similar residues is sufficient to confer DNA-binding specificity for Pqtip, we constructed VqmA_Vc^Q205E and VqmA_Vc^L209M and tested their DNA-binding functions as above. VqmA_Vc^Q205E and VqmA_Vc^L209M, like WT VqmA_Vc, activated PvqmR-lux but failed to activate Pqtip-lux (S6A and S6B Fig, respectively). We make the following four conclusions from these findings: 1) There are at least four residues (G201, A202, E207, and M211) required for VqmA_Phage to recognize Pqtip DNA. 2) Because the VqmA_Phage G201D, G201R, A202V, E207K, E207V, and M211K variants exhibit WT binding to PvqmR, the substitutions at these four residues must not significantly affect PvqmR recognition. 3) Because these residues are conserved or similar between VqmA_Phage and VqmA_Vc, one would expect VqmA_Vc to have the capacity to bind Pqtip. 4) However, because VqmA_Vc in fact does not bind Pqtip, VqmA_Vc likely possesses an additional feature that resides elsewhere in the protein that prevents Pqtip binding from occurring.

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Fig 4. VqmA_Phage residues G201, A202, E207, and M211 are required for binding to Pqtip.

(A) Growth curves of Δtdh ΔvqmA_Vc V. cholerae harboring phage VP882 vqmA_Phage::Tn5 and the indicated 3xFLAG-VqmA_Phage alleles in medium lacking (Control) or containing 0.2% arabinose (Induced). (B and C) Normalized reporter activity from Δtdh E. coli harboring (B) PvqmR-lux or (C) Pqtip-lux and the indicated arabinose-inducible 3xFLAG-VqmA_Phage alleles. Black, no arabinose; white 0.2% arabinose. Data are represented as mean ± SD (error bars) with n = 3 biological replicates. (D) Close up views of the DBD from the crystal structure of DPO-VqmA_Vc bound to PvqmR (PDB: 6ide, protein in gray with the HTH motif in yellow, and the DNA in orange). The color scheme for VqmA_Vc residues G199, A200, Q205, and L209 mirrors that used in panel A.

https://doi.org/10.1371/journal.pgen.1009550.g004

The restrictive element that prevents VqmA_Vc from binding Pqtip is located in its HTH motif and the adjacent C-terminal region of 25 residues

To test the hypothesis that a feature in the VqmA_Vc DBD restricts its DNA-binding capacity to PvqmR, we performed a genetic screen aimed at identifying VqmA_Vc mutants capable of activating Pqtip-lux expression. To do this, we constructed a library of random vqmA_Vc DBD alleles containing, on average, 1–2 substitutions, and we cloned them into a plasmid under an arabinose-inducible promoter. The library was transformed into the Δtdh E. coli strain harboring the Pqtip-lux reporter and transformants were propagated on plates containing arabinose. We screened ~10,000 transformants for colonies that produced light indicating that they contained VqmA_Vc proteins that activated Pqtip-lux. This strategy yielded no such transformants. Several possibilities could explain our result: We did not screen sufficient numbers of mutants, the mutagenesis did not yield the crucial change, or no alteration of a single residue can enable VqmA_Vc binding to Pqtip.

We expanded our search for the DNA-binding restrictive element present in VqmA_Vc by assessing whether a particular region in the VqmA_Vc DBD constrains promoter binding to PvqmR. To do this, we constructed five VqmA_Vc mosaic proteins by replacing ~20–30 residues in the V. cholerae VqmA_Vc DBD with the corresponding residues from the phage VP882 VqmA_Phage DBD. We call these proteins VqmA_Vc*^126–149, VqmA_Vc*^150–170, VqmA_Vc*^171–199, VqmA_Vc*^200–224, and VqmA_Vc*^225–246 (see S1 Fig for relevant protein segments). Each superscript denotes the VqmA_Vc amino acid residues that have been replaced by the corresponding residues from VqmA_Phage. In all the mosaics, either the intact VqmA_Vc HTH or the intact VqmA_Phage HTH was present. For reference, the VqmA_Vc HTH motif consists of residues 173 to 198 and the VqmA_Phage HTH spans residues 175 to 200. We tested the mosaic VqmA_Vc proteins for activation of the PvqmR-lux and Pqtip-lux reporters. The DNA specificity of all the VqmA_Vc mosaics mimicked WT VqmA_Vc as PvqmR-lux was expressed but Pqtip-lux was not (Fig 5A and 5B, respectively). We confirmed that the mosaic VqmA_Vc proteins are expressed at levels similar to WT VqmA_Vc (S7 Fig). Our results suggest that the feature that prevents V. cholerae VqmA_Vc from binding to Pqtip is larger than the regions delineated by any of the VqmA_Vc mosaics, or it could be that multiple patches in the VqmA_Vc DBD that are not contiguous in amino acid sequence are responsible.

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Fig 5. The VqmA_Vc HTH motif and the immediate C-terminal 25 residues, together, constrain binding to PvqmR.

(A-D) Normalized reporter activity from Δtdh E. coli harboring (A and C) PvqmR-lux or (B and D) Pqtip-lux and arabinose-inducible VqmA_Vc, VqmA_Phage, or the indicated VqmA_Vc allele. Data are represented as mean ± SD (error bars) with n = 3 biological replicates. Black, no arabinose; white, 0.2% arabinose. (E) Growth curves of Δtdh ΔvqmA_Vc V. cholerae harboring phage VP882 vqmA_Phage::Tn5 and VqmA_Vc, VqmA_Phage, or VqmA_Vc*^171–224 in medium lacking (Control) or containing 0.2% arabinose (Induced).

https://doi.org/10.1371/journal.pgen.1009550.g005

Pinpointing non-contiguous regions that could, together, contain the VqmA_Vc restrictive element is challenging. However, testing for a larger contiguous expanse that could contain the putative restrictive element is straightforward. Thus, we constructed two additional V. cholerae VqmA_Vc mosaic proteins. In one construct, called VqmA_Vc*^150–199, we introduced the VqmA_Phage HTH along with the immediate N-terminal 25 amino acids in place of the corresponding VqmA_Vc region. Second, in a construct called VqmA_Vc*^171–224, we introduced the VqmA_Phage HTH together with the immediate C-terminal 25 amino acid stretch in place of that VqmA_Vc region. VqmA_Vc*^150–199 and VqmA_Vc*^171–224 activated PvqmR-lux to approximately WT levels, whereas only VqmA_Vc*^171–224 activated Pqtip-lux, albeit weakly (Fig 5C and 5D, respectively). Consistent with this result, VqmA_Vc*^171–224 induced partial lysis in the V. cholerae phage VP882 lysogen (Fig 5E). VqmA_Vc*^171–224 was produced at levels similar to WT VqmA_Vc, eliminating the possibility that the observed binding to Pqtip was a consequence of overexpression (S7 Fig). We conclude that the region encompassing both the HTH motif and the C-terminal 25 residues are required to restrict the VqmA_Vc DBD from binding Pqtip.

Discussion

The DPO-VqmA QS AI-receptor pair controls lifestyle transitions in the pathogen V. cholerae and in the vibriophage VP882. Here, we studied the DNA-binding function of VqmA. VqmA proteins are cytoplasmic transcription factors composed of N-terminal PAS domains responsible for binding the DPO ligand and C-terminal DBDs containing HTH motifs. Most of the key residues required for binding the DPO ligand and for binding to PvqmR DNA are conserved between the two VqmA proteins. Indeed, both VqmA_Vc and VqmA_Phage bind DPO and activate transcription of vqmR. By contrast, only VqmA_Phage activates the phage gene qtip. Here, we investigated this asymmetric DNA-binding pattern. Our work shows that, in both proteins, the DBD determines promoter recognition. We have previously shown that DPO binding enhances VqmA transcriptional activity [9]. This earlier work, together with our present results, suggest a model in which the PAS domain specifies DNA-binding affinity (between the apo- and holo- states), and the DBD specifies DNA-binding selectivity.

The main goal of the present work was to discover features of the VqmA proteins that confer specificity in transcriptional activity. We propose that phage VP882 VqmA_Phage possesses a feature that relaxes its DNA-binding specificity and V. cholerae VqmA_Vc possesses a feature that restricts its DNA-binding capability. Regarding VqmA_Vc, our genetic analyses support the hypothesis that the VqmA_Vc DBD harbors elements that prevent it from binding Pqtip. This hypothesis stems from our finding that residues G201, A202, E207, and M211 are crucial for VqmA_Phage recognition of Pqtip. These residues are conserved between VqmA_Vc and VqmA_Phage. Specifically, in VqmA_Vc they are: G199, A200, Q205, and L209, respectively. More broadly, sequence alignments of VqmA proteins among Vibrios reveal that the residue at the 207^th position in VqmA_Phage (205^th position in VqmA_Vc) is most frequently either a Glu or a Gln [5]. Similarly, the residue at the 211^th position in VqmA_Phage (209^th position in VqmA_Vc) is commonly a hydrophobic residue, like Met, Leu, Ile, or Val. Thus, E207 and M211 are not unique to VqmA_Phage, but rather occur in most VqmA proteins. We propose that because the key residues for Pqtip binding are conserved in VqmA_Phage, VqmA_Vc, and other Vibrio VqmA proteins, VqmA_Vc is likely restricted from binding Pqtip by additional features elsewhere in its DBD. Regarding VqmA_Phage, the DPO-VqmA_Phage structure was reported during review of this manuscript [15]. Superimposition of this new structure (7DWM) onto the DPO-VqmA_Vc and DPO-VqmA_Vc-PvqmR structures (6KJU and 6IDE, respectively, and [9,11,14]) reveals two insights (S8 Fig). First, the conformations of the three PAS domains are similar except for the orientations of the first 20 N-terminal residues in each protein, indicating that the PAS domains do not confer the differences in promoter DNA specificity. Second, the DPO-VqmA_Phage DBDs adopt a conformation that is intermediate between that of the more open DBDs in the DPO-VqmA_Vc structure and the closed DBDs in the DPO-VqmA_Vc-PvqmR structure. Additionally, the interaction interface between the VqmA_Phage DBDs is less extensive, and thus more relaxed than that of the VqmA_Vc DBDs [15]. Likely, the more relaxed conformation exhibited by the VqmA_Phage DBDs underpins its promiscuity for promoter binding with respect to PvqmR and Pqtip.

In the case of VqmA_Phage, the residues G201, A202, E207, and M211 identified in our mutagenesis screen as necessary for Pqtip binding are, surprisingly, not in the HTH motif, nor do the corresponding VqmA_Vc residues make direct contacts with DNA in the DPO-VqmA_Vc-PvqmR crystal structure (Fig 4D). Thus, we wonder how the G201, A202, E207, and M211 residues could govern recognition of Pqtip. Our in vivo analyses showed that substitutions in VqmA_Phage at these residues enable activation of vqmR expression to WT levels, whereas only residual activation of qtip expression occurs (Fig 4A–4C). Surprisingly, the purified VqmA_Phage mutant proteins maintained some capability to bind Pqtip in vitro. A representative experiment using the VqmA_Phage^G201D protein is shown in S9A Fig.

We consider several possibilities to explain our findings:

First, the VqmA_Phage G201, A202, E207, and M211 residues could mediate interactions with an additional bacterial factor involved in transcription. Importantly, the failure of these VqmA_Phage variants to activate Pqtip expression in V. cholerae lysogens also occurred in E. coli, eliminating the possibility that these residues interact with a phage-specific or Vibrio-specific factor. Rather, these residues could be important for coordinating interactions with a conserved factor, such as RNA polymerase. If so, these mutant VqmA_Phage proteins, while capable of binding promoter DNA, are incapable of activating transcription. This situation would be analogous to the positive control mutants of the lambda phage cI repressor (cI_lambda). So called pc mutants bind DNA and exhibit repressor activity, but are deficient in positive transcriptional regulation due to the inability of the mutant cI_lambda proteins to productively interact with RNA polymerase [16,17]. In our case, the VqmA_Phage mutants maintain the capacity to activate vqmR expression so they must successfully interact with RNA polymerase at least at PvqmR. For this reason, we consider it unlikely that these VqmA_Phage mutants are analogous to lambda pc mutants.

Second, a global transcriptional regulator could be involved that is present in both V. cholerae and E. coli. One candidate is the histone-like nucleoid structuring protein (H-NS) that functions as a universal repressor of transcription [18]. In Vibrio harveyi, the QS master regulator, LuxR, displaces H-NS at promoter DNA to activate expression of QS-controlled genes [19]. Perhaps, the VqmA_Phage G201, A202, E207, and M211 mutants cannot successfully compete with H-NS for binding at Pqtip in vivo, whereas in an EMSA assay, since H-NS is not present, binding to Pqtip DNA occurs. To address this possibility, we examined whether WT VqmA_Phage and VqmA_Phage^G201D competed with H-NS for binding to Pqtip using EMSA assays. There was no difference between WT VqmA_Phage and VqmA_Phage^G201D binding to Pqtip DNA in the presence of purified H-NS (S9C and S9D Fig). These experiments suggest that it is unlikely that H-NS competition underlies our findings.

Third, the binding of the VqmA_Phage G201, A202, E207, and M211 mutants to Pqtip in vitro, while demonstrating loss of activity in vivo, could be a consequence of the unnaturally high DNA: VqmA_Phage stoichiometry in the EMSA, similar to what we observed for the GST-DBD_Vc construct (Fig 1G). Thus, the EMSA is not sufficiently sensitive to distinguish between the strength of DNA binding of WT VqmA_Phage and the residual binding by the VqmA_Phage G201, A202, E207, and M211 mutants. If this is the case, we propose that VqmA_Phage G201, A202, E207, and M211 could play allosteric roles in correctly positioning the VqmA_Phage HTH for proper contact with particular DNA nucleotides. Here, we compare this possibility to how site-specific recognition is accomplished by cI_lambda. Genetic and biochemical studies revealed that residues outside of the cI_lambda HTH motif are crucial for site-specific DNA recognition [20–24]. The crystal structure of the cI_lambda repressor bound to DNA shows that charged residues adjacent to those in the HTH interact with the DNA sugar phosphate backbone [25]. Additionally, the N-terminal arm of cI_lambda wraps around the DNA and makes contacts on the backside of the helix [25]. It is presumed that the backbone contacts function to position the HTH residues to contact specific DNA nucleotides. Thus, while the VqmA_Phage residues that we identified as important for Pqtip recognition (G201, A202, E207, and M211) do not function perfectly analogously to those in cI_lambda because they do not make contact with the DNA backbone, their role in site-specific recognition could be similar. A caveat of our interpretation is that, as noted, we do not have a structure of VqmA_Phage bound to Pqtip and we mapped the residues identified in our VqmA_Phage mutagenesis to the DPO-VqmA_Vc-PvqmR crystal structure. Therefore, it remains possible that the residues we identified here do indeed make contacts with DNA. A further possibility is that the residues we identified foster increased plasticity to the VqmA_Phage DBDs, perhaps, allowing VqmA_Phage to bind the longer palindrome that exists in Pqtip, which we discuss below. The recently reported DPO-VqmA_Phage crystal structure [15], together with the existing DPO-VqmA_Vc structures, could enable modeling to predict the roles played by particular residues in conferring a relaxed conformation to the VqmA_Phage DBDs. To our knowledge, no region analogous to the one we discovered in VqmA_Phage has been shown to confer promoter specificity to a transcription factor. Going forward, determining the structure of VqmA_Phage bound to Pqtip DNA should reveal the mechanism enabling recognition of Pqtip and the role that these residues play, individually and collectively, in determining DNA-binding specificity.

Previous work demonstrated that VqmA_Vc recognizes a key GG-N₆-CC palindrome in PvqmR [2,11]. Our sequence alignment of PvqmR and Pqtip showed that Pqtip does not possess this palindrome. Rather, the corresponding sequence in Pqtip is GG-N₆-TA (Fig 2A). The most obvious divergence between the two sequences is in the central six nucleotides: “-AAAATA-” in Pqtip and “-TTTCCC-” in PvqmR (Fig 2A). We hypothesized that this nucleotide stretch could be responsible for conferring the asymmetric DNA-binding patterns to the two VqmA proteins. Indeed, exchanging these nucleotides in Pqtip and PvqmR reversed the promoter binding preferences of the VqmA proteins. We verified our conclusion that this core 6 nucleotide stretch drives VqmA DNA-binding preference using our VqmA chimeric proteins (_VcN-C_Phage and _PhageN-C_Vc), a representative mosaic protein (VqmA_Vc*^171–224), and a representative protein containing a point mutation (VqmA_Phage^G201D) (S9A, S9B, and S10 Figs). While the present manuscript was under review, Gu et al. reported that a GG-N₉-CC palindrome in Pqtip is the key sequence for VqmA_Phage recognition [15]. According to our DNA sequence alignment, the GG-N₆-CC palindrome required for VqmA_Vc binding is only present in PvqmR, while the key GG-N₉-CC palindrome required for VqmA_Phage binding exists in both Pqtip and PvqmR (Fig 2A). Together, our results and those of Gu et. al. [15] explain, at the level of the promoter DNA, why VqmA_Phage binds both Pqtip and PvqmR while VqmA_Vc recognizes only PvqmR.

Genomic sequencing data have revealed the presence of many QS receptor-transcription factors encoded in phage genomes [26]. In general, however, their transcriptional outputs are uncharacterized, with the exception of VqmA_Phage, which is promiscuous with respect to binding to PvqmR and Pqtip, the only two promoters tested to our knowledge. It remains possible that VqmA_Phage regulates additional genes specifying bacterial and or/phage functions. Given that VqmA_Phage can regulate biofilm formation through its control of V. cholerae vqmR, probing the host regulon controlled by VqmA_Phage under various growth conditions could reveal unanticipated roles of QS in phage-Vibrio interactions.

Finally, we found that the VqmA_Vc^A192E variant exhibited modest, but detectable binding to Pqtip, whereas the VqmA_Vc quadruple mutant, and the VqmA_Vc*^171–199 mosaic protein did not. Western blot and PvqmR-lux assays eliminated the possibility that any of the mutant proteins were not expressed or were misfolded. Rather, we infer that a particular regional conformation in the VqmA proteins is required for this key residue to function properly. Our results also show that exchanging both the VqmA_Vc HTH motif and C-terminal 25 residues with the corresponding residues from VqmA_Phage enables some but not WT-level binding to Pqtip. This finding supports the notion that a set of non-contiguous amino acids or a particular conformation of the VqmA_Vc DBD prevents binding to Pqtip. This arrangement is perhaps not surprising given that V. cholerae would pay a significant penalty if VqmA_Vc bound the phage VP882 qtip promoter, as the consequence would be the launch of the phage lytic program and death of the host cell. To our knowledge, VqmA_Vc binds to only one promoter, PvqmR [3]. Thus, even in the context of the V. cholerae genome, VqmA_Vc transcriptional activity is tightly constrained. It is possible that other negative ramifications stem from non-specific VqmA_Vc binding in the V. cholerae genome. Distinct mechanisms are employed to restrict other QS receptor/transcription factors from promiscuously binding to DNA. For example, LuxR-type QS receptors can typically bind >100 promoters, but their solubilization, stability, and DNA-binding capabilities strictly rely on being bound to an AI whose availability is, in turn, highly regulated [27–31]. Therefore, precise control of gene expression is maintained in many QS circuits by confining QS receptor activity to the ligand-bound form coupled with discrete affinities of the ligand-receptor complexes for target promoters. By contrast, VqmA_Vc is expressed constitutively, and its DNA-binding capabilities are not limited by the presence of an AI. Thus, exquisitely tight control over promoter DNA-binding specificity by VqmA_Vc—restricting it to one and only one promoter—is apparently crucial for proper regulation of gene expression and survival.

Materials and methods

Bacterial strains, plasmids, primers, and reagents

Strains, plasmids, primers, and gBlocks used in this study are listed in S1–S4 Tables, respectively. In all experiments, Δtdh V. cholerae and Δtdh E. coli strains were used except in the experiment assaying expression of PvqmR-lux and Pqtip-lux in response to the DBD_Vc, DBD_Phage, GST-DBD_Vc, and GST-DBD_Phage proteins. In that case, the E. coli strain contained the WT tdh gene. V. cholerae and E. coli were grown aerobically in lysogeny broth (LB) at 37°C. Antibiotics and inducers were used at the following concentrations: 50 units mL^-1 polymyxin B, 200 μg mL^-1 ampicillin, 5 μg mL^-1 chloramphenicol, 100 μg mL^-1 kanamycin, 0.2% arabinose, and 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG).

Primers were obtained from Integrated DNA Technologies. Gibson assembly, intramolecular reclosure, and traditional cloning methods were employed for all cloning. PCR with Q5 High Fidelity Polymerase (NEB) was used to generate insert and backbone DNA. Gibson assembly relied on HiFi DNA assembly mix (NEB). All enzymes used in cloning were obtained from NEB. Mutageneses of the VqmA_Phage and VqmA_Vc DBDs were accomplished using the GeneMorph II EZClone Domain Mutagenesis Kit (Agilent) according to the manufacturer’s instructions. Transfer of plasmids carrying vqmA genes into the V. cholerae phage VP882 lysogen employed conjugation followed by selective plating on polymyxin B, chloramphenicol, and kanamycin, based on previously described protocols [32].

Genetic screens for VqmA_Phage and VqmA_Vc DNA-binding mutants

E. coli carrying a library of plasmid-borne vqmA_Phage mutants was mated with V. cholerae harboring a phage VP882 mutant (vqmA_Phage::Tn5) and the PvqmR-lux reporter integrated at the lacZ locus. Exconjugant V. cholerae colonies were collected and streaked onto LB agar plates supplemented with polymyxin B, chloramphenicol, kanamycin, and arabinose. PvqmR-lux activity of surviving exconjugants was assayed using an ImageQuant LAS4000 imager (GE). V. cholerae colonies that produced light were harvested for plasmid DNA preparation. Isolated plasmid DNA was subsequently transformed into E. coli strains carrying Pqtip-lux or PvqmR-lux to validate activity.

A library of plasmid-borne vqmA_Vc mutants was transformed into E. coli carrying the Pqtip-lux reporter. Transformants were plated on LB agar supplemented with ampicillin, kanamycin, and arabinose. Pqtip-lux activity was assayed using an ImageQuant LAS4000 imager.

Growth, lysis, and bioluminescence assays

To measure growth of V. cholerae phage VP882 lysogens or activation of the PvqmR-lux and Pqtip-lux reporters in bacterial strains, overnight cultures of V. cholerae or E. coli were back-diluted 1:1000 into LB medium supplemented with appropriate antibiotics prior to being dispensed (200 μL) into 96-well plates (Corning Costar 3904). Arabinose was added as specified. The plates were shaken at 37°C and a Biotek Synergy Neo2 Multi-Mode reader was used to measure OD₆₀₀ and bioluminescence. For bioluminescence assays, relative light units (RLU) were calculated by dividing bioluminescence by the OD₆₀₀ after 5 h.

Protein expression, purification, and electrophoretic mobility shift assay (EMSA)

Protein expression and purification were performed as described [9,19]. EMSAs were performed as described [8] with the following modifications: Following electrophoresis, 6% DNA retardation gels were stained with SYBR Green (Thermo) and visualized using an ImageQuant LAS 4000 imager with the SYBR Green settings. Unless specified otherwise, the highest concentration of VqmA assessed was 600 nM. 25 nM PvqmR or Pqtip DNA was used in all EMSAs. The percentage of promoter DNA bound was calculated using the gel analyzer tool in ImageJ and the estimated EC₅₀ values were derived from EC₅₀ analyses in Prism.

Western blot analysis

Western blot analyses probing for abundances of 3xFLAG-tagged proteins were performed as reported [3] with the following modifications: E. coli and V. cholerae carrying N-terminal 3xFLAG-tagged VqmA_Vc and N-terminal 3xFLAG-tagged VqmA_Phage alleles were back-diluted 1:1000 in LB supplemented with appropriate antibiotics and harvested after 6 h and 4 h of growth at 37°C, respectively. Cells were resuspended in Laemmli sample buffer at a final concentration of 0.006 OD/μL. Following denaturation for 15 min at 95°C, 5 μL of each sample was subjected to SDS-PAGE gel electrophoresis. RpoA was used as the loading control (Biolegend Inc.). Signals were visualized using an ImageQuant LAS 4000 imager.

Sequence alignments

Protein and DNA sequences in FASTA format were aligned in the BioEdit Sequence Alignment Editor using the default setting under the ClustalW mode. Figs 2A and S1 were prepared via the ESPript 3.0 online server [33].

Statistical methods

All statistical analyses were performed using GraphPad Prism software. Error bars correspond to standard deviations of the means of three biological replicates.

Supporting information

S1 Fig. Sequence alignment of VqmA proteins.

Protein sequence alignment (ClustalW) showing VqmA_Vc and VqmA_Phage. Black and white boxes designate identical and conserved residues, respectively. The PAS domain and HTH motif are indicated. The site used to fuse domains for chimera constructions is indicated by the red box. Key residues required for DPO binding are designated with black triangles. Conserved HTH residues are designated by black circles and open circles show residues with different charges in the HTH motifs of the two proteins. The residue in each HTH motif that contributes to Pqtip specificity is designated by the striped circle. The residues identified in the VqmA_Phage screen and the equivalent residues altered by site-directed mutagenesis in VqmA_Vc are designated by asterisks.

https://doi.org/10.1371/journal.pgen.1009550.s001

(TIF)

S2 Fig. VqmA_Phage has higher affinity for Pqtip DNA than for PvqmR DNA.

(A) EC₅₀ analysis of the designated VqmA proteins for binding to PvqmR and Pqtip. Data are representative of two independent experiments. The percentage of DNA bound was calculated using the gel analyzer tool in ImageJ and the estimated EC₅₀ values were derived from Prism. (B) Competitive VqmA_Phage and _VcN-C_Phage EMSA analysis. 25 nM PvqmR and Pqtip DNA were used and no protein (designated -) or 2-fold serially-diluted protein was added to the lanes. The lowest and highest protein (dimer) concentrations are 4.7 nM and 1200 nM, respectively.

https://doi.org/10.1371/journal.pgen.1009550.s002

(TIF)

S3 Fig. The VqmA_Vc and VqmA_Phage DBDs are non-functional.

(A and B) Normalized reporter activity from WT E. coli harboring (A) PvqmR-lux or (B) Pqtip-lux and arabinose-inducible VqmA_Vc, DBD_Vc, VqmA_Phage, and DBD_Phage. Black, no arabinose; white, 0.2% arabinose. Data are represented as mean ± SD (error bars) with n = 3 biological replicates. (C) EMSAs of DBD_Vc and DBD_Phage proteins binding to PvqmR and Pqtip. 25 nM PvqmR or Pqtip DNA was used in all EMSAs with no protein (designated -) or 2-fold serial dilutions of proteins. The lowest and highest protein (dimer) concentrations are 18.75 nM and 600 nM, respectively. (D) Gel filtration chromatogram showing UV₂₈₀ traces for the purification of (left) VqmA_Vc, DBD_Vc, and GST-DBD_Vc and (right) VqmA_Phage, DBD_Phage, and GST-DBD_Phage proteins. (E) EMSA of GST protein binding to PvqmR and Pqtip DNA as in panel C.

https://doi.org/10.1371/journal.pgen.1009550.s003

(TIF)

S4 Fig. The VqmA_Phage and VqmA_Vc variants are produced at levels similar to WT.

Western blot showing the designated (A and C) 3xFLAG-VqmA_Phage and (B) 3xFLAG-VqmA_Vc proteins produced by Δtdh E. coli and Δtdh ΔvqmA_Vc V. cholerae. A contaminating band below VqmA_Phage and VqmA_Vc is present in all Δtdh E. coli samples. The RNAPα subunit (RpoA) was used as the loading control. Data are representative of two independent experiments.

https://doi.org/10.1371/journal.pgen.1009550.s004

(TIF)

S5 Fig. VqmA_Phage mutants possessing WT activity induce phage lysis on agar plates supplemented with 0.2% arabinose.

Shown is growth of Δtdh ΔvqmA_Vc V. cholerae harboring phage VP882 vqmA_Phage::Tn5 as a lysogen and arabinose-inducible 3xFLAG-VqmA_Phage streaked onto agar plates with no arabinose (Control) or 0.2% arabinose (Induced).

https://doi.org/10.1371/journal.pgen.1009550.s005

(TIF)

S6 Fig. VqmA_Vc^Q205E and VqmA_Vc^L209M do not bind Pqtip.

(A and B) Normalized reporter activity from Δtdh E. coli harboring (A) PvqmR-lux or (B) Pqtip-lux and arabinose-inducible 3xFLAG-VqmA_Vc, 3xFLAG-VqmA_Phage, or the indicated 3xFLAG-VqmA_Vc allele. Black, no arabinose; white, 0.2% arabinose. Data are represented as mean ± SD (error bars) with n = 3 biological replicates.

https://doi.org/10.1371/journal.pgen.1009550.s006

(TIF)

S7 Fig. VqmA_Vc mosaic proteins are produced at levels similar to WT VqmA_Vc.

Western blot showing the designated 3xFLAG-VqmA_Vc mosaic proteins produced by Δtdh E. coli and Δtdh ΔvqmA_Vc V. cholerae. RpoA was used as the loading control. Data are representative of two independent experiments.

https://doi.org/10.1371/journal.pgen.1009550.s007

(TIF)

S8 Fig. Structural comparisons of the VqmA_Phage and VqmA_Vc proteins.

Previously reported crystal structures of DPO-VqmA_Vc-PvqmR (blue, PDB: 6IDE) and DPO-VqmA_Vc (green, PDB: 6KJU) superimposed onto the recently published crystal structure of DPO-VqmA_Phage (yellow, PDB: 7DWM) based on the orientations of the PAS domains. DNA in the DPO-VqmA_Vc-PvqmR structure was omitted for simplicity.

https://doi.org/10.1371/journal.pgen.1009550.s008

(TIF)

S9 Fig. EMSA analyses of the VqmA_Phage^G201D protein binding to DNA.

(A) EMSA showing binding of VqmA_Phage^G201D to PvqmR and Pqtip DNA. 25 nM DNA was used in all EMSAs with no protein (designated -) or 2-fold serial dilutions of proteins. The lowest and highest protein (dimer) concentrations are 18.75 nM and 600 nM, respectively. (B) As in panel A for PvqmR* and Pqtip* DNA. (C) EMSA showing WT VqmA_Phage and VqmA_Phage^G201D binding to Pqtip DNA in the presence of H-NS (300 nM). (D) EMSA showing H-NS binding to Pqtip DNA in the presence of WT VqmA_Phage or VqmA_Phage^G201D (each protein at 300 nM).

https://doi.org/10.1371/journal.pgen.1009550.s009

(TIF)

S10 Fig. EMSA analyses of mosaic and chimeric VqmA proteins binding to PvqmR* and Pqtip* DNA.

(A) EMSA showing binding of VqmA_Vc*^171–224 to PvqmR and Pqtip DNA. 25 nM DNA was used in all EMSAs with no protein (designated -) or 2-fold serial dilutions of proteins. The lowest and highest protein (dimer) concentrations are 18.75 nM and 600 nM, respectively. (B) As in panel A for PvqmR* and Pqtip* DNA. (C) As in panel A for _VcN-C_Phage binding to PvqmR* and Pqtip* DNA. (D) As in panel C for _PhageN-C_Vc.

https://doi.org/10.1371/journal.pgen.1009550.s010

(TIF)

S1 Table. Bacterial strains used in this study.

https://doi.org/10.1371/journal.pgen.1009550.s011

(DOCX)

S2 Table. Plasmids used in this study.

https://doi.org/10.1371/journal.pgen.1009550.s012

(DOCX)

S3 Table. Primers used in this study.

https://doi.org/10.1371/journal.pgen.1009550.s013

(DOCX)

S4 Table. gBlocks used in this study.

https://doi.org/10.1371/journal.pgen.1009550.s014

(DOCX)

S1 Data. Numerical data for Figs 1A, 1B, 1C, 1E, 1F, 2C, 2D, 3A, 3B, 3C, 3D, 3E, 3F, 4A, 4B, 4C, 5A, 5B, 5C, 5D, 5E, S2A, S3A, S3B, S3D, S6A and S6B.

https://doi.org/10.1371/journal.pgen.1009550.s015

(XLSX)

Acknowledgments

We thank members of the Bassler laboratory for insightful discussions.

References

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Figures

Abstract

Author summary

Introduction

Results

VqmA promoter-binding selectivity is conferred by the DNA-binding domain

VqmA DNA-binding preferences can be inverted by exchanging key DNA sequences in PvqmR and Pqtip

Protein sequence-guided mutagenesis reveals that residue E194 in phage VP882 VqmAPhage and the equivalent A192 residue in V. cholerae VqmAVc contribute to specificity for Pqtip

Random mutagenesis of the VqmAPhage DBD reveals that residues G201, A202, E207, and M211 are required for VqmAPhage to bind Pqtip but are dispensable for binding PvqmR

The restrictive element that prevents VqmAVc from binding Pqtip is located in its HTH motif and the adjacent C-terminal region of 25 residues

Discussion

Materials and methods

Bacterial strains, plasmids, primers, and reagents

Genetic screens for VqmAPhage and VqmAVc DNA-binding mutants

Growth, lysis, and bioluminescence assays

Protein expression, purification, and electrophoretic mobility shift assay (EMSA)

Western blot analysis

Sequence alignments

Statistical methods

Supporting information

S1 Fig. Sequence alignment of VqmA proteins.

S2 Fig. VqmAPhage has higher affinity for Pqtip DNA than for PvqmR DNA.

S3 Fig. The VqmAVc and VqmAPhage DBDs are non-functional.

S4 Fig. The VqmAPhage and VqmAVc variants are produced at levels similar to WT.

S5 Fig. VqmAPhage mutants possessing WT activity induce phage lysis on agar plates supplemented with 0.2% arabinose.

S6 Fig. VqmAVcQ205E and VqmAVcL209M do not bind Pqtip.

S7 Fig. VqmAVc mosaic proteins are produced at levels similar to WT VqmAVc.

S8 Fig. Structural comparisons of the VqmAPhage and VqmAVc proteins.

S9 Fig. EMSA analyses of the VqmAPhageG201D protein binding to DNA.

S10 Fig. EMSA analyses of mosaic and chimeric VqmA proteins binding to PvqmR* and Pqtip* DNA.

S1 Table. Bacterial strains used in this study.

S2 Table. Plasmids used in this study.

S3 Table. Primers used in this study.

S4 Table. gBlocks used in this study.

S1 Data. Numerical data for Figs 1A, 1B, 1C, 1E, 1F, 2C, 2D, 3A, 3B, 3C, 3D, 3E, 3F, 4A, 4B, 4C, 5A, 5B, 5C, 5D, 5E, S2A, S3A, S3B, S3D, S6A and S6B.

Acknowledgments

References

Protein sequence-guided mutagenesis reveals that residue E194 in phage VP882 VqmA_Phage and the equivalent A192 residue in V. cholerae VqmA_Vc contribute to specificity for Pqtip

Random mutagenesis of the VqmA_Phage DBD reveals that residues G201, A202, E207, and M211 are required for VqmA_Phage to bind Pqtip but are dispensable for binding PvqmR

The restrictive element that prevents VqmA_Vc from binding Pqtip is located in its HTH motif and the adjacent C-terminal region of 25 residues

Genetic screens for VqmA_Phage and VqmA_Vc DNA-binding mutants

S2 Fig. VqmA_Phage has higher affinity for Pqtip DNA than for PvqmR DNA.

S3 Fig. The VqmA_Vc and VqmA_Phage DBDs are non-functional.

S4 Fig. The VqmA_Phage and VqmA_Vc variants are produced at levels similar to WT.

S5 Fig. VqmA_Phage mutants possessing WT activity induce phage lysis on agar plates supplemented with 0.2% arabinose.

S6 Fig. VqmA_Vc^Q205E and VqmA_Vc^L209M do not bind Pqtip.

S7 Fig. VqmA_Vc mosaic proteins are produced at levels similar to WT VqmA_Vc.

S8 Fig. Structural comparisons of the VqmA_Phage and VqmA_Vc proteins.

S9 Fig. EMSA analyses of the VqmA_Phage^G201D protein binding to DNA.