A transcription factor-sRNA-mediated double-negative feedback loop confers pathogen-specific control of quorum-sensing genes

A transcription factor-sRNA-mediated double-negative feedback loop confers pathogen-specific control of quorum-sensing genes | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results A transcription factor-sRNA-mediated double-negative feedback loop confers pathogen-specific control of quorum-sensing genes View ORCID Profile Ameya A. Mashruwala , Kaitlin Decker , Chenyi Fei , Julie Valastyan , View ORCID Profile Bonnie L. Bassler doi: https://doi.org/10.1101/2025.08.22.671807 Ameya A. Mashruwala 1 Department of Molecular Biology, Princeton University , Princeton, New Jersey 08544, USA 2 The Howard Hughes Medical Institute , Chevy Chase, MD 20815, USA 3 The Stowers Institute for Medical Research , Kansas City, MO, 64110 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ameya A. Mashruwala Kaitlin Decker 1 Department of Molecular Biology, Princeton University , Princeton, New Jersey 08544, USA 2 The Howard Hughes Medical Institute , Chevy Chase, MD 20815, USA 5 Rutgers University , Piscataway, NJ, 08854 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Chenyi Fei 1 Department of Molecular Biology, Princeton University , Princeton, New Jersey 08544, USA 2 The Howard Hughes Medical Institute , Chevy Chase, MD 20815, USA 4 Massachusetts Institute of Technology , Cambridge, MA 02139 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Julie Valastyan 1 Department of Molecular Biology, Princeton University , Princeton, New Jersey 08544, USA 2 The Howard Hughes Medical Institute , Chevy Chase, MD 20815, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Bonnie L. Bassler 1 Department of Molecular Biology, Princeton University , Princeton, New Jersey 08544, USA 2 The Howard Hughes Medical Institute , Chevy Chase, MD 20815, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Bonnie L. Bassler For correspondence: bbassler{at}princeton.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract The cell-to-cell communication process called quorum sensing enables bacteria to synchronize collective behaviors. Quorum sensing relies on the production, release, and detection of signaling molecules called autoinducers. In Vibrio cholerae , the VqmA transcription factor, following binding of the DPO autoinducer, activates expression of the gene encoding the VqmR small regulatory RNA. VqmR controls traits including biofilm formation. Here, we identify repressors of DPO-VqmA-VqmR signaling. We focus on one identified repressor, the LuxT transcription factor. We show that LuxT represses vqmR transcription. VqmR post-transcriptionally represses luxT translation. This arrangement forms a double-negative feedback loop between the two regulators. Reciprocal control hinges on the N-terminal 8 amino acids of LuxT. The nucleotide sequence encoding this LuxT region serves as the VqmR binding site in the luxT mRNA and the amino acids specified by this same N-terminal region are required for LuxT to bind the vqmR promoter. This same LuxT N-terminal region also expands the DNA motifs to which LuxT can bind. We show this regulatory circuit is unique to V. cholerae and closely related species and absent from other vibrios. We define the set of LuxT-controlled genes in V. cholerae and show that LuxT promotes biofilm formation, a key requirement for successful colonization of eukaryotic hosts. Importance Bacterial quorum sensing enables control of collective behaviors. In Vibrio cholerae , the DPO-VqmA-VqmR quorum-sensing circuit governs key processes, including biofilm formation. Here, we identify a double-negative feedback loop between the transcription factor LuxT and the small RNA VqmR. This regulatory circuit depends on an eight amino acid N-terminal region that exists only in V. cholerae LuxT and LuxT from its close relatives. This short peptide sequence confers three distinct functions: It enables LuxT to repress vqmR , renders luxT mRNA susceptible to VqmR repression, and governs which DNA motifs LuxT can bind. Our findings reveal a pathogen-specific regulatory module that links small RNA targeting of mRNAs to transcription factor DNA binding specificity. The results show how evolution tailors bacterial regulatory circuits to adapt to different environments. Introduction Quorum sensing (QS) is a process of cell-to-cell communication that bacteria use to coordinate group behaviors ( 1 , 2 ). QS involves the production, release, and population-wide detection of extracellular signal molecules called autoinducers (AIs) ( 1 , 2 ). At low cell density, AI concentration is below the threshold for detection and genes required to perform individual behaviors are expressed ( 2 , 3 ). As population density increases, AIs accumulate and interact with their partner receptors ( 2 – 4 ). AI-bound receptors drive the transition from the low cell density to the high cell density gene expression pattern, and consequently, bacteria enact group behaviors ( 5 – 10 ). In the human pathogen Vibrio cholerae , the causative agent of the cholera disease, QS regulates traits including virulence factor production and biofilm formation ( 11 – 14 ). Regarding V. cholerae biofilms, they form at low cell density, and at high cell density, QS represses biofilm formation and promotes biofilm dispersal ( 11 , 15 , 16 ). V. cholerae possesses multiple QS systems that operate in parallel ( 14 , 17 – 21 ). The current work focuses on the QS system that relies on the AI called DPO (3,5-dimethylpyrazin-2-ol), which is produced via a threonine dehydrogenase (Tdh)-dependent mechanism ( 18 , 22 ). DPO is bound by the VqmA transcription factor ( 18 ). At high cell density, the DPO-VqmA complex activates expression of vqmR, encoding the VqmR small RNA (sRNA) ( 18 , 23 ). VqmR post-transcriptionally regulates gene expression, including repressing genes required for biofilm formation ( 18 , 23 ). In addition to extracting information encoded in DPO, VqmA also acts as an information processing hub that enables V. cholerae to integrate cues derived from the environment and the human host, such as oxygen levels and bile salts ( 24 ). VqmA indirectly perceives oxygen through cysteine residues that form redox-responsive disulfide bonds. In the presence of both oxygen and DPO, VqmA forms a C134-C134 intermolecular disulfide bond that enhances binding to the vqmR promoter. Indeed, the VqmA C134A protein, which cannot form this disulfide bond, exhibits diminished DNA binding capacity ( 24 ). Here, we perform a genetic screen to identify additional regulators of the DPO-VqmA-VqmR QS circuit. We focus on the LuxT transcription factor revealed in our analysis. We define the mechanism by which LuxT represses vqmR expression, we show that VqmR represses LuxT production, and we demonstrate how reciprocal regulation shapes QS-controlled behaviors. Our assessment of the distribution of this LuxT-VqmR double-negative feedback loop shows it is restricted to V. cholerae and its close relatives due to the presence of a 24 base pair (bp) sequence in the V. cholerae luxT mRNA to which VqmR binds, a sequence that is lacking in other vibrios ( Figure 1 ). Our findings indicate that reciprocal LuxT-VqmR regulation could provide functions specific to niches inhabited by V. cholerae . Download figure Open in new tab Figure 1. Simplified scheme for a double negative feedback loop comprised of the VqmR sRNA and the LuxT transcription factor. This regulatory loop exists in V. cholerae and closely related vibrios but not in more distant relatives. In the left panel, the designation 8-AA N-term on the LuxT protein and the red portion of the luxT mRNA show the regions required for the double negative feedback loop. See text for details. Results A genetic screen in V. cholerae reveals repressors of DPO-VqmA-VqmR QS signal transduction QS information, cues from the local environment, and human host-derived stimuli are all integrated by VqmA to modulate its transcriptional activity ( 24 ). Given this understanding, we wondered whether additional inputs also regulate VqmA-directed QS signaling. An earlier screen revealed that VqmA activates vqmR expression ( 23 ). Here, we carry out the opposite screen: a transposon mutagenesis screen for repressors of vqmR . Our strategy exploited a partially impaired VqmA protein, VqmA C134A , that exhibits reduced DNA binding capacity at the vqmR promoter ( 24 ). Using the VqmA C134A allele enabled us to conduct a simple, visual blue-white colony screen using a transcriptional reporter in which the vqmR promoter drives lacZ expression (hereafter P vqmR-lacZ , with the P prefix denoting promoter). Because basal P vqmR-lacZ activity is low in V. cholerae carrying VqmA C134A, colonies are white/pale blue, unlike V. cholerae colonies possessing wildtype (WT) VqmA, which are bright blue (Supplementary Figure S1). We reasoned that V. cholerae vqmA C134A P vqmR-lacZ mutants that had obtained transposon insertions in genes encoding repressors of DPO-VqmA-VqmR signaling would drive higher P vqmR-lacZ expression and be identifiable as blue colonies on petri plates (Supplementary Figure S1). Based on this logic, we mutagenized Δ tdh vqmA C134A-FLAG P vqmR - lacZ V. cholerae and screened for blue colonies in the presence of X-gal. We have previously shown that the vqmA C134A-FLAG allele accurately reflects VqmA protein abundance and function ( 24 ). Using a strain lacking tdh for the screen ensured that hits would be restricted to genes encoding components that act on signal relay ( vqmA or vqmR ) not signal production ( tdh ). We did not supply DPO during the screen. The strategy was to keep the endogenous P vqmR-lacZ activity as low as possible to enhance detection of insertion mutants exhibiting even modest increases. We assessed ∼20,000 colonies and identified 17 mutants, representing 14 unique transposon insertion sites. The mutated genes encoded 6 regulatory proteins, 4 components involved in transport and metabolism, and 4 genes with undefined functions. To confirm the hits from our screen, we engineered P vqmR - lux as a reporter to quantify vqmR promoter activity ( 23 , 24 ). We introduced the P vqmR - lux fusion onto the chromosomes of Δ tdh V. cholerae possessing either vqmA FLAG or vqmA C134A-FLAG . In the absence of DPO, P vqmR-lux activity was ∼27-fold higher in the Δ tdh vqmA FLAG strain than in the Δ tdh vqmA C134A-FLAG strain, showing that VqmA C134A-FLAG is, as expected, impaired in activity. Supplementation with DPO increased vqmR-lux activity ∼4-fold in the Δ tdh vqmA FLAG strain and ∼37-fold in the Δ tdh vqmA C134A-FLAG strain ( Figure 2A, B , respectively). Thus, the P vqmR-lux reporter responds properly to DPO-mediated VqmA activation, and exploiting the VqmA C134A-FLAG mutant expands the dynamic range of the AI response assay. Download figure Open in new tab Figure 2. A genetic screen identifies repressors of the DPO-VqmA-VqmR QS circuit. (A) Light production over time from Δ tdh vqmA FLAG P vqmR-lux and (B) Δ tdh vqmA C134A-FLAG P vqmR-lux V. cholerae strains following administration of the specified concentrations of DPO. (C) As in panel B in the strain containing the additional designated deletions. DPO was added at 10 µM. Maximum fold-changes relative to that in the parent for Δ vc0122 , Δ vc2614 , Δ vca0566 , and Δ vca0917 were 9-fold at 11 h, 13.5-fold at 11 h 20 min, 2.8-fold at 17 h, and 3.3-fold at 14 h, respectively. RLU denotes relative light units, which are bioluminescence per OD 600 . Scale bars represent color:intensity. Also see Supplementary Figure S1. To verify that components identified in our screen influence DPO-VqmA-VqmR signaling, we deleted each of the identified genes from the chromosome of the Δ tdh vqmA C134A-FLAG P vqmR-lux V. cholerae strain. Deletions of four of the candidate genes, vc0122, vc2614, vca0566, and vca0917 resulted in 9-, 13.5-, 2.8-, and 3.3-fold increases in P vqmR-lux activity, respectively ( Figure 2C ; growth curves for the strains are provided in Supplementary Figure S2 and the phenotypes following complementation of the deleted genes are shown in Supplementary Figure S3). Some temporal differences occur across strains. For simplicity, throughout this work we report maximal fold changes and OD 600 values during early-exponential growth. The remaining ten genes did not significantly alter P vqmR-lux output (Supplementary Figure S4). It is possible that these ten genes were false positives or that they only influence vqmR expression on solid medium, etc. We did not study them further. The remainder of the present work is focused on vc0122, vc2614, vca0566, and vca0917 . These genes encode Acy, Crp, WigR, and LuxT, respectively. LuxT represses vqmR transcription The putative repressors identified in our screen could decrease DPO-VqmA-VqmR signaling by repressing expression of either vqmA or vqmR . To distinguish between these two possibilities, we engineered a Δ tdh Δ vqmA V. cholerae strain carrying P BAD -vqmA at a neutral chromosomal locus. The strain also harbors the chromosomal P vqmR-lux fusion. We refer to this strain simply as the P BAD -vqmA strain. We introduced deletions of acy , crp , wigR , and luxT into the P BAD -vqmA strain and measured P vqmR-lux output. Our rationale is as follows: VqmA is required to activate vqmR expression. In the P BAD -vqmA strain, following the addition of arabinose, VqmA is produced and activates P vqmR-lux . Since vqmA is driven by a synthetic promoter, it is not subject to native regulation. Thus, if deletion of a gene(s) identified in our screen results in increased P vqmR-lux activity, then vqmR must be the target of that particular repressor. By contrast, if no change in P vqmR-lux occurs, then we can infer that repression must occur through changes in vqmA transcription, VqmA activity, or VqmA turnover. Figure 3A shows that deletion of crp and luxT from the P BAD -vqmA strain resulted in ∼13.2-fold and 2.5-fold increases in P vqmR-lux activity suggesting that crp and luxT modulate VqmR levels. By contrast, no change in P vqmR-lux activity occurred when acy and wigR were deleted, suggesting they modify vqmA expression or VqmA abundance or activity ( Figure 3A ; growth curves for the strains are provided in Supplementary Figure S5). We quantified VqmA FLAG abundance in the Δ tdh V. cholerae strain when acy or wigR was deleted. We note that, here, vqmA FLAG is driven by its endogenous promoter. We used the Δ tdh Δ crp and Δ tdh Δ luxT strains as controls for comparison. Our logic was that if Acy and WigR modify vqmA transcription or VqmA abundance, then in the Δ acy and Δ wigR strains, VqmA levels would differ from those in the parent strain. Neither mutant displayed any change in VqmA FLAG abundance (Supplementary Figure S6). Acy and WigR could affect VqmA function; we have not examined that mechanism. Download figure Open in new tab Figure 3. LuxT represses vqmR transcription. (A) Transcriptional output over time from P vqmR-lux following introduction of the indicated gene deletions in the Δ tdh Δ vqmA P BAD - vqmA V. cholerae strain carrying P vqmR-lux . Maximum fold changes relative to the parent of the Δ crp and Δ luxT strains were 13.2-fold at 10 h and 2.5-fold at 8 h 20 min, respectively. RLU and scale bar as in Figure 2 . (B) EMSA showing 6X-His-LuxT Vc (designated LuxT Vc ) binding to a P vqmR Vc probe (−111 to +49 relative to the vqmR Vc transcriptional start site) and the same probe with the putative LuxT binding site scrambled (P vqmR Vc scrambled ). The 20-nucleotide motif and its scrambled version are shown. All lanes contained 15 ng of promoter DNA. Bound and unbound DNA probes are designated. For the remainder of this study, we characterize the role of LuxT in vqmR regulation. V. cholerae LuxT ( LuxT Vc ) is a transcription factor, the mRNA of which was previously identified as a direct target of repression by the VqmR sRNA ( 23 ). Our identification of LuxT Vc as a repressor of vqmR suggests that VqmR and LuxT Vc constitute a double-negative feedback loop. To probe whether LuxT regulates vqmR by transcriptional repression, we analyzed the vqmR promoter for a consensus LuxT binding sequence. Vibrio harveyi LuxT (LuxT Vh ) shares 75% amino acid identity with LuxT Vc , making it likely that both proteins bind similar DNA sequences. A prior study in V. harveyi identified a LuxT (LuxT Vh ) binding site in the promoter of qrr 1 ( 25 ). Inspection of the V. cholerae vqmR promoter (P vqmR Vc ) using the site in the qrr 1 promoter as a guide, revealed a potential LuxT binding site located at −28 to −9 upstream of the vqmR Vc transcriptional start site. To determine if LuxT Vc binds this motif, we purified 6X-His-LuxT Vc and combined it in an electrophoretic mobility shift assay (EMSA) with a probe containing V. cholerae P vqmR Vc (−111 to +49) or, as a control, the same probe containing a scrambled version of the putative LuxT binding sequence (P vqmR Vc scrambled ). 6X-His-LuxT Vc bound to P vqmR Vc but not P vqmR Vc scrambled , suggesting that LuxT directly represses vqmR expression ( Figure 3B ). We refer to the LuxT binding motif in P vqmR Vc as Motif 1. Since we already know that the VqmR sRNA represses luxT , we conclude that LuxT and VqmR interact in a double-negative feedback loop. LuxT-VqmR feedback loops are present only in V. cholerae and V. cholerae -like vibrio species Given that both LuxT and VqmR are present across vibrios, we wondered whether their regulatory interactions are likewise conserved across the genus. Using bioinformatic analyses, we compared the DNA sequences encoding P vqmR and the luxT region targeted by vqmR from representative strains across sequenced vibrio species. First, we discuss LuxT repression of P vqmR . Vibrio P vqmR sequence comparisons are shown in a pair-wise sequence similarity color-map ( Figure 4A , blue indicating highest similarity). We compared a 50-nucleotide region from −48 to +2 relative to the vqmR transcriptional start sites. This sequence space contains 20 nucleotides corresponding to the LuxT Vc binding site (−28 to −9), together with 20 upstream and 10 downstream nucleotides. We analyzed one representative genome across sixty-three different vibrios that harbor both luxT and vqmR . For simplicity, only the LuxT consensus binding site is shown in the figure, and it varies across P vqmR sequences. To test LuxT tolerance for variation in binding site, we chose a few similar and quite different putative LuxT binding sites from the set of vqmR promoters and performed EMSA analyses using 6X-His-LuxT Vc . The test sites come from P vqmR in Vibrio metoecus , Vibrio splendidus , and V. harveyi (denoted P vqmR Vm , P vqmR Vs , and P vqmR Vh , respectively, and are displayed in Figure 4A ). The V. metoecus P vqmR sequence is in the same cluster as the site bound by LuxT Vc in P vqmR Vc , differing by only 3 bp (sequence differences are highlighted in red in Figure 4A ). Not surprisingly, like P vqmR Vc , LuxT Vc bound P vqmR Vm ( Figure 4B ). By contrast, the LuxT binding sequences from V. splendidus and V. harveyi , which differ from that in P vqmR Vc by ∼40-50%, were not bound by LuxT Vc ( Figure 4B ). These findings suggest that LuxT repression of P vqmR occurs only via DNA sequences present in V. cholerae and closely related vibrios. Download figure Open in new tab Figure 4. LuxT binds P vqmR in V. cholerae and in its close relative V. metoecus , but not P vqmR in more distantly related vibrios. (A) Pair-wise sequence similarity color-map of vqmR promoter regions from one representative genome from sixty-three vibrio species that harbor both luxT and vqmR . Hierarchical clustering was performed using the UPGMA algorithm (Unweighted Pair Group Methods with Arithmetic Mean). Putative LuxT binding sites for the designated groups are displayed below the heatmap with red indicating differences from the V. cholerae sequence. (B) EMSA of 6X-His-LuxT Vc (designated LuxT Vc ) binding to P vqmR DNA probes, similar in length to that in Figure 3B , containing putative LuxT binding sequences from V. cholerae (P vqmR Vc ), V. metoecus (P vqmR Vm ), V. splendidus (P vqmR Vs ), and V. harveyi (P vqmR Vh ). (C) Heatmap showing pair-wise sequence similarity of the vqmR target sites in the luxT transcript across the same genomes as in panel A. Hierarchical clustering was performed using the UPGMA algorithm. A V. cholerae -like group ( Vc ) and a V. harveyi -like group ( Vh ) are highlighted, with representative sequences from each group shown below the heatmap. Red indicates differences from the V. cholerae sequence. The predicted TTG ( V. cholerae ) and ATG ( V. harveyi ) start codons are underlined. To further probe this hypothesis, we examined LuxT regulation of vqmR in V. harveyi by qPCR. In this case, we predict that LuxT does not bind the vqmR promoter. As a positive control for LuxT-dependent regulation, we measured expression of swrZ , a known V. harveyi LuxT target ( 26 ). vqmR expression was the same in the V. harveyi parent and Δ luxT strains (Supplementary Figure S7). By contrast, swrZ expression was ∼100-fold higher in the V. harveyi Δ luxT strain than the parent. Together, these results support the conclusion that LuxT does not regulate vqmR in V. harveyi , consistent with the absence of a LuxT binding site in the V. harveyi vqmR promoter. Now we discuss VqmR repression of luxT . We have previously shown that the VqmR sRNA binds to a 24 bp region encoding the first 8-amino acids of LuxT Vc ( 23 ). To determine whether this N-terminal 8 amino acid extension is present in other vibrio LuxT proteins, we compared this 24 bp region in luxT DNA sequences across vibrio genomes. A pair-wise sequence similarity color-map shows that across vibrios, luxT genes separate into multiple clusters. A majority cluster into a group that includes V. harveyi , and there is a smaller cluster possessing luxT genes resembling that of V. cholerae ( Figure 4C ). Notably, the V. cholerae start codon (TTG) in the luxT sequence is absent in vibrios that are not closely related to V. cholerae ( Figure 4C ). Rather, in V. harveyi and its relatives, luxT transcription is predicted to initiate at a downstream, ATG start codon. Consequently, we predict that V. harveyi LuxT Vh and vibrios in the LuxT Vh cluster will produce a shorter LuxT isoform that lacks the 8 N-terminal amino acids present in V. cholerae LuxT. These findings imply that VqmR might only repress production of LuxT Vc but not LuxT in vibrios that contain LuxT proteins that cluster with LuxT Vh . The N-terminal 8 amino acids in V. cholerae LuxT expand the DNA motifs to which LuxT can bind Our bioinformatic results suggest that the 24 bp region encoding the first 8 amino acids of LuxT Vc is specific to V. cholerae . We wondered whether having that N-terminal amino acid extension is accompanied by functional consequences on the ability of LuxT Vc to bind DNA. To explore this idea, we purified 6X-His-LuxT Vc and 6X-His-LuxT Vc Δ8-AA N-term , which lacks the first 8 N-terminal amino acids, and tested binding to P vqmR Vc . As expected, the probe was bound by LuxT Vc , however, LuxT Vc Δ8-AA N-term displayed minimal binding ( Figure 5A ). These results show that the first 8 amino acids in LuxT Vc are essential for binding to P vqmR Vc . We also examined whether the absence of the first eight amino acids in LuxT affected binding at other target DNA promoter sequences. As mentioned, in V. harveyi , LuxT Vh regulates swrZ (P swrZ Vh ) encoding a GntR type transcriptional regulator ( 26 ). As no LuxT-controlled genes other than vqmR were known in V. cholerae , we assessed LuxT Vc binding to P swrZ Vh as a surrogate (see note on a V. cholerae LuxT-controlled gene, hapR , reported during our ongoing work, in the Discussion ( 27 )). Both LuxT Vc and LuxT Vc Δ8-AA N-term bound with similar affinities to P swrZ Vh , demonstrating that while the first 8 amino acids of LuxT are necessary to bind P vqmR Vc , they are dispensable for binding to P swrZ Vh ( Figure 5B ). Below, we explore LuxT Vc regulation of V. cholerae genes beyond vqmR . Download figure Open in new tab Figure 5. LuxT Vc binds distinct motifs in P vqmR Vc and P swrZ Vh and the 8 N-terminal amino acids in LuxT govern which sequence is bound. (A, B) EMSA analyses for 6X-His-LuxT Vc and 6X-His-LuxT Vc Δ8-AA N-term (denoted LuxT Vc and LuxT Vc Δ8-AA N-term , respectively) binding to the P vqmR Vc probe DNA or to the V. harveyi P swrZ Vh −110 to +20 probe. (C) Heatmap showing a multi-sequence alignment of P swrZ Vh sequences across vibrio species. The consensus sequence logo is shown on top, with taller letters indicating higher conservation. The nucleotide-level alignment is provided in Supplementary Figure 9. The two conserved sequences, shown by the black bars underneath, differ by four bps and are designated Motif 2L (for Left) and Motif 2R (for Right). DNA sequences for Motif 2L and Motif 2R are provided in Supplemental Figure 10. (D) EMSA analyses for LuxT Vc binding to the designated V. harveyi P swrZ Vh regions. Leftmost panel: the probe spans −110 to +20 and contains Motif 2L and Motif 2R sequences. In the three right panels, the −110 to +20 probe has one or both of Motif 2L and Motif 2R scrambled, as designated. Supplementary Figure S11 shows EMSAs for V. harveyi LuxT binding to the same fragments. To pinpoint the sequence in the −110 to +20 P swrZ Vh probe that is bound by LuxT Vc , we assayed seven overlapping DNA fragments spanning the region. Comparison of the binding profiles suggested that multiple, non-continuous sequences are bound by LuxT Vc (Supplementary Figure S8). Consistent with this result, alignment of the P swrZ sequences across vibrio species revealed two conserved sequence segments separated by 18 bp. These sequences are ∼15 bp in length, divergently oriented, and differ by four bp. The first sequence is TAATCAGTACGCTGT (designated Motif 2L (for Left); −73 to −59), and the second is AACTACGTACTGTTT (designated Motif 2R (for Right); −41 to −27) ( Figure 5C , Supplementary Figures S9 and S10). Motif 2L and Motif 2R share a GTAC core with the Motif 1 sequence, however their flanking regions differ. Notably, neither Motif 2L or 2R exist in P vqmR Vc . To test whether Motif 2L and Motif 2R support LuxT Vc binding, we assayed a probe spanning P swrZ Vh −110 to +20 (containing both Motif 2L and Motif 2R) and compared it to probes in which one or both motifs were scrambled. Probes containing at least one intact motif supported LuxT Vc binding, while binding did not occur when both motifs were scrambled ( Figure 5D ). Incubation of LuxT Vc with the P swrZ Vh probe containing both motifs resulted in two band shifts, whereas probes containing only a single motif yielded one band shift. These results suggest that LuxT can bind Motif 2L and Motif 2R individually and simultaneously. Other TetR family proteins, such as QacR, also bind to motifs located in close proximity ( 28 ). We also assessed binding of LuxT Vh to various versions of P swrZ Vh −110 to +20 fragments. The LuxT Vh binding profile was similar to that of LuxT Vc (Supplementary Figure S11). To examine sufficiency of Motif 2L and Motif 2R for binding by LuxT, we used 15 bp probes containing exclusively Motif 2L or Motif 2R. Both LuxT Vc and LuxT Vh bound to these fragments, albeit with lower affinity than to the corresponding full-length probes. Thus, the Motif 2L and Motif 2R DNA sequences are sufficient for LuxT binding, and the flanking residues promote stronger binding (Supplementary Figure S12). We conclude that LuxT Vc can recognize Motif 1, Motif 2L, and Motif 2R. Moreover, the N-terminal 8 amino acids in LuxT Vc are dispensable for LuxT Vc to bind P swrZ Vh . Given that these N-terminal 8 amino acids are required for LuxT Vc to bind Motif 1 in P vqmR Vc , we infer that harboring these amino acids expands the possibilities for V. cholerae LuxT to control gene expression. LuxT Vc promotes biofilm formation in V. cholerae at low cell density A previous RNA-Seq study in V. harveyi identified that the LuxT Vh regulon includes swrZ and ∼10 genes specifying GGDEF or EAL domain-containing proteins that synthesize and degrade, respectively, the second messenger c-di-GMP molecule ( 26 ). In V. cholerae, c-di-GMP levels influence biofilm formation ( 29 , 30 ). Thus, we wondered whether LuxT Vc regulates biofilm formation. To assess this possibility, we monitored colony biofilm morphologies of the V. cholerae Δ tdh and Δ tdh Δ luxT strains. In V. cholerae , biofilm formation drives a change in colony appearance from smooth to wrinkled, with the latter coinciding with an increase in colony height ( 31 – 34 ). Thus, three-dimensional colony height profiles track with biofilm formation. Figure 6 shows images of biofilm colonies and their corresponding height profiles, quantitation of which are displayed as heatmaps. Both the Δ tdh and Δ tdh Δ luxT strains failed to form biofilms. Download figure Open in new tab Figure 6. LuxT Vc promotes V. cholerae colony biofilm formation. Representative images of biofilms formed by the designated V. cholerae strains and companion quantitative 3D colony height profiles after 4 d. Height profiles are color mapped according to the scale provided. The master high cell density QS regulator HapR is a key repressor of V. cholerae biofilm formation ( 11 , 29 ). Thus, we reasoned that LuxT Vc -dependent biofilm phenotypes might be revealed in the absence of hapR . Indeed, Figure 6 shows that while the Δ tdh Δ hapR strain formed colony biofilms with heights reaching up to ∼550 µm, heights of Δ tdh Δ hapR Δ luxT colonies reached only ∼250 µm. As expected, complementation of the Δ hapR Δ luxT strain with luxT on a plasmid restored biofilm formation (Supplementary Figure S13) Thus, V. cholerae forms colony biofilms only in the absence of HapR and, in this low cell density context, LuxT Vc promotes biofilm formation. Production of the VqmR sRNA at high cell density suppresses colony biofilm formation ( 18 , 23 ). To probe whether LuxT Vc modulates biofilm formation via control of vqmR , we compared colony morphologies of the Δ tdh Δ hapR Δ vqmA and Δ tdh Δ hapR Δ vqmA Δ luxT strains. VqmA is required for activation of P vqmR Vc , thus in its absence, vqmR is not expressed ( 23 ). The Δ tdh Δ hapR Δ vqmA strain formed colony biofilms similar to those of the Δ tdh Δ hapR strain with heights reaching up to ∼550 µm, while colony biofilms of the Δ tdh Δ hapR Δ vqmA Δ luxT strain had heights of ∼250 µm ( Figure 6 ). Thus, LuxT Vc activates biofilm formation. Moreover, in our assays, endogenous levels of VqmA and VqmR do not influence colony biofilm formation so LuxT Vc must drive biofilm formation by a mechanism that is independent of VqmA and VqmR. LuxT Vc controls a regulon of V. cholerae genes at high cell density Given that LuxT Vc binds to distinct DNA motifs and it regulates V. cholerae biofilm formation independently of VqmA-VqmR, we reasoned that LuxT Vc may control additional genes. To define the V. cholerae LuxT Vc -controlled regulon, we conducted RNA-Seq on the V. cholerae Δ tdh Δ luxT strain carrying plasmid-borne luxT Vc under control of the arabinose promoter (p-P BAD - luxT Vc ). Following growth to high cell density in the presence and absence of arabinose, approximately 20 genes displayed changes in expression of >2-fold ( Figure 7 ). As anticipated, luxT Vc was the most upregulated gene in the dataset. Other LuxT Vc -regulated genes included ones involved in galactose and sulfate metabolism, which were activated and repressed ∼2-4 fold, respectively. Alteration of expression of a gene specifying an orphan histidine kinase ( vc1088 , ∼3-fold) occurred. vc1088 resides immediately adjacent to an 8-gene operon ( vc1080 - vc1087 ) encoding genes involved in nitric oxide sensing and biofilm formation ( 35 – 37 ). Additionally, several genes specifying hypothetical proteins or those with domains of unknown function were differentially expressed. Other than vc1088 , which, given its genomic context, we hypothesize is a regulator of biofilm genes, the regulon did not harbor genes known to be involved in biofilm formation. This result was expected because the strain used in the RNA-Seq experiment possesses HapR, and as shown in Figure 6 , HapR represses biofilm formation at high cell density. Also, as expected, we did not identify vqmR because our sample preparation was not optimized for enrichment of sRNAs. The RNA-Seq data, together with the other findings from the above studies, suggest that LuxT acts broadly to regulate signaling and metabolic pathways in V. cholerae , ranging from QS to biofilm formation to sulfate metabolism. Download figure Open in new tab Figure 7. Transcriptomic analyses reveal the V. cholerae LuxT Vc -controlled regulon. Volcano plot displaying gene expression patterns in the Δ tdh Δ luxT V. cholerae strain carrying luxT Vc expressed from an arabinose inducible promoter with 0.1% arabinose (p-P BAD - luxT Vc - FLAG . Sulfate metabolism genes are highlighted in black, galactose metabolism genes in orange, vc1088 in red, and luxT in green. Data represent fold-changes for the indicated strain grown with 0.1% arabinose compared to the no arabinose condition. Genes repressed and activated by LuxT are denoted. The horizontal dotted line represents a P-value of 0.05. Left and right vertical dotted lines represent log 2 fold-changes of −1 and 1, respectively. Samples are from n = 3 biological replicates. Complete datasets are provided in Dataset S1. Discussion Here, we used a genetic screen to identify repressors of the V. cholerae DPO-VqmA-VqmR QS circuit. Our screen revealed four genes: acy , crp , wigR , and luxT . We first interpret our findings regarding acy , crp , wigR in the context of QS regulation and then we discuss luxT , the gene we focused on in the current study. The acy and crp genes encode adenylate cyclase (Acy) and the cAMP receptor protein (Crp), respectively. Acy and Crp typically function together ( 38 – 40 ). Acy synthesizes cAMP, which is bound by Crp. The Crp-cAMP complex regulates transcription of target genes, particularly those involved in utilization of different carbon sources. Given this well-established partnership, it is surprising that we find that Acy and Crp apparently work independently of each other to control the DPO-VqmA-VqmR QS circuit with Acy affecting VqmA activity, and Crp modifying P vqmR expression. V. cholerae possesses at least two additional Crp family proteins, neither of which have been studied. One or both of them could partner with Acy to regulate VqmA activity. WigR is a response regulator that, with its partner kinase WigK, constitutes a two-component system that is proposed to monitor cell wall damage ( 41 , 42 ). Our finding suggests the possibility that cell wall damage feeds into QS control of gene expression. LuxT is a TetR-family transcriptional regulator. TetR-family proteins frequently bind small-molecule ligands and a target DNA motif ( 43 ). We demonstrate that V. cholerae LuxT binds DNA; it remains to be determined whether it binds a ligand. luxT is conserved across vibrio species. In V. harveyi , LuxT represses expression of the gene encoding the QS sRNA called Qrr1 at low cell density, a function conserved in several vibrio species but not in V. cholerae ( 25 ). By contrast, we show that in V. cholerae , LuxT represses expression of the gene encoding the VqmR sRNA, and this feature is not conserved in V. harveyi. Thus, as a broader regulatory theme, LuxT represses genes encoding sRNAs that function at the hearts of QS systems. In the context of LuxT regulation of QS components, we note that, after we concluded our experiments, a study was published reporting that LuxT Vc binds P hapR Vc . The motif identified is similar to that in P vqmR Vc ( Figure 5D ) ( 27 ). Our findings also reveal that VqmR post-transcriptionally represses LuxT by targeting the mRNA region encoding its first eight amino acids. The eight N-terminal amino acid residues are required for LuxT to bind the vqmR promoter. Furthermore, while LuxT Vc Δ8-AA N-term fails to bind the vqmR promoter, it nonetheless retains binding to another promoter, P swrZ . LuxT in other vibrios lacks this N-terminal extension. We propose that the acquisition of the LuxT N-terminal 8 amino acids delivers three activities: it allows repression of luxT by VqmR, it enables LuxT binding to the vqmR promoter to establish the VqmR-LuxT double-negative feedback loop, and it promotes regulon expansion by broadening the DNA motifs to which LuxT can bind ( Figure 1 ). To our knowledge, this is the first example in which the mRNA sequence that is targeted by a sRNA regulator encodes a portion of a protein that governs that protein’s DNA-binding capabilities. Double negative feedback loops are common in biological systems requiring tight regulation of state transitions. For instance, in bacteriophage λ, the cI and Cro proteins mutually repress each other to control the switch between lysogenic and lytic cycles ( 44 ). Similarly, in Sinorhizobium meliloti , a double negative feedback loop between the NtrBC two-component system and the sRNA NfeR1 modulates nitrogen metabolism ( 45 ). In V. cholerae , the transition from low cell density to high cell density QS modes involves upregulation of genes encoding energetically costly processes such as motility and the type six secretion system ( 7 , 46 , 47 ). Thus, the double negative feedback between LuxT and VqmR may serve as a buffer that prevents inappropriate transitions between states until conditions are optimal. In V. cholerae , we find that LuxT promotes biofilm formation in a strain genetically locked into expressing low cell density QS behaviors. The mechanism by which LuxT mediates this effect remains undefined but is independent of HapR. Preliminary analyses of promoter regions of canonical biofilm genes do not reveal recognizable LuxT binding motifs, raising the possibility that LuxT binds to an uncharacterized DNA sequence in these promoters or, alternatively, it indirectly regulates biofilm formation genes. That LuxT regulates biofilm formation is particularly intriguing in light of recent work in V. harveyi , where LuxT was shown to be a global regulator of gene expression at low cell density ( 25 , 26 ). Here, we primarily characterized LuxT functions in V. cholerae at high cell density. Studies to identify the full V. cholerae LuxT regulon across different cell densities are required to delineate the scope of its regulatory roles. Biofilm formation is required for V. cholerae to colonize the anaerobic intestinal lumen of infant mice and to form communities on human host cells ( 27 , 48 – 50 ). Given that luxT and the DPO-VqmA-VqmR QS circuit are responsive to oxygen levels and human-host produced bile salts, future studies could examine the roles of LuxT, the DPO-VqmA-VqmR QS circuit, and the VqmR-LuxT double negative feedback during the V. cholerae infectious lifecycle ( 24 , 27 ). The regulatory logic uncovered here highlights how a modest genetic innovation, the eight amino acid N-terminal extension in V. cholerae LuxT, generates a pathogen-specific signaling architecture that links sRNA-mediated post-transcriptional control to transcription factor DNA-binding specificity. This dual-function sequence represents, to our knowledge, the first example in which a sRNA binding site also encodes a domain required for the encoded protein’s regulatory activity. Moreover, the LuxT-VqmR feedback loop is restricted to V. cholerae and its close relatives, illustrating how QS circuits can be rewired to produce species-specific behaviors. These findings provide conceptual insight into evolution of new regulatory modules and help us understand how QS networks can be tailored to particular ecological niches. Materials and Methods Bacterial growth, strain construction and reagents Escherichia coli Top10 and Saccharomyces cerevisiae were used as hosts for cloning, while E. coli S17-1 λ pir was used for conjugations. Cultures of V. cholerae and E. coli were grown in LB medium at 37°C with shaking. When required, media were supplemented with streptomycin, 200 μg/mL; kanamycin, 50 μg/mL; polymyxin B, 50 μg/mL; chloramphenicol, 1 μg/mL; spectinomycin, 200 μg/mL. Bioluminescence-reporter assays were conducted as previously described ( 24 ). Where indicated, relative light units (RLU) denote bioluminescence output divided by the culture optical density. Chromosomal alterations were introduced into V. cholerae using multiplexed genome editing (MuGENT) or the pRE112 suicide vector harboring the counter-selectable sacB gene as previously described ( 24 , 51 ). Unless otherwise specified, chromosomal DNA from V. cholerae C6706 was used as the template for PCR reactions. Plasmids were constructed using pBAD-pEVS or pRE112 as backbones and assembled using NEB Hi-Fi reagent or yeast-recombination-assisted assembly as previously described ( 52 ). Strains, plasmids, and oligonucleotides used in the study are listed in Supplementary Tables 1, 2, and 3, respectively. Gel purification and plasmid preparation kits, iProof DNA polymerase, and Deoxynucleotide Mix were purchased from Qiagen, Bio-Rad, and New England Biolabs, respectively. Sequence analyses Sequence-similarity based identification of luxT and vqmR genes across vibrio genomes has been described ( 25 , 53 ). A custom MATLAB (MathWorks; 2022) algorithm was used to search for luxT and vqmR genes in the genomes of 134 vibrio strains, each representing a distinct species. The initial search identified 63 vibrio species harboring both luxT and vqmR genes. We further analyzed these species for sequence conservation within each gene as well as their promoters. Multiple sequence alignments were performed and the output analyzed in MATLAB. A standard scoring matrix NUC44 (see ftp.ncbi.nih.gov/blast/matrices/ ) was used to compute similarity scores between DNA sequences. The unweighted pair group method with arithmetic mean (UPGMA) was subsequently used to cluster vibrio species with similar sequences. Protein purification Genes encoding 6X-His-tagged proteins were cloned into pET15b and transformed into E. coli BL21. Strains for protein production were cultured in LB with 100 µg/mL ampicillin and incubated at 37°C with aeration. When cultures reached an OD 600 of 0.5, 400 µM IPTG was added to induce protein production. The cultures were further incubated at 18°C overnight. Cells were harvested via centrifugation at 4,000 rpm for 10 min. The resulting cell pellets were resuspended in 1/100 volume of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8) containing 5 µM benzonase, 3 µM imidazole, 250 µg/mL lysozyme, and BugBuster Protein Extraction Reagent (Novagen). The lysate was subjected to centrifugation at 13,000 rpm for 20 min, and the clarified supernatant was combined with Ni-NTA Superflow resin (Qiagen) equilibrated with 3 µM imidazole. After allowing the resin and protein to incubate for 1 h at 4°C, the resin was subjected to a series of washes with imidazole at concentrations from 3 µM to 40 µM. Protein was eluted from the resin with buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8) containing 300 µM imidazole. Purified protein was dialyzed overnight at 4°C with Slide-A-Lyzer modules (Thermo Fisher) in buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8), concentrated, flash-frozen, and stored at −80°C. Bioluminescence measurements Strains harboring luciferase reporters were cultured overnight at 37°C in LB and diluted into fresh LB at a final OD 600 = 0.0025. Where indicated, the medium was supplemented with 640 nM DPO. 150 µL of the cultures were transferred to 96-well plates with transparent bottoms. Where indicated expression of the pBAD promoter was induced with 0.007% to 0.2% arabinose. Plates were incubated at 37°C. Bioluminescence production and OD 600 were measured using a BioTek Synergy Neo2 HTS multimode microplate reader. Reporter activity is presented as Relative Light Units (RLUs), which is bioluminescence divided by OD 600 . Transposon mutagenesis screen and identification of insertion sites Mutagenesis was performed as previously described using a Tn 5 transposon system carrying kanamycin resistance ( 54 ). Transposons were introduced into the Δ tdh vqmA C134A-FLAG P vqmR-lux V. cholerae strain by conjugation from an E. coli donor. Conjugation was limited to 2 h to reduce the likelihood of recovering sibling insertion events. Exconjugants were selected on LB agar supplemented with kanamycin, polymyxin B, and X-gal ( 54 ). Following overnight growth at 37°C, colonies were examined for changes in color. Mutants displaying increased blue color relative to the plate average were purified by restreak on LB agar containing kanamycin and polymyxin B. Transposon insertion sites were mapped using arbitrary PCR, as described previously ( 54 ). Electromobility gel shift assays (EMSAs) DNA probes were purchased as G-Blocks from IDT (∼200 bp) and were used as templates for PCR amplification. The resulting PCR products were gel purified using the QIAquick Gel Purification kit (Qiagen), eluted with sterile water, and stored at 4°C until use. Alternatively, duplex DNA oligomers (∼20-30 bp) were purchased from IDT and used in assays. To initiate EMSA assays, 0.6 to 6 µM of purified protein in buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8) was combined with DNA probes normalized to 5-15 ng. Mixtures were reacted for 20 min at room temperature and subsequently subjected to electrophoresis on Novex 6% DNA Retardation Gels (Thermo Fisher) in 1X Tris-buffered EDTA (TBE) at 4°C ( 24 ). DNA probes were visualized with Sybr Green stain (Thermo Fisher) and imaged with an ImageQuant 800 imaging system (Cytiva) ( 24 ). Immunoblotting V. cholerae strains were cultured overnight in M9 minimal medium. The next day, the cultures were diluted 1:1000 into fresh M9 medium containing 0.01% arabinose and grown to an OD 600 of 1. Cells were pelleted by centrifugation and resuspended in 20 µL of ice-cold phosphate-buffered saline (PBS) at an OD 600 of 3.5. Cells were lysed, protein extracts were separated on SDS-PAGE gels, and immunoblotting was performed as described previously ( 24 ). Protein levels were assessed using ImageJ software. Biofilm assays and image analyses V. cholerae strains were cultured overnight in LB medium at 37°C with aeration. Cultures were subjected to vortex for 5 min with 4 mm glass beads added to disrupt cell aggregates. The samples were diluted to OD 600 = 0.5 in 1X PBS. The cultures were again subjected to vortex for 5 min without beads. 1 µL of each culture was spotted onto an LB plate containing 50 mg/mL polymyxin B. When strains carried plasmids, plates contained 40 mg/mL polymyxin B, 25 mg/mL kanamycin, and, when specified, 0.01% arabinose. The plates were incubated at 37°C and biofilms were imaged every 24 h with a Leica M125 stereomicroscope. Colony height profiles were captured using a Keyence VK-X3000 laser microscope ( 31 ). Images were analyzed using the manufacturer provided MultiFileAnalyzer application. Lateral topographic cross sections were profiled in triplicate. For each cross section, the program identified the minimum and maximum heights. qPCR Overnight cultures of V. harveyi were diluted to OD 600 = 0.0025 and grown to OD 600 = 0.1 in LM medium. The cultures were treated with RNAprotect (Qiagen) and RNA was purified using the RNeasy Mini Kit (Qiagen). gDNA was degraded and cDNA was generated using SuperScript IV VILO Master Mix with ezDNAse (ThermoFisher) using the manufacturer’s recommended procedures. RNA was quantified using PerfeCTa SYBR Green FastMix with Low ROX (QuantaBio). Relative RNA quantities were calculated by ΔΔCt compared to WT and the reference gene, hfq . RNA sequencing Overnight cultures of V. cholerae were diluted to OD 600 = 0.0025 and grown to OD 600 = 0.05 in LB. Cultures were divided in half: 0.1% arabinose was administered to one half and the other half received LB as a control. The treated cultures were grown to OD 600 = 0.25. RNA Protect solution (Qiagen) was added at 2:1 v/v reagent:culture and RNA was purified using the RNeasy Mini Kit (Qiagen) followed by treatment with DNase (Ambion AM 1907) using the manufacturers’ recommended procedures with minor modifications are described previously ( 7 ). RNA samples were sequenced by SeqCenter (Pittsburgh, PA). Author contributions A.A.M., K.D., J.V., C.F., and B.L.B. designed experiments and analyzed data; A.A.M., K.D., and J.V. constructed strains and performed wet-lab experiments; C.F., A.A.M., and B.L.B. designed bioinformatic experiments and C.F. performed bioinformatic experiments and analyses; K.D., C.F., and A.A.M., wrote custom scripts for data analyses and visualization; A.A.M., K.D., and B.L.B. wrote the original draft; A.A.M., K.D., J.V., C.F., and B.L.B. reviewed and edited subsequent manuscript versions; B.L.B. provided oversight, resources, and funding. Acknowledgments We thank Bassler group members for thoughtful discussions. This work was supported by the Howard Hughes Medical Institute and NSF grant MCB-2508324 to B.L.B. A.A.M. was supported as a Howard Hughes Medical Institute Fellow of the Life Sciences Research Foundation and, subsequently, as a member of the Stowers Institute for Medical Research. Footnotes Summary of changes in revised version Genetic complementation experiments have been added for key mutants (acy, wigR, and luxT), confirming restoration of the observed phenotypes (Supplementary Figure S3). Additional experiments were performed to test species specificity of LuxT mediated regulation. These data show that LuxT does not regulate vqmR in Vibrio harveyi, consistent with the absence of a conserved binding site (Supplementary Figure S7). The analysis of LuxT DNA-binding has been expanded. New experiments refine the identification and interpretation of multiple binding motifs in the vqmR and swrZ promoters, clarifying the role of the N terminal extension (Figure 5 and Supplementary Figures S8-S12). Quantitative analyses have been added or expanded. These include biological replicate quantification of VqmA protein levels (Supplementary Figure S6), growth curves for relevant mutants (Supplementary Figures S2 and S5), and statistical analysis of biofilm phenotypes with complementation (Supplementary Figure S13). Luminescence data presentation has been clarified. All reporter measurements are now explicitly defined as normalized to cell density (OD600), with updated figure legends and Methods. A schematic outlining the transposon mutagenesis screen has been added (Supplementary Figure S1). The Methods section has been expanded to include details on transposon mutagenesis, reporter assays, and data analysis. The complete RNA seq dataset has been included (Supplementary Dataset S1). Text revisions were made throughout. The Discussion has been revised to more clearly articulate the conceptual significance of the LuxT and VqmR regulatory mechanism. References 1. ↵ Bassler BL . 1999 . How bacteria talk to each other: regulation of gene expression by quorum sensing . Current Opinion in Microbiology 2 : 582 – 587 . OpenUrl CrossRef PubMed Web of Science 2. ↵ Schauder S , Bassler BL . 2001 . The languages of bacteria . Genes Dev 15 : 1468 – 1480 . OpenUrl FREE Full Text 3. ↵ Papenfort K , Bassler BL . 2016 . Quorum sensing signal–response systems in Gram-negative bacteria . Nat Rev Microbiol 14 : 576 – 588 . OpenUrl CrossRef PubMed 4. ↵ Svenningsen SL , Waters CM , Bassler BL . 2008 . A negative feedback loop involving small RNAs accelerates Vibrio cholerae’s transition out of quorum-sensing mode . Genes Dev 22 : 226 – 238 . OpenUrl Abstract / FREE Full Text 5. ↵ van Kessel JC , Rutherford ST , Shao Y , Utria AF , Bassler BL . 2013 . Individual and Combined Roles of the Master Regulators AphA and LuxR in Control of the Vibrio harveyi Quorum-Sensing Regulon . J Bacteriol 195 : 436 – 443 . OpenUrl Abstract / FREE Full Text 6. Rutherford ST , van Kessel JC , Shao Y , Bassler BL . 2011 . AphA and LuxR/HapR reciprocally control quorum sensing in vibrios . Genes Dev 25 : 397 – 408 . OpenUrl Abstract / FREE Full Text 7. ↵ Mashruwala AA , Qin B , Bassler BL . 2022 . Quorum-sensing- and type VI secretion-mediated spatiotemporal cell death drives genetic diversity in Vibrio cholerae . Cell 185 : 3966 – 3979 .e13. OpenUrl CrossRef PubMed 8. Shao Y , Bassler BL . 2014 . Quorum regulatory small RNAs repress type VI secretion in Vibrio cholerae . Mol Microbiol 92 : 921 – 930 . OpenUrl CrossRef PubMed 9. Suckow G , Seitz P , Blokesch M . 2011 . Quorum Sensing Contributes to Natural Transformation of Vibrio cholerae in a Species-Specific Manner▿ . J Bacteriol 193 : 4914 – 4924 . OpenUrl Abstract / FREE Full Text 10. ↵ Tsou AM , Frey EM , Hsiao A , Liu Z , Zhu J . 2008 . Coordinated regulation of virulence by quorum sensing and motility pathways during the initial stages of Vibrio cholerae infection . Commun Integr Biol 1 : 42 – 44 . OpenUrl CrossRef PubMed 11. ↵ Hammer BK , Bassler BL . 2003 . Quorum sensing controls biofilm formation in Vibrio cholerae : Biofilms in Vibrio cholerae . Molecular Microbiology 50 : 101 – 104 . OpenUrl CrossRef PubMed Web of Science 12. Zhu J , Mekalanos JJ . 2003 . Quorum Sensing-Dependent Biofilms Enhance Colonization in Vibrio cholerae . Developmental Cell 5 : 647 – 656 . OpenUrl CrossRef PubMed Web of Science 13. Zhu J , Miller MB , Vance RE , Dziejman M , Bassler BL , Mekalanos JJ . 2002 . Quorum-sensing regulators control virulence gene expression in Vibrio cholerae . Proc Natl Acad Sci USA 99 : 3129 – 3134 . OpenUrl Abstract / FREE Full Text 14. ↵ Higgins DA , Pomianek ME , Kraml CM , Taylor RK , Semmelhack MF , Bassler BL . 2007 . The major Vibrio cholerae autoinducer and its role in virulence factor production . Nature 450 : 883 – 886 . OpenUrl CrossRef PubMed Web of Science 15. ↵ Bridges AA , Fei C , Bassler BL . 2020 . Identification of signaling pathways, matrix-digestion enzymes, and motility components controlling Vibrio cholerae biofilm dispersal . Proc Natl Acad Sci U S A 117 : 32639 – 32647 . OpenUrl Abstract / FREE Full Text 16. ↵ Bridges AA , Bassler BL . 2019 . The intragenus and interspecies quorum-sensing autoinducers exert distinct control over Vibrio cholerae biofilm formation and dispersal . PLoS Biol 17 : e3000429 . OpenUrl CrossRef PubMed 17. ↵ Miller MB , Skorupski K , Lenz DH , Taylor RK , Bassler BL . 2002 . Parallel Quorum Sensing Systems Converge to Regulate Virulence in Vibrio cholerae . Cell 110 : 303 – 314 . OpenUrl CrossRef PubMed Web of Science 18. ↵ Papenfort K , Silpe JE , Schramma KR , Cong J-P , Seyedsayamdost MR , Bassler BL . 2017 . A Vibrio cholerae autoinducer–receptor pair that controls biofilm formation . Nat Chem Biol 13 : 551 – 557 . OpenUrl CrossRef PubMed 19. Chen X , Schauder S , Potier N , Van Dorsselaer A , Pelczer I , Bassler BL , Hughson FM . 2002 . Structural identification of a bacterial quorum-sensing signal containing boron . Nature 415 : 545 – 549 . OpenUrl CrossRef PubMed Web of Science 20. Jung SA , Chapman CA , Ng W-L . 2015 . Quadruple Quorum-Sensing Inputs Control Vibrio cholerae Virulence and Maintain System Robustness . PLoS Pathog 11 : e1004837 . OpenUrl CrossRef PubMed 21. ↵ Barrasso K , Watve S , Simpson CA , Geyman LJ , van Kessel JC , Ng W-L . 2020 . Dual-function quorum-sensing systems in bacterial pathogens and symbionts . PLoS Pathog 16 : e1008934 . OpenUrl PubMed 22. ↵ Lacey DM , D’Agostino GD , Shine EE , Bassler BL . 2026 . Multiple Adenylate-Forming Enzymes Contribute to Biosynthesis of the DPO Quorum-Sensing Autoinducer . ACS Chem Biol 21 : 380 – 391 . OpenUrl PubMed 23. ↵ Papenfort K , Förstner KU , Cong J-P , Sharma CM , Bassler BL . 2015 . Differential RNA-seq of Vibrio cholerae identifies the VqmR small RNA as a regulator of biofilm formation . Proc Natl Acad Sci USA 112 . 24. ↵ Mashruwala AA , Bassler BL . 2020 . The Vibrio cholerae Quorum-Sensing Protein VqmA Integrates Cell Density , Environmental, and Host-Derived Cues into the Control of Virulence 11 . 25. ↵ Eickhoff MJ , Fei C , Huang X , Bassler BL . 2021 . LuxT controls specific quorum-sensing-regulated behaviors in Vibrionaceae spp. via repression of qrr1, encoding a small regulatory RNA . PLoS Genet 17 : e1009336 . OpenUrl PubMed 26. ↵ Eickhoff MJ , Fei C , Cong J-P , Bassler BL . 2022 . LuxT Is a Global Regulator of Low-Cell-Density Behaviors, Including Type III Secretion , Siderophore Production, and Aerolysin Production, in Vibrio harveyi 13 . 27. ↵ Li Y , Yan J , Li J , Xue X , Wang Y , Cao B . 2023 . A novel quorum sensing regulator LuxT contributes to the virulence of Vibrio cholerae . Virulence 14 : 2274640 . OpenUrl PubMed 28. ↵ Schumacher MA , Miller MC , Grkovic S , Brown MH , Skurray RA , Brennan RG . 2002 . Structural basis for cooperative DNA binding by two dimers of the multidrug-binding protein QacR . EMBO J 21 : 1210 – 1218 . OpenUrl Abstract / FREE Full Text 29. ↵ Waters CM , Lu W , Rabinowitz JD , Bassler BL . 2008 . Quorum Sensing Controls Biofilm Formation in Vibrio cholerae through Modulation of Cyclic Di-GMP Levels and Repression of vpsT . J Bacteriol 190 : 2527 – 2536 . OpenUrl Abstract / FREE Full Text 30. ↵ Krasteva PV , Fong JCN , Shikuma NJ , Beyhan S , Navarro MVAS , Yildiz FH , Sondermann H . 2010 . Vibrio cholerae VpsT Regulates Matrix Production and Motility by Directly Sensing Cyclic di-GMP . Science 327 : 866 – 868 . OpenUrl Abstract / FREE Full Text 31. ↵ Yan J , Fei C , Mao S , Moreau A , Wingreen NS , Košmrlj A , Stone HA , Bassler BL . 2019 . Mechanical instability and interfacial energy drive biofilm morphogenesis . eLife 8 : e43920 . OpenUrl CrossRef PubMed 32. Casper-Lindley C , Yildiz FH . 2004 . VpsT is a transcriptional regulator required for expression of vps biosynthesis genes and the development of rugose colonial morphology in Vibrio cholerae O1 El Tor . J Bacteriol 186 : 1574 – 1578 . OpenUrl Abstract / FREE Full Text 33. Fong JCN , Karplus K , Schoolnik GK , Yildiz FH . 2006 . Identification and characterization of RbmA, a novel protein required for the development of rugose colony morphology and biofilm structure in Vibrio cholerae . J Bacteriol 188 : 1049 – 1059 . OpenUrl Abstract / FREE Full Text 34. ↵ Fong JCN , Yildiz FH . 2007 . The rbmBCDEF gene cluster modulates development of rugose colony morphology and biofilm formation in Vibrio cholerae . J Bacteriol 189 : 2319 – 2330 . OpenUrl Abstract / FREE Full Text 35. ↵ Plate L , Marletta MA . 2012 . Nitric oxide modulates bacterial biofilm formation through a multi-component cyclic-di-GMP signaling network . Mol Cell 46 : 449 – 460 . OpenUrl CrossRef PubMed Web of Science 36. Anantharaman S , Guercio D , Mendoza AG , Withorn JM , Boon EM . 2023 . Negative regulation of biofilm formation by Nitric Oxide Sensing Proteins . Biochem Soc Trans 51 : 1447 – 1458 . OpenUrl CrossRef PubMed 37. ↵ Hossain S , Heckler I , Boon EM . 2018 . Discovery of a Nitric Oxide Responsive Quorum Sensing Circuit in Vibrio cholerae . ACS Chem Biol 13 : 1964 – 1969 . OpenUrl CrossRef PubMed 38. ↵ Liang W , Pascual-Montano A , Silva AJ , Benitez JA . 2007 . The cyclic AMP receptor protein modulates quorum sensing, motility and multiple genes that affect intestinal colonization in Vibrio cholerae . Microbiology 153 : 2964 – 2975 . OpenUrl CrossRef PubMed Web of Science 39. Liang W , Sultan SZ , Silva AJ , Benitez JA . 2008 . Cyclic AMP post-transcriptionally regulates the biosynthesis of a major bacterial autoinducer to modulate the cell density required to activate quorum sensing . FEBS Letters 582 : 3744 – 3750 . OpenUrl CrossRef PubMed 40. ↵ Manneh-Roussel J , Haycocks JRJ , Magán A , Perez-Soto N , Voelz K , Camilli A , Krachler A-M , Grainger DC . 2018 . cAMP Receptor Protein Controls Vibrio cholerae Gene Expression in Response to Host Colonization . mBio 9 : e00966 – 18 . OpenUrl PubMed 41. ↵ Dörr T , Alvarez L , Delgado F , Davis BM , Cava F , Waldor MK . 2016 . A cell wall damage response mediated by a sensor kinase/response regulator pair enables beta-lactam tolerance . Proc Natl Acad Sci USA 113 : 404 – 409 . OpenUrl Abstract / FREE Full Text 42. ↵ Cheng AT , Ottemann KM , Yildiz FH . 2015 . Vibrio cholerae Response Regulator VxrB Controls Colonization and Regulates the Type VI Secretion System . PLoS Pathog 11 : e1004933 . OpenUrl CrossRef PubMed 43. ↵ Cuthbertson L , Nodwell JR . 2013 . The TetR Family of Regulators . Microbiol Mol Biol Rev 77 : 440 – 475 . OpenUrl Abstract / FREE Full Text 44. ↵ Lee S , Lewis DEA , Adhya S . 2018 . The Developmental Switch in Bacteriophage λ: A Critical Role of the Cro Protein . J Mol Biol 430 : 58 – 68 . OpenUrl CrossRef PubMed 45. ↵ García-Tomsig NI , García-Rodriguez FM , Guedes-García SK , Millán V , Becker A , Robledo M , Jiménez-Zurdo JI . 2023 . A double-negative feedback loop between NtrBC and a small RNA rewires nitrogen metabolism in legume symbionts . mBio 14 : e0200323 . OpenUrl PubMed 46. ↵ Herzog R , Peschek N , Fröhlich KS , Schumacher K , Papenfort K . 2019 . Three autoinducer molecules act in concert to control virulence gene expression in Vibrio cholerae . Nucleic Acids Research 47 : 3171 – 3183 . OpenUrl CrossRef PubMed 47. ↵ Mashruwala AA , Bassler BL . 2024 . Quorum sensing orchestrates parallel cell death pathways in Vibrio cholerae via Type 6 secretion-dependent and −independent mechanisms . Proc Natl Acad Sci U S A 121 : e2412642121 . OpenUrl PubMed 48. ↵ Vidakovic L , Mikhaleva S , Jeckel H , Nisnevich V , Strenger K , Neuhaus K , Raveendran K , Ben-Moshe NB , Aznaourova M , Nosho K , Drescher A , Schmeck B , Schulte LN , Persat A , Avraham R , Drescher K . 2023 . Biofilm formation on human immune cells is a multicellular predation strategy of Vibrio cholerae . Cell 186 : 2690 – 2704 .e20. OpenUrl CrossRef PubMed 49. Gallego-Hernandez AL , DePas WH , Park JH , Teschler JK , Hartmann R , Jeckel H , Drescher K , Beyhan S , Newman DK , Yildiz FH . 2020 . Upregulation of virulence genes promotes Vibrio cholerae biofilm hyperinfectivity . Proceedings of the National Academy of Sciences 117 : 11010 – 11017 . OpenUrl Abstract / FREE Full Text 50. ↵ Conner JG , Teschler JK , Jones CJ , Yildiz FH . 2016 . Staying Alive: Vibrio cholerae ’s Cycle of Environmental Survival, Transmission, and Dissemination . Microbiol Spectr 4 : 4.2.02 . OpenUrl 51. ↵ Dalia AB , McDonough E , Camilli A . 2014 . Multiplex genome editing by natural transformation . Proc Natl Acad Sci USA 111 : 8937 – 8942 . OpenUrl Abstract / FREE Full Text 52. ↵ Joska TM , Mashruwala A , Boyd JM , Belden WJ . 2014 . A universal cloning method based on yeast homologous recombination that is simple, efficient, and versatile . Journal of Microbiological Methods 100 : 46 – 51 . OpenUrl CrossRef PubMed 53. ↵ Duddy OP , Silpe JE , Fei C , Bassler BL . 2023 . Natural silencing of quorum-sensing activity protects Vibrio parahaemolyticus from lysis by an autoinducer-detecting phage . PLOS Genetics 19 : e1010809 . OpenUrl 54. ↵ Jemielita M , Wingreen NS , Bassler BL . 2018 . Quorum sensing controls Vibrio cholerae multicellular aggregate formation . Elife 7 : e42057 . OpenUrl PubMed View the discussion thread. Back to top Previous Next Posted April 17, 2026. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following A transcription factor-sRNA-mediated double-negative feedback loop confers pathogen-specific control of quorum-sensing genes Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share A transcription factor-sRNA-mediated double-negative feedback loop confers pathogen-specific control of quorum-sensing genes Ameya A. Mashruwala , Kaitlin Decker , Chenyi Fei , Julie Valastyan , Bonnie L. Bassler bioRxiv 2025.08.22.671807; doi: https://doi.org/10.1101/2025.08.22.671807 Share This Article: Copy Citation Tools A transcription factor-sRNA-mediated double-negative feedback loop confers pathogen-specific control of quorum-sensing genes Ameya A. Mashruwala , Kaitlin Decker , Chenyi Fei , Julie Valastyan , Bonnie L. Bassler bioRxiv 2025.08.22.671807; doi: https://doi.org/10.1101/2025.08.22.671807 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7636) Biochemistry (17703) Bioengineering (13897) Bioinformatics (41960) Biophysics (21458) Cancer Biology (18597) Cell Biology (25524) Clinical Trials (138) Developmental Biology (13381) Ecology (19905) Epidemiology (2067) Evolutionary Biology (24325) Genetics (15612) Genomics (22512) Immunology (17738) Microbiology (40421) Molecular Biology (17187) Neuroscience (88627) Paleontology (667) Pathology (2834) Pharmacology and Toxicology (4825) Physiology (7645) Plant Biology (15158) Scientific Communication and Education (2046) Synthetic Biology (4302) Systems Biology (9825) Zoology (2271)