Quorum sensing regulation by the nitrogen phosphotransferase system in Pseudomonas aeruginosa

Quorum sensing regulation by the nitrogen phosphotransferase system in Pseudomonas aeruginosa | 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 Quorum sensing regulation by the nitrogen phosphotransferase system in Pseudomonas aeruginosa Samalee Banerjee , Nicole E. Smalley , Pradtahna Saenjamsai , View ORCID Profile Anthony Fehr , Ajai A. Dandekar , Matthew T. Cabeen , View ORCID Profile Josephine R. Chandler doi: https://doi.org/10.1101/2025.02.01.636002 Samalee Banerjee a Department of Molecular Biosciences, University of Kansas , Lawrence, KS Find this author on Google Scholar Find this author on PubMed Search for this author on this site Nicole E. Smalley b Department of Microbiology, University of Washington , Seattle, WA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Pradtahna Saenjamsai a Department of Molecular Biosciences, University of Kansas , Lawrence, KS Find this author on Google Scholar Find this author on PubMed Search for this author on this site Anthony Fehr a Department of Molecular Biosciences, University of Kansas , Lawrence, KS Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Anthony Fehr Ajai A. Dandekar b Department of Microbiology, University of Washington , Seattle, WA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Matthew T. Cabeen c Department of Microbiology and Molecular Genetics, Oklahoma State University , Stillwater, Oklahoma Find this author on Google Scholar Find this author on PubMed Search for this author on this site Josephine R. Chandler a Department of Molecular Biosciences, University of Kansas , Lawrence, KS Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Josephine R. Chandler For correspondence: jrchandler{at}ku.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT In the opportunistic pathogen Pseudomonas aeruginosa , the nitrogen-related phosphotransferase system (PTS Ntr ) influences multiple virulence behaviors. The PTS Ntr is comprised of three enzymes: first PtsP, then the PtsO phosphocarrier, and the final PtsN phosphoacceptor. We previously showed that ptsP inactivation increases LasI-LasR quorum sensing, a system by which P. aeruginosa regulates genes in response to population density. LasI synthesizes a diffusible autoinducer that binds and activates the LasR receptor, which activates a feedback loop by increasing lasI expression. In this study, we examined the impact of the PTS Ntr on quorum sensing. Disruption of ptsP increased the expression of some, but not all, tested quorum-controlled genes, including lasI, phzM (pyocyanin biosynthesis), hcnA (hydrogen cyanide biosynthesis), and, to a lesser extent, rsaL (quorum sensing regulator). Expression of these genes remained dependent on LasR and the autoinducer, whether provided endogenously or exogenously. Increased lasI expression in Δ ptsP (or Δ ptsO ) cells was partly due to the presence of unphosphorylated PtsN, which alone was sufficient to elevate lasI expression. However, we observed residual increases in Δ ptsP or Δ ptsO cells even in the absence of PtsN, suggesting that PtsP and PtsO can regulate gene expression independent of PtsN. Indeed, genetically disrupting the PtsO phosphorylation site impacted gene expression in the absence of PtsN, and transcriptomic evidence suggested that PtsO and PtsN have distinct regulons. Our results expand our view of how the PTS Ntr components function both within and apart from the classic phosphorylation cascade to regulate key virulence behaviors in P. aeruginosa . IMPORTANCE Pseudomonas aeruginosa often causes severe and difficult-to-treat infections. P. aeruginosa virulence requires the nitrogen-related phosphotransferase system (PTS Ntr ), which comprises the phosphocarrier proteins PtsP and PtsO and the final phosphoacceptor, PtsN. The PTS Ntr is known to modulate quorum sensing, but little is known about the mechanism of regulation. Here, we examined quorum sensing regulation by the PTS Ntr . We showed that the PTS Ntr increases quorum sensing-mediated activation of certain genes through the additive effects of both PtsO and PtsN. We also used transcriptomics to determine the regulons of PtsO and PtsN and found that they are largely nonoverlapping. The results position PtsO and PtsN as independent effectors in the Nitro-PTS and shed new light on virulence regulation in this important pathogen. INTRODUCTION Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen found in many habitats, particularly those linked with human activity ( 1 ). P. aeruginosa causes severe and sometimes fatal infections in people with cystic fibrosis, acute leukemia, burn wounds and organ transplants. It is also commonly contracted in healthcare settings ( 2 ). There is a significant global health burden from P. aeruginosa infections, which are thought to be responsible for over $700 million in health-related costs annually ( 3 ). P. aeruginosa infections are particularly difficult to treat due to the prevalence of multidrug-resistant strains, an arsenal of virulence factors, and its ability to adapt and survive in diverse environments ( 4 – 6 ). In P. aeruginosa, several key virulence factors are regulated by the nitrogen-related phosphotransferase system (PTS Ntr )( 7 ). This system was first described in Escherichia coli as important for regulating changes in metabolism in response to the available ratio of carbon and nitrogen ( 8 ). The PTS Ntr regulates diverse behaviors in different bacteria; for example, in E. coli , the PTS Ntr regulates metabolism ( 9 ) and potassium transport ( 10 ), and the Pseudomonas putida PTS Ntr regulates toluene degradation ( 11 ) and polyhydroxyalkanoates ( 12 ). The PTS Ntr is paralogous to the canonical sugar PTSs that phosphorylate and import saccharides. The first enzyme of the PTS Ntr is PtsP (“enzyme I” or EI, which is analogous to the sugar-PTS EI enzymes). PtsP transfers phosphate, thought to be taken from phosphoenolpyruvate (PEP), to the second enzyme, PtsO (“NPr,” analogous to the sugar PTS histidine protein HPr). PtsO then transfers phosphate to the final enzyme PtsN (“enzyme IIA” or EIIA, analogous to EIIA of the sugar PTS). A hallmark of the PTS Ntr system as compared to sugar PTSs is that the EI has a GAF domain (named after some of the proteins it is found in; c G MP-specific phosphodiesterases, a denylyl cyclases and F hlA) ( 13 ); GAF domains directly bind small molecule ligands and subsequently effect a response ( 14 ). In P. aeruginosa and other bacteria, the ptsO and ptsN genes are located downstream of the nitrogen-related sigma factor gene rpoN, while the ptsP gene is located elsewhere ( 15 ). The P. aeruginosa PTS Ntr is important for virulence, although its virulence effects are not well understood. Deleting the first gene, ptsP, attenuates P. aeruginosa pathogenicity in infections of mice, Caenorhabditis elegans and plant leaves ( 16 , 17 ). In mice, ptsP null mutations decrease resistance to host innate immunity ( 18 ). In laboratory evolution experiments, ptsP mutations increase resistance to the clinically important antibiotic tobramycin ( 19 – 21 ) through an unknown mechanism. In addition, the PTS Ntr system impacts P. aeruginosa biofilm formation by modulating the Pel polysaccharide ( 22 ). Studies of biofilms also suggest that PtsO acts as a “specificity factor,” to ensure that PtsN is not phosphorylated by another PTS EI, FruB (from the fructose PTS system), and that modulation of PtsN phosphorylation impacts the expression of dozens of genes, including many that are virulence associated ( 7 ). ptsP disruption also activates transcription of the quorum sensing signal synthase gene lasI ( 21 , 23 ). Quorum sensing is a population density-dependent communication system (for reviews, see refs. ( 24 , 25 )). In P. aeruginosa, LasI synthesizes the acyl-homoserine lactone (AHL) signal molecule N- (3-oxo)-dodecanoyl L-homoserine lactone (3OC12-HSL), which is detected by the signal receptor LasR. Upon binding, LasR activates transcription of dozens of genes, including lasI, which creates a positive feedback loop. In addition to the LasR-LasI system, there is a second AHL signal-receptor pair in P. aeruginosa, RhlR-RhlI, which synthesizes and responds to N- butanoyl L-homoserine lactone (C4-HSL). Together, these systems activate the production of virulence factors such as pyocyanin, protease, rhamnolipids, hydrogen cyanide, biofilm matrix proteins, lectin and alkaline protease, and they have been shown to be important for virulence in numerous infection models ( 16 , 26 – 29 ). Although a regulatory link between quorum sensing and the PTS Ntr has been established ( 21 , 23 ), studies to clarify the role of ptsP mutation and the other two PTS Ntr enzymes on quorum sensing have not been done. Using transcriptional reporters, we determined that ptsP disruption influences only a subset of quorum sensing-regulated genes. By adding exogenous 3OC12-HSL and using a synthetically inducible lasI strain, we showed that ptsP disruption increases LasR-dependent gene activation in response to both endogenous and exogenous 3OC12-HSL, suggesting the effects of Δ ptsP are not due to changes in 3OC12-HSL biosynthesis. We also demonstrated independent, but additive, effects of PtsO and PtsN on lasI expression; the conserved phosphorylation sites of each protein were important for differential regulation. Transcriptomics studies of strains in which the PtsO enzyme phosphorylation state varied uncovered a PtsO-dependent, distinct regulon that did not overlap with that of PtsN. The results implicate PtsO and PtsN as independent outputs of the PTS Ntr , highlighting the complexity of this important virulence determinant in P. aeruginosa . RESULTS ptsP deletion increases expression of some, but not all, LasR-controlled genes To better understand the role of ptsP- null mutations on LasR activity, we used a pP lasI -gfp reporter plasmid ( 30 ), which contains the promoter of the LasR-responsive lasI gene fused to a gene encoding GFP. We transformed the pP lasI -gfp plasmid into P. aeruginosa Δ ptsP and ΔlasR and compared fluorescence intensities over time ( Fig. 1A ). These deletion mutants grew identically to the wild type strain, PA14 (Fig. S1). Consistent with our prior studies, at the end of the experiment (14 h), we observed ∼3-fold higher P lasI -gfp activation in Δ ptsP compared with wild type ( Fig. 1A , unpaired t- test, p<0.001); there was no activation in the absence of LasR. This difference was observed after cultures reached an OD 600 of ∼0.6, which correlates with the initiation of stationary phase (Fig. S1). We also measured the 3OC12-HSL concentration in wild type and Δ ptsP cultures after 18 h growth. We found that 3OC12-HSL levels were almost 5-fold higher for the Δ ptsP strain compared with that of the wild type; 279 nM for Δ ptsP and 57 nM for the wild type (unpaired t- test, p < 0.0001), consistent with the observed difference in lasI transcription in these two strains. Download figure Open in new tab Fig. 1. Δ ptsP influence on quorum sensing gene transcription. GFP fluorescence was measured in strains harboring reporters to the lasI (A), phzM (B), lasB (C), rsaL (D), hcnA (E), and rhlA (F) promoters in PA14, PA14 ΔptsP and PA14 Δ lasR. Fluorescence output was measured over a time course using a BioTek plate reader. Data points are the means of three replicates, and the error bars represent standard deviation. Significance by t-test of wild type compared with Δ ptsP from the final time point (OD 600 -adjusted fluorescence values); ***, p < 0.0005; **, p < 0.005; *, p < 0.05; ns, not significant. We sought to test if ptsP disruption similarly regulates expression of other quorum sensing-regulated genes. We selected promoters for the following genes: phzM , encoding a key enzyme in phenazine biosynthesis, based on prior studies showing PTS Ntr regulation of phenazine production ( 21 , 23 ); lasB (elastase protease), rsaL (repressor of LasR-I system), hcnA (hydrogen cyanide biosynthesis), and rhlA (rhamnolipid surfactant)( 31 ). This suite of genes as a group tests the activity of both RhlR and LasR quorum sensing. We engineered plasmid-based reporters of each of these gene promoters (pP phzM -gfp, pP lasB -gfp, pP rsaL -gfp, pP hcnA -gfp, and pP rhlA -gfp ). As expected, the expression of each of these genes was dependent on LasR in our time course experiments ( Fig. 1 ). Compared with wild type, in the Δ ptsP strain we observed higher activation of the phzM, hcnA, and, to a lesser degree rsaL reporters. These results show disrupting ptsP increases expression of some, but not all, quorum sensing-regulated genes. Δ ptsP -dependent lasI regulation requires LasR and 3OC12-HSL We sought to gain further insight into the relationship of PtsP to lasI regulation. We first asked if LasR is required for Δ ptsP- dependent activation of lasI transcription. We deleted lasR from a Δ ptsP mutant and measured lasI expression levels using our pP lasI -gfp reporter. In the absence of lasR, we observed no significant difference in lasI activation in a Δ ptsP mutant compared with wild type ( Fig. 2A ), supporting the idea that elevated lasI expression in a Δ ptsP mutant requires LasR. Download figure Open in new tab Fig. 2. Role of LasR and exogenous 3OC12-HSL on Δ ptsP- dependent lasI activation. Growth-adjusted GFP fluorescence in strains harboring the P lasI - GFP reporter plasmid. Fluorescence was measured after 18 h growth in test-tube grown cultures. For panels B and C, strains carried a chromosomally inserted CTX-1 cassette (CTX), CTX plus ptsP, or CTX plus the arabinose-inducible lasI gene (Para- lasI ). For (C), black bars indicate strains with the native ptsP gene intact and white bars indicate strains with Δ ptsP ; 0.2% arabinose was added to all cultures. Data points are the means of three replicates, and the error bars represent standard deviation. Significance by t-test; ***, p < 0.0005; ns, not significant. Next, we considered whether the Δ ptsP mutation increases biosynthesis or production of 3OC12-HSL, which could increase lasI expression via activation of LasR. We used two approaches to test this hypothesis. First, we deleted lasI from our wild type and Δ ptsP mutant strains and grew these strains with varying concentrations of exogenously added 3OC12-HSL ( Fig. 2B ). With the pP lasI -gfp reporter, the minimum 3OC12-HSL concentration we could detect a response was 1 nM. The response was saturated at concentrations of 25 nM and higher. In this concentration range, we observed a ∼3-fold increase in GFP levels in the Δ ptsP strain. We could restore GFP levels to the wildtype level this strain by inserting an intact copy of the ptsP gene into the neutral att site in the chromosome. We also engineered strains to constitutively express lasI using an arabinose-inducible promoter (P ara - gfp ), which we inserted into the neutral att site in the chromosome of our Δ lasI and Δ lasI- Δ ptsP mutants. This approach removes lasI expression from LasR control ( 32 ). With these strains, we also observed a ∼3-fold increase in P lasI -gfp activation due to the Δ ptsP mutation. The Δ lasI and Δ lasI- Δ ptsP strains harboring P ara - lasI produced 3OC12-HSL at concentrations of 254 nM and 198 nM, respectively, which is sufficient to maximally induce the GFP reporter ( Fig. 2B ). From these results, we conclude that deleting ptsP increases LasR sensitivity to 3OC12-HSL, rather than modulating 3OC12-HSL biosynthesis. Deleting the PtsP GAF domain does not impact lasI expression In E. coli, the phosphorylation state of PtsP appears to depend on the cellular ratio of nitrogen to carbon, which is detected by direct binding of glutamine and α-ketoglutarate by PtsP through the GAF domain ( Fig. 3A ). In P. aeruginosa, the GAF domain appears to be required for PtsP to transfer phosphates to PtsO and PtsN ( 7 ). We thus asked whether the PtsP GAF domain is required for its role in lasI regulation. Therefore, we constructed a PA14 strain in which we deleted the GAF domain from ptsP at its native site in the genome ( ptsP ΔGAF). We introduced our pP lasI -gfp reporter plasmid to this ptspΔ GAF strain and compared lasI transcription activation with that of the wild type and a full ptsP deletion mutant. As expected, lasI expression increased in the Δ ptsP strain; however, deleting only the GAF domain did not similarly increase lasI expression ( Fig. 3B ). We also tested whether the PtsP GAF domain is required for PtsP’s role in regulating the phzM or hcnA promoters by using the pP phzM -gfp and pP hcnA -gfp reporter plasmids, respectively. As with lasI, we did not observe a significant role for the PtsP GAF domain in regulating phzM or hcnA expression (Fig. S2). These results show the GAF domain is dispensable for PtsP’s role in regulating lasI, phzM and hcnA . Download figure Open in new tab Fig. 3. The PtsO and PtsN enzymes, but not the PtsP GAF domain, contribute to lasI regulation. (A) Illustration of the PTS Ntr phosphorylation cascade. Phosphate is transferred by PtsP (EI Ntr) from phosphoenolpyruvate (PEP) to PtsO (Npr) and in turn to PtsN (EII Ntr), which has unknown regulatory targets. The GAF domain of PtsP is thought to regulate phosphorylation by detecting changes in the ratio of glutamine and α-ketoglutarate. (B) and (C) Transcription from the lasI promoter was monitored as GFP fluorescence in cells transformed with the pP lasI -gfp reporter plasmid. ptsP ΔGAF is an allelic replacement of ptsP with a deletion of the sequence encoding the GAF domain. Δ ptsP-O-N is the Δ ptsP- Δ ptsO -Δ ptsN triple mutant. Data shown are growth-adjusted fluorescence (B) or growth-adjusted fluorescence normalized to wild type (C) after 18 h growth. Data are means of at least three replicates, and error bars represent SD. Statistical significance by one-way ANOVA compared with wild type; ****, p < 0.0001; **, p < 0.005; ns, not significant. lasI is regulated by other enzymes in the PTS Ntr pathway Next, we examined the role of PtsO and PtsN, the other two enzymes in the PTS Ntr phosphotransfer system ( Fig. 3A ), for lasI regulation. As a first step, we introduced the P lasI -gfp reporter plasmid to wild-type PA14 and single, double and triple deletion mutant(s) of the three PTS Ntr genes ptsP , ptsO and ptsN ( Fig. 3C ). Deleting ptsO from the wild-type genome increased lasI expression to nearly the same level as the Δ ptsP mutant (∼2-fold for Δ ptsO and ∼3-fold for Δ ptsP ) ( Fig. 3C ). In the Δ ptsO mutant, we could restore lasI expression to that of the wild type by introducing a functional copy of ptsO into the genome (Fig. S3). These results show that lasI is regulated, in part, by PtsO. Deleting ptsN from the wild-type genome did not significantly alter lasI expression ( Fig. 3C ); however, deleting ptsN from the Δ ptsP mutant decreased lasI expression but not to wild type levels. Thus, PtsN regulates transcription from the lasI promoter, but only in the absence of PtsP. In a strain in which all three of the PTS Ntr enzymes are deleted (Δ ptsP-ΔptsO-ΔptsN ), we observed an increase in lasI expression as compared to wild type. Together, the results support the idea that PTS Ntr may have regulatory effects on lasI expression that are both activating (in the absence of PtsP or PtsO) and suppressing (when PtsN, or PtsO and PtsN, are absent in a Δ ptsP background). Unphosphorylated PtsN activates lasI expression PtsN is unphosphorylated in the absence of PtsP ( 7 ), leading us to posit that lasI activation in a Δ ptsP mutant may be at least partially due to unphosphorylated PtsN. This model is consistent with our finding that deleting ptsN from a Δ ptsP mutant reduces lasI expression in this strain ( Fig. 3C ). To test this hypothesis, we utilized a PtsN allele that harbors a single amino acid substitution (H68A) that changes its phosphorylation site to alanine. This substitution has been shown to effectively block PtsN phosphorylation in P. aeruginosa ( 7 ). We moved the unmutated PtsN or the PtsN H68A genes in single copy to the chromosome of the Δ ptsN mutant and used the pP lasI -gfp reporter plasmid to compare lasI expression in these strains with that of the Δ ptsN and wild type strains ( Fig. 4A and B ). Consistent with our earlier result, lasI expression was similar in the wild type and Δ ptsN mutants. In the Δ ptsN strain, we found that PtsN H68A , but not the wild-type PtsN, increased lasI expression by ∼3-fold; this difference was observed after cultures reached stationary phase, with no significant effects on growth (OD 600 ∼0.6; Fig. S4). We also assessed the role of PtsN and PtsN H68A in regulating lasI expression in the Δ ptsP-ΔptsO-ΔptsN mutant, where we could evaluate regulation effects in the absence of the other PTS Ntr enzymes. In this genetic background, we expected lasI expression to be activated by ectopically expressing PtsN H68A , as observed when we expressed this allele in the Δ ptsN strain. We also expected lasI expression to be similarly activated with wild-type PtsN in this strain, because PtsN will be unphosphorylated in the absence of ptsP. Consistent with our expectations, PtsN H68A increased lasI expression levels ∼2.4-fold compared with no PtsN alleles. However, wild-type PtsN increased levels to a lesser degree than that of the PtsN H68A allele, by only ∼1.4-fold ( Fig. 4C ). We speculate that PtsN may be partially phosphorylated in this strain by FruB, the fructose EI enzyme, which can phosphorylate PtsN in the absence of PtsO ( 7 ). Thus we also examined the role of PtsN and PtsN H68A on lasI expression in a Δ ptsP ΔptsN mutant, where PtsO is intact. In this strain, transcription from the lasI promoter was increased to the same level by ectopically expressing either PtsN H68A or PtsN (Fig. S4). Together, these results support the conclusion that unphosphorylated PtsN activates lasI expression, and disrupting ptsP results in elevated transcription levels due to an accumulation of unphosphorylated PtsN. Download figure Open in new tab Fig. 4. Mutating the PtsN phosphorylation site increases lasI expression. Transcription from the lasI promoter was monitored as GFP fluorescence in cells transformed with the pP lasI -gfp reporter plasmid. Panels A, C and D show growth-adjusted fluorescence normalized to wild type after 18 h of growth, and panel B shows fluorescence output measured over a time course using a BioTek plate reader. Strains carried a chromosomally inserted CTX-1 cassette (CTX), CTX plus the wild type ptsN, or CTX plus the H68A variant of PtsN ( ptsN (H68A)). Data are means of at least three replicates, and error bars represent SD (error bars are too small to be seen in panel B). Statistical significance by one-way ANOVA compared to wild type (or as indicated) for panels A-C and compared to Δ ptsP-ΔptsN + ptsN- H68A for panel D; ****, p < 0.0001; ***, p<0.001; **, p < 0.005; ns, not significant. Statistical analysis of data shown in panel B were of OD-adjusted fluorescence values from the final time point. In the absence of PtsN, lasI expression is repressed by both PtsP and PtsO In our experiments, lasI reporter activity was significantly lower in the Δ ptsN mutant than in the Δ ptsP-ΔptsN mutant ( Fig. 3C ), suggesting there is another mechanism of PTS Ntr regulation that may be through PtsP or PtsO. Thus, we sought to unravel this additional regulation mechanism. We reasoned that if lasI is repressed by PtsP only, we should observe a PtsP-dependent reduction of lasI reporter activity in the absence of both PtsO and PtsN. To test this hypothesis, we moved ptsP in single copy into the chromosome of the Δ ptsP-ΔptsO-ΔptsN triple mutant and transformed these strains with the P lasI - gfp plasmid reporter to measure transcription from the lasI promoter. We found that ptsP had no effect on lasI expression in this strain ( Fig. 5A ), showing that PtsP alone does not modulate lasI expression. However, in the Δ ptsP-ΔptsN mutant (where ptsO is intact), ptsP significantly decreased lasI expression ( Fig. 5A ); differences in expression, but not growth, were observed after cultures reached stationary phase (OD 600 ∼0.6; Figs. 5B and S5A). These results suggest that PtsP can repress lasI expression but only in the presence of PtsO. To determine whether PtsO can regulate lasI on its own, we compared lasI expression in the Δ ptsP-ΔptsO-ΔptsN triple mutant with that of the Δ ptsP-ΔptsN double mutant (where ptsO is intact) ( Fig. 5A ). However, lasI expression in these two strains was indistinguishable ( Fig. 5A ), indicating PtsO alone does not modulate lasI expression. From these data we conclude that lasI suppression in the absence of PtsN requires both PtsP and PtsO. Download figure Open in new tab Fig. 5. lasI regulation by PtsP and PtsO. Transcription from the lasI promoter was monitored as GFP fluorescence in cells transformed with the pP lasI -gfp reporter plasmid. (A and C) Growth-adjusted fluorescence normalized to wild type after 18 h of growth. (B and D) Fluorescence output measured over a time course using a BioTek plate reader. Strains carried a chromosomally integrated CTX-1 cassette (CTX), CTX plus the wild type ptsO, or CTX plus the H15A variant of PPtsO ( ptsO (H15A)). Data are means of three replicates, and error bars represent SD (error bars are too small to be seen in panel B). Statistical significance by one-way ANOVA compared with the wild type (A and B) or Δ ptsOΔptsN (C and D) *, p < 0.05; ns, not significant. Statistical analysis of data shown in panel B and D were of OD-adjusted fluorescence values from the final time point. PtsO represses lasI transcription through its conserved phosphorylation site The requirement for both PtsP and PtsO in suppressing lasI transcription implied that PtsO phosphorylation, which depends on PtsP, might have a regulatory role. Thus, we considered the possibility that phosphorylated PtsO represses lasI expression. To test this hypothesis directly, we ectopically expressed ptsO from the neutral attB site in the chromosome of the Δ ptsO-ΔptsN mutant (where PtsP is present). In this mutant, introducing ptsO caused a small but significant decrease in lasI expression ( Fig. 5C ), which correlated with stationary phase in time-course experiments ( Fig. 5D and S5B). Next, we constructed and tested a ptsO allele in which the conserved phosphorylation site (His15) is substituted with an alanine. PtsO has a high degree of conservation across many bacterial species, and the His15 phosphorylation site is universally conserved across this family ( 33 ). Mutation of this residue to alanine blocks phosphotransfer to PtsN and disrupts PTS Ntr -controlled phenotypes in the related species Pseudomonas putida ( 34 ). To test the hypothesis that the PtsO His15 residue is important for it to repress lasI , we moved the PtsO H15A gene into the chromosome of the Δ ptsO-ΔptsN strain and assessed activation from the lasI promoter using the P lasI - gfp reporter plasmid. We did not observe any decrease in transcription from the lasI promoter in the strain with the PtsO H15A allele as we did for PtsO ( Fig. 5C and D ). There was also no significant effect on growth or PtsO levels in whole-cell lysates that could explain this result (Fig. S5). Although there are other potential explanations, such as misfolding of the PtsO H15A protein, these results are consistent with the idea that PtsO represses lasI expression due to phosphorylation at its His15 residue. In our experiments, the ability of PtsO to repress lasI transcription did not require PtsN, suggesting that PtsO repression is independent of its role in phosphotransfer to PtsN, the terminal acceptor. There are two other enzymes in P. aeruginosa with terminal phosphoacceptor activity analogous to that of PtsN; these are the carbohydrate PTS enzymes FruB and NagF. Thus, we sought to test the hypothesis that FruB or NagF could serve as alternative phosphor-recipients from PtsO. We expected that if FruB or NagF are important for PtsO to regulate lasI, then deleting fruB or nagF from the wild-type genome would increase lasI expression similar to deleting ptsO . However, deleting fruB and nagF had no measurable effects on lasI expression (Fig. S6). These results support the idea that PtsO represses lasI expression independently of other PTS enzymes. PtsO regulates a distinct subset of genes PtsO is not known to modulate gene transcription independent of its role in phosphotransfer to PtsN in P. aeruginosa. We hypothesized that it might and sought to determine if PtsO similarly regulates genes by a PtsN-independent mechanism. First, we examined phzM and hcnA because expression from these promotors was affected by the Δ ptsP mutation ( Fig. 1 ). For this experiment, we introduced the P phzM- gfp and P hcnA -gfp reporter plasmids to the Δ ptsO-ΔptsN mutant with ectopically expressed PtsO, PtsO H15A , or neither, and compared fluorescence intensities over a time course. We observed a small but significant reduction of phzM reporter activity due to expression of PtsO that was not observed with PtsO H15A ( Fig. 6A ) and not explained by differences in growth (Fig. S7A); however, we observed no PtsO-dependent differences in hcnA reporter activation (Fig. S8). These results support the idea that phosphorylated PtsO decreases transcription from the phzM promoter, but not the hcnA promoter. We also assessed whether phzM transcription is regulated by PtsN. To test this, we introduced the P phzM -gfp reporter to the Δ ptsN strain with ectopically expressed PtsN, PtsN H68A , or neither. We found that PtsN H68A , but not PtsN, increased phzM expression >3-fold compared with (Fig. S7B). Thus both PtsO and PtsN modulate transcription from the phzM promoter in a manner that is similar to that of lasI . Download figure Open in new tab Fig. 6. PtsO regulates a unique set of genes independent of PtsN. (A) PtsO decreases transcription from the phzM promoter due to its phosphorylation site. Fluorescence output measured over a time course in 96-well plates using a BioTek plate reader. (B) Venn diagram showing overlap of genes downregulated in the first strain compared with the second strain listed, as indicated, with genes >2-fold differentially expressed for each comparison. Genes for the wild type vs. Δ ptsN + ptsN- H68A were from re-analyzing data from Underhill et al. ( 7 ). Our finding that PtsO regulates both phzM and lasI independent of the effects of PtsN led us to ask whether PtsO regulates other genes in a similar manner. To this end, we took advantage of previously conducted transcriptomic studies aimed at elucidating the PtsN regulon ( 7 ). This prior analysis included wild type, Δ ptsN and Δ ptsP- Δ ptsN strains, and we added an unpublished Δ ptsO transcriptome that was collected and analyzed with the strains from the original experiment. This combination of strains allowed us to infer whether PtsO has a distinct regulon from PtsN. We included genes regulated >2-fold to capture relatively small regulatory effects such as those we observed with lasI . To determine the PtsO regulon, we first compared differentially expressed genes in wild type and Δ ptsO strains to identify genes impacted by the presence of PtsO (presumed to be phosphorylated in the wild-type strain) vs. no PtsO. Note that PtsN phosphorylation also differs between these strains, as PtsN is unphosphorylated in Δ ptsO ( 7 ). We identified 653 genes that were downregulated in the wild type compared with Δ ptsO. We then compared differentially expressed genes in Δ ptsN and Δ ptsP- Δ ptsN as another way to deduce genes regulated by PtsO; in this case the strains were presumed to have different phosphorylation states of PtsO (phosphorylated in Δ ptsN vs. unphosphorylated in ΔptsP-ΔptsN). The absence of PtsN in these strains eliminates any regulatory effects due to PtsN phosphorylation, but we note that the presence of PtsP differs between these strains. We identified 211 downregulated genes in Δ ptsN compared with Δ ptsP-ΔptsN, and more importantly, 52 genes downregulated in both the first and second strain comparisons ( Fig. 6B and Table S3). Because PtsO is phosphorylated in the first strain in both comparisons, this list of 52 genes includes genes downregulated by phosphorylated PtsO. In principle, the 52 genes identified above could also include genes downregulated by both phosphorylated PtsP and PtsN, which also differ in the first and second comparison, respectively. To this end, we included a third comparison that served to exclude genes downregulated by phosphorylated PtsN. This third comparison was of wild type and Δ ptsN complemented with the PtsN H68A phosphorylation site mutant. In this comparison, we uncovered 449 genes downregulated by phosphorylated PtsN ( Fig. 6B ), with a majority (69%) of these genes also downregulated in the wild type vs. Δ ptsO comparison, presumably due to the difference in PtsN phosphorylation, which is common to both comparisons. Importantly, 50 of the 52 genes from our first two strain comparisons were excluded from those identified to be regulated by PtsN. These results lend confidence in these 50 genes as candidates for repression by phosphorylated PtsO. Among the 50 candidate PtsO-repressed genes, the most highly regulated were those encoding tRNAs (tRNA-Leu, tRNA-Glu and tRNA-Gly), 5S rRNA, and the aminoglycoside-specific MexXY efflux pump. Notably, the mexXY genes were more strongly downregulated in Δ ptsN vs. Δ ptsP -Δ ptsN (∼45-fold) than in the wild type compared with Δ ptsO comparison (∼3-fold), suggesting these genes may have differing responses to different PTS Ntr enzymes. Of note, neither lasI nor phzM were among this list of 50 genes; however, their absence might be explained by differences in growth conditions (lysogeny broth for reporter experiments vs. synthetic cystic fibrosis sputum medium for transcriptomic studies). Nevertheless, the results of our transcriptomic analysis support the idea that PtsO and PtsN can have independent effects on gene regulation. Discussion In this study, we examined the regulatory link between PTS Ntr and quorum sensing in P. aeruginosa . We identified several quorum sensing-regulated genes that are also regulated by PTS Ntr . By examining regulatory effects on lasI, the gene encoding the synthase of the LasR-specific signal, we delineated the role of each of the three PTS Ntr enzymes in gene regulation and showed that modulating phosphorylation of either PtsO or PtsN is sufficient to impact lasI gene expression. Our results suggest a model where blocking phosphotransfer through the Nitro-PTS Ntr mediates PtsN-dependent effects through unphosphorylated PtsN and PtsN-independent effects through PtsO ( Fig. 7 ). These data support the notion that the constituent enzymes of PTS Ntr have roles beyond mere phosphotransfer to PtsN. Consistent with this idea, we also identified a PtsO-specific regulon that is distinct from that of PtsN. Download figure Open in new tab Fig. 7. Model of PTS Ntr regulation. (A) When PtsP is blocked, PtsO and PtsN is unphosphorylated, and unphosphorylated PTsN activates quorum sensing genes (e.g. lasI ). (B) When PtsP is not blocked there is phosphate flow leading to phosphorylation of PtsO and PtsN, and phosphorylated PtsO represses quorum sensing genes. Collectively, our study adds to the growing body of knowledge of the PTS Ntr in P. aeruginosa and provides new information on its connection to quorum sensing regulation. In the absence of PtsP, additive effects of both PtsN-dependent and PtsN-independent regulation increase lasI expression ( Figs. 2A , 3C ). The increase in lasI expression causes a corollary increase in the LasI-dependent quorum sensing signal, 3OC12-HSL ( Fig. 2 ), although this increase in 3OC12-HSL is not sufficient to activate quorum sensing more generally, as not all the LasR-LasI-dependent genes are transcriptionally activated in a Δ ptsP mutant (e.g. lasB; Fig. 1 ). The mechanism by which the PTS Ntr interacts with the Las quorum-sensing system warrants further inquiry. A major finding of our study is that PtsO has distinct regulatory effects that are independent of its role in phosphotransfer to PtsN. Evidence from other species corroborates this finding: for example, PtsO regulates lipid A biosynthesis ( 35 , 36 ) and envelope stress responses ( 37 ) in E. coli. The conservation of PtsN-independent regulation by PtsO across other species suggests there may be a benefit of PtsO and PtsN acting as distinct regulatory outputs of PTS Ntr . One possible benefit is to enable a greater range or sensitivity to different inputs. For example, PtsN can be phosphorylated by the PTS Fru enzyme FruB in both P. aeruginosa ( 7 ) and P. putida ( 38 ). In principle, FruB phosphorylation of PtsN might lead to differential phosphorylation states of PtsN and PtsO. This type of regulation might enable the cells to respond independently to changes in fructose concentrations and to other inputs of the PTS Ntr system. In our transcriptomic analyses, the presence of phosphorylated PtsO was associated with suppressed synthesis of tRNAs that can be charged with lysine, leucine, selenocysteine, glutamate and glycine. These results suggest phosphorylated PtsO might alter synthesis of certain proteins based on their amino acid composition. The depletion of certain tRNAs could also slow down translation or cause ribosome stalling, which could serve to induce certain stress responses (e.g., the stringent response). It is notable that the mexXY genes, which are known to be induced by ribosome stalling ( 39 – 41 ), were also downregulated by phosphorylated PtsO, which could be an indirect effect from the effects on translation. These genes were also more strongly repressed in the Δ ptsN vs. Δ ptsP ΔptsN comparison than in the wild type vs. Δ ptsO comparison suggesting mexXY may have an additional level of repression by PtsP, which differs in the first comparison and not in the second. Further studies of the link between the PTS Ntr and MexXY, antibiotic resistance, or both, are needed to better understand this connection. To date, few direct PTS Ntr targets have been identified. The protein targets of the PTS Ntr phosphoenzymes are assumed to be regulated through protein-protein interactions. In E. coli, TrkA, a K + transporter, was identified as the direct target of PtsN ( 42 ), and the P. putida PtsN directly targets the PDH enzyme complex, which converts pyruvate to acetyl-CoA in the TCA cycle ( 43 , 44 ). To our knowledge, no direct targets have been studied in P. aeruginosa. Nonetheless, our studies of quorum sensing support the idea that PtsO and PtsN may have unique direct targets that each regulate different downstream genes. MATERIALS AND METHODS Bacterial culture conditions and reagents Bacteria were routinely grown in Lysogeny Broth (LB) (if Escherichia coli ) or LB buffered to pH 7 with 50 mM 3-(morpholino)-propanesulfonic acid (MOPS) (if Pseudomonas aeruginosa ), or on LB agar (LBA; 1.5% w/v Bacto-Agar; for both E. coli and P. aeruginosa ). All P. aeruginosa broth cultures were grown in 18 mm test tubes (for 2 ml cultures) at 37°C with shaking at 250 rpm, 125 ml baffled flasks (for 10 ml cultures), or 250 ml baffled flasks (for 50 ml cultures), unless otherwise specified. For E. coli , 10-20 µg ml -1 gentamicin (depending on the strain), 10 µg ml -1 tetracycline and 100 µg ml -1 ampicillin were used. For P. aeruginosa , 50–200 µg ml -1 gentamicin (on LBA), 15 µg ml -1 gentamicin (in LB broth), and 200 µg ml -1 tetracycline were used. The CTX-2-P ara - lasI strains were grown in either 0% or 0.5% arabinose to induce plasmid expression. N-3-oxo-dodecanoyl-L-homoserine lactone (3OC12-HSL) was purchased from Cayman Chemicals (Ann Arbor, MI, USA), dissolved in acidified ethyl acetate (ethyl acetate mixed with 0.1 ml l −1 glacial acetic acid) ( 45 ), and added to culture tubes and dried using nitrogen gas prior to adding cultures. Genomic or plasmid DNA was extracted using Qiagen Puregene Core A kit (Hilden, Germany) or IBI Scientific plasmid purification mini-prep kit (IA, USA) while PCR products were purified using IBI Scientific PCR clean-up/gel extraction kits, according to the manufacturer’s protocol. Gentamicin antibiotics was purchased from GoldBio (MO, USA), tetracycline was purchased from Fisher Scientific (PA, USA), and ampicillin was purchased from Sigma Aldrich (MO, USA). To measure luminescence (β-galatosidase) activity, the Galacto-Light Reaction Tropix kit from ThermoFisher Scientific (PA, USA) was used. Bacterial strains and strain construction All bacterial strains, plasmids, and primers used in this study are listed in Tables 1-3. P . aeruginosa strain UCBPP-PA14 (‘PA14’)( 46 ) and PA14 derivatives were used for these studies. Allelic exchange was used to make markerless deletions in specific loci of P . aeruginosa PA14 as described elsewhere ( 47 ). For allelic exchange, DNA fragments carrying the mutated or deleted gene allele plus 500 bp flanking DNA were synthesized by GenScript and inserted into the pEXG2 suicide vector. During the process of allelic exchange, the plasmids were moved to P. aeruginosa by conjugation using an E. coli donor strain and transformants were selected on Pseudomonas Isolation Agar (PIA) using gentamicin (200 μg ml -1 ) and counterselected on NaCl-free LB agar containing 15% sucrose. Putative mutants were verified through antibiotic sensitivity tests and gene-targeted Sanger sequencing. CTX plasmids were also moved to P. aeruginosa strains via conjugation from an E. coli donor strain. DNA fragments from CTX that incorporated at the neutral chromosomal attB locus were selected on PIA-Tetracycline (200 μg ml -1 ) and verified by PCR ( 48 , 49 ). All replicating plasmids were introduced to P. aeruginosa via electroporation ( 30 ), selected on LB agar using gentamicin at 50–200 μg ml -1 and routinely grown with gentamicin (50 μg ml -1 for agar and 15 μg ml -1 for broth) for plasmid maintenance. Reporter activity measurements The influence of the PTS Ntr system on lasI, phzM, rhlA, lasB, rsaL, and hcnA gene expression levels in P. aeruginosa was measured using transcriptional reporter plasmids with each gene promoter fused to the promoterless gfp reporter in the pPROBE plasmid. Each plasmid was moved to P. aeruginosa by electroporation, and single transformant colonies were used to inoculate 2 mL LB-MOPS Gm 15 to start the experiments. For end-point measurements of activity, cells were grown for 18 h, washed with phosphate buffered saline (PBS), suspended in PBS, and fluorescence and optical density were measured using a BioTek Synergy 2 plate reader. For time-course measurements, overnight cultures were diluted 1:100 in fresh medium and grown to an optical density at 600 nm (OD 600 ) of ∼0.1. This culture was then diluted to an OD 600 of ∼0.004 and dispensed in a black 96-well clear flat-bottom plate with 200 µl per well. Plates were incubated with double orbital shaking at 37 °C in a Biotek Synergy H1 plate reader with OD 600 and GFP fluorescence (excitation 485 nm, emission 528 nm) measured every 15 min for 14 h. To account for background, the empty vector pPROBE P empty -gfp was also measured and subtracted from the measured values of each reporter strain. The final fluorescence values were plotted with respect to OD 600 using GraphPad Prism. Quorum sensing signal measurements We used a bioassay to measure 3OC12-HSL produced by different strains of P. aeruginosa. To prepare samples for analysis, we used 5 ml cultures grown 18 h in LB-MOPS. The cells were removed from the culture fluid by centrifugation, the culture fluid was extracted twice with acidified ethyl acetate, and the ethyl acetate fraction was evaporated to dryness under a stream of nitrogen gas. The residue was dissolved in 0.5 ml acidified ethyl acetate, and the ethyl acetate solutions were used in bioassay. For the bioassay, we used strain E. coli DH5a pSC11 pJ105L-LasR with P lasI-lacZ and lasR on different plasmids ( 30 ). Details of the bioassay procedure have been described elsewhere. Briefly, overnight E. coli cultures grown in LB-MOPS were diluted 1:100 into fresh LB-MOPS and grown to an OD 600 of ∼0.2-0.3 prior to adding arabinose for induction of lasR. The culture was then grown to an OD 600 of ∼0.5-0.6, and aliquots (0.5 ml) were dispensed into 2 ml Eppendorf tubes with evaporated culture fluid extracts, synthetic 3OC12-HSL standards or no signal and grown an additional 3 h. β-galactosidase was detected using the Galacto-light Reaction Tropix Kit (Thermo Fisher) and measured using a Biotek Synergy 2 plate reader. A standard curve was generated from the synthetic signal samples and signal levels in the culture fluid extracts were determined by comparing with the standard curve. RNA extraction and sequencing RNA extraction and sequencing was as described previously ( 7 ). Briefly, cultures grown to an OD600 of 0.3 in SCFM2 were harvested and RNA was extracted according to the NEB (New England Biolabs, MA, USA) Monarch RNA extraction kit. rRNA was depleted using the Illumina RiboZero kit (Illumina, CA, USA) and samples were sequenced by 150-bp paired-end Illumina sequencing at the University of Oklahoma Health Sciences Center core facility in Oklahoma City, OK, USA. Sequence mapping and analysis were performed at the Oklahoma University Health Sciences Center Laboratory for Molecular Biology and Cytometry Research using CLC software. Genes were classified as differentially expressed in pair-wise comparisons based on the FDR-adjusted P-value 1, and a summary of the overlap in differentially expressed genes between pair-wise comparisons was generated with the R package ggVennDiagram v 1.5.2 in R v 4.4.1 ( 50 , 51 ). 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