Combinatorial action of regulatory systems generates colistin heteroresistance

Combinatorial action of regulatory systems generates colistin heteroresistance | 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 Combinatorial action of regulatory systems generates colistin heteroresistance View ORCID Profile Jacob E. Choby , Emily K. Crispell , Muqing Ma , Linda M. Vu , Mary-Kate Key , View ORCID Profile Robert K. Ernst , View ORCID Profile Minsu Kim , View ORCID Profile David S. Weiss doi: https://doi.org/10.1101/2025.08.11.668767 Jacob E. Choby 1 Emory Antibiotic Resistance Center , Atlanta, GA, USA 2 Emory Vaccine Center , Atlanta, GA, USA 3 Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine , Atlanta, GA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jacob E. Choby Emily K. Crispell 1 Emory Antibiotic Resistance Center , Atlanta, GA, USA 2 Emory Vaccine Center , Atlanta, GA, USA 3 Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine , Atlanta, GA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Muqing Ma 1 Emory Antibiotic Resistance Center , Atlanta, GA, USA 4 Department of Physics, Emory University , Atlanta, GA 30322 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Linda M. Vu 5 University of Maryland-Baltimore, Department of Microbial Pathogenesis , Baltimore, MD 21201, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mary-Kate Key 1 Emory Antibiotic Resistance Center , Atlanta, GA, USA 2 Emory Vaccine Center , Atlanta, GA, USA 3 Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine , Atlanta, GA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Robert K. Ernst 5 University of Maryland-Baltimore, Department of Microbial Pathogenesis , Baltimore, MD 21201, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Robert K. Ernst Minsu Kim 1 Emory Antibiotic Resistance Center , Atlanta, GA, USA 4 Department of Physics, Emory University , Atlanta, GA 30322 6 Graduate Division of Biological and Biomedical Sciences, Emory University , Atlanta, GA 30322, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Minsu Kim For correspondence: david.weiss{at}emory.edu minsu.kim{at}emory.edu David S. Weiss 1 Emory Antibiotic Resistance Center , Atlanta, GA, USA 2 Emory Vaccine Center , Atlanta, GA, USA 3 Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine , Atlanta, GA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for David S. Weiss For correspondence: david.weiss{at}emory.edu minsu.kim{at}emory.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Heteroresistance is a form of antibiotic resistance in which a minor subpopulation of resistant cells coexists with a majority susceptible population. Colistin heteroresistance is common among Enterobacter cloacae clinical isolates, threatens its utility as a last-line therapeutic, and has become a model with which to understand the fundamental bases of heteroresistance. Despite numerous insights, the mechanism by which phenotypic heterogeneity is generated within the population and leads to colistin heteroresistance has been unclear. Here, using a transposon-based mutagenesis screen, we identify the sigma factor σ E as the source of heterogeneity in population-wide colistin resistance levels. Single-cell tracking experiments revealed that σ E is active in only one percent of the population at baseline, and only those cells with active σ E survive colistin exposure. However, σ E expression and population heterogeneity are insufficient for survival, as a mutant lacking the PhoPQ two-component system controlling lipid A modifications necessary for colistin resistance retains heterogeneity but loses colistin resistance. These findings lead to a new paradigm in heteroresistance, where the combinatorial action of multiple regulatory systems, encompassing a heterogeneity generator and a distinct resistance generator, are required to give rise to colistin heteroresistance. INTRODUCTION Colistin is a last-resort antibiotic reserved for treatment of multi-drug resistant Gram-negative bacteria, but its usefulness is challenged by emerging resistance. A member of the polymyxin class, colistin is a cationic antimicrobial peptide (CAMP) that targets the Gram-negative outer membrane constituent lipopolysaccharide (LPS) and disrupts the outer and inner membranes, killing the bacterium ( 1 ). Gram-negative bacteria can resist colistin killing by changing the composition of the outer membrane, in particular the lipid A portion of LPS ( 2 ). Generally, carbapenem-resistant Enterobacterales, a WHO Critical Priority ( 3 ) and CDC Urgent Threat ( 4 ), are designated colistin susceptible. However, conventional colistin resistance can occur when an isolate has intrinsic or horizontally-acquired LPS-modifying enzymes. Equally or more common, but challenging to detect ( 5 – 8 ), is a form of phenotypic colistin resistance termed heteroresistance ( 9 ). Heteroresistance (HR) is a type of phenotypic heterogeneity in which cells in a population exhibit differences in relative antibiotic resistance; a low frequency subpopulation of cells is markedly more resistant than the majority susceptible population. The resistant subpopulation can be extremely rare (as few as 1 per 10 6 cells) and is thus often undetected by conventional antimicrobial susceptibility testing. The subpopulation is also dynamic: the resistant cells are enriched in the presence of a given antibiotic but return to their baseline frequency in its absence. For these reasons, HR can contribute to failure of antibiotic therapy, when a patient is treated with an antibiotic to which the infecting organism exhibits undetected HR ( 10 – 13 ). Colistin HR among carbapenem-resistant Enterobacterales is especially common in the Enterobacter genus ( 5 , 8 ). The Enterobacter are diverse, and many pathogenic species are often collectively referred to as the Enterobacter cloacae complex ( 14 ). They are leading nosocomial, opportunistic pathogens and cause pneumonia, bloodstream infections, and urinary tract infections ( 15 ). A challenge for treating infections caused by E. cloacae complex isolates is the high rate of antibiotic resistance, especially to cephalosporins and carbapenems, making colistin an important last-line treatment option. We previously described colistin HR in a cephalosporin-resistant Enterobacter clinical isolate ( 16 ). That work, and analyses of other colistin HR Enterobacter isolates, have resulted in colistin HR serving as a model for Gram-negative antibiotic HR. The resistant subpopulation in colistin HR Enterobacter harbors 4-amino-4-deoxy--arabinose (ʟ-Ara4N) modifications on the lipid A moiety of LPS, increasing cellular surface charge and thus reducing binding of cationic colistin and other CAMPs ( 8 , 16 – 19 ). The arnBCADTEF gene cassette encodes the ʟ-Ara4N modification machinery and its expression is activated by the two-component system PhoPQ ( 2 ). Different alleles of phoP , phoQ, their promoter ( 8 , 17 ), the PhoPQ-regulating mgrB ( 8 ), or ecr ( 20 ), affect PhoPQ/ arn activation and the subsequent frequency of the resistant subpopulation. However, the underlying basis for the phenotypic heterogeneity exhibited within the cellular population of a colistin HR isolate has remained unresolved. In this work, we used a transposon-based mutagenesis screen and single-cell tracking to identify the alternative sigma factor σ E as the critical determinant of phenotypic heterogeneity in colistin HR in an Enterobacter clinical isolate. σ E responds to envelope stress and activates transcription of a large stress response regulon ( 21 ). We observed that the resistant subpopulation had distinctly high σ E activity which resulted in those cells surviving colistin treatment. σ E increased ʟ-Ara4N modification of lipid A and responded to antimicrobial peptide exposure. However, while σ E was the critical determinant of phenotypic heterogeneity, it was insufficient to cause HR, since a phoPQ mutant strain maintained heterogeneity but lost HR. Thus, we have identified σ E as the generator of heterogeneity in the population that, in tandem with the resistance enhancement conferred by PhoPQ and the Arn machinery, establishes a rare, colistin resistant subpopulation. This establishes a novel mechanistic paradigm where the distinct but combinatorial activity of two regulatory systems generates heteroresistance, an enigmatic yet increasingly recognized source of antibiotic failure in the clinic. RESULTS Colistin heteroresistance requires lipid A modification Enterobacter cloacae strain RS, a bloodstream isolate from a renal transplant patient, exhibits colistin HR and caused colistin treatment failure in a murine model of infection ( 16 ). Population analysis profile (PAP; Supp. Figure 1 ), the gold standard test to sensitively detect HR, which involves plating a range of dilutions of a bacterial strain at a range of concentrations of a given antibiotic, revealed that ∼1% of the total RS population (−2 log) was resistant to 4 μg/mL colistin, the CLSI breakpoint concentration used to discriminate susceptible from resistant strains. In contrast, the ColR2 strain demonstrated homogeneous resistance; every cell in the population survived at high colistin concentrations. ColS1 was susceptible to the colistin breakpoint concentration with no cells surviving ( Figure 1a , Supp. Figure 2 ). When exposed to colistin, the resistant subpopulation of cells in strain RS survived and were enriched ( Figure 1b ). However, characteristic of HR, when colistin was removed, the frequency of the resistant subpopulation returned to the baseline level within one passage, demonstrating that the phenotypically resistant subpopulation can be transiently enriched and is not the result of stable genetic changes that confer resistance ( Figure 1b ). Additionally, when the genome of the resistant subpopulation was compared to that of the majority susceptible population, no sequence variants were detected ( 16 ) and no gene copy number variation was observed (Supplemental Table 1), consistent with phenotypic differences between the resistant and susceptible cells that are not due to genetic changes. Download figure Open in new tab Figure 1. 4-amino-4-deoxy-ʟ-arabinose modification of lipid A is required for the resistant subpopulation in Enterobacter cloacae RS. (a) Population analysis profile (PAP) of strains RS, ColS1, and ColR2 plated on Mueller-Hinton agar (MHA) containing colistin; the proportion of surviving colonies is quantified relative to MHA containing no colistin, from three independent experiments with n=8 total biological replicates. (b) Quantification of the colistin resistant subpopulation of RS in media alone to exponential phase (0), subcultured into 100 μg/mL colistin and grown for 22 h (100), and subcultured into fresh media without colistin and grown to exponential phase (0). At each point, an aliquot was diluted and plated onto MHA containing 100 μg/mL colistin to quantify the resistant subpopulation, from two independent experiments with n=10 total biological replicates. **** indicates p<0.0001 by RM one-way ANOVA with Dunnet’s multiple comparisons correction, F (1.001, 9.009) = 283.8. (c) Model of the lipid A modifications in colistin-resistant cells, in which the arn operon is activated by PhoPQ, resulting in 4-amino-4-deoxy-ʟ-arabinose modification of lipid A and colistin resistance. (d-e) MALDI-TOF-MS spectra of strain RS following growth in (d) broth alone or (e) or 100 μg/mL colistin, a single spectra representative of 3 biological replicates is shown. (f) PAP of strain RS wildtype, Δ phoPQ , and Δ arnB mutants on Mueller-Hinton agar (MHA) containing colistin; from three independent experiments with n=9 total biological replicates for RS and Δ arnB , n=12 total biological replicates for Δ phoPQ . (g) P arn gfp expression intensity in strain RS wildtype, from three independent experiments; data were determined from 700-1000 cells for each experiment. While the majority susceptible population of cells did not have detectable ւ-Ara4N modifications to lipid A ( Figure 1d , Supp. Table 2 ), which can confer colistin resistance, these modifications were prominent after the resistant subpopulation was enriched in broth containing colistin ( Figure 1e , Supp. Table 2 ). Correspondingly, genetic deletion of arnB which encodes UDP-4-amino-4-deoxy-ʟ-arabinose-oxoglutarate aminotransferase, converts UDP-4-ketopentose to UDP-ʟ-Ara4N, and is required for ւ-Ara4N addition to lipid A ( 22 ), resulted in loss of the colistin resistant subpopulation ( Figure 1f ). Similarly, deletion of phoPQ , encoding the two-component system that activates expression of the arn operon ( 16 , 17 ), resulted in elimination of the highly resistant subpopulation ( Figure 1f , Supp. Figure 3a-b ). Further, mutants lacking arnB or phoQ exhibited no ւ-Ara4N modifications to lipid A ( Supp. Figure 3c-d ). Thus, the colistin resistant subpopulation is dependent on PhoPQ and ւ-Ara4N modification of lipid A. Activation of the arn operon promoter is not bimodal Since the arn genes are critical for the colistin resistant subpopulation, we hypothesized that heterogeneous activation of the arn promoter might generate the phenotypic heterogeneity in colistin HR in strain RS. If correct, we predicted ∼1% of the population would have high arn expression and demonstrate resistance, while ∼99% of the cells would have low arn expression and be susceptible. To test this hypothesis, we generated a chromosomally-encoded transcriptional reporter by fusing the arn promoter to gfp (P arn gfp ). We imaged RS::P arn gfp and found that, counter to our hypothesis, P arn gfp expression was not bimodal but rather unimodal with normal distribution ( Figure 1g ). These data demonstrate that while arn expression is required for colistin HR, its expression is not sufficient to generate population heterogeneity. Activation of the alternative sigma factor σ E pathway increases colistin resistance To attempt to identify the factors that generate the phenotypic heterogeneity in colistin HR, we first performed a transposon screen for mutants that do not survive on colistin at wildtype levels. Of ∼16,000 colonies screened, we identified 42 transposon mutants that demonstrated reduced frequency of the colistin resistant subpopulation. Validating our screen, we identified a mutant in arnD , part of the arnBCADTEF operon that is required for colistin resistance ( Supp. Table 3 ). We noticed that transposon mutants were identified in clpP , which encodes a protease adaptor involved in many cellular pathways, and sspB , encoding the stringent starvation protein B, which acts in conjunction with ClpP in the activation of the alternative sigma factor σ E (encoded by rpoE ) ( Figure 2a ). We therefore pursued the hypothesis that σ E may contribute to colistin HR. Clean deletion mutants of either clpP or sspB displayed increased susceptibility to colistin, further confirming the accuracy of the transposon screen, although the degree of susceptibility of the clpP mutant was much greater than that of sspB ( Figure 2b , Supp. Figure 4a ). An rpoE mutation was not identified in our screen, and we were unable to generate an rpoE deletion, consistent with its reported essentiality in E. coli ( 23 ). To further test the involvement of σ E , we instead deleted rseA , encoding its anti-sigma factor which restrains its activity, and rseB , which encodes a periplasmic protein that enhances RseA inhibition of σ E ( 24 , 25 ). Inactivation of RseA, and to a lesser extent RseB, is known to increase σ E activity ( 24 , 25 ). The frequency of the colistin resistant subpopulation increased in both the Δ rseA and Δ rseB mutants relative to wildtype ( Figure 2c ). In the case of the Δ rseA mutant, the frequency of the resistant cells reached almost 100% even when exposed to 100 μg/mL colistin ( Figure 2c , Supp. Figure 4b-c ). Taken together, the genetic inactivation of proteins which promote σ E activation (Δ clpP , Δ sspB ) resulted in a lower frequency of the colistin resistant population, and inactivation of proteins which restrain σ E activation (Δ rseA , Δ rseB ) increased the frequency of cells resistant to colistin. These results demonstrate the role of the σ E pathway in colistin HR. Download figure Open in new tab Figure 2. Activation of alternative sigma factor σ E pathway increases colistin resistance. (a) Model of σ E activation: following proteolytic degradation of its anti-sigma factor RseA and accessory protein RseB by DegS and RseP, σ E is liberated from the membrane. The protease ClpP with the adaptor protein SspB degrade the remaining RseA domain to allow σ E to transcriptionally regulate its large regulon. (b) Population analysis profile (PAP) of strain RS and Δ sspB and Δ clpP mutants. (c) PAP of strain RS and Δ rseA and Δ rseB mutants; (b-c) are from the same two independent experiments with n=6 total biological replicates for RS and Δ rseB and n=8 total biological replicates for the rest of the strains. (d) The micA promoter (σ E -controlled) was used to monitor σ E activity: P micA gfp expression intensity in wildtype strain RS, from three independent experiments; data were determined from 700-1000 cells for each experiment. σ E promoter activation is bimodal Having identified the σ E pathway as an important regulator of colistin HR, we returned to our hypothesis that bimodality in gene expression could generate HR. We therefore generated a chromosomally-encoded reporter of σ E activity using the promoter for a σ E -regulated gene, micA (RS::P micA gfp ) ( 26 ). We then microscopically imaged individual cells of the RS::P micA gfp strain and measured their fluorescence. We observed that σ E activity demonstrated bimodality, in which ∼1% of the population showed high P micA gfp expression ( Figure 2d ), which matches the measured percentage of colistin resistant cells when RS is grown in the absence of colistin ( Figure 1a ). Together, these data suggest that active σ E in a rare subpopulation of cells could generate the colistin resistant subpopulation. Rare σ E high cells constitute the colistin resistant subpopulation To investigate the relationship between σ E activity and survival on colistin, we next assessed the P micA gfp σ E reporter strain before ( Figure 3a ) and after addition of 8 μg/mL colistin ( Figure 3b, c ) and performed single-cell, time-lapse microscopy to track the fate of each cell upon colistin exposure (Supplemental Video). We designated each cell as susceptible or resistant based on its ability to replicate in the presence of colistin. Intriguingly, cells that did not express the P micA gfp reporter did not replicate in the presence of colistin, whereas only cells with high reporter expression prior to colistin exposure were able to grow and divide ( Figure 3d ). Further, when we encoded the P micA gfp reporter in the Δ rseA strain, which is made up almost exclusively of colistin resistant cells ( Figure 2c ), we observed a corresponding and robust increase in P micA gfp expression as compared to the parental wild-type RS strain ( Figure 3e ). Together, these data suggest that the resistant subpopulation in RS is generated by pre-existing, bimodal σ E activity prior to colistin exposure. Download figure Open in new tab Figure 3. σ E promoter activation is bimodal and predictive of colistin resistance. (a-c) Representative images of P micA gfp expression (a) prior to exposure to 8 μg/mL colistin, (b) after 2 h of colistin exposure and (c) after 4 h of exposure. (d-e) P micA gfp expression intensity in strain (d) RS wildtype or (e) the Δ rseA mutant prior to colistin exposure. The cell fate was monitored over time after adding 8 µg/mL colistin, and each cell was designated as susceptible or resistant. (d-e) are from three independent experiments; data were determined from 700-1000 cells for each experiment. Colistin heteroresistance conferred by σ E activation requires ւ-Ara4N modification It was unclear how activation of the σ E pathway led to colistin HR and if lipid A modifications were required. We performed PAP of a strain lacking both rseA and arnB and observed that deletion of arnB rendered the highly resistant Δ rseA strain susceptible to colistin ( Figure 4a , Supp. Figure 5 ). Next, we directly characterized the lipid A of the Δ rseA and Δ arnB rseA strains grown in the absence of colistin. Compared to wildtype RS, the Δ rseA strain demonstrated extensive modifications with ւ-Ara4N, consistent with its high frequency of colistin resistant cells. However, these modifications were absent in the Δ arnB rseA strain ( Figure 4b ). Thus, the colistin resistance conferred by constitutive activation of σ E requires arn -mediated lipid A modification. Since arn plays a critical role in lipid A modification and colistin resistance, we examined its expression at the single-cell level and its relationship with survival on colistin using the P arn gfp reporter in wildtype RS. While arn expression overall was unimodal ( Figure 1g ), we observed a trend where the cells with higher arn expression were more likely to survive on colistin ( Figure 4c ), although there was overlap in arn expression between the resistant and susceptible cells. This was in contrast to the results for micA expression which was bimodal and where there was no overlap between the resistant and susceptible populations. Download figure Open in new tab Figure 4. σ E confers colistin resistance by increasing arn levels. (a) Population analysis profile (PAP) of strain RS wildtype, and the Δ rseA , Δ arnB , and Δ arnB rseA mutants, from two independent experiments with n=7 total biological replicates. (b) MALDI-TOF-MS spectra of strain RS, Δ rseA, and Δ arnB rseA following growth in broth alone; a single spectra representative of 3 biological replicates is shown. Spectra of RS at top of B is reproduced from Figure 1d for the purpose of comparison. (c) P arn gfp expression intensity in strain RS wildtype or prior to colistin exposure. The cell fate was monitored over time after adding 8 µg/mL colistin, and each cell was designated as susceptible or resistant, from three independent experiments; data were determined from 700-1000 cells for each experiment. The σ E pathway generates heterogeneity but is not sufficient for heteroresistance PhoPQ is involved in arn induction and colistin HR in RS, yet it was not clear if or how its activity impacted σ E activation. The heterogeneous activation of σ E , as measured by the micA reporter, was maintained in the phoPQ mutant ( Figure 5a , compared to Figure 2d ), indicating that PhoPQ is not required for this phenomenon. This finding is also supported by the PAP of the phoPQ mutant, where a subpopulation of roughly 1 in 100,000 cells with a higher MIC than the main population was present at 0.25, 0.5, and 1 ug/ml colistin, demonstrating heterogeneity ( Figure 1f ). However, none of the cells in the phoPQ mutant were classified as colistin resistant, since none of them survived at the clinical breakpoint concentration of 4 μg/ml. This is consistent with vastly reduced arn reporter expression in the phoPQ mutant ( Figure 5b , compared to Figure 1g ). This highlights that 1) overall population phenotypic heterogeneity (heterogeneous survival) and heteroresistance (resistance of a subpopulation at the clinical breakpoint) are separable traits in strain RS, and 2) that PhoPQ is not required for phenotypic heterogeneity, but is required for the overall increase in MIC of a subpopulation to and beyond the clinical breakpoint, thus making RS heteroresistant to colistin. These results demonstrate that the σ E pathway is the generator of phenotypic heterogeneity in RS and PhoPQ is a generator of resistance, with the combined activity of these two regulatory systems being required to generate colistin heteroresistance. Download figure Open in new tab Figure 5. PhoPQ does not affect σ E pathway heterogeneity. (a) P micA gfp expression intensity and (b) P arn gfp expression intensity in strain RS Δ phoPQ , from three independent experiments; data were determined from 700-1000 cells for each experiment. DISCUSSION Clinical isolates are conventionally categorized as susceptible or resistant to a given antibiotic, classifications which omit the phenotypic heterogeneity observed in heteroresistance. Colistin HR is a frequent feature of clinical Enterobacter isolates, is often undetected by conventional antibiotic susceptibility testing, and can cause treatment failure in murine models of infection. However, it was still unclear what generated the phenotypic heterogeneity in this system. Here, we performed a transposon screen and identified a critical role for the alternative sigma factor σ E in colistin HR ( Figure 2 ). In a population of wildtype cells of strain RS, prior to colistin exposure, ∼1% of the population exhibited increased σ E activity, making up a distinct second peak in the bimodal distribution of σ E activity ( Figure 2d ). Single-cell tracking revealed that exclusively these cells with high σ E activity survived colistin exposure ( Figure 3d ). Although PhoPQ has been strongly linked to colistin HR, we were surprised to discover that this system is not the generator of phenotypic heterogeneity, which was maintained in a phoPQ mutant strain ( Figure 5a ). However, the heterogeneity was lost, as evidenced by a unimodal curve of σ E activity, when σ E was constitutively activated via deletion of the σ E inhibitory anti-sigma factor, rseA ( Figure 3e ), further demonstrating that the σ E pathway is the generator of heterogeneity. While phenotypic heterogeneity was dependent on the activity of the σ E pathway, HR to colistin nonetheless required PhoPQ as none of the cells of the phoPQ mutant survived at the colistin clinical breakpoint ( Figure 1f ). Importantly, the cells surviving colistin exposure also had higher arn expression ( Figure 4c ), which required PhoPQ ( Figure 5b ). These data highlight that PhoPQ is not required for phenotypic heterogeneity, but is required for the overall increase in MIC of a subpopulation to the clinical breakpoint, thus generating colistin HR. Interestingly, these data demonstrate that overall population phenotypic heterogeneity (heterogeneous survival) and heteroresistance (resistance of a subpopulation at the clinical breakpoint) are separable traits in strain RS. Further, these results reveal that the σ E pathway is the generator of phenotypic heterogeneity in RS, and PhoPQ is a generator of resistance, with the combined activity of these two regulatory systems being required to generate colistin heteroresistance. Together, these data elucidate a novel mechanistic paradigm for heteroresistance via the combinatorial activity of distinct regulatory pathways ( Figure 6 ). Download figure Open in new tab Figure 6. Proposed model for bimodality in σ E generating a colistin-resistant subpopulation. (a) A proposed model for the activity of signaling pathways active in the subpopulation of cells with pre-existing colistin resistance. PhoPQ directly activates transcription of the arn operon, while activation of σE additionally directly or indirectly increases arn transcription, resulting in lipid A modification that prevents colistin entry. (b) Prior to colistin exposure, arn promoter activity follows a broad unimodal expression, while σ E promoter activity is distinctly bimodal. High σ E cells have higher arn promoter activation and are colistin-resistant, while cells with moderately high P arn activity but low σ E activation remain colistin-susceptible. This combinatorial regulatory model of HR is quite distinct from the best understood mechanism of HR in which one event, an increase in copy number of a given single gene, generates HR. The clearest and most commonly observed example thus far has been the tandem gene amplification of a DNA segment including a beta-lactamase gene and leading to overproduction of the beta-lactamase in the cells in the population with increased gene copy number. This mechanism can generate a broad continuum of cellular subpopulations resistant to a given beta-lactam antibiotic ( 27 ). It should be noted that gene amplification is capable of causing colistin HR as this was observed in one instance in S. Typhimurium ( 28 ), although this does not appear to be a common mechanism at least among Enterobacter isolates. Interestingly, using the rseA mutant in which σ E activity is constitutive, we observed that the σ E pathway can also impact arn expression. In the rseA mutant, the entire population demonstrated a shift to increased arn expression ( Supp. Figure 6g ) and colistin resistance ( Figure 2c ). However, phoPQ deletion did not significantly reduce arn expression in the rseA mutant ( Supp. Figure 6c , Supp. Figure 7 ) and only led to a modest reduction in the frequency of the resistant subpopulation to ∼1 in 10 cells ( Supp. Figure 6a ). This was in contrast to the phenotype of the rseA/arnB double mutant where no resistant cells remained ( Figure 4a ). This highlights that in the setting of robust, constitutive σ E activation, arn plays a more critical role in colistin HR than PhoPQ, and that σ E activation can induce arn . Since many host antimicrobial peptides are highly cationic, similar to colistin, we hypothesized that antimicrobial peptide treatment would activate the σ E pathway. When strain RS was treated with the human antimicrobial peptide LL-37, we observed an increase in transcript abundance of rpoE and rseA , consistent with activation of the σ E pathway ( Supp. Figure 8 ). These conditions also activated the canonical PhoPQ response to LL-37, as measured by expression of the PhoPQ-activated mgtA transcript and arnB ( Supp. Figure 8a ). Consistent with a σ E pathway response to LL-37, the Δ rseA strain was more resistant to LL-37 killing than wildtype ( Supp. Figure 8b ). Thus, these data demonstrate that σ E is involved not only to survival in colistin, but also in response to host antimicrobial peptides. It is not clear why lipid A is not modified by ʟ-Ara4N in every cell, constitutively., leading to homogeneous resistance to colistin and other CAMPs. There is a fitness cost associated with constitutive σ E activation, which is toxic in E. coli (reviewed in ( 21 ). Thus, HR may serve as a form of bet-hedging, in which a dynamic pre-existing subpopulation can co-exist with other phenotypically diverse subpopulations, and can be transiently enriched when the selective pressure is present. The mammalian host environment is one such environment, as the colistin resistant subpopulation in strain RS is enriched during murine infection by the selective pressures of lysozyme, NADPH oxidase, and the antimicrobial peptide mCRAMP, even in the absence of colistin treatment ( 16 ). Thus, bimodality in σ E activation may confer on the population an advantage in resistance to antibiotic and host antimicrobial stresses, without a commitment of the entire population to costly LPS modifications. Our model of combinatorial regulatory signaling converging on the arn resistance determinant is consistent with work on colistin HR in other Enterobacter , where inactivating PhoPQ/ arn eliminates the resistant subpopulation, and manipulating PhoPQ activity changes the frequency of the resistant subpopulation, but not the baseline heterogeneity in the population ( 8 , 17 ). In sum, we have identified a transcriptional regulator that generates phenotypic heterogeneity to colistin in the population. This heterogeneity facilitates HR when the PhoPQ two-component system increases the overall resistance level of all the cells in the population. Thus, this combinatorial regulatory model is a new paradigm in the mechanistic generation of HR, a phenomenon which is increasingly recognized as a cause of antibiotic treatment failure in the clinic. Funding This work was supported by funding from the National Institutes of Health [AI158080 (DSW and MK), AI141883 (DSW)] and the National Institutes of Health’s Office of the Director, Office of Research Infrastructure Programs, P51 OD011132. DSW is supported by a Burroughs Wellcome Fund Investigators in the Pathogenesis of Infectious Disease award. JEC was supported by the Cystic Fibrosis Foundation and National Institutes of Health [T32 DK108735]. Author contributions Conceptualization: JEC, EKC, MM, MK, DSW. Investigation: JEC, EKC, MM, LMV, MKK. Visualization: JEC, MM, LMV. Funding acquisition: MK, RKE, DSW. Supervision: MK, RKE, DSW. Writing-original draft: JEC and DSW. Writing-review and editing: all authors Competing interests The authors declare that they have no competing interests Data and materials availability all data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Source data is provided as a Supplementary File. MATERIALS AND METHODS Statistical analysis and data presentation The number of experiments and number of biological replicates are detailed in the figure legends. PAP graphs show the mean and standard deviation; alternative graphs showing individual data points are in Supplemental Figures. Other graphs show each data point and mean, and error bars indicate standard deviation as applicable. Violin plots show median and quartiles. Statistical analysis was performed in Graphphad Prism version 10.4.2 and details of each test are in figure legends. Isolate information Enterobacter cloacae complex strain RS was isolated from a blood sample from a renal transplant recipient at Emory University Hospital, Atlanta, GA and described previously ( 16 , 29 ). The RS genome is available in NCBI Genbank, accession numbers CP010512 and CP010513 . E. cloacae ColS1 and ColR2 are clinical isolates and described previously ( 29 ). Strains and plasmids in this paper are detailed in Supp. Table 4 . Reagents Mueller-Hinton agar (MHA; BD Difco) and Mueller-Hinton broth (MHB; BD Difco) were used throughout as indicated. For growth of E. cloacae RS before plating on MHA containing colistin, bacteria were grown at 37°C with shaking, 1.5 mL volume in 12x75 mm polypropylene aeration tubes (Globe Scientific #110438). YESCA was made by autoclaving 1 g/L yeast extract (Fisher BP1422) and 10 g/L casamino acids (Acros #61204) in water. YESCA agar included 20 g/L agar (Sigma A1296). Antibiotics used were: colistin sulfate (Sigma #C4461), kanamycin sulfate (Teknova #K2150; 90 µg/mL for strain RS), tetracycline (Alfa Aesar #J61714; 20 µg/mL for RS), chloramphenicol (Fisher #BP904, 50 µg/mL for RS) and spectinomycin dihydrochloride pentahydrate (Sigma #S4014; 50 µg/mL for E. coli , 300 µg/mL for RS). Medium gel pads used for cell imaging under microscope: UltraPure TM agarose (Invitrogen #16500100) and MHB was used to prepare MHB 1.5% agarose gel pad. Population analysis profile Population analysis profile (PAP) was performed as indicated in Supp. Figure 1 . A given strain was grown overnight from a single colony streaked to MHA from -80°C glycerol stocks. After approximately 16-20 h of growth, the culture was diluted 1:1000 (except for strains with rseA or clpP mutation, which were diluted 1:250 to account for diminished growth yield of these strains) into fresh media and grown until OD reached ∼0.3. The cultures were serially diluted in PBS in a 96-well plate (Falcon) and 7.5 or 10 µl of each dilution was plated on MHA containing colistin as indicated. Colonies were enumerated after 24-48 h of growth. For many experiments shown, the same protocol was followed but the cultures were plated only on MHA containing 0 or a single colistin concentration indicated. The surviving colonies are enumerated and the isolate is classified as resistant if at least 50% of the total colonies grow at 1 or 2X breakpoint. An isolate is considered susceptible if less than 0.0001% (−6 logs) of the cells grow at any concentration shown. An isolate is considered heteroresistant if there is greater than 0.0001% survival at 1X and 2X breakpoint and an ≥8-fold difference between the resistance of the subpopulation and resistance of the main population. As an example for RS, <50% of the population survives at 0.125 µg/mL, thus the MIC susceptible population is 0.125 µg/mL. The subpopulation survives up to 100 µg/mL, so the MIC resistant population of the resistant population is > 100 µg/mL. For RS the fold change of the MIC resistant population relative to MIC susceptible population is >8. The limit of detection is approximately -5.5-6 logs but varies based on the density of the culture, in the figures the y-axis is set to the approximate limit of detection for each graph. In defining the features of heteroresistance in the isolates of this work, based on guidelines set forth by the field ( 6 , 9 , 30 ): Clonality: these isolates demonstrate monoclonal heteroresistance, they are purified isolates and single colonies are used throughout experiments. Level of resistance: the MIC of the resistant subpopulation in wildtype RS is ≥8X the MIC of the main population, when comparing the amount of killing by the lowest concentration of colistin used throughout, and the growth of the resistant subpopulation at 100 µg/mL. Frequency of the resistant subpopulation: We consider the frequency of the subpopulation at 2X the CLSI breakpoint, for wildtype RS the frequency is ∼0.1-1%. Instability in the frequency of the resistant subpopulation: wildtype RS demonstrated unstable heteroresistance. As shown in the passage experiments after selection, there was a significant reduction in the resistant population frequency within a single passage (∼10 generations per passage) in antibiotic free media. Copy number variation sequencing analysis Illumina sequencing of samples of RS previously grown in Mueller Hinton broth containing 0 or 100 μg/mL colistin ( 16 ) were accessed from NCBI SRA. Reads were mapped to the respective reference genome with CNV analysis. Quality control and adapter trimming was performed with bcl2fastq( 31 ). Reads were mapped to their respective references via bwa mem( 32 ). PCR and optical duplicates were marked and excluded from the analysis using PicardTools’ ‘MarkDuplicates’( 33 ) functionality. Aligned read counts were imported into R’s CNOGpro( 34 )package. CNV events were called via CNOGpro using a bootstrapping method, which calculates an average gene number event giving a possible upper and lower bound. Genome sequencing and analysis were performed by SeqCenter ( https://seqcenter.com ). Supplemental Table 1 details analysis results as well as SRA accessions for each sample. Resistance stability assays RS was streaked from -80°C glycerol stocks to MHA for isolation and a single colony was used to start overnight cultures in 1.5 mL of MHB and grown 15-20 h. 15 µl of the cultures were diluted into 1.5 mL of MHB and grown until OD reached ∼0.3-0.4. These cultures were diluted and plated MHA containing 0 or 100 µg/mL colistin, the baseline. 15 µL of the cultures were added to 1.5 mL fresh MHB containing 100 µg/mL colistin and grown for 20 h. Following growth, the cultures were diluted and plated MHA containing 0 or 100 µg/mL colistin. 1.5 µL of the cultures were added to 1.5 mL fresh MHB and grown until OD reached ∼0.3-0.4. Following growth, the cultures were diluted and plated MHA containing 0 or 100 µg/mL colistin. Lipid A mass spectrometry Sample growth: strain RS and indicated mutants were grown overnight in 1.5 mL MHB and 120 µL was added to 12 mL fresh MHB in a 50 mL conical tube and grown for 4 h, then harvested by centrifugation. For RS grown in colistin, 100 µL of overnight culture was added to 10 mL MHB containing 100 µg/mL colistin in a 50 mL conical tube and grown for 9 h, then harvested by centrifugation. Cells were washed in PBS and stored in -80°C. Fast lipid analysis technique (FLAT)( 35 ) was adapted as follows: bacteria were scraped from frozen pellets and spotted directly onto a steel MALDI plate. 1uL of FLAT extraction buffer (0.2 M anhydrous citric acid, 0.1 M trisodium citrate dihydrate) was spotted on top of the plated bacteria. The MALDI plate was placed into a humidified 100 °C “panini-press” chamber for 30 minutes. Afterwards, the plate was rinsed with endotoxin-free water and air dried. 1uL of Norharmane matrix (10 mg/mL in 2:1 (v/v) chloroform-methanol solution) was spotted on each sample. Samples were analyzed in linear, negative ion mode on a Bruker Microflex LRF. Data was processed with flexAnalysis software. Imaging of cells and GFP expression RS wildtype or mutants encoding att :: P arn gfp+ or att :: P micA gfp+ were used to visualize population-level variation in promoter activation prior to colistin exposure, and to assess viability following treatment. Imaging of cells and GFP expression was performed as previously described ( 36 , 37 ). When a cell culture reached an OD 600 of approximately 0.1, cells were transferred onto a 35 mm glass-bottom Petri dish (Cellvis) and overlaid with Mueller-Hinton Broth (MHB) gel pad containing 1.5% agarose. Cells were imaged using an Olympus IX83 inverted microscope with a 60× oil immersion phase-contrast objective, housed within a temperature-controlled (37 °C) incubation chamber (InVivo Scientific). GFP fluorescence was detected using a GFP filter cube. Snapshot imaging of cells was performed before exposure to colistin. Time-lapse images of cells were captured at 30-minute intervals after adding colistin to the agarose pad covering cells. Image analysis Fluorescence intensity of GFP was measured using a plug-in for Fiji ImageJ software, named MicrobeJ ( 38 ) (version 5.13 I ( 22 )). Reported fluorescence intensities represent intracellular GFP signals after subtracting both extracellular background and the autofluorescence of wildtype RS cells. Cell viability under colistin treatment was determined through time-lapse imaging. Continuous cellular division and colony formation represented survival. Killing of cells by colistin was defined as visible lysis or permanent arrest of cell growth. Transposon screen 750 µl of a culture of a RS att ::P arn gfp + was combined with 750 µl E. coli BW19851 carrying pFD1, collected by centrifugation and resuspended in 50 µl YESCA (yeast extract casamino acids broth). The suspension was plated on a 0.45 µM nitrocellulose membrane placed on top of a YESCA agar plate and incubated at 37°C for 2.5 h. The nitrocellulose was washed in a 15 ml tube with YESCA containing 1 mM IPTG and 300 µg/mL spectinomycin, grown for 3 h at 37°C, diluted and plated on MHA containing 300 µg/mL spectinomycin and 90 µg/mL kanamycin. After 24 h of growth at 37°C, colonies were replica-plated using sterile velvet onto MHA containing kanamycin or kanamycin and 100 µg/mL colistin. Colonies that appeared on kanamycin but not kanamycin+colistin were restreaked for isolation. To identify the transposon insertion site, colony PCR was performed using primers 71/72 to yield the DNA flanking the transposase. 2 µl of the PCR product from JCP71/72 was the template for subsequent nested PCR with JCP73/74. PCR products were Sanger sequenced (Azenta) using JCP74 and aligned to the RS genome. Strain construction: gene deletions Lambda-red based allelic exchange was used to replace the coding sequence of genes with a kanamycin resistance gene as indicated. The kanamycin resistance gene from pEXR6K_kanFRT was cloned using Promega GoTaq 2X master mix with Flp recognition sequence and homology to regions flanking the gene to be replaced using primers JCP171/172 ( phoPQ ), EKC202/203 ( arnB ), EKC301/302 ( sspB ) JCP59/60 ( clpP ) EKC309/310 ( rseA ) and JCP56/57 ( rseB ), detailed in Supp. Table 5 . The purified PCR product was electroporated into competent RS (or mutant) carrying pKD46-tet and transformants were selected on kanamycin. Transformants were re-streaked to kanamycin for isolation and subject to PCR for successful allelic exchange as indicated by a product size change using primers flanking the gene to be replaced: primers JCP173/174 ( phoPQ ), EKC208/209 ( arnB ), EKC303/304 ( sspB ) JCP61/62 ( clpP ) EKC311/312 ( rseA ) and JCP58/EKC311 ( rseB ) detailed in Supp. Table 5 . As indicated, the kanamycin resistance gene was removed with Flp recombinase( 39 , 40 ) to create unmarked in-frame deletions The mutants were then made electrocompetent and electroporated with pCP20( 40 ). Transformants were selected at 30°C on chloramphenicol, patched to chloramphenicol at 30°C and grown for 24 h, then patched to MHA and MHA+kanamycin. Kanamycin-sensitive mutants were streaked for isolation and PCR was used with the same flanking primers to screen for the loss of the kanamycin resistance gene. Strain construction: chromosomally integrated plasmids For P rmicA gfp , pCD13PSK was digested with BamHI, the micA promoter region was an ultramer JCP41, and promoterless gfp+ was cloned with JCP31/42 from the vector pZEP07 ( 41 ) as template. For P arn gfp, pCD13PSK gfp + was amplified with JCP556/557 and the arn promoter cloned with JCP558/559. For pCD13PSK phoPQ , P phoP phoPQ was cloned with EKC223/235 and assembled with pCD13PSK digested with XhoI and XbaI. For pCD13PSK arnBCADTEF , the entire arn operon with native promoter was cloned with JCP67/68 and assembled with pCD13PSK digested with BamHI and SacI. For each construct, PCR and plasmid components were assembled with NEB Hifi assembly according to manufacturer’s instructions and transformed. Plasmids were propagated in E. coli PIR2 with spectinomycin, and Sanger sequenced (Azenta) using primers JCP49/50. RS wildtype or indicated mutants were made electrocompetent, transformed with pINT-ts-tet and selected for with tetracycline. Strains carrying pINT-ts-tet were made electrocompetent and transformed with appropriate pCD13PSK plasmid. Of note, following 1 h of recovery at 37°C, the culture was moved to 42°C for 30 min before plating on spectinomycin. Integration of pCD13PSK at the attB site was confirmed by colony PCR using JCP51/53. To additionally confirm, the region of the chromosome encoding the cloned genes and pCD13PSK with amplified with JCP51/539 and Sanger sequenced (Azenta) using primers JCP49/50. For experiments, spectinomycin was not used because the integration is stable ( 42 ). Strain construction: plasmid-borne complementation P rpoE rpoE-rseA was cloned with JCP554/582 and assembled with pBAV amplified with JCP280/552 Plasmids were propagated in E. coli NEB5α, Sanger sequenced (Azenta) using primers JCP339/340, and electroporated into appropriate strain with kanamycin selection. Quantitative PCR For LL-37 treatment: RS was grown overnight (16-18h) in MHB, subsequently 6 µl of culture was added to fresh 3 mL MHB and grown to OD 600 of approximately 0.3. 50 µg/mL human cathelicidin LL-37 (Elabscience, E-PP-1466) was added for 30 minutes. 100 µl aliquots were taken before and after LL-37 exposure and plated on MHA following serial dilution. RNA was isolated using RNeasy Mini Kit (Qiagen #74104) then treated with DNAse (BioLab #M0303S) according to manufacturer’s instructions. RNA was stored at -80 °C.Reverse transcriptase quantitative PCR was performed with Power SYBR Green RNA to CT 1-Step Kit (Applied Biosystems #4389986). cys G served as the housekeeping gene for normalization. cysG (JCP241/242), mgtA (mgtA-1F/R) , arnB (arnB-3F/R) , rseA (rseA-2F/R), and rpoE (JCP608/609) were detected by primers detailed in Supp. Table 5 . Fold change was determined for each biological replicate by comparison to the same replicate without LL-37 exposure. Fold change was calculated using the 2 -ΔΔCT method( 43 ). LL-37 killing assay Strains RS and the Δ rseA mutant were streaked to MHA from -80°C glycerol stocks. After overnight growth, a colony was inoculated into 1.5 mL MHB. After approximately 16-20 h of growth, the culture was diluted 1:1000 for RS and 1:500 for Δ rseA into 1 mL fresh MHB, and an aliquot was taken, serially diluted, and plated on MHA for enumeration. After 3.5 h of growth an aliquot was taken for enumeration, then 100 μg/mL of LL-37 (Elabscience, E-PP-1466) was added. At 0.5, 1, 2, and 3 h following treatment, aliquots were taken for CFU enumeration. SUPPLEMENTAL MATERIAL Download figure Open in new tab Supplemental Figure 1. Overview of heteroresistance and population analysis profile (PAP). (a) A depiction of the cells grown from a single colony of an isolate exhibiting conventional resistance, heteroresistance, or susceptibility to a given antibiotic. (b) Population dynamics of a heteroresistant isolate following antibiotic treatment. (c) Colistin PAP: a clinical isolate is isolated and grown in broth, then subcultured into fresh broth and grown to exponential phase. The culture is then serially diluted, plated on agar with increasing concentrations of colistin, and incubated. (d) The surviving colonies are enumerated and the proportion of total colonies that survive on a given concentration is calculated: . The isolate is classified as resistant if at least 50% of the total colonies grow at 1 or 2X breakpoint. An isolate is considered susceptible if less than 0.0001% (−6 logs) of the cells grow at any concentration shown. An isolate is considered heteroresistant if there is greater than 0.0001% survival at 1X and 2X breakpoint and an ≥8-fold difference between the resistance of the subpopulation and resistance of the main population. As an example, if 16 µg/mL, making the fold change ≥32. Along the x-axis, the breakpoints based on CLSI are shown in gray, and the corresponding concentration of colistin is in black. Because the culture density of exponential phase cultures is generally lower, in data presented in this manuscript the limit of detection is often between -5.5 to -6.5 logs. Throughout, the y-axis is set to a limit of detection for each experiment presented. Download figure Open in new tab Supplemental Figure 2. Alternative presentations of PAP of strain RS, ColS1, ColR2 from Figure 1a showing each data point, from three independent experiments with n=8 total biological replicates. Download figure Open in new tab Supplemental Figure 3. PhoPQ and ArnB are required for heteroresistance E. cloacae strain RS. (a) Alternative presentations of PAP of strain RS wildtype, Δ phoPQ , and Δ arnB mutants from Figure 1f on Mueller-Hinton agar (MHA) containing colistin; from three independent experiments with n=9 total biological replicates for RS and Δ arnB , n=12 total biological replicates for Δ phoPQ . (b) Proportion of the population surviving on 100 µg/mL colistin of the strains indicated, from two independent experiments n=6 total biological replicates. (c-d)) MALDI-TOF-MS spectra of strain (c) Δ phoQ, and (d) Δ arnB following growth in broth alone; a single spectra representative of 3 biological replicates is shown. Download figure Open in new tab Supplemental Figure 4. Activation of σ E confers colistin resistance. (a) Alternative presentation of PAP data from strain RS and Δ sspB and Δ clpP mutants in Figure 2b . (b) Alternative presentation of PAP data from strain RS and Δ rseA and Δ rseB mutants in Figure 2c ; (a-b) are from the same two independent experiments with n=6 total biological replicates for RS and Δ rseB and n=8 total biological replicates for the rest of the strains. (c) Proportion of the population surviving on 100 µg/mL colistin of the strains indicated, from two independent experiments with n=6 total biological replicates. Download figure Open in new tab Supplemental Figure 5. Resistance conferred by rseA deletion requires arn . (a) Alternative presentation of PAP of strain RS wildtype, and the Δ rseA , Δ arnB , and Δ arnB rseA mutants from Figure 4a , from two independent experiments with n=7 total biological replicates. (b) Proportion of the population surviving on 100 µg/mL colistin of the strains indicated, from two independent experiments with n=6 total biological replicates. Download figure Open in new tab Supplemental Figure 6. PhoPQ independent colistin resistance in Δ rseA background. (a) PAP of strain RS wildtype and the Δ rseA , Δ phoPQ , and Δ rseA Δ phoPQ mutants; from three independent experiments with n=9 total biological replicates for RS and Δ arnB , n=11 for Δ rseA , and n=12 total biological replicates for Δ rseA Δ phoPQ and Δ phoPQ . Data for RS and Δ phoPQ are also shown in Figure 1f . (b-g) gfp expression intensity, driven by the promoter indicated, in strain backgrounds as indicated, among cells prior to colistin exposure. The cell fate was monitored over time after adding colistin at the concentrations indicated, and each cell was designated as susceptible or resistant, from three independent experiments; data were determined from 700-1000 cells for each experiment. Panels d,e,f, and g are reproduced from Figures 3d , 3e, 4c and 4d, respectively, for the purpose of comparison. Download figure Open in new tab Supplemental Figure 7. Colistin resistance in Δ rseA Δ phoPQ . (a) Alternative presentation of PAP in Supp. Fig. 8a , of strain RS wildtype and the Δ rseA , Δ phoPQ , and Δ rseA Δ phoPQ mutants; from three independent experiments with n=9 total biological replicates for RS and Δ arnB , n=11 for Δ rseA , and n=12 total biological replicates for Δ rseA Δ phoPQ and Δ phoPQ . Data for RS and Δ phoPQ are also shown in Supplemental Figure 3a. (b) Proportion of the population surviving on 100 µg/mL colistin of the strains indicated, from two independent experiments with n=6 total biological replicates, except for n=7 for Δ rseA Δ phoPQ att :: pBAV . Download figure Open in new tab Supplemental Figure 8. The σ E pathway is activated by LL-37 and confers resistance. (a) Quantitative PCR of transcripts indicated after 30 minutes of 50 µg/mL LL-37 exposure in exponential phase cultures of strain RS wildtype, from two independent experiments with n=7 total biological replicates. *** indicates p=0.0008, ** ( mgtA ) indicates 0.0077, ( rpoE ) 0.0089, ( rseA ) 0.0084 by RM one-way ANOVA with Dunnet’s multiple comparisons correction, F (1.964, 11.78) = 18.82. (b) Culture density of strain RS wildtype and the Δ rseA mutant in broth. Stationary phase cultures were diluted into fresh broth and grown for 3.5 h. 100 µg/mL LL-37 was added (indicated by arrow) and culture density was measured over time. ** indicates p=0.0011, **** p<0.0001, and *** p=0.0001 by ordinary one-way ANOVA with Šídák’s multiple comparisons test, comparing strains at each time point, F ( 9 , 60) = 12.49. From two independent experiments with n=7 total biological replicates. View this table: View inline View popup Download powerpoint Supplemental Table 2: theoretical m/z units of lipid A and modified lipid A. View this table: View inline View popup Download powerpoint Supplemental Table 3. Transposon screen mutant identification View this table: View inline View popup Download powerpoint Supplemental Table 4: Strains and plasmids View this table: View inline View popup Download powerpoint Supplemental Table 5: Primers ACKNOWLEDGMENTS pBAV1K-T5-gfp was a gift from Ichiro Matsumura (Addgene plasmid # 26702; http://n2t.net/addgene:26702 ; RRID:Addgene_26702). We thank Edgar Sherman for generating the Δ arnB rseA strain. Funder Information Declared National Institute of Allergy and Infectious Diseases, https://ror.org/043z4tv69 , AI158080 , AI141883 National Institutes of Health , P51 OD011132 Burroughs Wellcome Fund, https://ror.org/01d35cw23 Cystic Fibrosis Foundation, https://ror.org/00ax59295 National Institute of Diabetes and Digestive and Kidney Diseases, https://ror.org/00adh9b73 , T32 DK108735 REFERENCES 1. ↵ A. Sabnis et al. , Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane . Elife 10 , ( 2021 ). 2. ↵ B. W. Simpson , M. S. Trent , Pushing the envelope: LPS modifications and their consequences . Nat Rev Microbiol 17 , 403 – 416 ( 2019 ). 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Wanner , Conditional-replication, integration, excision, and retrieval plasmid-host systems for gene structure-function studies of bacteria . J Bacteriol 183 , 6384 – 6393 ( 2001 ). OpenUrl Abstract / FREE Full Text View the discussion thread. Back to top Previous Next Posted August 12, 2025. 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 Combinatorial action of regulatory systems generates colistin heteroresistance 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. 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