Molecular bases for the loss of type VI secretion system activity during enteroaggregative E. coli experimental evolution

Molecular bases for the loss of type VI secretion system activity during enteroaggregative E. coli experimental evolution | 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 Molecular bases for the loss of type VI secretion system activity during enteroaggregative E. coli experimental evolution View ORCID Profile Boris Taillefer , View ORCID Profile Jonas B. Desjardins , View ORCID Profile Eric Cascales doi: https://doi.org/10.1101/2025.02.08.637259 Boris Taillefer 1 Laboratoire d’Ingénierie des Systèmes Macromoléculaires (LISM), Institut de Microbiologie, de la Méditerranée (IMM) , Aix-Marseille Univ – CNRS, UMR7255, 31 Chemin Joseph Aiguier CS7071, 13402 Marseille Cedex 09, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Boris Taillefer Jonas B. Desjardins 1 Laboratoire d’Ingénierie des Systèmes Macromoléculaires (LISM), Institut de Microbiologie, de la Méditerranée (IMM) , Aix-Marseille Univ – CNRS, UMR7255, 31 Chemin Joseph Aiguier CS7071, 13402 Marseille Cedex 09, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jonas B. Desjardins Eric Cascales 1 Laboratoire d’Ingénierie des Systèmes Macromoléculaires (LISM), Institut de Microbiologie, de la Méditerranée (IMM) , Aix-Marseille Univ – CNRS, UMR7255, 31 Chemin Joseph Aiguier CS7071, 13402 Marseille Cedex 09, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Eric Cascales For correspondence: cascales{at}imm.cnrs.fr Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT The type VI secretion system (T6SS) is a nanoweapon deployed by Gram-negative to inject effectors into target cells and hence involved in pathogenesis and bacterial competition. While T6SS gene clusters are found in all recently isolated commensals or pathogenic Escherichia coli strains, they are absent from classical laboratory strains. These E. coli strains, which have been used since decades for bacterial genetics, were usually grown in pure cultures, suggesting that T6SS might have been lost during evolution in absence of competitors. Here, we conducted a 640-generation experimental evolution by passaging the enteroaggregative E. coli (EAEC) 17-2 strain under controlled competition conditions against susceptible or immune recipient cells. EAEC T6SS activity was almost abolished when grown in the presence of immune recipients, while no difference with the ancestral strain was observed for EAEC grown in the presence of susceptible cells. Whole genome sequencing of 18 clones identified adaptative mutations responsible for the loss of T6SS activity, including mutations of the T6SS promoter and within genes encoding T6SS subunits, and revealed a novel regulatory mechanism involving RfaH-dependent antitermination. We further found that the RfaH binding site ops element is present in T6SS gene clusters of several species, suggesting a conserved RfaH-dependent regulation of T6SSs across enterobacteria. This work exemplifies the power of experimental evolution to understand T6SS genetic adaptation and to unravel new players for its function. INTRODUCTION The type VI secretion system (T6SS) is a nanoweapon that delivers effectors into prokaryotic and eukaryotic cells, participating to interbacterial competition and virulence ( 1 ). The T6SS belongs to the broad family of contractile injection systems, which use a spring-like mechanism to propel an inner tube loaded with effectors. The T6SS is comprised of 14 core proteins that assemble two subcomplexes with distinct evolutionarily histories. In Proteobacteria, the membrane complex is constituted of the TssJLM proteins that recruits and anchors a cytoplasmic phage-like tubular structure. This tubular structure is constituted of a baseplate composed of TssEFGK wedges polymerized around the VgrG-PAAR spike complex, which serves as assembly platform for the inner tube wrapped by the contractile tube. Once assembled, the T6SS sheath contracts, propelling an effector-loaded needle towards the target cell. The sheath proteins are then disassembled and recycled by the dedicated ClpV ATPase ( 2 ). Effectors delivered by the T6SS are either released in the environment, such as metallophores that chelate metals, or directly injected into target cells. We distinguish toxins with eukaryotic-specific (e.g., actin cross-linking), antibacterial-specific (e.g., peptidoglycan hydrolases, FtsZ-, 23S RNA- or EF-Tu-targeting ADP-ribosyltransferases), and trans-kingdom activities (e.g., DNases, proteases, phospholipases, NADH glycosylhydrolases) ( 3 ). T6SS + bacteria species delivering antibacterial toxins protect themselves by the production of immunity proteins that usually bind and inhibit their cognate toxins. T6SS genes are clustered in 20-70-kb large operons that are comprised of genes encoding the structural subunits of the apparatus, the toxins with the cognate immunities, and accessory proteins ( 4 – 8 ). These accessory proteins might be transcriptional or post-translational regulators, chaperones, adaptor proteins or proteins that facilitate the assembly or the mechanism of action of the T6SS. T6SS gene clusters are widespread in Gram-negative bacteria with an overrepresentation in Bacteroidetes and Proteobacteria ( 6 , 9 – 11 ). It is not only found in pathogens, but also in environmental species or commensal and symbionts strains. T6SS gene clusters are also abundant in pathogenic or commensal Escherichia coli species including all pathovar types or clinical isolates and categorize in the T6SS i -1, -2 and -3 families ( 12 ). Strikingly they are absent of all classical laboratory strains of E. coli such as MC4100, MG1655, W3110 or DH5α. Most of these strains derive from strains W1485 and 58-161 from Joshua Lederberg and Edward Tatum, which themselves originate from strain NCTC86 or K-12 isolated by Theodor Escherich in 1885, or J.E. Blair in 1922, respectively ( 13 ). Interestingly, the genome sequence of strain NCTC86 showed that it carries one copy of a T6SS gene cluster, typical of the T6SS i -2 group found in E. coli strains ( 14 ). The observation that E. coli laboratory strains are devoid of T6SS genes whereas original strains from which they derive and recently isolated strains carry one or several copies of clusters raised the question of how T6SS genes were lost upon years or decades of pure subcultures in rich media ( 15 ). To reveal the mechanisms underlying loss of T6SS, we conducted an experimental evolution of the enteroaggregative E. coli (EAEC) strain 17-2 in a medium in which the Sci1 T6SS is active. This approach, which consists of subjecting a population to serial passages in a specific and controlled condition, allows to identify mutations that are responsible for a fitness gain or for adaptation ( 16 ). Here, we passaged the EAEC strain for 640 generations in the presence of susceptible or immune cells. Whereas the T6SS activity of the population evolved with susceptible cells was comparable to the ancestral strain, the T6SS activity of the population evolved with immune cells was significantly decreased, with a loss of 99% of the activity compared to the ancestral strain. To identify the genetic changes responsible for decrease or lost activity, we sequenced 18 clones with intermediate and null T6SS phenotypes identifying single-nucleotide polymorphisms (SNPs) in metabolic pathways ( glpK , rne , rpoC , and aqpZ ), in Sci1 T6SS promoter and genes ( tssM , tssK and tssF ), as well as in the rfaH and csgE genes. While the metabolic mutations correspond to adaptation to the medium, genetically reconstructed strains of T6SS and rfaH mutations confirmed their importance for T6SS activity. We then defined the molecular basis for the loss of activity of the T6SS and delineated the impact of the RfaH antiterminator in T6SS regulation. RESULTS Experimental design To study the EAEC 17-2 T6SS Sci1 adaptation in the absence of target cell to eliminate, we first engineered a strain carrying an insertion of a kanamycin cassette in replacement of the lacZ gene (17-2 Δ lacZ Ω kan ) for selection. This strain was then serially passaged for a total of approximatively 640 generations in sedimentation conditions in Sci1 T6SS inducing medium (SIM, M9 supplemented with glycerol and 10% LB) in the presence of susceptible W3110 E. coli K-12 cells or of immune EAEC 17-2 cells. Competition conditions were carried out for 10 generations, followed by a selection of the 17-2 Δ lacZ Ω kan strain on selective (kanamycin) X-Gal agar plates and archived prior to being subjected to a new passage ( Figure 1 ). Download figure Open in new tab Figure 1. Schematic representation of the experimental evolution design. (1) The ancestral EAEC Δ lacZ Ω kan (EAEC-Km, T6SS + ) strain and the susceptible (W3110, prey) and immune (EAEC) recipient were grown overnight from a single-colony. (2) EAEC-Km was mixed with W3110 (EP condition) or with EAEC (E condition) in a 1:4 ratio and (3) co-cultured in SIM at room temperature without agitation for 10 generations in sedimentation conditions. (4) The evolved EAEC-Km was isolated from the coculture by selection on kanamycin-LB plates, (5) resuspended in a liquid culture and (6) subjected to a new passage. The experiment was repeated 64 times (∼ 640 generations) to yield the EP640 and E640 populations. Created with Biorender. Experimental evolution in presence of immune cells led to a significant decrease of T6SS activity The antibacterial activity of the resulting EAEC populations, named hereafter EP640 (evolved with W3110 recipient) and E640 (evolved with EAEC 17-2 recipient), were tested against the susceptible W3110 strain, and compared to the antibacterial activity of the ancestral strain or its Δ sci1 derivative (deleted of the sci1 T6SS gene cluster). Figure 2A shows that the EP640 population exhibited an antibacterial activity similar to that of the ancestral strain. In contrast, the E640 population was significantly impaired for T6SS activity, with a decrease of ∼ 99% compared to the ancestral strain ( Figure 2A ). These results indicate that most EAEC cells evolved with immune cells have lost T6SS activity, whereas this activity was conserved when evolved with susceptible cells, demonstrating that the T6SS provides a fitness advantage in the presence of sensible target cells. Download figure Open in new tab Figure 2. Loss of T6SS activity during experimental evolution of EAEC with immune cells. A. Competition assay between the EAEC ancestral (WT) strains or evolved E640 or EP640 populations or the T6SS-deletion mutant Δ sci1 and the susceptible W3110 strain. W3110 mortality was measured by the SGK method. Data are the mean of three independent replicates. B. Representative antibacterial activity of selected clones from the EP640 ( n =144) and E640 ( n =192) populations using the LAGA assay (left). The ancestral EAEC (WT) and Δ tagA , Δ clpV and Δ sci1 derivative strains were used as a control for full, intermediate plus, intermediate minus and null T6SS activity, respectively. The color intensity of CPRG (yellow to purple) correlates with the antibacterial activity. Clone phenotype percentage from EP640 and E640 population with wild-type, intermediate or null T6SS activity (left). This screening was performed on in a single replicate C. Antibacterial activity of selected evolved clones (light grey bars EP640, n =4; dark grey bars E640, n =22) using the SGK method. The ancestral strain (WT) and derivatives Δ tagA , Δ clpV and Δ sci1 strains (colored bars) were used as controls. Data are the mean of at least three independent biological replicates. To better characterize the EP640 and E640 populations, the antibacterial activity of 150-200 independent clones isolated from each population was assessed. Among the EP640 population, 97% of the 144 isolated clones presented a T6SS activity comparable to that of the ancestral strain, and 3% displayed a slightly reduced activity ( Figure 2B ). Conversely, ∼85% of the 192 E640 clones had no measurable T6SS activity, and 4% presented significantly reduced activity ( Figure 2B ). Twenty-six clones, including the 9 from E640 and 4 from EP640 population with intermediate T6SS activity, and 13 from E640 population with null activity, were selected for further analysis. Using a quantitative competition assay, we confirmed the intermediate antibacterial activity of the 9 E640 selected clones, with 4 presenting an activity between the WT and Δ tagA strains, and 5 others between the Δ tagA and Δc lpV mutant strains, and the absence of antibacterial activity for the 13 other E640 clones ( Figure 2C ). To test whether the absence of antibacterial activity is not linked to an impaired metabolism, the growth rate of all selected clones was measured. Although some clones displayed a reduced growth compared to the ancestral strain (longer lag time or lower biomass), only three of them presented significant growth defects and were not included for further analyses ( Figure S1 ). Whole genome sequencing of evolved clones revealed mutations in genes involved in T6SS function To understand how the phenotypes observed are linked to genetic changes, the ancestral strain and 18 evolved clones were subjected to whole genome sequencing using the Illumina technology. The genomes of selected clones were aligned and compared to the ancestral strain using the MAUVE software. The identified mutations, listed in Table 1 , and illustrated in Figure S2 , can be classified in three categories: metabolic adaptations ( glpK , rne , rpoC , and aqpZ encoding a glycerol transporter, a ribonuclease, the RNA polymerase and the aquaporin Z, respectively); T6SS mutations ( sci1 promoter, tssF , tssK and tssM genes); other functions ( rfaH and csgE ). Mutations detected in evolved clones were either base substitutions (SNPs) or deletions (gap). We found 2 major lineages with metabolic adaptations [ rpoC ] and [ glpK , aqpZ ] ( Table S3). The metabolic adaptation in rne gene was acquired independently in both lineages. These lineages appeared independently in the E640 and EP640 populations, in agreement with the fact that these metabolic mutations are adaptative mutations basically arising during experimental evolution in minimal media ( 17 – 20 ). View this table: View inline View popup Download powerpoint Table 1. Mutations appeared during the EE in the selected evolved clones Experimental evolution on minimal medium led to metabolic adaptation As mentioned above, the metabolic adaptation mutations were already identified in independent studies where E. coli strains were evolved in starved conditions or in glycerol synthetic media ( 17 ). The D73K substitution in the glycerol-3-phosphate transporter GlpK, close to the E69 position identified here, increases GlpK catalytic activity and improve glycerol catabolism ( 17 , 19 ). The 9-bp deletion in the rpoC gene is identical to that found during evolution in glycerol ( 17 ) and is suggested to impact the global transcriptome in order to optimize growth in starved conditions ( 21 ). The synonymous mutation found in the aqpZ gene corresponds to residue A188 located in the loop E, which is involved in the substrate specificity ( 22 – 24 ). The next residue, R189, is important for water permeability ( 25 ). The substitutions changes the GCG codon for the less frequent GCA codon, and it is proposed that such a change in the bias codon may improve the overall fitness ( 26 , 27 ). Finally, two different mutations were found in the rne gene, producing a distinct substitution in the same amino acid (G172S ou G172A). The G172S substitution was already identified in E. coli MG1655 grown in minimal media supplemented with glycerol ( 17 ) and has been shown to improve growth rate. Since these mutations likely corresponds to i) adaptation to the SIM medium, ii) are found in clones with very distinct antibacterial activities, iii) appeared in both the E640 and EP640 populations, and iv) are combined with other mutations in the different clones ( Table S3 ), we did not test their impact on T6SS function. Experimental evolution targeted T6SS promoter and structural components All the E640 clones with no detectable T6SS antibacterial activity carried mutations within the Sci1 promoter or within genes encoding key structural components of the secretion apparatus, TssM, TssK and TssF. All these mutations were re-introduced in the genome of the ancestral strain, and antibacterial activity assays confirmed that the phenotype was linked to these mutations. Psci1 Two different mutations were identified in the sci1 promoter (P sci1 ): a 34-bp deletion and a base substitution ( Table 1 ). The 34-bp deletion encompasses the -10 transcriptional element ( 28 ) and is likely to prevent proper binding of the RNA polymerase. The other mutation, an A➔T substitution, is located 13-bp upstream the tssB start codon, and near the tssB mRNA ribosome-binding site (RBS) ( Figure 3A ). Structural predictions suggest that the A➔T substitution near the RBS changes the overall 5’UTR mRNA architecture ( Figure 3B ), which may impact mRNA translation. Alternatively, it has been recently shown that a single substitution near the RBS deny the binding and its translation by the ribosome ( 29 , 30 ). In agreement with a potential impact on translation, we observed that the mutation induces a 2-fold reduction of fluorescence in a strain expressing the GFP inserted between the tssC and tssK genes (transcriptional reporter C-GFP-K, Figure 3C ). Download figure Open in new tab Figure 3. P sci1 substitution may impact T6SS expression or translation. A. Alignment of the tssB -mRNA 5’-UTR sequence of the WT and P sci1 * evolved clone (RBS*A>T). The consensus RBS sequence (AGGAGA) is underlined. The tssB start codon is shown in pink whereas the SNP is highlighted in yellow. B. Prediction of the secondary structure of the WT (left) and RBS*A>T (right) tssB mRNA using the Vienna RNA Websuite ( 31 ). The WT sequence shows interaction between the -13A (arrow) and an upstream nucleotide, whereas the variant -13U (arrow) interacts with the -6A of the RBS, modifying the secondary structure and RBS exposition. C. Mean fluorescence of a EAEC 17-2 C-GFP-K population in the WT compared to the RBS*A>T variant ( n =1100 cells) (left). Representative fluorescence microscopy image of EAEC 17-2 C-GFP-K WT or RBS*A>T (right). TssM TssM is an inner membrane protein that assembles, together with TssL and TssJ, the T6SS membrane complex in EAEC ( 32 , 33 ). The C-terminal domain of TssM interacts with the TssJ outer membrane lipoprotein in the periplasm ( 34 ). The identified mutation is a 1-bp deletion of nucleotide 869, hence producing a frameshift and a premature stop codon removing part of the cytoplasmic domain, the third transmembrane helix and the periplasmic domain ( Figure 4A ). Interestingly, a recent comparative genomic study on multi-drug-resistant E. coli lineages identified tssM as the target of an insertion sequence in one isolate, likely disrupting TssM function ( 15 ). Using bacterial two-hybrid, we showed the loss of interaction between TssM and TssJ when introducing the mutation found in the E640 evolved clone ( Figure 4B ). By avoiding this essential interaction, this mutation prevents T6SS sheath assembly ( Figures 4C ) and antibacterial activity ( Figure 4D ). Download figure Open in new tab Figure 4. T6SS tssM , tssK and tssF mutations affect the T6SS assembly and activity. A. Schematic representation of the TssM protein and its truncated variant (TssM*)(green), and the TssJ OM-associated lipoprotein (orange). IM, inner membrane; OM, outer membrane. B. Representative fluorescence microscopy fields of the WT, tssM* and tssK* strains producing TssB-sfGFP. C. Statistical analyses of WT, tssM* and tssK* cells with polymerized and dynamic sheaths ( n =1000 cells). D. Competition assay between the WT, tssM* and tssK* strains and the susceptible W3110 strain using the SGK assay (3 independent replicates). E. Side (left) and top (right) views of the EAEC TssK trimer structure (pdb: 5M30). Each monomer is shown with a different color, and the position of residue A430 is shown as a red sphere. F. Bacterial two hybrid assay. WT TssK or its A430E variant (TssK*) fused to the T18 domain of the adenylate cyclase were tested against TssK, TssFG or the cytoplasmic domain of TssL (TssL C ) fused to the T25 domain. G. Side view of the EAEC TssFGK wedge complex (pdb: 6GIY). TssG is colored green, the two TssK trimers are colored grey whereas the two TssF copies are colored in orange and blue. The position of residue D43 is shown as a red sphere. H, I. Bacterial two hybrid assay. WT TssF or its D43H variant (TssF*) were tested against TssG ( H ), and VgrG, TssK and TssE ( I ). TssK TssK is a trimeric cytoplasmic protein that assembles, together with TssE, -F and -G the T6SS baseplate wedges ( 35 – 38 ). TssK resembles phage-like receptor-binding proteins and anchors the baseplate to the membrane complex via specific interactions between its C-terminal domain and the cytoplasmic domain of the TssL inner membrane protein ( 35 , 39 – 41 ). The mutation identified in this study corresponds to a substitution of Ala-430, located in one of the loops of the TssK C-terminal domain ( Figure 4E ), downstream residue Pro-429 that establishes intimate contacts with TssL ( 41 ). In agreement with these latter study, the TssK A430E substitution did not impact TssK oligomer formation or interaction with TssFG but prevented TssKL complex formation ( Figure 4F ), significantly decreasing T6SS sheath assembly ( Figures 4C ) and antibacterial activity ( Figure 4D ). TssF TssF forms with TssG the trifurcating unit skeleton of the T6SS baseplate wedge ( 36 – 38 ). The mutation that appeared during experimental evolution corresponds to the substitution of Asp-43 by a histidine residue ( Figure 4G ), which does not impact formation of the TssFG complex ( Figure 4H ) but prevented recruitment of TssE to the TssFG unit ( Figure 4I ). Mutations outside the T6SS gene cluster targeted the transcriptional RfaH antiterminator, affecting proper sci1 transcription Two mutations located outside the T6SS gene cluster were also identified, affecting the csgE and rfaH genes. A 1-bp deletion producing a frameshift and a premature stop codon in the csgE gene was selected. csgE belongs to the csgDEFG operon, which, together with the csgABC operon, encode the components of surface-associated curli ( 42 ). Curli are amyloid fibers that are involved in the adhesion to host epithelial cells by binding fibronectin and MHC-1 ( 43 ) and biofilm formation ( 44 , 45 ). CsgE is a periplasmic protein that interacts with the outer membrane protein CsgG to form the secretion canal that exports and assembles the extracellular CsgA and CsgB curli subunit to the cell surface ( 46 – 49 ). Reconstruction of the csgE frameshift in the EAEC 17-2 ancestral strain did not reproduce the phenotype observed in the evolved strain, suggesting that csgE is not involved in the T6SS activity but rather could be a metabolic adaptation or be required for T6SS-related function in other conditions ( Figure S3 ). The mutation that appeared in the rfaH gene is a 4-bp deletion frameshift inducing a premature stop codon. RfaH is a transcriptional regulator that unlocks the RNAP pausing at specific sites named the ops elements (consensus GGCGGTAGCGTG) ( 50 ). The RNAP- ops complex is recognized by RfaH, which binds and stabilizes the RNAP by its N-terminal domain and recruits the ribosome by its C-terminal domain, enhancing the transcription and translation efficiency ( 51 , 52 ). Ops elements are usually found in large operons such as those encoding the capsule biosynthesis and secretion machine ( wza , 21 kb) or secretions systems (T4SS, 33 kb; T1SS, 23 kb) ( 53 ). In EAEC, we found the ops consensus and derivative sequences in genes related to diverse functions such as transposition, exopolysaccharide production, membrane transporter, LPS biosynthesis or metabolism ( Table S4 ). Interestingly, we identified two ops elements within the EAEC T6SS sci1 gene cluster, one with a consensus sequence located in the intergenic region between the hcp and clpV genes ( ops1 ), and a second, less consensus, but with the most conserved GGTAGNNT motif, located in the intergenic region between the accessory genes EC042_4547 and EC042_4548 ( ops2 ) ( Figure 5A ). Mutagenesis of the ops1 and ops2 elements indeed impaired T6SS activity ( Figure 5 ). Mutation in ops1 element completely abolished T6SS assembly ( Figure 5C ) and, consequently, reduced antibacterial activity ( Figure 5D ). This result is consistent with the ops1 role in governing the proper transcription of the downstream essential structural genes, suggesting a defect in the proper amount of T6SS proteins translated from genes downstream the ops1 element. In agreement with the position of the ops2 element, located in the T6SS accessory gene operon, the ops2 mutation impacted T6SS sheath assembly without abolishing it ( Figure 5C ). To better understand the contribution of ops elements and rfaH in the transcription of T6SS genes, transcriptional fluorescent reporter strains were engineered, in which the mCherry-coding sequence was inserted at different positions in the T6SS gene cluster in the WT and rfaH , ops1 and ops2 mutants: upstream the ops1 element (between the tssC and tssK genes, C-mCh-K), between the ops1 and ops2 elements (between the clpV and EC042_4531 genes, ClpV-mCh-31), and downstream the ops2 element (between the EC042_4548 and EC042_4549 genes, 48-mCh-49). Fluorescence measurements showed that the C-mCh-K reporter remained unchanged in all the strains ( Figure 5E ). In contrast, the transcription of the ClpV-mCh-31 reporter drastically decreased in the ops1 and rfaH strains, and that of the 48-mCh-49 reporter decreased in ops1 , ops2 , and rfaH strains ( Figure 5E ). Taken together, these results demonstrate that RfaH is required for the proper transcription of the T6SS gene cluster in EAEC, likely by solving transcriptional pause at ops sites located within the cluster. Download figure Open in new tab Figure 5. RfaH regulates proper sci1 expression via conserved ops elements. A. Schematic representation of the EAEC sci1 T6SS gene cluster, highlighting the position and the conserved sequence of the two ops elements. The most conserved residues are shown in bold whereas the critical residues for RfaH binding are shown underlined. B. Representative fluorescence microscopy fields of the WT, ops1 * and ops2 * strains producing TssB-sfGFP. C. Statistical analyses of WT ( n =2500), ops1* ( n =2500) and ops2* ( n =1300) cells with polymerized and dynamic sheaths. D. Competition assay between the WT, ops1* and reconstituted ops1 + strains and the susceptible W3110 strain using the SGK assay (3 independent replicates). E. mCherry fluorescence measurements in WT (blue bars), ops1* (orange bars), ops2* (grey bars) and rfaH (yellow bars) cells with mCherry insertions between the tssC and tssK (C-mCh-K), clpV and EC042_4531 (ClpV-mCh-31), or EC042_4548 and EC042_4549 (48-mCh-49) genes. The mean fluorescence of each construction was normalized to the WT strain. Standard deviation bars represent the error between 3 biological replicates. F. Syntheny of the ops1 element in T6SS gene clusters in different enterobacteria. The position of a consensus ops element is indicated by the red arrow. As T6SS genes are usually clustered in large operon, we asked whether RfaH-dependent regulation of the T6SS is a conserved mechanism. Genomic analyses on representative enterobacteria showed that ops1 elements is highly conserved in T6SS clusters ( Figure 5F ). This concerns various Enterobacteriaceae such as human pathogens ( Citrobacter pasteurii and Klebsiella aerogenes ), entomopathogens ( Photorhabdus laumondii and Serratia marcescens ) and plant pathogens ( Erwinia amylovora ). These ops elements possess a high synteny with EAEC ops1 as they locate in the hcp - clpV intergenic region in all strains with the exception of P. laumondii in which the ops element is found between tssF and tssG genes ( Figure 5F ). The presence of T6SS ops sites in diverse clusters suggests that RfaH-dependent transcription of T6SS gene clusters is a conserved mechanism across enterobacteria. DISCUSSION Experimental evolution is a powerful technique to understand the dynamics of emergence of mutations that optimize a process in a specific laboratory-controlled condition ( 16 ). It is also a powerful tool to identify new key elements that regulate or modulate a process. Here we conducted an experimental evolution of the EAEC strain 17-2 in competition experiments against susceptible or immune cells in SIM medium, which is the inducing medium for the antibacterial Sci1 T6SS. We observed that while the population evolved in the presence of susceptible cells retains antibacterial activity over 640 generations, about 90% of the cells of the population evolved in the presence of immune cells (EP640) presented a significant attenuation or total loss of T6SS activity. This result, which demonstrates that cells have a tendency to eliminate the T6SS in conditions in which it is not necessary, i.e., in presence of immune cells or in pure culture, supports the hypothesis that classical laboratory strains of E. coli , which are devoid of T6SS gene clusters (or only remnants of them), have evolved over decades of pure cultures in laboratory conditions. Why losing T6SS activity when not necessary? One obvious hypothesis will be that T6SS production, assembly, function or recycling is energetically costly for the cell, as previously shown for the T3SS in Salmonella enterica Typhimurium. An experimental evolution of the S. Typhimurium hns mutant, which is highly impaired in growth under laboratory conditions, led to the systematic deletion of a large portion of the costly SPI-1 T3SS gene cluster which is induced in the hns mutant, allowing to counteract the fitness defect of the hns deletion ( 54 ). However, while the T6SS SPI-6 gene cluster is also induced in the hns mutant ( 55 ), no mutations impacting the T6SS were identified, suggesting a lower energetic cost for this apparatus. Indeed, recent experiments did not show any significant impact of the T6SS on growth in EAEC ( 56 – 59 ). Thus, why bacteria gain and loss the T6SS over the course of evolution is still elusive. To identify the genetic modifications that conducted to decreased or loss of T6SS activity, selected clones from the two populations were subjected to whole genome sequencing and the genomes compared to the ancestral strain. Clones with both reduced or no T6SS activity were selected, as we reasoned that they might provide different information. Clones with no activity are likely to bear mutations that directly affect genes that are essential for T6SS assembly or function. In contrast, clones with decreased activity might impact genes encoding accessory subunits of the T6SS or processes that indirectly affect T6SS expression, production, assembly or function ( 60 ). First, we identified mutations common in both conditions suggesting no direct link to competition but rather corresponds to adaptative changes to the medium. Indeed, two mutations targeting the RNA polymerase rpoC and ribonuclease rne genes, are known adaptative conditions in minimal medium and increase the general fitness in nutrient starvation. Two other mutations, targeting the glycerol transporter glpK and the aqpZ aquaporin genes, were also found during experimental evolution of E. coli or Salmonella enterica Typhimurium grown in minimal media with glycerol as carbon source, such as the SIM medium used in our study ( 17 ). The other mutations we specifically identified in the population evolved in the presence of immune cells were found within the sci1 promoter, within T6SS gene coding sequences ( tssF , tssK , tssM ) or within genes that are not known to be related to T6SS regulation or function, rfaH and csgE . To verify that these mutations were responsible for the loss or decrease of T6SS activity, they were introduced into the ancestral strain. With the exception of the csgE mutation, which did not show any impact on T6SS activity when reconstructed, all the other mutations phenocopied the activity of their cognate evolved clones. The two mutations found in the promoter consist to a large deletion encompassing the -10 transcriptional element, likely responsible for an expression defect of the T6SS gene cluster, and to a SNP in the 5’-UTR region, at very close proximity to the RBS. We did not characterize the first deletion since the impact of the -10 transcriptional element deletion on T6SS expression looks obvious regarding the regulation of EAEC Sci1 ( 61 ). The second mutation near the RBS is responsible for a decrease in T6SS expression. While we do not have experimental evidence of the molecular mechanism, one may hypothesize that this substitution impacts the mRNA secondary structures that may decrease ribosome access to the RBS, based on predictive tools ( 29 , 30 ). Thus, further analyses are required to understand the impact of this mutation on mRNA structure and stability. The mutations in the genes encoding the TssF, TssK and TssM subunits were shown to impact the TssF-TssE, TssK-TssL and TssM-TssJ interactions, respectively, which are essential for proper assembly of the T6SS ( 38 ). Interestingly, recent phylogenetic analysis reported gain and loss of the T6SS activity during the evolution ( 15 , 62 – 65 ). Vibrio cholerae evolved as pandemic and non-pandemic strains via point mutations in T6SS clpV or tssM genes ( 62 , 64 – 66 ). Comparative genomics in Bacteroidetes revealed frameshifts caused by independent 2- or 5-bp deletions that affect the tssC or vgrG genes, suggesting that these strains are in the process of naturally losing the T6SS ( 67 ). More generally, laboratory experimental evolutions impose a strong selective pressure on highly conserved protein domain which it is hypothesized to be easily reversed ( 68 ). It would be interesting to test whether the mutations we identified in our study could be reversed by subjecting them to a novel experimental evolution in the presence of susceptible recipient cells. More generally, using experimental evolution with diverse strains could be a basis to compare what is observed by comparative genomics and to test whether this approach could reflect the evolution in nature. We have to note that our conditions targeted the antibacterial Sci1 T6SS of EAEC whereas comparative genomic studies can show evolution of other T6SS types potentially targeting other biological forms such as host eukaryotic cells, fungi or protists. Thus, experimental evolution on other T6SS in the appropriate conditions should be perform to support hypothesis on genetic basis for T6SS evolution found in comparative studies. The most interesting mutation acquired during the experimental evolution in the presence of immune cells is the frameshift within the rfaH gene. The RfaH antiterminator is responsible for RNA polymerase stabilization for the transcription of long operons, by resolving RNAP pausing at specific ops sequences and suppressing premature termination ( 51 ). Interestingly, we found two functional ops elements within the Sci1 T6SS gene cluster, located in intergenic regions within the main ( ops1 ) and accessory ( ops2 ) operons. We further showed that these two elements are important for the transcription of downstream genes, suggesting that they can enhance RNA polymerase transcription processivity. Therefore, RfaH and the two ops elements are new regulators of T6SS expression in EAEC. A comparative genomic analysis suggested that the RfaH-dependent regulation of the T6SS is likely to be conserved in enterobacteria, as consensus ops sequences are found in intergenic regions in pathogenic and commensal E. coli , Salmonella , Citrobacter , Enterobacter , Klebsiella , Serratia or Photorhabdus species. In agreement with this observation, it has recently been showed that the rfaH deletion impacted T6SS gene expression in Erwinia amylovora ( 69 ) and that RfaH solves RNA polymerase pausing and counteracts H-NS silencing of T6SS genes in uropathogenic E. coli ( 70 ). One may hypothesize that this additional counter-silencer role of RfaH is conserved as T6SS gene clusters in E. coli EDL933, S. Typhimurium and Edwardsiella piscicida , which are regulated by H-NS ( 71 ), carry ops elements. Further investigations are necessary to understand how RfaH is involved in the regulation of the T6SS, notably by studying its own regulation and going deeper in the biochemical interaction with its DNA, RNA polymerase and ribosome partners. The observation of ops elements in several loci on EAEC genome suggests that the T6SS could be co-regulated with other systems and be part of a specific program governed by RfaH. We uncovered a new layer of regulation among transcriptional and post-translational checkpoint in the T6SS field that we need to better characterize to understand how and when T6SS are employed. Taken together, this study highlights the power of experimental evolution to understand the molecular basis of the evolution of T6SS in bacterial strains but also to unveil new regulatory elements that optimize its expression or function. Further work aiming at characterizing more evolved clones or the archived intermediate evolved populations will likely provide additional information such as new accessory genes or how loss-of-function mutations emerge. MATERIAL AND METHODS Strains and growth conditions Bacterial strains and plasmids used in this study are listed in Table S1 . Escherichia coli strains were routinely grown in LB or T6SS inducing medium (SIM: M9 minimal medium supplemented with 0.2% glycerol, 1 μg/ml vitamin B1, 40 μg/ml casaminoacids and 10% (v/v) LB medium) ( 28 ) at 37°C. When necessary cultures were supplemented with kanamycin (50 μg/mL), ampicillin (100 μg/mL) or chloramphenicol (40 μg/mL). Experimental evolution procedure The EAEC 17-2 ancestral strain was engineered by replacing the lacZ gene by the kanamycin resistance (Kan R ) cassette from pKD4 by λ-red-mediated recombination ( 72 ) using plasmid pKOBEG ( 73 )to yield strain EAEC 17-2 Δ lacZ Ω kan . Serial passage experiments were conducted in two conditions, in the presence of the W3110 p mCherry -ampR susceptible recipient (EP) or of the EAEC 17-2 immune recipient (E). Strains were cultured in SIM at 37°C with agitation until OD 1 prior to be mixed in 1:4 (v/v) donor:recipient ratio, diluted 1/1000 in 10 mL of fresh SIM, cultured at 37°C with shaking until absorbance at λ=600 nm ( A 600 ) of 0.25, sedimented and further incubated without shaking until an A 600 of 1. Then 200 µL of the co-culture were isolated on LB agar supplemented with kanamycin to select the evolved donor and X-gal to check its phenotype. After ON incubation at 37°C, evolved donor cells were recovered in SIM, the A 600 was adjusted to 1, and cells were archived by storage at -80°C in glycerol and used to seed a new co-coculture by 1/1000 dilution with new susceptible (EP) or immune € cells ( Fig. 1 ). Bacterial competition assay The LAGA and SGK methods were employed to test T6SS activity ( 74 ). The LAGA assay is based on the degradation of Chloro-Phenol-Red-β-D-galactopyrannoside (CPRG, yellow color), a membrane-impermeant substrate of the β-galactosidase, which is degraded in Chloro-Phenol-Red (CPR, purple color) by the β-galactosidase released during recipient lysis. The color is therefore a qualitative indication of recipient cell lysis. The SGK assay is based on the lag time necessary for surviving recipient cells to reach a certain A 600 , which is inversely proportional to the level of surviving cells, and hence proportional to the antibacterial activity of the donor. For both, donor and recipient strains were precultured in LB overnight and diluted 1/100 in SIM until A 600 reaches 0.8. Donor and recipient were mixed in a 4:1 ratio (v/v) and 10 µL of the mix was spotted on SIM agar plate in triplicate. Spots were dried and incubated at 37°C for 4 hours. For the LAGA qualitative assay, 10 µL of CPRG 2 mM were added on each competition spot. For the SGK assay, each spot were resuspended in 1 mL of selective medium for the recipient and 100 µL of 1/10 dilutions were added to 100 µL of selective medium in a 96-well microplate (Thermofisher, Nunc). Cultures were grown for 15 hours at 37°C with agitation and A 600 was measured every 5 minutes using a microplate reader (TECAN). Serial dilutions of the recipient alone were cultured to establish a standard curve. The time of emergence (Te, time necessary to reach an A 600 of 0.4) of each standard samples was associated with a log difference and a linear regression with equation Δ log = a × Te + b was generated. The log difference between each strain was then calculated with their Te using the linear regression equation. All values of each replicate of three biological replicates were compared between strains with a Wilcoxon statistical test using the R software. For both assays, controls with the recipient alone, the donor alone, or the recipient with the ancestral EAEC 17-2 Δ lacZ Ω kan strain, were systematically performed. Whole genome sequencing and bioinformatic analysis Eighteen evolved clones were selected based on their antibacterial activity and the absence of major growth defects for whole genome sequencing. Genomic DNA was extracted with the DNA MiniKit (Qiagen) and subjected to Illumina sequencing (MicrobesNG, Birmingham, UK). The sequencing files were first merged in fasta format using the Galaxy online plateform ( 75 ) and implemented in MAUVE ( 76 , 77 ) to identify SNPs and gaps. mRNA secondary structure predictions were performed by inferring the 5’-UTR- tssB mRNA sequence to the RNAfold webserver from Vienna RNA Websuite ( 31 ). Molecular biology Oligonucleotides used in this study are listed in Table S2 . SNPs and gaps were reintroduced in the ancestral EAEC 17-2 wild-type strain by chromosomal directed mutagenesis using the thermosensitive pKO3 suicide vector ( 78 ). Briefly, a 1-kb DNA fragment comprising the site to be mutagenized was amplified by PCR and cloned into the pJET1.2/blunt vector. The region was then mutagenized by Quick-change mutagenesis using the Q5 Pfu polymerase and complementary oligonucleotides bearing the desired mutation or deletion. After verification by DNA sequencing (Eurofins genomics), the mutated fragment was amplified by PCR and cloned into the pKO3 plasmid. After verification by DNA sequencing, the pKO3 recombinant suicide vector was introduced into EAEC 17-2 by electroporation. The first event of recombination was selected on chloramphenicol LB agar plates at 37°C, and excision of the plasmid by a second recombination event was selected on LB agar plates supplemented with Sucrose 10%. The mutation was checked by DNA sequencing after PCR amplification of the region of interest (Eurofins Genomic). Fluorescent reporter (mCherry or sfGFP) cassettes were amplified by PCR using vectors pKD4-Nter-sfGFPr and pKD4-Nter-mCherry ( 39 ) and oligonucleotides carrying 5’ 50-bp extensions corresponding to sequences upstream and downstream the site of insertion, and inserted on the chromosome by λ-red-mediated recombination using plasmid pKOBEG. Microscopy analysis EAEC 17-2 producing the fluorescent reporters were precultured in 3 mL LB overnight, diluted in SIM to an A 600 of 0.8. Cells were concentrated to an A 600 of 8 by centrifugation and resuspension, and 1 µL of the suspension was spotted on SIM-agarose pads casted using a gene frame stuck on a microscopy slide. A high precision cover slip was added prior to the observation using a Nikon Ti2 microscope. A mask was generated from microscopy images using the MiSiC program ( 79 ), implemented into the Fiji software ( 80 ), and cells were segmented using MicrobeJ plugin ( 81 ). Fluorescence intensity for each cell were extracted and the presence of a sheath was determined for a ratio of max fluorescence/mean fluorescence >2 since sheaths are brighter objects in a cell. Data availability All data are shown in the manuscript or in the supplemental material. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper. ACKNOWLEDGEMENTS We thank members of the Cascales laboratory for discussions, and Moly Ba, Isabelle Bringer, and Annick Brun for technical assistance. This work was supported by the CNRS, the Aix-Marseille Université and by grants from the Agence Nationale de la Recherche (ANR-20-CE11-0011), the Fondation pour la Recherche Médicale (DEQ20180339165) and the Fondation Bettencourt-Schueller to EC. REFERENCES 1. ↵ Cianfanelli , F.R. , Monlezun , L. and Coulthurst , S.J . ( 2016 ) Aim, Load, Fire: The Type VI Secretion System, a Bacterial Nanoweapon . Trends in Microbiology , 24 , 51 – 62 . OpenUrl CrossRef PubMed 2. ↵ Cherrak , Y. , Flaugnatti , N. , Durand , E. , Journet , L. and Cascales , E . ( 2019 ) Structure and Activity of the Type VI Secretion System . Microbiology Spectrum , 7 , 7.4.11 . OpenUrl CrossRef 3. ↵ Jurėnas , D. and Journet , L . ( 2021 ) Activity, delivery, and diversity of Type VI secretion effectors . Mol Microbiol , 115 , 383 – 394 . OpenUrl CrossRef PubMed 4. ↵ García-Bayona , L. , Coyne , M.J. and Comstock , L.E . ( 2021 ) Mobile Type VI secretion system loci of the gut Bacteroidales display extensive intra-ecosystem transfer, multi-species spread and geographical clustering . PLoS Genet , 17 , e1009541 . OpenUrl CrossRef PubMed 5. Robinson , L. , Liaw , J. , Omole , Z. , Corcionivoschi , N. , Hachani , A. and Gundogdu , O . ( 2022 ) In silico investigation of the genus Campylobacter type VI secretion system reveals genetic diversity in organization and putative effectors: This article is part of the “Genomics, Epidemiology and Evolution of Campylobacter, Helicobacter and Related Organisms” collection . Microbial Genomics , 8 . 6. ↵ Unni , R. , Pintor , K.L. , Diepold , A. and Unterweger , D . ( 2022 ) Presence and absence of type VI secretion systems in bacteria: This article is part of the Bacterial Cell Envelopes collection . Microbiology , 168 . 7. Aoun , N. , Georgoulis , S.J. , Avalos , J.K. , Grulla , K.J. , Miqueo , K. , Tom , C. and Lowe-Power , T.M . ( 2024 ) A pangenomic atlas reveals eco-evolutionary dynamics that shape type VI secretion systems in plant-pathogenic Ralstonia . mBio , 15 , e00323 – 24 . OpenUrl PubMed 8. ↵ Habich , A. , Chaves Vargas , V. , Robinson , L.A. , Allsopp , L.P. and Unterweger , D . ( 2025 ) Distribution of the four type VI secretion systems in Pseudomonas aeruginosa and classification of their core and accessory effectors . Nat Commun , 16 , 888 . OpenUrl CrossRef PubMed 9. ↵ Boyer , F. , Fichant , G. , Berthod , J. , Vandenbrouck , Y. and Attree , I . ( 2009 ) Dissecting the bacterial type VI secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources? BMC Genomics , 10 , 104 . OpenUrl CrossRef PubMed 10. Coyne , M.J. , Roelofs , K.G. and Comstock , L.E . ( 2016 ) Type VI secretion systems of human gut Bacteroidales segregate into three genetic architectures, two of which are contained on mobile genetic elements . BMC Genomics , 17 , 58 . OpenUrl CrossRef PubMed 11. ↵ Peng , M. , Lin , W. , Zhou , A. , Jiang , Z. , Zhou , F. and Wang , Z . ( 2024 ) High genetic diversity and different type VI secretion systems in Enterobacter species revealed by comparative genomics analysis . BMC Microbiol , 24 , 26 . OpenUrl CrossRef PubMed 12. ↵ Journet , L. and Cascales , E . ( 2016 ) The Type VI Secretion System in Escherichia coli and Related Species . EcoSal Plus , 7 , ecosalplus.ESP-0009-2015 . OpenUrl 13. ↵ Bachmann , B.J . ( 1972 ) Pedigrees of some mutant strains of Escherichia coli K-12 . Bacteriol Rev , 36 , 525 – 557 . OpenUrl FREE Full Text 14. ↵ Dunne , K.A. , Chaudhuri , R.R. , Rossiter , A.E. , Beriotto , I. , Browning , D.F. , Squire , D. , Cunningham , A.F. , Cole , J.A. , Loman , N. and Henderson , I.R . ( 2017 ) Sequencing a piece of history: complete genome sequence of the original Escherichia coli strain . Microbial Genomics , 3 . 15. ↵ Cummins , E.A. , Moran , R.A. , Snaith , A.E. , Hall , R.J. , Connor , C.H. , Dunn , S.J. and McNally , A . ( 2023 ) Parallel loss of type VI secretion systems in two multi-drug-resistant Escherichia coli lineages . Microbial Genomics , 9 . 16. ↵ Remigi , P. , Masson-Boivin , C. and Rocha , E.P.C . ( 2019 ) Experimental Evolution as a Tool to Investigate Natural Processes and Molecular Functions . Trends in Microbiology , 27 , 623 – 634 . OpenUrl CrossRef PubMed 17. ↵ Herring , C.D. , Raghunathan , A. , Honisch , C. , Patel , T. , Applebee , M.K. , Joyce , A.R. , Albert , T.J. , Blattner , F.R. , van den Boom , D. , Cantor , C.R. , et al. ( 2006 ) Comparative genome sequencing of Escherichia coli allows observation of bacterial evolution on a laboratory timescale . Nat Genet , 38 , 1406 – 1412 . OpenUrl CrossRef PubMed Web of Science 18. Conrad , T.M. , Frazier , M. , Joyce , A.R. , Cho , B.-K. , Knight , E.M. , Lewis , N.E. , Landick , R. and Palsson , B.Ø . ( 2010 ) RNA polymerase mutants found through adaptive evolution reprogram Escherichia coli for optimal growth in minimal media . Proc. Natl. Acad. Sci. U.S.A ., 107 , 20500 – 20505 . OpenUrl Abstract / FREE Full Text 19. ↵ Applebee , M.K. , Joyce , A.R. , Conrad , T.M. , Pettigrew , D.W. and Palsson , B.Ø . ( 2011 ) Functional and Metabolic Effects of Adaptive Glycerol Kinase (GLPK) Mutants in Escherichia coli . Journal of Biological Chemistry , 286 , 23150 – 23159 . OpenUrl Abstract / FREE Full Text 20. ↵ Knöppel , A. , Knopp , M. , Albrecht , L.M. , Lundin , E. , Lustig , U. , Näsvall , J. and Andersson , D.I . ( 2018 ) Genetic Adaptation to Growth Under Laboratory Conditions in Escherichia coli and Salmonella enterica . Front. Microbiol ., 9 , 756 . OpenUrl CrossRef PubMed 21. ↵ Fong , S.S. , Joyce , A.R. and Palsson , B.Ø . ( 2005 ) Parallel adaptive evolution cultures of Escherichia coli lead to convergent growth phenotypes with different gene expression states . Genome Res ., 15 , 1365 – 1372 . OpenUrl Abstract / FREE Full Text 22. ↵ Calamita , G. , Bishai , W.R. , Preston , G.M. , Guggino , W.B. and Agre , P . ( 1995 ) Molecular Cloning and Characterization of AqpZ, a Water Channel from Escherichia coli . Journal of Biological Chemistry , 270 , 29063 – 29066 . OpenUrl Abstract / FREE Full Text 23. Wallace , I.S. , Wills , D.M. , Guenther , J.F. and Roberts , D.M . ( 2002 ) Functional selectivity for glycerol of the nodulin 26 subfamily of plant membrane intrinsic proteins . FEBS Letters , 523 , 109 – 112 . OpenUrl CrossRef PubMed Web of Science 24. ↵ Wang , Y. , Schulten , K. and Tajkhorshid , E . ( 2005 ) What Makes an Aquaporin a Glycerol Channel? A Comparative Study of AqpZ and GlpF . Structure , 13 , 1107 – 1118 . OpenUrl CrossRef PubMed 25. ↵ Jiang , J. , Daniels , B.V. and Fu , D . ( 2006 ) Crystal Structure of AqpZ Tetramer Reveals Two Distinct Arg-189 Conformations Associated with Water Permeation through the Narrowest Constriction of the Water-conducting Channel . Journal of Biological Chemistry , 281 , 454 – 460 . OpenUrl Abstract / FREE Full Text 26. ↵ Frumkin , I. , Lajoie , M.J. , Gregg , C.J. , Hornung , G. , Church , G.M. and Pilpel , Y . ( 2018 ) Codon usage of highly expressed genes affects proteome-wide translation efficiency . Proc. Natl. Acad. Sci. U.S.A ., 115 . 27. ↵ Bailey , S.F. , Alonso Morales , L.A. and Kassen , R . ( 2021 ) Effects of Synonymous Mutations beyond Codon Bias: The Evidence for Adaptive Synonymous Substitutions from Microbial Evolution Experiments . Genome Biology and Evolution , 13 , evab141 . OpenUrl CrossRef PubMed 28. ↵ Brunet , Y.R. , Bernard , C.S. , Gavioli , M. , Lloubès , R. and Cascales , E . ( 2011 ) An Epigenetic Switch Involving Overlapping Fur and DNA Methylation Optimizes Expression of a Type VI Secretion Gene Cluster . PLoS Genet , 7 , e1002205 . OpenUrl CrossRef PubMed 29. ↵ Masachis , S. , Tourasse , N.J. , Chabas , S. , Bouchez , O. and Darfeuille , F . ( 2018 ) FASTBAC-Seq: Functional Analysis of Toxin–Antitoxin Systems in Bacteria by Deep Sequencing . Methods in Enzymology , Elsevier , Vol. 612 , pp. 67 – 100 . OpenUrl CrossRef PubMed 30. ↵ Wong , J.L.C. , David , S. , Sanchez-Garrido , J. , Woo , J.Z. , Low , W.W. , Morecchiato , F. , Giani , T. , Rossolini , G.M. , Beis , K. , Brett , S.J. , et al. ( 2022 ) Recurrent emergence of Klebsiella pneumoniae carbapenem resistance mediated by an inhibitory ompK36 mRNA secondary structure . Proc. Natl. Acad. Sci. U.S.A ., 119 , e2203593119 . OpenUrl CrossRef PubMed 31. ↵ Gruber , A.R. , Lorenz , R. , Bernhart , S.H. , Neubock , R. and Hofacker , I.L . ( 2008 ) The Vienna RNA Websuite . Nucleic Acids Research , 36 , W70 – W74 . OpenUrl CrossRef PubMed Web of Science 32. ↵ Aschtgen , M.-S. , Gavioli , M. , Dessen , A. , Lloubès , R. and Cascales , E . ( 2010 ) The SciZ protein anchors the enteroaggregative Escherichia coli Type VI secretion system to the cell wall . Molecular Microbiology , 75 , 886 – 899 . OpenUrl CrossRef PubMed 33. ↵ Durand , E. , Nguyen , V.S. , Zoued , A. , Logger , L. , Péhau-Arnaudet , G. , Aschtgen , M.-S. , Spinelli , S. , Desmyter , A. , Bardiaux , B. , Dujeancourt , A. , et al. ( 2015 ) Biogenesis and structure of a type VI secretion membrane core complex . Nature , 523 , 555 – 560 . OpenUrl CrossRef PubMed 34. ↵ Felisberto-Rodrigues , C. , Durand , E. , Aschtgen , M.-S. , Blangy , S. , Ortiz-Lombardia , M. , Douzi , B. , Cambillau , C. and Cascales , E . ( 2011 ) Towards a Structural Comprehension of Bacterial Type VI Secretion Systems: Characterization of the TssJ-TssM Complex of an Escherichia coli Pathovar . PLoS Pathog , 7 , e1002386 . OpenUrl CrossRef PubMed 35. ↵ Zoued , A. , Durand , E. , Bebeacua , C. , Brunet , Y.R. , Douzi , B. , Cambillau , C. , Cascales , E. and Journet , L . ( 2013 ) TssK Is a Trimeric Cytoplasmic Protein Interacting with Components of Both Phage-like and Membrane Anchoring Complexes of the Type VI Secretion System . Journal of Biological Chemistry , 288 , 27031 – 27041 . OpenUrl Abstract / FREE Full Text 36. ↵ English , G. , Byron , O. , Cianfanelli , F.R. , Prescott , A.R. and Coulthurst , S.J . ( 2014 ) Biochemical analysis of TssK, a core component of the bacterial Type VI secretion system, reveals distinct oligomeric states of TssK and identifies a TssK–TssFG subcomplex . Biochemical Journal , 461 , 291 – 304 . OpenUrl Abstract / FREE Full Text 37. Brunet , Y.R. , Zoued , A. , Boyer , F. , Douzi , B. and Cascales , E . ( 2015 ) The Type VI Secretion TssEFGK-VgrG Phage-Like Baseplate Is Recruited to the TssJLM Membrane Complex via Multiple Contacts and Serves As Assembly Platform for Tail Tube/Sheath Polymerization . PLoS Genet , 11 , e1005545 . OpenUrl CrossRef PubMed 38. ↵ Cherrak , Y. , Rapisarda , C. , Pellarin , R. , Bouvier , G. , Bardiaux , B. , Allain , F. , Malosse , C. , Rey , M. , Chamot-Rooke , J. , Cascales , E. , et al. ( 2018 ) Biogenesis and structure of a type VI secretion baseplate . Nat Microbiol , 3 , 1404 – 1416 . OpenUrl CrossRef PubMed 39. ↵ Zoued , A. , Cassaro , C.J. , Durand , E. , Douzi , B. , España , A.P. , Cambillau , C. , Journet , L. and Cascales , E . ( 2016 ) Structure–Function Analysis of the TssL Cytoplasmic Domain Reveals a New Interaction between the Type VI Secretion Baseplate and Membrane Complexes . Journal of Molecular Biology , 428 , 4413 – 4423 . OpenUrl CrossRef PubMed 40. Nguyen , V.S. , Logger , L. , Spinelli , S. , Legrand , P. , Huyen Pham , T.T. , Nhung Trinh , T.T. , Cherrak , Y. , Zoued , A. , Desmyter , A. , Durand , E. , et al. ( 2017 ) Type VI secretion TssK baseplate protein exhibits structural similarity with phage receptor-binding proteins and evolved to bind the membrane complex . Nat Microbiol , 2 , 17103 . OpenUrl CrossRef PubMed 41. ↵ Vanlioğlu , E. , Santin , Y.G. , Filella-Merce , I. , Pellarin , R. and Cascales , E . ( 2022 ) Coevolution-guided mapping of the Type VI secretion membrane complex-baseplate interface . Journal of Molecular Biology , 167918 . 42. ↵ Brombacher , E. , Baratto , A. , Dorel , C. and Landini , P . ( 2006 ) Gene Expression Regulation by the Curli Activator CsgD Protein: Modulation of Cellulose Biosynthesis and Control of Negative Determinants for Microbial Adhesion . J Bacteriol , 188 , 2027 – 2037 . OpenUrl Abstract / FREE Full Text 43. ↵ Olsén , A. , Wick , M.J. , Mörgelin , M. and Björck , L . ( 1998 ) Curli, Fibrous Surface Proteins ofEscherichia coli, Interact with Major Histocompatibility Complex Class I Molecules . Infection and Immunity , 66 , 944 – 949 . OpenUrl Abstract / FREE Full Text 44. ↵ Prigent-Combaret , C. , Brombacher , E. , Vidal , O. , Ambert , A. , Lejeune , P. , Landini , P. and Dorel , C . ( 2001 ) Complex Regulatory Network Controls Initial Adhesion and Biofilm Formation in Escherichia coli via Regulation of thecsgD Gene . Journal of Bacteriology , 183 , 7213 – 7223 . OpenUrl Abstract / FREE Full Text 45. ↵ Carter , M.Q. , Louie , J.W. , Feng , D. , Zhong , W. and Brandl , M.T . ( 2016 ) Curli fimbriae are conditionally required in Escherichia coli O157:H7 for initial attachment and biofilm formation . Food Microbiology , 57 , 81 – 89 . OpenUrl CrossRef PubMed 46. ↵ Chapman , M.R. , Robinson , L.S. , Pinkner , J.S. , Roth , R. , Heuser , J. , Hammar , M. , Normark , S. and Hultgren , S.J . ( 2002 ) Role of Escherichia coli Curli Operons in Directing Amyloid Fiber Formation . Science , 295 , 851 – 855 . OpenUrl Abstract / FREE Full Text 47. Robinson , L.S. , Ashman , E.M. , Hultgren , S.J. and Chapman , M.R . ( 2006 ) Secretion of curli fibre subunits is mediated by the outer membrane-localized CsgG protein . Molecular Microbiology , 59 , 870 – 881 . OpenUrl CrossRef PubMed Web of Science 48. Fronzes , R. , Remaut , H. and Waksman , G . ( 2008 ) Architectures and biogenesis of non-flagellar protein appendages in Gram-negative bacteria . The EMBO Journal , 27 , 2271 – 2280 . OpenUrl Abstract / FREE Full Text 49. ↵ Nenninger , A.A. , Robinson , L.S. , Hammer , N.D. , Epstein , E.A. , Badtke , M.P. , Hultgren , S.J. and Chapman , M.R . ( 2011 ) CsgE is a curli secretion specificity factor that prevents amyloid fibre aggregation . Molecular Microbiology , 81 , 486 – 499 . OpenUrl CrossRef PubMed 50. ↵ Bailey , M.J.A. , Hughes , C. and Koronakis , V . ( 1997 ) RfaH and the ops element, components of a novel system controlling bacterial transcription elongation . Molecular Microbiology , 26 , 845 – 851 . OpenUrl CrossRef PubMed Web of Science 51. ↵ Artsimovitch , I. and Landick , R . ( 2002 ) The Transcriptional Regulator RfaH Stimulates RNA Chain Synthesis after Recruitment to Elongation Complexes by the Exposed Nontemplate DNA Strand . Cell , 109 , 193 – 203 . OpenUrl CrossRef PubMed Web of Science 52. ↵ Zuber , P.K. , Schweimer , K. , Rösch , P. , Artsimovitch , I. and Knauer , S.H . ( 2019 ) Reversible fold-switching controls the functional cycle of the antitermination factor RfaH . Nat Commun , 10 , 702 . OpenUrl CrossRef PubMed 53. ↵ Wang , B. , Gumerov , V.M. , Andrianova , E.P. , Zhulin , I.B. and Artsimovitch , I . ( 2020 ) Origins and Molecular Evolution of the NusG Paralog RfaH . mBio , 11 , e02717 – 20 . OpenUrl PubMed 54. ↵ Ali , S.S. , Soo , J. , Rao , C. , Leung , A.S. , Ngai , D.H.-M. , Ensminger , A.W. and Navarre , W.W . ( 2014 ) Silencing by H-NS Potentiated the Evolution of Salmonella . PLOS Pathogens , 10 , e1004500 . OpenUrl CrossRef PubMed 55. ↵ Brunet , Y.R. , Khodr , A. , Logger , L. , Aussel , L. , Mignot , T. , Rimsky , S. and Cascales , E . ( 2015 ) H-NS Silencing of the Salmonella Pathogenicity Island 6-Encoded Type VI Secretion System Limits Salmonella enterica Serovar Typhimurium Interbacterial Killing . Infect Immun , 83 , 2738 – 2750 . OpenUrl Abstract / FREE Full Text 56. ↵ Robitaille , S. , Simmons , E.L. , Verster , A.J. , McClure , E.A. , Royce , D.B. , Trus , E. , Swartz , K. , Schultz , D. , Nadell , C.D. and Ross , B.D . ( 2023 ) Community composition and the environment modulate the population dynamics of type VI secretion in human gut bacteria . Nat Ecol Evol , 7 , 2092 – 2107 . OpenUrl CrossRef PubMed 57. Septer , A.N. , Sharpe , G. and Shook , E.A. ( 2023 ) The Vibrio fischeri type VI secretion system incurs a fitness cost under host-like conditions . 58. Taillefer , B. , Giraud , J.F. and Cascales , E . ( 2023 ) No fitness cost entailed by type VI secretion system synthesis, assembly, contraction, or disassembly in enteroaggregative Escherichia coli . J Bacteriol , 205 , e00357 – 23 . OpenUrl PubMed 59. ↵ Zhang , C. , Datta , S. , Ratcliff , W.C. and Hammer , B.K . ( 2024 ) Constitutive expression of the Type VI Secretion System carries no measurable fitness cost in Vibrio cholerae . Ecology and Evolution , 14 , e11081 . OpenUrl CrossRef 60. ↵ Santin , Y.G . ( 2019 ) Uncovering the In Vivo Proxisome Using Proximity-Tagging Methods . BioEssays , 41 , 1900131 . OpenUrl CrossRef 61. ↵ Brunet , Y.R. , Bernard , C.S. , Gavioli , M. , Lloubès , R. and Cascales , E . ( 2011 ) An Epigenetic Switch Involving Overlapping Fur and DNA Methylation Optimizes Expression of a Type VI Secretion Gene Cluster . PLoS Genet , 7 , e1002205 . OpenUrl CrossRef PubMed 62. ↵ Kostiuk , B. , Santoriello , F.J. , Diaz-Satizabal , L. , Bisaro , F. , Lee , K.-J. , Dhody , A.N. , Provenzano , D. , Unterweger , D. and Pukatzki , S . ( 2021 ) Type VI secretion system mutations reduced competitive fitness of classical Vibrio cholerae biotype . Nat Commun , 12 , 6457 . OpenUrl CrossRef PubMed 63. Baseggio , L. , Silayeva , O. , Engelstädter , J. and Barnes , A.C . ( 2022 ) The Evolution of a Specialized, Highly Virulent Fish Pathogen through Gene Loss and Acquisition of Host-Specific Survival Mechanisms . Appl Environ Microbiol , 88 , e00222 – 22 . OpenUrl CrossRef PubMed 64. ↵ Drebes Dörr , N.C. , Proutière , A. , Jaskólska , M. , Stutzmann , S. , Bader , L. and Blokesch , M. ( 2022 ) Single nucleotide polymorphism determines constitutive versus inducible type VI secretion in Vibrio cholerae . ISME J , 16 , 1868 – 1872 . OpenUrl CrossRef PubMed 65. ↵ Ng , S.L. , Kammann , S. , Steinbach , G. , Hoffmann , T. , Yunker , P.J. and Hammer , B.K . ( 2022 ) Evolution of a cis -Acting SNP That Controls Type VI Secretion in Vibrio cholerae . mBio , 13 , e00422 – 22 . OpenUrl CrossRef PubMed 66. ↵ Unterweger , D. , Kitaoka , M. , Miyata , S.T. , Bachmann , V. , Brooks , T.M. , Moloney , J. , Sosa , O. , Silva , D. , Duran-Gonzalez , J. , Provenzano , D. , et al. ( 2012 ) Constitutive Type VI Secretion System Expression Gives Vibrio cholerae Intra- and Interspecific Competitive Advantages . PLoS ONE , 7 , e48320 . OpenUrl CrossRef PubMed 67. ↵ García-Bayona , L. , Coyne , M.J. and Comstock , L.E . ( 2021 ) Mobile Type VI secretion system loci of the gut Bacteroidales display extensive intra-ecosystem transfer, multi-species spread and geographical clustering . PLoS Genet , 17 , e1009541 . OpenUrl CrossRef PubMed 68. ↵ Cohen , Y. and Hershberg , R . ( 2022 ) Rapid Adaptation Often Occurs through Mutations to the Most Highly Conserved Positions of the RNA Polymerase Core Enzyme . Genome Biology and Evolution , 14 , evac105 . OpenUrl CrossRef PubMed 69. ↵ Klee , S.M. , Sinn , J.P. , Held , J. , Vosburg , C. , Holmes , A.C. , Lehman , B.L. , Peter , K.A. and McNellis , T.W . ( 2022 ) Putative transcription antiterminator RfaH contributes to Erwinia amylovora virulence . Molecular Plant Pathology , 23 , 1686 – 1694 . OpenUrl CrossRef PubMed 70. ↵ Hustmyer , C.M. , Wolfe , M.B. , Welch , R.A. and Landick , R . ( 2022 ) RfaH Counter-Silences Inhibition of Transcript Elongation by H-NS–StpA Nucleoprotein Filaments in Pathogenic Escherichiacoli . mBio , 13 , e02662 – 22 . OpenUrl PubMed 71. ↵ Kim , S.K. , Lee , J.B. and Yoon , J.W . ( 2022 ) Characterization of transcriptional activities at a divergent promoter of the type VI secretion system in enterohemorrhagic Escherichia coli O157:H7 . J Microbiol ., 60 , 928 – 934 . OpenUrl CrossRef PubMed 72. ↵ Datsenko , K.A. and Wanner , B.L . ( 2000 ) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products . Proc. Natl. Acad. Sci. U.S.A ., 97 , 6640 – 6645 . OpenUrl Abstract / FREE Full Text 73. ↵ Chaveroche , M.-K . ( 2000 ) A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans . Nucleic Acids Research , 28 , 97e – 997 . OpenUrl CrossRef PubMed 74. ↵ Taillefer , B. , Grandjean , M. , Herrou , J. , Robert , D. , Mignot , T. , Sebban-Kreuzer , C. and Cascales , E . ( 2023 ) Qualitative and Quantitative Methods to Measure Antibacterial Activity Resulting from Bacterial Competition . BIO-PROTOCOL , 13 . 75. ↵ Blankenberg , D. , Kuster , G.V. , Coraor , N. , Ananda , G. , Lazarus , R. , Mangan , M. , Nekrutenko , A. and Taylor , J . ( 2010 ) Galaxy: A Web-Based Genome Analysis Tool for Experimentalists . CP Molecular Biology , 89 . 76. ↵ Darling , A.C.E. , Mau , B. , Blattner , F.R. and Perna , N.T . ( 2004 ) Mauve: Multiple Alignment of Conserved Genomic Sequence With Rearrangements . Genome Res ., 14 , 1394 – 1403 . OpenUrl Abstract / FREE Full Text 77. ↵ Rissman , A.I. , Mau , B. , Biehl , B.S. , Darling , A.E. , Glasner , J.D. and Perna , N.T . ( 2009 ) Reordering contigs of draft genomes using the Mauve Aligner . Bioinformatics , 25 , 2071 – 2073 . OpenUrl CrossRef PubMed Web of Science 78. ↵ Link , A.J. , Phillips , D. and Church , G.M . ( 1997 ) Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization . J Bacteriol , 179 , 6228 – 6237 . OpenUrl Abstract / FREE Full Text 79. ↵ 79. Panigrahi , S. , Murat , D. , Le Gall , A. , Martineau , E. , Goldlust , K. , Fiche , J.-B. , Rombouts , S. , Nöllmann , M. , Espinosa , L. and Mignot , T. ( 2021 ) Misic, a general deep learning-based method for the high-throughput cell segmentation of complex bacterial communities . eLife , 10 , e65151 . OpenUrl CrossRef PubMed 80. ↵ Schindelin , J. , Arganda-Carreras , I. , Frise , E. , Kaynig , V. , Longair , M. , Pietzsch , T. , Preibisch , S. , Rueden , C. , Saalfeld , S. , Schmid , B ., et al. ( 2012 ) Fiji: an open-source platform for biological-image analysis . Nat Methods , 9 , 676 – 682 . OpenUrl CrossRef PubMed Web of Science 81. ↵ Ducret , A. , Quardokus , E.M. and Brun , Y.V . ( 2016 ) MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysis . Nat Microbiol , 1 , 16077 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted February 09, 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 Molecular bases for the loss of type VI secretion system activity during enteroaggregative E. coli experimental evolution 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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