Quorum sensing dependent repression of the Staphylococcus aureus transcription termination factor Rho is necessary for pathogenicity

Quorum sensing dependent repression of the Staphylococcus aureus transcription termination factor Rho is necessary for pathogenicity | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Quorum sensing dependent repression of the Staphylococcus aureus transcription termination factor Rho is necessary for pathogenicity Laura Barrientos , Marion Blum , Florence Couzon , View ORCID Profile François Vandenesch , View ORCID Profile Karen Moreau , View ORCID Profile Pascale Romby , View ORCID Profile Isabelle Caldelari doi: https://doi.org/10.1101/2025.07.24.666493 Laura Barrientos 1 Université de Strasbourg, CNRS, Architecture et Réactivité de l’ARN , UPR9002, F-67000 Strasbourg, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Marion Blum 1 Université de Strasbourg, CNRS, Architecture et Réactivité de l’ARN , UPR9002, F-67000 Strasbourg, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Florence Couzon 2 CIRI, Centre International de Recherche en Infectiologie, Université de Lyon , Inserm U1111, Université Claude Bernard Lyon 1 , CNRS UMR5308, ENS de Lyon, Lyon, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site François Vandenesch 2 CIRI, Centre International de Recherche en Infectiologie, Université de Lyon , Inserm U1111, Université Claude Bernard Lyon 1 , CNRS UMR5308, ENS de Lyon, Lyon, France 3 Centre National de Référence des Staphylocoques, Institut des agents infectieux, Hospices Civils de Lyon , Lyon, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for François Vandenesch Karen Moreau 2 CIRI, Centre International de Recherche en Infectiologie, Université de Lyon , Inserm U1111, Université Claude Bernard Lyon 1 , CNRS UMR5308, ENS de Lyon, Lyon, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Karen Moreau Pascale Romby 1 Université de Strasbourg, CNRS, Architecture et Réactivité de l’ARN , UPR9002, F-67000 Strasbourg, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Pascale Romby Isabelle Caldelari 1 Université de Strasbourg, CNRS, Architecture et Réactivité de l’ARN , UPR9002, F-67000 Strasbourg, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Isabelle Caldelari For correspondence: caldelari{at}ibmc-cnrs.unistra.fr Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT In Staphylococcus aureus , the transcription termination Rho factor has been identified as an attenuator of virulence, highlighting this universal protein as a new player in S. aureus pathophysiology. Moreover, Rho is required for the repression of “pervasive” transcription, which results from transcription at cryptic promoters or from transcriptional readthrough. In our study, we demonstrated that the rho transcript is specifically repressed by the quorum sensing-dependent RNAIII during the stationary phase of growth, allowing exotoxins to be activated at this stage. Rho-dependent RNA targets were identified using RIP-seq and differential transcriptomic analyses, revealing that several of these targets are involved in adhesion, dissemination, and the host responses. Our data provide a molecular explanation for the functions of this well-conserved termination factor, which have evolved in this human pathogen. INTRODUCTION In bacteria, transcription termination is an essential step of gene expression and is also required for maintenance of genome integrity 1 , 2 . Transcription termination occurs through two mechanisms long considered independent from one another: intrinsic termination and factor-dependent termination. Intrinsic termination requires the formation of a terminator hairpin at the 3’ end of the nascent RNA followed by a stretch of U residues. This secondary structure alone promotes the dissociation of the transcription elongation complex. Conversely, Rho-dependent termination involves the transcription termination factor Rho, an ATP-dependent RNA-DNA helicase, that binds specific features on the nascent RNA named rut sites - composed of non-structured cytidine-rich regions - and releases the RNA polymerase from the DNA-RNA hybrid 3 . The Rho factor has been extensively studied in Escherichia coli since this protein is essential and mediates the widespread termination of about half of transcription events. In E. coli , the transcription elongation factor NusG has been found to stimulate Rho-dependent termination in vitro by directly interacting with the termination factor and accelerating RNA release at termination sites and stalled elongation complexes 4 – 6 . About one third of Rho-dependent terminators are NusG-dependent in E. coli . It is believed that the interaction of NusG with Rho induces a rapid conformational change to the active form of Rho, helping to stabilize the Rho-RNA interaction at suboptimal rut sites 7 . NusG is universally conserved among bacteria and its absence has global effects on gene expression and bacterial physiology, like motility in Bacillus subtilis 8 . In addition, NusG, together with NusA, enhance intrinsic termination at weak terminators by inducing transcriptional pausing 8 , 9 . More recently, it has been shown that there is an interplay between intrinsic termination and Rho to enhance termination at the 3’ ends of genes carrying inefficient intrinsic terminators in E. coli, Mycobacterium tuberculosis , and B. subtilis to prevent readthrough at these locations 10 , 11 . The significance of Rho beyond simple transcription termination is also well established. This factor is important for regulation of gene expression through transcriptional polarity, riboswitch-mediated attenuation, and sRNA-mediated regulation 12 , 13 , 14 . For instance, Rho regulates the transcription of several genes, such as the tna operon in E. coli or the mgtA riboswitch in Salmonella 15 . By terminating transcription in the leader region of these operons, Rho prevents transcription of the downstream genes through a mechanism known as attenuation. Some sRNAs, such as ChiX from Salmonella can influence Rho-dependent termination by affecting the accessibility of the rut sites on target mRNAs 16 . Additionally, Rho serves as a guardian of genome integrity as it limits the expression of exogenous DNA, which could be deleterious, and recycles stalled RNA polymerase to facilitate DNA repair and other essential DNA-dependent processes for cell survival 17 , 18 . Most notably, Rho suppresses pervasive transcription, which frequently produces antisense (asRNAs) that can interfere with gene expression, genome stability, and replication 6 . The spurious transcription of asRNAs leads to the formation of asRNA-mRNA duplexes, which are then targeted for degradation by the double-stranded endonuclease RNase III 19 , 20 . Even though Rho is well conserved among bacteria, with over 90% of genomes containing a Rho homologue, the factor is not always required for cell viability 21 . Most notably, Rho is not essential in the Gram-positive B. subtilis and the opportunistic human pathogen Staphylococcus aureus, but it accomplishes relevant biological function 21 , 22 . In B. subtilis , Rho regulates motility, sporulation, and cell fate determination 23 . Rho is also necessary for the adaption of B. subtilis to stationary phase, a phase characterized by physiological remodeling for bacterial adaptation to limited resources 24 . In S. aureus , the mechanism of action of Rho is not well studied. Rho decreases antisense transcription consistent with its conserved role in suppressing pervasive transcription 25 . Furthermore, the rho mutant appears more virulent than its cognate WT strain in a murine model of bacteremia showing that Rho acts as a suppressor of virulence 26 . This effect might be due to the deregulation of the SaeRS regulon, which is induced in an S. aureus rho deletion mutant 26 . The Sae two-component system (TCS) responds to several stimuli, especially induced by the host defenses, and controls the expression of multiple virulence genes encoding toxins, adhesins, and immune evasion proteins 27 . This system is composed of four genes: the sensor histidine kinase SaeS, the response regulator SaeR and two auxiliary proteins, SaeP and SaeQ, necessary for the activation of the phosphatase activity of SaeS 27 . Interestingly, the activation of the Sae regulon was shown to be directly dependent on the transcription termination activity of Rho, since its inhibition by bicyclomycin, an antibiotic that specifically blocks ATPase activity of Rho, has the same virulence-inducing effect 26 . The physiological significance of Rho in different bacterial species supposes the existence of tight regulatory mechanisms to ensure the correct expression of this protein. For instance, E. coli rho is autogenously regulated by transcriptional attenuation of the rho mRNA, leading to premature termination 28 , 29 , 30 . Conversely, the sRNA SraL binds to the 5’UTR of the rho mRNA and counteracts premature transcription termination by competing with Rho for the rut sites 31 . Another layer of regulation has been recently described, where Rho can be transiently inactivated upon stress by undergoing oligomerization 32 . This sequestration mechanism is reversible and allows modulation of Rho activity during stress conditions, where the latter could be detrimental 32 . Similarly, in the Gram-negative Acinetobacter baumanii , where rho is essential, Rho levels are maintained constant but its activity is modulated by external factors to decrease Rho-dependent termination, promoting a switch between virulent and avirulent states 33 . Similarly, in B. subtilis , Rho amounts remain low 34 , and both rho mRNA and protein levels decrease upon entry intro stationary phase, suggesting the existence of regulatory pathways controlling rho expression 24 . In S. aureus , the level of rho mRNA is at its lowest during stationary phase, indicating that the rho gene regulation is crucial once the bacterial population grows, when significant physiological remodeling takes place and exotoxin activation occurs 25 . Regulation of virulence factors in S. aureus involves complex regulatory networks including TCS, transcription factors, and regulatory RNAs (sRNAs). The accessory gene regulator (Agr) quorum-sensing system responds to cellular density and is a global regulator of virulence 35 . The system senses an auto-inducing peptide (AIP) and only when its extracellular concentration reaches a certain threshold, the system is activated. The agr locus is composed of two divergent operons initiating the transcription of divergent transcripts named RNAII and RNAIII. The RNAII mRNA encompasses the four agrBDCA genes that encode the TCS machinery, AgrC being the sensor and AgrA, the response regulator 35 . One of the main effector of the system is RNAIII, which is a 514 nt-long bifunctional sRNA that encodes ∂-hemolysin ( hld ) 36 . RNAIII post-transcriptionally regulates many mRNA targets by direct RNA-RNA interactions, affecting stability and/or translation of the target mRNAs 37 , 38 , 39 . For instance, it activates α-hemolysin (Hla) synthesis by favoring its mRNA translation and represses the translation of other mRNAs such as rot , encoding the transcriptional repressor of exotoxins, or spa , the mRNA encoding the immunoglobin binding protein A 40 , 41 . All-in-one, RNAIII coordinates the switch from the adhesion mode of early infection to a disseminative state necessary during later stage of infection. As mentioned above, in S. aureus, rho mRNA levels decrease during stationary phase when the expression of exotoxins is activated. Rho has been described as a suppressor of this phenomenon by inhibiting the expression of the Sae regulon during bacterial proliferation. These observations suggest the existence of fine-tuning mechanisms for the levels of Rho to ensure proper virulence gene expression. In this work, we show that the rho gene undergoes post-transcriptional regulation during stationary phase, mainly by RNAIII to allow its repression. We also identified RNA targets of Rho, highlighting its direct involvement in repressing the saePQRS locus and virulence genes. Our findings place Rho within the main regulatory network of virulence in S. aureus and add transcription termination to the vast repertoire of sophisticated mechanisms this human pathogen has developed to adapt and colonize its host. RESULTS Expression of rho mRNA is quorum-sensing dependent In S. aureus, Rho is most likely involved in the regulation of virulence pathways under the control of the quorum sensing system 25 . Therefore, we first analysed the steady-state levels of rho mRNA during cell growth. The expression of the rho mRNA was monitored at different time points during growth of HG001 WT strain ( Fig. 1A ). Culture samples were taken at early exponential (2 h of growth), mid exponential (4 h) and early stationary phases (6 h) for RNA extraction and visualization on Northern blot. We observed an accumulation of the rho mRNA during early exponential phase, followed by a strong decline during stationary phase ( Fig. 1A ). Download figure Open in new tab Figure 1. rho mRNA is regulated by the quorum sensing agr system. A. Northern blot analysis of rho mRNA in HG001 WT and HG001 △ agr strains. Total RNA was extracted at 2, 4, and 6 h of growth in BHI medium at 37°C and loaded on a 1% agarose gel. DIG-labeled RNA probes against rho and the loading control 5S rRNA (5S) were used. The 5S rRNA was probed on the same membrane. B . Northern blot analysis of rho mRNA and RNAIII in HG001 pCN51, △ rnaIII pCN51 and △ rnaIII pCN51::RNAIII strains. Total RNA was extracted at 2, 4 and 6 h of growth in BHI medium supplemented with 10 µg/mL erythromycin and loaded on a 1% agarose gel. DIG-labeled RNA probes against rho, RNAIII, and the loading control 5S rRNA (5S) were used. RNAIII and 5S rRNA were probed from the same gel as rho mRNA. Given that rho expression of is growth-phase dependent, we examined the levels of rho mRNA in various HG001 mutant strains, each lacking one of the two-component systems (TCS) that detect and connect environmental changes to physiological responses (Fig. S1) 42 . Among the tested TCS, we found that the agr mutant strain alleviates the repression of rho at the late stationary phase of growth (Fig. S1, Fig. 1A ). Since the regulatory bi-functional RNAIII is one of the main intracellular effectors of the agr system, we analysed the yields of rho mRNA in the deletion mutant Δ rnaIII HG001 strain, as well as in the same mutant strain complemented with a plasmid expressing RNAIII. We found that in the Δ rnaIII mutant strain, expression of rho becomes constitutive while the phase-dependent repression of rho was restored in the complemented strain expressing RNAIII similar to what is observed in the WT HG001 strain ( Fig. 1B ). Note that RNAIII transcribed from the plasmid is slightly longer than the chromosomal copy, most probably due to the weakness of its terminator, leading to the usage of the terminator sequence encoded on the plasmid. However, this longer RNAIII is still able to regulate key virulence factors, as the activation of hla was restored in the complemented mutant strain (Fig. S2). The RNAIII-dependent repression of rho was further confirmed in S. aureus RN6390 WT strain, in which RNAIII expression is stronger than in HG001 (Fig. S3). All in all, our results show that the quorum sensing agr system, through its main effector RNAIII, represses rho during stationary phase. RNAIII binds to rho mRNA at multiple sites RNAIII is a long, highly structured sRNA with 14 hairpins and contains the coding region for the ∂-hemolysin gene ( hld ) 43 . To assess the existence of a rho -RNAIII interaction in vivo , we first applied the MAPS technique (MS2 affinity purification coupled with RNA sequencing) which was previously used to determine the RNA targetome of several staphylococcal sRNAs 44 , 45 , 46 . Briefly, RNAIII was tagged with an MS2 tag at its 5’ end and expressed in the Δ rnaIII mutant strain from a plasmid under the control of its own P3 promoter. This tagged version of RNAIII exhibited very similar steady-state levels as compared to the WT RNAIII (Fig. S4). After purification by affinity chromatography, RNAs from each fraction were extracted and analyzed by Northern blot using specific probes against RNAIII and rho mRNA. The MS2-RNAIII was specifically retained by the MS2-MBP fusion protein and the rho mRNA was significantly enriched in the elution fractions of MS2-RNAIII ( Fig. 2 ). Download figure Open in new tab Figure 2. rho mRNA was pooled down with RNAIII in vivo . MS2-affinity purification of MS2-RNAIII was performed in a △ rnaIII background using HG001 strain expressing the MS2-tag alone as a control. HG001 pCN51::P3 MS2 and △ rnaIII pCN51::P3 MS2-RNAIII strains were grown in BHI medium supplemented with 10 µg/µL erythromycin for 6 h. Cells were harvested, lysed and the supernatant was subjected to affinity purification. RNAs from each fraction (CE: crude extract, FT: flowthrough, W: wash, E: elution) were precipitated and loaded on two separate 1% agaroses gel to perform Northern blot. Specific DIG-labeled RNA probes against RNAIII and rho mRNA were used. The data of two different replicates are shown (1, 2). In silico predictions of base-pairing interactions between RNAIII and rho mRNA were then performed using IntaRNA 47 – 50 . The CU-rich unpaired region of hairpin 9 of RNAIII was predicted to interact with two distant regions of the coding sequence of the rho mRNA. These two regions included either nucleotides +46 to +82 ( Fig. 3A ) or nucleotides +1167 to +1224 near the 3’-end of rho mRNA (Fig. S5A). To determine whether RNAIII and rho mRNA can form a stable complex, we conducted gel retardation assays using in vitro transcribed rho mRNA and the full length RNAIII labeled at its 5’ end. Two different fragments of rho mRNA were used respectively, the 5’-end containing nucleotides 1 to 468 and the 3’ end of the mRNA encompassing nucleotides 965 to 1417. A stable interaction with the 5’ part of rho could be visualized in vitro ( Fig. 3B ) and quantification indicated that the dissociation constant (Kd) is around 150 nM. Unexpectedly, the deletion of hairpin 9 of RNAIII (RNAIII △H9) had only a minor effect on the binding to rho mRNA in gel retardation assay ( Fig. 4A ) suggesting that there is an alternative binding site on RNAIII ( Fig. 3A ). Download figure Open in new tab Figure 3. RNAIII binds to rho mRNA in vitro . A . Electrophoretic mobility shift assay shows the formation of the complex between 5’-end radiolabeled RNAIII and a 468 nt-long fragment of the rho mRNA from the 5’ end comprising the region predicted to interact with RNAIII. The 5’-end radiolabeled RNAIII was incubated with increasing concentrations of cold rho mRNA (50 to 400 nM) (left side). On the right side is the predicted interactions in hairpins 7 and 9. B . The RNAIII- rho duplex is cleaved by RNase III in vitro. (Left) 5’-end radiolabeled RNAIII is incubated with the rho mRNA fragment (468 nts from the 5’ end) without or with increasing concentrations of purified S. aureus RNase III. Lane 1: incubation control of RNAIII in the absence of RNase III; Lane 2: RNase III (200 nM) cleavage assay of RNAIII; Lane 3: incubation control of RNAIII with 400 nM rho in the absence of RNase III; Lanes 4-7: hydrolysis of the RNAIII- rho complex in the presence of increasing concentrations of RNase III (25 nM, 50 nM, 100 nM and 200 nM). Reactions were performed in the presence of Mg 2+ required for RNase III activity. Lane 8: reaction control using Ca 2+ instead of Mg 2+ to inhibit RNase III activity. Lanes T1, L: RNase T1 and alkaline ladders, respectively, under denaturing conditions (left side). The secondary structure model of RNAIII is from Benito, et al 43 showing deletions of hairpin 7 (ΔH7) and hairpin 9 (ΔH9) and the identified strong RNase III cleavage in hairpin loop H7 (arrow) (right side). The RNAIII regions predicted by IntaRNA complementarity to rho are colored in red. Download figure Open in new tab Figure 4: RNAIII binds to rho mRNA at multiple sites A . Electrophoretic mobility shift assays show the formation of complexes between RNAIII and the 5’-end radiolabeled 468 nt-long fragment of the rho mRNA. Several truncated versions of RNAIII were deleted of either one hairpin H7 (ΔH7) or H9 (ΔH9), or the two hairpins H7 and H9 (ΔH7ΔH9). The 5’-end radiolabeled rho was incubated with increasing concentrations of cold RNAIII (50 to 300 nM). B . Effect of deletion of H7 and H9 of RNAIII on RNaseIII hydrolysis in the presence of rho mRNA. In vitro transcribed RNAIII, RNAIII△H7, RNAIII△H9 and RNAIII△H7△H9 (200 nM) were incubated with rho mRNA with or without 200 nM of RNase III in the presence of Mg 2+ or Ca 2+ . Lane 1: incubation control of RNAIII in the absence of RNase III; Lane 2: Hydrolysis of RNAIII in the presence of 200 nM RNase III; Lane 3: incubation control of RNAIII with 400 nM rho in the absence of RNase III; 4: Hydrolysis of RNAIII bound to rho mRNA (400 nM) in the presence of RNase III (200 nM); Lane 5: Reaction control using Ca 2+ instead of Mg 2+ to inhibit RNase III activity; Lanes A, G, C, TA are DNA sequencing ladders. White arrows denote the major RNase III cleavage in rho mRNA bound to RNAIII. RNase III has previously been shown to specifically cleave various RNAIII-mRNA complexes, making it a useful tool for identifying the targeted C-rich motifs 51 . We thus tested its ability to cleave the RNAIII- rho mRNA complex in vitro ( Fig. 3B ). The hybrid formed in vitro was incubated with increasing concentrations of purified S. aureus RNase III. Appropriate incubation controls were done and the cleavage positions were assigned using 5’ end-labeled RNAIII subjected to RNase T1 cleavage and alkaline hydrolysis. A strong RNase III cleavage was detected in the hairpin loop 7 of RNAIII at the C residues while no cleavage was observed in hairpin 9 ( Fig. 3B ). This cleavage is highly specific, as substituting Mg 2+ with Ca 2+ ions—an action that inhibits enzyme activity—abolishes the cleavage signal (lane 8 in Fig. 3B ). This strongly indicates that hairpin 7 is also part of the rho binding site, which contains several G-rich motifs complementary to the C-rich sequence of hairpin loop 7 of RNAIII ( Fig. 3B ). To evaluate the contribution of hairpins 7 and 9 in the formation of a stable complex with rho mRNA, we have synthesized RNAIII mutants lacking either hairpin 9 (RNAIII △H9), hairpin 7 (RNAIII △H7) or both hairpins (RNAIII △H7△H9). We then performed gel retardation assays using the 5’-end labeled rho mRNA incubated with increasing concentrations of each RNAIII variants. Deleting one hairpin had only a minor effect on rho binding compared to the WT RNAIII ( Fig. 4A ). However, deletion of both hairpins significantly reduced the interaction with rho mRNA, although some weak binding was still detectable. We then assessed how these mutations in RNAIII affected the RNase III cleavage patterns. Each RNAIII mutant were incubated with or without rho mRNA, and the formed complexes were subjected to RNase III cleavage. To detect the cuts, reverse transcription was performed using a 5’ end radiolabeled primer complementary to RNAIII ( Fig. 4B ). Deletion of hairpin 9 did not change the cleavage observed in hairpin 7 of RNAIII bound to rho ( Fig. 4B ), suggesting that hairpins 7 and 9 have independent binding sites on rho . All these data point to a direct interaction of RNAIII with the rho mRNA and that RNAIII interacts at several sites of rho mRNA. Binding of RNAIII to rho mRNA inhibits its translation To determine whether RNAIII affects translation of rho mRNA, we first examined if RNAIII fixation impacts the formation of a simplified initiation ribosomal complex on rho mRNA using toe-printing assays ( Fig. 5A ). Binding of the initiator tRNA fMet and of the S. aureus 30S subunits to rho mRNA causes a typical pause of the reverse transcriptase at position +16, the so-called toe-print signal, which strongly decreased even at the lowest concentration of RNAIII. This result indicates that RNAIII hinders the ribosome binding site ( Fig. 5A ). In contrast, the addition of RNAIII Δ7Δ9 only reduced the toe-print signal at its highest concentration (150 nM). Interestingly, we observed two strong reverse transcriptase stops at G+73 and G+72 of rho mRNA caused by RNAIII binding in the absence or presence of the 30S subunit, which were not detected when RNAIII Δ7Δ9 was added in the assays. This observation correlates well with the predicted base-pairing interactions between the coding region of rho mRNA (region +46 to +82 from the AUG codon) and hairpin 9 of RNAIII. Download figure Open in new tab Figure 5. RNAIII affects translation of the rho mRNA in vitro . A. Toe-print assays showing the binding of S. aureus ribosomes on rho mRNA in the absence or presence of increasing concentrations of RNAIII WT or RNAIII△H7△H9. Lanes 1 & 2, incubation controls of mRNA alone with or without 30S subunit respectively; lanes 3 & 5, toe-printing assays on rho mRNA in the absence of RNAIII; lane 4, incubation control of rho mRNA with RNAIII WT (150 nM); lanes 6-8, toe-printing in the presence of increasing concentrations of RNAIII (40, 80, 150 nM); lane 9, incubation control of rho mRNA with RNAIII△H7△H9 (150 nM); lane 10, toe-printing assay on rho mRNA in the absence of RNAIIIΔH7ΔH9; lanes 10-13, toe-printing assays performed with increasing concentrations of RNAIII△H7△H9 (40, 80, 150 nM). Lanes A, T, C, G : DNA sequencing ladders. The Shine and Dalgarno (SD) sequence, the start site of translation (ATG), the toe-printing signal (+16), and the reverse transcription stops caused by RNAIII binding (H9) are indicated. (*) Note that less amount of radioactivity material was present in lane 5. B. In vitro translation assays of rho-flag mRNA in the absence or presence of increasing amounts of RNAIII WT or RNAIII△H7△H9 in the presence of S. aureus 70S ribosomes. Reactions were loaded on a 12% polyacrylamide-SDS gel. For the Western blot, membranes were incubated with anti-FLAG mouse antibodies followed by the incubation with a goat anti-mouse peroxidase (HRP) antibody. Coomassie staining of the membrane was used as loading control. We then performed in vitro translation assays using S. aureus ribosomes and the first 468 nucleotides of rho mRNA with a Flag-tag sequence at the 3’ end. Western analysis with anti-FLAG mouse antibodies revealed the presence of the Rho protein ( Fig. 5B ). The addition of increasing concentrations of RNAIII significantly decreased Rho protein levels, while the addition of the mutant RNAIII Δ7Δ9 only slightly inhibited rho translation at the highest concentration ( Fig. 5B ). These data strongly suggested that binding of RNAIII to rho mRNA represses its translation in vitro . Identification of Rho-dependent pathways determined by RNAseq Previous tiling-array studies have shown that rho deletion lead to an overall increase levels of antisense RNAs and transcripts belonging to the SaeRS regulon 26 . To get a genome-wide analysis of Rho functions, we constructed a deletion mutant of rho in HG001 by homologous recombination. We then performed RNAseq on total RNA extracts prepared from cultures of HG001 WT and the isogenic HG001 △rho mutant grown in BHI medium at 37°C until early exponential phase, when rho mRNA is mostly expressed. The experiments were done in duplicates with high reproducibility. We have considered a gene to be regulated by Rho when a two-fold expression change was observed between both strains with adjusted p-values (padj) below a 0.05 threshold (Table S1). Most differentially expressed genes in the △rho mutant are depicted in the volcano plot ( Fig. 6A ) allowing the identification of different gene categories under the regulation of Rho. The data revealed significant changes in expression consisting primarily in the upregulation of virulence factors, antisense transcripts, and prophage genes in the Δ rho mutant strain (Table S1A). Hence, the deletion of rho causes pervasive and antisense transcripts while differential regulation was observed as the result of readthrough at putative Rho-dependent sites sometimes associated with Rho-independent terminators. Download figure Open in new tab Figure 6. Rho regulates the expression of numerous virulence genes. A. Volcano plot of differentially expressed genes between HG001 WT and HG001 △rho strains. RNAseq analysis was performed on purified RNAs from duplicates of each strain grown in BHI medium at 37°C until exponential phase. Each point represents one gene. Depicted in blue are strongly upregulated genes and in red downregulated genes in the △rho mutant strain. B. Comparison of the differential transcriptomics and RIP-Seq data for the genes belonging to the SaeRS regulon. For the RIPseq, the endogenously 3xFLAG-tagged Rho protein was immunoprecipitated from cultures grown in BHI medium at 37°C until exponential phase, and co-immunoprecipitated RNAs were isolated and sequenced. In blue are genes presenting FC>2 with padj2 with padj<0.05 in the RIPseq data. Several genes were downregulated upon deletion of rho and concomitantly increased levels of antisense RNAs were observed (Table S1B). This is particularly the cases of genes associated to prophages and all the genes involved in nitrate respiration ( narG , narH , narI and narJ ), transport ( narT ), and reduction ( nirB , nirD ), and the nreABC operon encoding TCS involved in the regulation of nitrate/nitrite reduction (Fig. S6A). Another strong enhanced RNA antisense was observed in the Δ rho mutant strain with a concomitant decreased of the yield of the corresponding operon mbtS , BSR_RS14940, and BSR_RS14945 (Fig. S6B). Interestingly, MbtS is a membrane bound transcriptional factor regulating the cysteine protease staphopain A and serine proteases SplA-F, all involved in host defenses 52 . In agreement with this observation, the mRNAs encoding the staphopain A (SspP) and its inhibitor SspC were upregulated (Table S1A). Other genes involved in anaerobic metabolism such as hmp encoding the flavohemoprotein, ldh1 or ldhD1 encoding lactate dehydrogenases, adh encoding alcohol dehydrogenase, pflA and pflB encoding respectively the pyruvate formate lyase and formate acetyl transferase enzymes, and srrAB encoding the TCS SrrAB (staphylococcal respiratory response), which promotes resistance to nitrosative stress and hypoxia 53 , are repressed in the Δ rho mutant without the obvious presence of antisense RNAs (Table S1B). Hence, downregulation of mbtS and srrAB mRNAs in the Δ rho mutant strain is expected to induce indirect regulatory effects. In the Δ rho strain, many upregulated genes encode virulence factors. Notably, a large proportion of these genes are part of the sae regulon, including the saeP and saeQRS transcripts. Specifically, 25 out of 39 SaeRS-dependent genes are significantly upregulated in the Δ rho mutant, including major exotoxins such as HlgA, HlgB, HlgC, Hla, and LukGH ( Fig. 6B ). Within the four genes in the saePQRS operon, saeP is the most upregulated ( Fig. 6B , S7). Among the most upregulated genes from the sae regulon, chp encodes the chemotaxis inhibitory protein 54 , sbi a major immunoglobulin-binding protein 55 , and hlb-1 the non-pore forming hemolysin beta 56 (Fig. S8, S9). For hlb-1 and sbi , the regulation is most probably occurring at the transcriptional level. For the chp gene, a readthrough generates a long transcript antisense to the downstream genes encoding a putative phage amidase (BSR30_RS10675) and the staphylokinase SAK (BSR30_RS10680) in the Δ rho strain (Fig. S9). Surprisingly, this antisense RNA has no significant effect on the level of the complementary mRNA (Fig. S9). Noteworthy, a Rho-independent terminator structures are predicted at the 3’-end of the chp gene and at the same position on the opposite strand at the 3’-end of BSR30_RS10675, creating “mirror” terminator hairpins on both strands. Similarly, in the Δrho mutant, a readthrough transcript was also produced from the BSR30_RS10675 gene on the opposite strand and terminated at the Rho-independent terminator of the scn gene (Fig. S9). This large transcript generated an antisense RNA to the chp gene comprising the sRNA SprD, a translational repressor of sbi mRNA (Fig. S9) 57 , 58 . The riboflavin synthesis operon comprising the genes ribD , ribE ribAB and ribH under the control of the transcriptional FMN riboswitch was upregulated in the Δ rho mutant (Fig. S10, Table S1A). Higher transcription levels of Teg74 and the downstream rib genes might result from transcriptional readthrough at Teg74 when Rho is absent (Fig. S10). Overall, differential RNA-seq provided a global view of the impact of Δ rho deletion, revealing several Rho-dependent pathways linked to metabolism and virulence. The RNA interactome of Rho studied by RIP-Seq In addition to the RNA-seq approach, we unveiled the RNA interactome of Rho by performing RIP-seq (RNA immunoprecipitation followed by RNA-seq). In this experiment, Rho was co-immunoprecipitated along with its RNA partners, which were then identified by deep sequencing. To perform RIP-seq, we constructed a strain expressing a chromosomally encoded tagged version of the Rho protein using homologous recombination. A triple FLAG-tag was added to the C-terminal of the protein. The steady-state levels of the modified rho mRNA were like those of the WT rho mRNA (Fig. S11A) and the Rho-3xFLAG protein was well expressed (Fig. S11B). The Rho-3xFLAG was immunoprecipitated from cultures grown until early exponential phase using anti-FLAG antibodies followed by elution of the recombinant Rho-RNA complexes using a 3xFLAG peptide. A control Co-IP was performed using the untagged Rho protein from a WT strain. Since the Rho-3xFLAG protein was successfully enriched in the elution fraction (Fig. S12), the co-immunoprecipitated RNAs were extracted for sequencing. By comparing the RNA reads from Rho-3xFLAG purification and the untagged Rho, we identified 328 RNAs enriched with Rho (at least two-fold enrichment with padj < 0.05) (Table S2). The majority of the enriched RNAs (152 out of 328) are antisense RNAs or overlapping genes, correlating with the major function of Rho in suppressing pervasive transcription. To identify putative direct targets of Rho, we cross-referenced our RNA-seq and RIP-seq datasets, yielding 121 RNAs significantly enriched in RIP-seq and whose expression was affected by rho deletion (Table S3). This list includes eight mRNAs known to be regulated by the SaeRS TCS, such as saeP , hlb-1 , sbi , lukGH , scn , and chp ( Fig. 5B ). Among other candidates, is ribH from the riboflavin synthesis operon, which is under the control of the FMN riboswitch (Table S3). Among the downregulated genes in the Δ rho mutant strain, only six transcripts co-immunoprecipitated with Rho, such as narT (nitrate transporter) and the operon including cydB , ktrA and BSR30_RS05090 genes. In summary, our RIP-Seq data correlates well with our differential transcriptomics with a high selectivity for RNAs repressed by Rho since the expression of the vast majority (115/121) of enriched RNAs was highly upregulated in the rho mutant. Rho-dependent termination of specific mRNA targets To monitor Rho effects on target RNAs, we constructed a GFP reporter with sequences containing the regulatory region of two target genes under the control of a constitutive promoter in the pCN57 vector 59 ( Fig. 7A ). All plasmids were transformed into the strains HG001 pMKX (wild-type strain containing an empty pMKX plasmid), HG001 △rho pMKX ( rho deletion mutant containing an empty pMKX plasmid), and HG001 △rho pMKX:: rho ( rho deletion mutant strain complemented with the pMKX:: rho plasmid expressing rho under the control of a xylose-inducible promoter) 25 . As previously published, we observed a leaky expression of the rho transcript in the complemented strain without promoter induction (Fig. S13) 25 . The strains were grown in BHI medium until exponential phase and cells were lysed to measure GFP expression. If Rho terminates transcription at the predicted locations of the constructs, elongation through the gfp gene should be affected. Conversely, Rho transcriptional readthrough should lead to increased elongation through the gfp gene, resulting in higher expression of the GFP protein. Download figure Open in new tab Figure 7. Rho terminates transcription of teg74 and potentially chp . A . Schematic representation of pCN57:: gfp reporter fusion plasmid. Target sequences of the chp and teg74 genes comprising their own Rho-independent terminator hairpin are cloned upstream the gfp sequence under the control of the constitutive promoter BlaZ. RBS is for ribosome binding site. B. GFP levels of cellular extracts from strains HG001 WT pMKX, HG001 △rho pMKX and HG001 △rho pMKX:: rho transformed with plasmids containing the sequences of chp and teg74. Cells were grown until exponential phase and lysed extracts were used for GFP measurement (n=6). Statistical analysis was performed with one-way ANOVA test. To analyze the effect of Rho on the teg74 riboswitch, we have first cloned the sequence of the riboswitch including the Rho-independent terminator in the reporter fusion construct. The data showed a significant increase of the GFP synthesis in the Δ rho mutant strain as compared to the WT and the complemented mutant strains ( Fig. 7B ). The same type of experiment was performed for the chp operon. In that case, we used a fragment comprising the 3’-end of the chp gene and the intergenic region downstream containing a Rho-independent terminator. We chose this region where transcriptional readthrough was observed (Fig. S9). The data revealed a tendency for a slight increase of GFP synthesis in the Δ rho mutant strain in comparison to the WT and complemented strains ( Fig. 7B ). These data strongly support a direct role of Rho in transcription termination of specific mRNAs. Rho expression impacts S. aureus virulence Based on the Rho-dependent regulation identified above, different phenotypic assays were chosen to define the impact of Rho on the physiopathology of S. aureus . We have compared the behaviors of the WT HG001 strain, the isogenic Δ rho mutant strain, and the complemented strain. Since several mRNAs encoding exotoxins (Hla, HlgC) were upregulated in the Δ rho mutant strains, we first measured the amounts of the alpha (Hla) and gamma (HlgC) hemolysins by ELISA. A 2,8-fold increase in Hla and a 16-fold increase in HlgC were observed in the Δ rho mutant strain compared to the WT strain, and the mutant strain overexpressing Rho ( Fig. 8A ). These protein levels correlated with an increase in the cytotoxic activity of the Δ rho mutant strain on specific monocytes expressing gamma hemolysin receptors C5aR1 (3-fold increase) and CxCR2 (4-fold increase) ( Fig. 8B ) and on rabbit red blood cells sensitive to alpha hemolysin (4,6-fold increase) ( Fig. 8C ). Download figure Open in new tab Figure 8. Impact of Rho on S. aureus cytotoxicity. A. Quantification of HlgC and Hla proteins (in nanograms per milliliter) from supernatants of HG001 WT (HG001-pMKX), mutant deleted for rho gene (HG001Δ rho pMKX) and complemented strain (HG001Δ rho pMKX:: rho ) cultures grown during 18 h in CCY medium (n=3). B. Bacterial supernatant cytotoxicity was quantified by measuring propidium iodide incorporation into U937 monocytes, and into U937 monocytes expressing gamma hemolysin receptors C5aR1, and CXCR2 (n=9). C. Graphic representation of the Hla hemolytic activity of the supernatants from cultures grown during 18 h in CCY medium on rabbit red blood cells (n=3); the Hla activity value corresponds to the inverse of the exact dilution causing the lysis of 25% of the red blood cells. D. Isogenic strains of S. aureus examined for their binding capacity to fibrinogen coated on 96-well plates, at concentration of 5 µg/ml. The adherence of bacterial cells was quantified at 485 nm using TECAN plate reader. Mann-Whitney tests performed: * = p-value < 0.05, ** = p-value < 0.01, *** = p-value < 0.001, **** = p-value < 0.0001. Other mRNAs encoding several adhesin factors (Fibronectin binding proteins FnbAB, coagulase, and map) were also upregulated in the absence of Rho ( Fig. 6 ). We first tested the fibrinogen adhesion phenotype in vitro and observed an increased ability of the Δ rho mutant strain to adhere to fibrinogen compared to the WT strain ( Fig. 8D ). Additionally, we monitored the ability of the same strains to coagulate rabbit plasma and detected a higher coagulase titer in the Δ rho mutant strain compared to the WT strain (Fig. S14). Noticeably, a restoration of most of the phenotypes was observed in the HG001Δ rho ::pMKX- rho complemented strain, albeit with a reduced effect as compared to the WT strain. One cannot exclude that the over-expression of Rho from the pMKX plasmid resulted in a less active protein under the tested conditions. All-in-one, these data show that Rho decreases cell cytotoxicity and reduces cell adherence reinforcing that Rho is an attenuator of virulence 26 . DISCUSSION In this study, we investigated and characterized the regulation and the roles of the transcription termination factor Rho in S. aureus . This highly conserved protein is found in over 90% of bacterial genomes and accomplishes diverse biological functions, including regulation of specific operons, participation in sRNA-dependent mechanisms, and prevention of spurious transcription. In E. coli , Rho has a broad role, since it terminates transcription of up to 50% of all genes and often cooperates with NusA and NusG 21 . Rho can be part of the RNase E-based degradosome, demonstrating that Rho is a versatile and global regulator. 60 In S. aureus , however, besides its role in controlling pervasive transcription, the functions of Rho appear to be more specific to virulence regulation. Indeed, the deletion or inactivation of Rho leads to increased expression of the SaeRS dependent-targets, resulting in enhanced virulence, as tested in a murine model of infection 26 . While virulence is a tightly controlled and time-dependent phenotype, we demonstrate that Rho synthesis is growth phase-dependent and is post-transcriptionally control by the quorum sensing regulatory RNAIII. In addition, we identify new Rho-dependent targets highlighting the dual functions of Rho as both a suppressor of pervasive transcription and a regulator of virulence gene expression. Rho protein synthesis is under the control of the quorum sensing dependent RNAIII We showed that the expression of the rho mRNA levels decreases sharply at the entrance of the stationary phase. This regulation is under the control of the agr quorum-sensing system, specifically by its main effector, the 514-nt long RNAIII 37 – 41 . Indeed, the time-dependent regulation of the rho mRNA is alleviated in the deletion Δ rnaIII mutant strain. In vitro , RNAIII forms a stable complex with rho mRNA, which can be cleaved by RNase III ( Fig. 3B , 4B ). Our in vitro data support the existence of several interacting sites between RNAIII and rho mRNA, involving at least hairpin loops H7 and H9 of RNAIII ( Fig. 3 , S5C). While the deletion of either H9 or H7 only partially affected the binding to rho , their simultaneous deletion had a synergistic effect but did not completely abolish the binding. These data suggested that other compensatory binding sites between RNAIII and rho mRNA take place. Noteworthy, the 5’ UTR and the coding sequence of rho mRNA contain several redundant GGGA motifs potentially complementary to the UCCC motifs found in hairpin loops 7, 13 and 14 of RNAIII. This GGGA motif is also part of the binding sites of other RNAIII targets, such as coa and rot mRNAs, and a similar RNase III cleavage has been observed in the hairpin loop 7 at the C-rich motif due to a loop-loop interaction 39 , 41 , 61 . The importance of each hairpin of RNAIII for interaction with rho would need further clarification. We nevertheless propose that H9 of RNAIII binds to the coding region of rho (nts +46 to +82), forming at least 10 consecutive base-pairing interactions, further stabilized by hairpin loop H7 binding either to the 314 GGGAU 318 or to the 327 UGGGA 331 motif (Fig. S5C). We do not exclude that hairpin loops H13 and H14 of RNAIII may contribute to the overall stability of the complex by binding to the UGGG motifs in the 5’UTR of rho including its Shine and Dalgarno (SD) sequence. Since RNAIII covers a large region of rho mRNA next to the ribosome binding site, rho mRNA translation is expected to be repressed. Using toe-printing and in vitro translation assays, we showed that RNAIII binding to rho inhibits translation most likely by preventing the recruitment of the ribosome at the initiation step. We propose that the untranslated mRNA is more sensitive to degradation by ribonucleases. This would most probably contribute to the rapid depletion of rho mRNA at late exponential level of growth. Rho-dependent termination can enhance gene expression using different mechanisms Deletion of rho increased the expression of antisense transcripts, whose functions are still controversial; consequently, the global effect of this increased pervasive transcription is difficult to evaluate. In our study, we did not always observe a correlation between an enhanced expression of an antisense and downregulation of the corresponding sense transcript. For instance, the antisense produced in the Δ rho mutant strain after readthrough at the chp gene did not have any effect on the levels of the sense transcript (Fig. S9 and Table S1A). Intriguingly, we often detected enhanced antisense transcription in poorly transcribed regions and we cannot exclude that the effects of antisense transcription are masked by the low basal levels of sense transcription. Nevertheless, the co-immunoprecipitation of many antisense transcripts with Rho suggests that this factor is needed to suppress their expression most likely through premature transcription termination and is in accordance with the roles of Rho in suppressing pervasive transcription. Noteworthy, several genes involved in nitrate respiration were positively regulated by Rho. We frequently observed the generation of antisense transcripts to these genes, pointing to possible sense/antisense RNA regulation (Fig. S6). This hypothesis is supported by the fact that none of the relevant mRNAs co-immunoprecipitated with Rho in the RIP experiment, while several antisense transcripts to the affected genes were enriched (Table S1). Thus, Rho could inhibit the synthesis of these antisense transcripts to ensure the proper expression of genes implicated in nitrate respiration. However, we cannot exclude the possibility that the effect on these genes is indirect and occurs through other upstream pathways. In the RIP experiment, the enrichment of narT mRNA, which encodes a nitrate transporter, suggests a direct effect of Rho on this gene. In addition, a recent study indicates that the narGHJI operon is mostly expressed at the early exponential phase when rho mRNA is present 62 . Besides nitrate respiration, Rho activates the synthesis of proteins (e.g., the dehydrogenases Adh and Ldh-1, Hmp, and the SrrAB TCS) involved in anaerobia although rho transcription does not rely on SrrAB known to sense low O 2 levels (Fig. S1). All these genes have in common that they are transcriptionally controlled by the redox sensing regulator Rex 63 . When O 2 level is high, Rex is activated by NAD + and represses anaerobic genes such as dehydrogenases. Nonetheless, rex transcript is not differentially expressed in the Δ rho mutant nor pulled out in the RIP-seq experiment with Flagged-Rho. We can postulate that for a yet unknown reason, the NADH/NAD + ratio is perturbed in the Δ rho mutant which would interfere with Rex activity. The expression of anaerobic gene enables S. aureus to adapt to changes in oxygen concentration, which is especially relevant in an infection context. Since oxygen levels can vary within the human host, these variations can impact bacterial physiology and virulence factor production 64 . Therefore, regulation of anaerobic gene expression by Rho could influence the ability of S. aureus to adapt to different host tissues and its pathogenesis, particularly in low oxygen environments such as abscesses. Similarly to narT , the cydB , ktrA and BSR30_RS05090 mRNA operon involved in respiration and potassium transport were also co-immunoprecipitated with Rho. These data could indicate that for a reduced number of genes (mostly involved in metabolism/anaerobic respiration), Rho might be required for proper transcription termination. Rho contributes to FMN-responsive transcriptional riboswitch regulation In bacteria, homeostasis of riboflavin or vitamin B2 is regulated by the widely distributed class of flavine mononucleotide (FMN) riboswitches. The FMN riboswitches regulate the biosynthesis and uptake pathways of riboflavin, which is then converted into (FMN) and flavine adenine dinucleotide (FAD), two essential co-factors for bacterial growth as they are critical for many redox reactions of cellular metabolism 65 . FMN acts as the primary ligand regulating FMN riboswitches by binding to the aptamer when its concentration is high. Binding induces conformational changes to inhibit either the transcription or the translation of the downstream genes 66 . In S. aureus , inhibiting riboflavin biosynthesis does not reduce virulence, but simultaneously disrupting biosynthesis and uptake pathways with FMN riboswitch inhibitors like roseoflavin or ribocil-C efficiently impairs MRSA growth and virulence in a murine septicemia model 67 . Thus, specifically targeting FMN riboswitches in S. aureus could be a promising strategy to treat drug-resistant bacteria. S. aureus encodes two highly related but different FMN-responsive riboswitches. Teg74 regulates the riboflavin biosynthetic operon ( ribDEBAH ) at the transcriptional level while Teg66 controls the uptake transporter ( ribU ) at the translational level. Only, the FMN-responsive transcriptional riboswitch teg74 gene along with the downstream rib operon were upregulated in the Δ rho strain. The transcripts were also enriched upon RIP, although with a non-significant padj value, which may be explained by the relatively low expression of the operon in rich growth medium and early exponential phase. Using the reporter gene where teg74 was cloned upstream the gfp -encoding sequence, a significant increase of GFP synthesis was observed in the Δ rho mutant strain. These observations strongly suggest that Rho-dependent termination of teg74 is required for the repression of the downstream rib operon, linking Rho-dependent termination to riboswitch-mediated regulation in S. aureus . Although both riboswitches share 96% identity 67 , Teg66 and ribU were not affected upon rho deletion nor co-immunoprecipitated with Rho, suggesting the specific action of Rho on the transcriptional FMN riboswitch. Interestingly, the association of Rho to this particular riboswitch is conserved in different bacteria such as E. coli 68 , Clostridium botulinum 69 and Bacillus subtilis (E. Bidnenko, S. Durand, personal communication). Overall, Rho-dependent regulation of the transcriptional FMN riboswitch in S. aureus might contribute to modulate pathogenesis. Rho acts as an attenuator of virulence The RNA-seq analysis together with the RIP-seq revealed that the absence of Rho lead to an increased production of virulence factors, explaining the phenotypic assays ( Fig. 8 ). We mainly focused on virulence associated genes and only considered the direct targets of Rho that are repressed. Since Rho attenuates virulence in S. aureus , our rationale is that Rho induces premature transcription termination of these targets to reduce their expression. Since almost the entire sae regulon is upregulated in the absence of Rho, it was stipulated that the effect of Rho would occur through the two-component system SaeRS, which acts as a major regulator of virulence gene expression in staphylococci 26 . Our data indicates that Rho binds only to the saeP mRNA, one component of the sae operon. The saePQRS operon contains two promoters: P1 induces the expression of saeP and P3 the expression of saeR , and saeS . The P1 promoter is strongly autoregulated, and is activated by the quorum sensing effector AgrA, while it is repressed by Sigma factor B 70 . The saeP transcript can be produced by two independent mechanisms: one involves a specific RNase Y cleavage that generates an unstable saeP and a stable saeQRS mRNAs, and the other involves a termination event at a Rho-independent terminator that produces saeP 71 . Our data suggested that in the Δ rho mutant, a transcriptional readthrough event occurred at saeP allowing further transcription into the saeQRS genes (Fig. S7). Thus, saeP can be transcribed separately from saeQRS as previously hypothesized 71 . The fact that only saeP mRNA co-immunoprecipitated with Rho supports the idea that Rho might terminate the transcription of saeP . Hence, it is possible that premature termination and/or degradation of saeP alone could significantly impact the functioning of this system. Interestingly, in S. aureus , PNPase negatively alters the expression of the saePQRS operon 72 in the absence of maturation by RNase Y that forms protective 3’ extremities 72 . Thus, it is tempting to propose that Rho-dependent termination of saeP might induce degradation by PNPase affecting expression of the entire operon as observed in the differential transcriptome analyses (Table S1). Meanwhile, SaeRS autoinduces the transcription of the entire sae operon at P1 promoter. This feedback loop likely generates sufficient SaeRS TCS in the activated state. We propose that the Rho-dependent repression of saeP might play a critical role in disrupting this feedback loop, thereby significantly reducing the expression levels of the entire sae operon ( Fig. 9 ). Intriguingly, the deletion of saeP or the P1 promoter has been shown to enhance S. aureus cytotoxicity against human neutrophils by modulating the sae regulon 73 , 74 . Consequently, the deletion of either saeP or rho generates similar phenotypic effects due to the activation of the sae regulon. SaeP has been demonstrated to form a complex with SaeQ in vitro , which activates the phosphatase activity of the sensor kinase SaeS 75 . This could explain the seemingly indirect effects observed for some sae -regulated genes, such as sbi , where a major transcriptional effect was detected without any visible readthrough event (Fig. S8). Thus, overexpression of SaeRS targets in the Δ rho mutant, especially of those that were not enriched upon RIP, might be indirect through saeP . Most importantly, rho expression must be repressed in the stationary phase when SaeRS is active (see below and Fig. 9 ). Download figure Open in new tab Figure 9. Proposed model of regulation of rho during Staphylcoccus aureus growth. At low cell density, Rho play crucial roles by repressing the expression of the sae operon, suppressing pervasive antisense transcription, and activating the synthesis of proteins involved in anaerobic and nitrate metabolisms. At high cell density, the quorum sensing induced RNAIII represses the translation of rho mRNA through direct RNA-RNA pairing. The expression of sae operon is enhanced directly via AgrA, and indirectly via RNAIII. These regulatory mechanisms collectively promote the synthesis of exotoxins. Activation is represented by arrows and repression by bars: red color is for post-transcriptional regulation and black color for transcriptional control. Green arrow depicts the direct and indirect effect of Rho on anaerobia and nitrate respiration (see text for details). However, several of the SaeRS targets also co-immunoprecipitated with Rho pointing to a simultaneous indirect and direct effects of Rho ( Fig. 6B ). The genomic region including chp has retained our attention, because a readthrough was observed on both strands downstream the chp and BSR30_RS10675 genes, and because many of the depicted genes were enriched in the RIP-seq ( Fig. 6 and Table S1). We believe that Rho is involved in premature transcription termination of several loci in this region, for instance at the chp , BSR30_RS10675 and scn genes, containing Rho-independent terminators and also on the hlb_1 gene with no predicted intrinsic terminator hairpin. Using a transcriptional GFP reporter plasmid fused to the chp gene, we identify a readthrough event due to the absence of Rho in vivo , supporting the direct involvement of Rho in the regulation of the chp gene. All these genes are virulence factors and an increase in their mRNA levels may explain the hypervirulence phenotype of the deletion mutant of rho 26 . Considering the role of Rho as an attenuator of virulence, the repression mediated by quorum sensing-induced RNAIII at high cell density represents an important asset for pathogenicity. Besides RNAIII represses the translation of both rho mRNA and rot mRNA in a very similar manner 51 , the latter encoding a transcriptional repressor of SaeRS 76 . Additionally, the transcription of the sae operon was shown to be enhanced by AgrA. Taken together, these regulatory mechanisms contribute to optimize the synthesis of exotoxins facilitating the invasive state of the bacterial population ( Fig. 9 ). Moreover, a recent work showed that the RNAIII-mediated translational repression of Rot is essential to protect the bacteria from exogenous reactive oxygen species 77 . Interestingly, this long-lived protection was uncoupled from quorum sensing activation suggesting that the bacteria anticipate the ROS-mediated immune attack and the oxidative damages. As Rho protein regulates genes involved in riboflavin synthesis, nitrate respiration and anaerobia, it is tempting to propose that the RNAIII-dependent repression of rho might also be required under specific conditions encountered during the infection, even if RNAIII is not highly expressed. Indeed, it was previously shown that efficient repression of target genes occurred as soon as RNAIII is induced 78 . Rho protein is involved in phenotypic switches in different bacterial species Interestingly the growth phase dependent repression of Rho is also observed in B. subtilis, and is required for cell adaptation, although the mechanism of regulation is not known 24 , 79 . In Acinetobacter baumanii which can adopt a virulent and an avirulent phenotypes, it has been shown that Rho is necessary for premature termination of transcriptional regulators and their repression in the virulent status 33 . Conversely, when switching to an avirulent phenotype, Rho-dependent termination is reduced to allow correct expression of these regulators. Interestingly, levels of Rho remained unchanged in both phenotypes, suggesting the existence of mechanisms that inactivate the protein or reduce its binding to the mRNA targets 33 . As demonstrated in E. coli, Rho can be transiently and reversibly inactivated through oligomerization and aggregation 32 . Also, in Clostridium botulinum, Rho can adopt free or aggregated conformations due to the presence of a prion-like domain in the NTD which can impact the activity of the protein and act as a regulatory switch of Rho functions 80 . Further analyses will be needed to investigate if a similar phenomenon exists in S. aureus, since Rho also contains an NTD insertion that could mediate a similar regulatory switch. This might be relevant because the levels of the Rho protein slightly vary between exponential and stationary phase, suggesting that other mechanisms might exist to ensure the inactivation of Rho at high cell density. Nevertheless, the RNAIII-dependent regulation of rho is expected to enhance the synthesis of several exotoxins at this stage. This is also supported by the phenotypic assays showing that Rho decreases cell cytotoxicity. CONCLUSION The role of Rho in the control of genome surveillance through the regulation of antisense transcription has been conserved among bacteria. These pervasive transcripts are often associated to horizontally acquired genetic material or mobile elements. In B. subtilis and S. aureus accessory genes have been acquired for sporulation or virulence, respectively, which would explain why Rho seems to have more specialized regulatory functions in these bacteria. This ubiquitous termination factor has evolved for the adaptation of Bacillota to the physiological remodeling taking place in the transition to stationary phase. Our findings have also unveiled the rho mRNA as a novel target of RNAIII, adding another level to the complex networks controlling the activation of exotoxins during stationary phase. Overall, our results give a first glimpse of the roles and functions of Rho in S. aureus and placed Rho as another key component of the main regulatory networks of virulence of S. aureus . MATERIALS AND METHODS Strains and plasmids All strains and plasmids used and constructed in this study are described in Tables S4 and S5. The primers designed for cloning and mutagenesis are provided in Table S6. E. coli strain IM08B was used as an intermediate strain for plasmid amplification before electroporation in S. aureus . Plasmids were extracted from chemically transformed E. coli pellets with the NucleoSpin Plasmid kit according to the manufacturer’s protocol (Macherey-Nagel). Electrocompetent S. aureus strains were then transformed with the purified plasmids by electroporation (Bio-Rad Gene Pulser). The HG001 rho and HG001 rnaIII deletion mutants as well as the HG001 rho-3xflag strain were constructed by homologous recombination using plasmid pMAD in S. aureus. HG001Primers used are listed in Table S6. The pCN51-P3:: rnaIII and P3::MS2-RNAIII plasmids were constructed by ligating PCR-amplified fragments (see Table S6 for primers) containing the 514 nt-long sequence of rnaIII previously digested with Sph I/ Bam HI or the sequence of rnaIII fused to an MS2 tag at the 5’end digested with Pst I/ Bam HI into a pCN51-P3 plasmid digested with the same enzymes, respectively. The pCN51-P3:: rnaIII△H7 , pCN51-P3:: rnaIII△H9 and pCN51-P3:: rnaIII△H7△H9 plasmids containing deletion mutants of hairpins 7, 9 or both, of RNAIII were constructed by site directed mutagenesis using plasmid pCN51-P3:: rnaIII as a template. A PCR using Phusion High Fidelity DNA polymerase (Thermo Scientific) was performed using the primers listed in S6 to introduce the deletions. One denaturation step (30 s at 90°C) was followed by eighteen cycles of amplification of the fragment (30 s at 95°C, 1 min 55°C, 6 min at 68°C) and ultimately an elongation step (5 min at 72°C) was performed. PCR products were then treated with 10U/µL Dpn I to eliminate the template pCN51-P3:: rnaIII plasmid. Reactions were then transformed into E. coli TOP10 and positive clones were selected and sequenced. These plasmids were used as templates for PCR amplification to introduce the T7 promoter for templates in in vitro transcription assay. Plasmids pCN57::GFP- chp, pCN57::GFP- teg74, pCN57::GFP- saeP and pCN57::GFP- teg66 were constructed by ligating PCR-amplified fragments of the chp , teg74, saeP and teg66 genes (see Table S6 for primers) previously digested with Eco RI/ Bam HI into a pCN57::GFP plasmid digested with the same enzymes. Growth conditions E. coli strains were grown in Lysogeny-Broth (LB, Roth) medium supplemented with ampicillin (100 μg/mL). S. aureus strains were grown in Brain-Heart infusion (BHI, BD) media containing 10 μg/mL of erythromycin or chloramphenicol when needed. pMAD mutagenesis Strains HG001 △rho , HG001 △rnaIII and HG001 rho-3xflag were constructed by allelic recombination using the shuttle vector pMAD as previously described 81 . To generate the mutants, we amplified fragments of the flanking upstream and downstream regions of the targeted genes by PCR using chromosomal DNA of S. aureus HG001 as template. The final PCR products were obtained by flanking PCRs or double digestion/ligation steps (Table S6 for primers) and cloned into pMAD shuttle plasmid with Bam HI/ Bgl II or Bam HI/ Eco RI restriction enzymes. Plasmids were amplified into RN4220 after electroporation and then transferred in HG001 for homologous recombination steps as described in Arnaud et al , 2004 81 . Northern blot Isolation of total RNA of S. aureus cultures was performed accordingly to the FastRNA pro blue kit (MP Biomedicals) using the FastPrep machine (MP Biomedicals) for mechanical lysis of bacterial cells. Electrophoresis of 15 µg of total RNA was run in 1% agarose-TBE 1X gel containing 25 mM guanidium thiocyanate (Sigma). After migration at 150 V, RNAs were transferred on Hybond+ nitrocellulose membrane (Cytiva) by capillarity with saline-sodium citrate 2X buffer (SSC 2X) and then fixed by UV-crosslinking (Stratalinker 1800 Stratagene). RNA detection was performed by hybridization with specific digoxigenin (DIG)-labeled RNA probes complementary to each targeted RNA as described in Tomasini et al , 2017 46 . (see S3 for primers used for DIG-probe production). Western blot Total proteins were extracted from S. aureus cultures grown for 2, 4 or 6 h in BHI. After harvesting by centrifugation, bacterial pellets were washed once wit 1X PBS, then resuspended in 1X PBS and finally lysed using the Fastprep (MP Biomedicals). Quantification of proteins was performed with Bradford reagent following the manufacturer protocol (BioRad) and using BSA as standard. 10 µg of total protein extract were migrated on 12 % polyacrylamide-SDS gel and transferred onto a PVDF membrane using the Trans-blot Turbo Transfer system (BioRad). Membranes were incubated with anti-FLAG mouse antibodies (Sigma) at the final dilution of 1:2500 followed by the incubation with a goat anti-mouse peroxidase (HRP) antibody (BioRad) at the final dilution of 1:5000. Membranes were revealed using the ChemiDoc (BioRad) and were then stained by Coomassie blue as loading controls of samples. MAPS Cultures of HG001 pCN51::MS2 or △rnaIII pCN51::MS2-RNAIII strains were grown in BHI medium for 6 h and then centrifuged. Pellets were then harvested, lysed and purified by affinity chromatography as previously described 82 . RNA was isolated, purified from the collected fractions with PCI (Carl Roth, pH 4,5-5), precipitated with EtOH and used for Northern blot 82 . Transcriptomics Cultures of HG001 and HG001 △rho strains were grown in BHI medium until OD 600 =0.5 and harvested by centrifugation. Total RNA was extracted as described above and treated with DNase I (Sigma) prior to RNA-seq. RNAs were treated to deplete abundant rRNAs and the cDNA libraries were performed using the NEBNext Ultra II directional RNA kit. The libraries were sequenced using NextSeq2000 P2. Each RNAseq reaction was performed in duplicates. The reads were then processed to remove adapter sequences and poor-quality reads using Trimmomatric 83 , then converted to FASTQ format using FASTQ Groomer 84 , and aligned to the HG001 genome 85 using Bowtie2 86 . Finally, the number of reads mapping to each annotated feature was determined with HTSeq 87 . To perform the differential expression analysis, we used DESeq2 88 . All processing steps were performed on the Galaxy platform 89 . RIPseq Cultures of HG001 WT and HG001 rho-3xflag strains were grown in BHI medium until early exponential phase (OD 600 =0,5) and harvested by centrifugation. Pellets were resuspended in lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and protease inhibitors) and transferred into flacon tubes containing Lysing Matrix B silica beads (MP Biomedicals) to proceed to lysis of the cells using the Fastprep (MP Biomedicals). After centrifugation to remove cell debris, the supernatant was incubated for 2 h at 4°C on a rotator with pre-equilibrated ANTI-FLAG M2 affinity Gel (Sigma) as described by the manufacturer protocol. The suspension was centrifuged, the supernatant discarded, and the beads were washed twice with cold 1X TBS. The antibody-bound fusion protein Rho-3xFLAG was then eluted by competition with 100 µg/mL of triple FLAG peptide (Sigma) in 1X TBS. A sample of each fraction was used for Western blot to assess the enrichment of the tagged protein after elution. RNA was purified from the elution fraction with PCI precipitation (Carl Roth, pH 4.5-5) and treated with DNase I (Sigma) prior to RNA-seq analysis. RNAs were treated to deplete abundant rRNAs and the cDNAs were performed using the NEB Next Ultra II directional RNA kit. The libraries were sequenced using HiSeq 1000 Single read 50 bases. Each RNAseq was performed in duplicates. Treatment of RNAseq data was performed as described above. To identify co-precipitating asRNAs, the RNAseq analysis was analyzed as described above but in a reverse orientation. Preparation of RNAs for in vitro experiments Transcription of the different RNAs used (RNAIII, RNAIII△H7, RNAIII△H9, RNAIII △H7△H9, rho5’ and rho3’ ) was achieved using PCR products containing the sequence of the RNA downstream the T7 promoter, directly introduced with the primer (Table S6). These PCR products were used as templates for in vitro transcription using T7 RNA polymerase. In the case of RNAIII transcription, reactions were supplemented with 5 mM of ApG RNAs to avoid the 5’-end dephosphorylation step. RNAs were treated with DNase I and purified on an 8% polyacrylamide-8 M urea gel and eluted with 0,5 M ammonium acetate pH 6.1, 1 mM EDTA, and 0.1% SDS and precipitated in cold absolute ethanol. Precipitated RNAs where then labeled with T4 polynucleotide kinase (Fermentas) and [γ 32 P] ATP and ultimately purified on a 8% polyacrylamide-8M urea gel and eluted as described above. EMSA The 5’-end radiolabeled RNAs (10’000 cps/sample, <1pM), and cold RNAs were denatured separately by incubating at 90°C for 1 min in 100 mM Tris-HCl pH 7.5, 300 mM KCl, 200 mM NH 4 Cl, then cooled down for 1 min on ice and renatured at 20°C for 10 min after addition of 10 mM MgCl 2 . Complexes were formed at 37°C for 15 min. After the addition of 1 volume of glycerol blue, the samples were loaded on a native 6% polyacrylamide gel containing 10 mM MgCl 2 and migrated at 300 V and 4°C in 1X TB buffer with 10 mM MgCl 2. RNase III cleavage The 5’-end radiolabeled RNAIII (10’000 cps/sample, <1 pM) and cold rho mRNA were denatured separately at 90°C for 1 min, cooled down on ice for 1 min and renatured in 1X TMK buffer (Tris HCl 100 mM pH7, MgCl 2 100 mM, KCl 1M) at 20°C for 10 min. Cold RNAs are added to the labeled-RNA mix and RNA-RNA complexes are formed at 37°C for 15 min. RNAs are then incubated with increasing concentrations of purified S. aureus RNase III (25 nM, 50 nM, 100 nM and 200 nM) in presence of Mg 2+ or Ca 2+ and 1 µg of yeast total tRNA at 37°C for 15 min. RNAs are purified with PCI precipitation (Carl Roth, pH 4.5-5) and loaded on a 12 % polyacrylamide-8 M urea gel. For the alkaline ladder, radiolabeled RNA is incubated with Alkaline hydrolysis buffer 1X (Ambion) at 90°C for 10 min before addition of 1 volume of loading buffer (Ambion). For the T1 ladder, radiolabeled RNA is denatured at 90°C for 1 min in sequencing buffer 1X (Ambion). After renaturation at 20°C for 1 min, 1 U of RNase T1 (Sigma) is added and incubated at 37°C for 5 min before addition of one volume of loading buffer (Ambion). For RNase III cleavage experiments with RNAIII mutants, unlabeled rho and RNAIII variants to final concentrations of 400 nM and 200 nM, respectively, were used to form duplexes and RNase III cleavage was performed as described above. Cleavage signals on RNAIII were detected by primer extension. Precipitated RNAs were then hybridized with a 5’ end-labeled primer complementary to the RNAIII 3’ end (Table S6) and reverse transcription with AMV reverse transcriptase was performed at 37°C for 30 min. Sequencing reactions were performed using DNA template for each tested RNA and Vent (exo)-DNA polymerase (NEB) (25 cycles, 1 min denaturation at 95°C, 1 min annealing at 52°C and 1 min elongation at 72°C). Toe-printing The preparation of S. aureus 30S subunits, the formation of a simplified translational initiation complex with mRNA, and the extension inhibition conditions were performed as previously described 44 . Increasing concentrations of either RNAIII or RNAIIIΔ7Δ9 concentrations were used to monitor their effects on the formation of the initiation complex with rho . In vitro translation assays The translation assays were performed using the 5’- rho mRNA and the PURExpress In vitro Protein Synthesis kit (NEB) supplemented with 70S staphylococcal ribosomes purified as described 90 . The construct carries an additional sequence corresponding to the FLAG peptide, which was inserted at the 3′ end of rho . The reactions were performed at 37°C for 2 h following the kit protocol. Experiments were also carried out in the presence of increasing concentrations of RNAIII or of RNAIIIΔ7Δ9. Reactions were stopped by adding an equal volume of Laemmli buffer. The proteins were detected by western blot with an antibody against the FLAG tag (Sigma). GFP fusions S. aureus strains containing the pCN57 plasmids derivatives were grown in BHI medium supplemented with 10 μg/mL erythromycin and chloramphenicol until exponential phase. Total proteins were extracted and quantified as described above. For each sample, 200 µL of dilutions of 1 mg/mL, 0.5 mg/mL and 0.25 mg/mL of total proteins were loaded on a black uCLEAR 96 wells-plate (GreinerBioOne) and GFP signal was measured with the platereader Spectramax Id3. HlgC and Hla quantification by ELISA Bacterial strains were cultured in CCY broth supplemented with chloramphenicol at 10 μg/mL at 37°C overnight with shaking to stimulate toxins production 91 . Samples were centrifuged at 10’000 g for 10 min and supernatants were collected. HlgC and Hla quantification was performed by a sandwich ELISA using custom-made antibodies. 96-well Nunc MaxiSorp plate (Thermo Scientific) were coated with anti-HlgC monoclanal (R&D Biotech) or anti-Hla polyclonal (MedImmune) antibodies at 10 μg/mL and 2 μg/mL respectively, and incubated overnight at 20°C. After 5 consecutive washes with phosphate-buffered saline (PBS)–0.05% Tween (PBS-T), wells were saturated for 1 h 30 min at 20°C with a blocking solution containing PBS-T, low-fat milk (5 g/L), and bovine serum albumin (BSA) (1 g/L). Standard dilutions from 15 to 1’000 ng/mL of recombinant HlgC or from 0,3125 to 20 ng/mL of recombinant Hla or the culture supernatant were denatured for 1 h at 95°C, loaded in duplicate, and incubated for 2 h at 37°C. For HlgC quantification, after washing, polyclonal rabbit F(ab)′ 2 biotinylated antibody (1.55 μg/mL, R&D Biotech) was added to each well, and the plate was incubated for 1 h 30 at 37°C and washed. Then, ExtrAvidin-peroxidase antibody (Sigma) targeting the biotin molecule and conjugated with horseradish peroxidase (HRP) was added. The plate was incubated for 1 h at 20°C. For Hla quantification, the plate was washed, monoclonal anti-Hla antibody (2 μg/mL) was added, and the plate was incubated for 1 h 15 at 37°C. After washing, polyclonal goat F(ab)′ 2 ant-human Fcg-HRP conjugated with horseradish peroxidase (HRP) antibody (Jackson Immuno-Research) was added and the plate was incubated for 1 h 15 min at 37°C. For both, a final wash was performed and 75 μL of the substrate tetramethylbenzidine (KPL SureBlue; SeraCare) were added. The reaction was stopped with 1 N sulfuric acid. The plates were read at 450 nm in a Bio-Rad model 680 microplate reader. Red blood cells lysis Bacterial strains were cultured in CCY broth supplemented with chloramphenicol at 10 μg/mL at 37°C overnight with shaking, centrifuged at 10’000 g for 10 min and supernatant were collected. Activity of alpha-hemolysin was confirmed by cell permeability assays using rabbit erythrocytes. In 96-well round bottom plate, bacteria culture supernatants were diluted in PBS (1/2 to 1/2048) and mixed with 50 µl of washed rabbit erythrocytes solution (10 8 cells/ml; Atlantis). After 60 min of incubation at 37°C, the plate was centrifuged and the OD 415nm of the supernatants measured. The cut off (corresponding to lysis of 25% of red blood cells) was determined using the following formula: (OD lysis control – OD neg control)/2, where the lysis control is the total lysis of red blood cells with pure water and the negative control is the mix of red blood cells with PBS. For each condition, the HLA activity value corresponds to the inverse of the exact dilution causing the lysis of 25% of the red blood cells. Cytotoxicity assay Bacterial strains were cultured in CCY broth supplemented with chloramphenicol at 10 μg/mL at 37°C overnight with shaking, centrifuged at 10’000 g for 10 min and supernatant were collected. U937 Human monocytes expressing C5aR1 or CXCR2 receptor 92 were routinely cultured in Roswell Park Memorial Institute (RPMI ; Thermofisher) 1640 Medium supplemented with 10% foetal bovine serum at 37°C with 5% CO 2 . U937 cells were routinely cultured in DMEM growth medium supplemented with 10% foetal bovine serum at 37°C with 5% CO 2 . Cells were diluted at 1.10 6 cell/ml and Iodure Propidium (IP) was added for a final concentration at 25 µg/mL. A 96-well plate was seeded with 90 µl of this solution and 10 µl of culture supernatant diluted at ½ in DMEM medium were added. IP incorporation into cells was measured by determining OD at 635 nm after excitation at 535 nm using TECAN plate reader. The measure was done every 10 min for 24 h and the OD max was conserved to compare cytotoxic activities of the different bacterial supernatants. Adherence assay Adherence of bacterial cells to immobilized fibrinogen was performed as described by Hartford et al ,1997 93 . Briefly, 96-well plate was coated with 5 μg/mL of fibrinogen (SIGMA) in PBS and incubated overnight at 4 °C with shaking. Bacterial strains were cultured in BHI broth at 37°C overnight. The plate was incubated with bovine serum albumin solution (1% in PBS) for 1 h at 37 °C. 1 mL of bacterial cell adjusted at 10 8 CFU/mL were labelled with 2 µL of BacLight RedoxSensor Green Vitality (Thermofischer) for 15 min at 37°C with gentle shaking. 100 µL bacterial cell suspension was added per well in duplicate and the plate was incubated for 1 h at 37°C in the dark. After incubation, the plate was washed three times with PBS and the adherence of bacterial cells was quantified at 485 nm using TECAN plate reader. Coagulase quantification Bacterial strains were cultured 5 h at 37°C with agitation in Brain Heart Infusion (BHI; Difco) supplemented with chloramphenicol at 10 μg/mL and then centrifuged. Coagulase activity was assayed by adding 0.5 mL of two-fold serial dilutions of culture supernatant in PBS to 0.5 mL of rabbit plasma (Biorad) diluted 1 in 3 in distilled water. The titer was the reciprocal of the highest dilution of the supernatant that showed evidence of clotting after incubation for 18 h at 37°C. Statistical Analysis The statistical analyses were performed using GraphPad Prism 10 software. Data from 3 strains were compared using multiple comparisons of the means through a Brown-Forsythe and Welch ANOVA tests. The significance threshold was set at 0.05 for all tests. FUNDING This work was supported by the Centre National de la Recherche Scientifique (CNRS), by the Institut pour la Santé et la Recherche Médicale (INSERM), by the French National Research Agency ANR (ANR-18-CE12-0025-04 CoNoCo to [P.R.]). This work of the Interdisciplinary Thematic Institute IMCBio, as part of the ITI 2021-2028 program of the University of Strasbourg, CNRS and Inserm, was supported by IdEx Unistra (ANR-10-IDEX-0002) and by SFRI-STRAT’US (ANR 20-SFRI-0012), and EUR IMCBio (IMCBio ANR-17-EURE-0023) under the framework of the French Investments for the Future Program. LB were supported by the “Fondation pour la Recherche Médicale” (ECO202006011534 and FDT202304016346). ACKNOWLEDGMENTS We are deeply thankful to E. Bidnenko, V. Bidnenko, M. Boudvillain, S. Durand and C. Condon for fruitful discussions. We also thank Sandra Derozier and Cyprien Guerin for the visualisation of the transcriptomic data with Genoscapist. Funder Information Declared Centre National de la Recherche Scientifique (CNRS) Institut pour la Santé et la Recherche Médicale (INSERM) French National Research Agency ANR , ANR-18-CE12-0025-04 CoNoCo IdEx Unistra , ANR-10-IDEX-0002 SFRI-STRAT’US , ANR 20-SFRI-0012 EUR IMCBio , IMCBio ANR-17-EURE-0023 Fondation pour la Recherche Médicale , ECO202006011534 , FDT202304016346 REFERENCES 1. ↵ Porrua , O. , Boudvillain , M. & Libri , D . Transcription Termination: Variations on Common Themes . Trends Genet . 32 , 508 – 522 ( 2016 ). OpenUrl CrossRef PubMed 2. ↵ Aguilera , A. & García-Muse , T . R Loops: From Transcription Byproducts to Threats to Genome Stability . Mol. Cell 46 , 115 – 124 ( 2012 ). OpenUrl CrossRef PubMed Web of Science 3. ↵ Ray-Soni , A. , Bellecourt , M. J. & Landick , R . Mechanisms of Bacterial Transcription Termination: All Good Things Must End . Annu. Rev. Biochem . 85 , 319 – 347 ( 2016 ). OpenUrl CrossRef PubMed 4. ↵ Chalissery , J. , Banerjee , S. , Bandey , I. & Sen , R . Transcription Termination Defective Mutants of Rho: Role of Different Functions of Rho in Releasing RNA from the Elongation Complex . J. Mol. Biol . 371 , 855 – 872 ( 2007 ). OpenUrl CrossRef PubMed Web of Science 5. Burns , C. M. , Nowatzke , W. L. & Richardson , J. P . Activation of Rho-dependent Transcription Termination by NusG . J. Biol. Chem . 274 , 5245 – 5251 ( 1999 ). OpenUrl Abstract / FREE Full Text 6. ↵ Chalissery , J. et al. Interaction Surface of the Transcription Terminator Rho Required to Form a Complex with the C-Terminal Domain of the Antiterminator NusG . J. Mol. Biol . 405 , 49 – 64 ( 2011 ). OpenUrl CrossRef PubMed 7. ↵ Mitra , P. , Ghosh , G. , Hafeezunnisa , Md . & Sen , R . Rho Protein: Roles and Mechanisms . Annu. Rev. Microbiol . 71 , 687 – 709 ( 2017 ). OpenUrl CrossRef PubMed 8. ↵ Mandell , Z. F. et al. NusG is an intrinsic transcription termination factor that stimulates motility and coordinates gene expression with NusA . eLife 10 , e61880 . 9. ↵ Mondal , S. , Yakhnin , A. V. , Sebastian , A. , Albert , I. & Babitzke , P . NusA-dependent transcription termination prevents misregulation of global gene expression . Nat. Microbiol . 1 , 15007 ( 2016 ). 10. ↵ Ahmad , E. , Mahapatra , V. , Vanishree , V. M. & Nagaraja , V . Intrinsic and Rho-dependent termination cooperate for efficient transcription termination at 3’ untranslated regions . Biochem. Biophys. Res. Commun . 628 , 123 – 132 ( 2022 ). OpenUrl CrossRef PubMed 11. ↵ Nicolas , P. et al. Condition-Dependent Transcriptome Reveals High-Level Regulatory Architecture in Bacillus subtilis . Science 335 , 1103 – 1106 ( 2012 ). OpenUrl Abstract / FREE Full Text 12. ↵ Magán , A. et al. iRAPs curb antisense transcription in E. coli . Nucleic Acids Res . 47 , 10894 – 10905 ( 2019 ). OpenUrl CrossRef PubMed 13. ↵ Sedlyarova , N. et al. sRNA-Mediated Control of Transcription Termination in E. coli . Cell 167 , 111 – 121.e13 ( 2016 ). OpenUrl CrossRef PubMed 14. ↵ Sedlyarova , N. et al. Natural RNA Polymerase Aptamers Regulate Transcription in E. coli . Mol. Cell 67 , 30 – 43.e6 ( 2017 ). OpenUrl CrossRef PubMed 15. ↵ Turnbough , C. L . Regulation of Bacterial Gene Expression by Transcription Attenuation . Microbiol. Mol. Biol. Rev . 83 , e00019 – 19 ( 2019 ). OpenUrl CrossRef PubMed 16. ↵ Bossi , L. , Schwartz , A. , Guillemardet , B. , Boudvillain , M. & Figueroa-Bossi , N . A role for Rho-dependent polarity in gene regulation by a noncoding small RNA . Genes Dev . 26 , 1864 – 1873 ( 2012 ). OpenUrl Abstract / FREE Full Text 17. ↵ Cardinale , C. J. et al. Termination Factor Rho and Its Cofactors NusA and NusG Silence Foreign DNA in E. coli . Science 320 , 935 – 938 ( 2008 ). OpenUrl Abstract / FREE Full Text 18. ↵ Jain , S. , Gupta , R. & Sen , R . Rho-dependent transcription termination in bacteria recycles RNA polymerases stalled at DNA lesions . Nat. Commun . 10 , 1207 ( 2019 ). OpenUrl CrossRef PubMed 19. ↵ Wade , J. T. & Grainger , D. C . Pervasive transcription: illuminating the dark matter of bacterial transcriptomes . Nat. Rev. Microbiol . 12 , 647 – 653 ( 2014 ). OpenUrl CrossRef PubMed 20. ↵ Lasa , I. et al. Genome-wide antisense transcription drives mRNA processing in bacteria . Proc. Natl. Acad. Sci . 108 , 20172 – 20177 ( 2011 ). OpenUrl Abstract / FREE Full Text 21. ↵ D’Heygère , F. , Rabhi , M. & Boudvillain , M . Phyletic distribution and conservation of the bacterial transcription termination factor Rho . Microbiology 159 , 1423 – 1436 ( 2013 ). OpenUrl CrossRef PubMed Web of Science 22. ↵ Washburn , R. S. , Marra , A. , Bryant , A. P. , Rosenberg , M. & Gentry , D . R. rho Is Not Essential for Viability or Virulence in Staphylococcus aureus . Antimicrob. Agents Chemother . 45 , 1099 – 1103 ( 2001 ). OpenUrl Abstract / FREE Full Text 23. ↵ Bidnenko , V. et al. Termination factor Rho: From the control of pervasive transcription to cell fate determination in Bacillus subtilis . PLOS Genet . 13 , e1006909 ( 2017 ). OpenUrl CrossRef PubMed 24. ↵ Bidnenko , V. et al. Termination factor Rho mediates transcriptional reprogramming of Bacillus subtilis stationary phase . PLOS Genet . 19 , e1010618 ( 2023 ). OpenUrl PubMed 25. ↵ Mäder , U. et al. Staphylococcus aureus Transcriptome Architecture: From Laboratory to Infection-Mimicking Conditions . PLOS Genet . 12 , e1005962 ( 2016 ). OpenUrl CrossRef PubMed 26. ↵ Nagel , A. et al. Inhibition of Rho Activity Increases Expression of SaeRS-Dependent Virulence Factor Genes in Staphylococcus aureus, Showing a Link between Transcription Termination, Antibiotic Action, and Virulence . mBio 9 , e01332 – 18 ( 2018 ). OpenUrl PubMed 27. ↵ Liu , Q. , Yeo , W. & Bae , T . The SaeRS Two-Component System of Staphylococcus aureus . Genes 7 , 81 ( 2016 ). 28. ↵ Barik , S. , Bhattacharya , P. & Das , A . Autogenous regulation of transcription termination factor Rho . J. Mol. Biol . 182 , 495 – 508 ( 1985 ). OpenUrl CrossRef PubMed 29. ↵ Brown , S. , Albrechtsen , B. , Pedersen , S. & Klemm , P . Localization and regulation of the structural gene for transcription-termination factor rho of Escherichia coli . J. Mol. Biol . 162 , 283 – 298 ( 1982 ). OpenUrl CrossRef PubMed 30. ↵ Matsumoto , Y. , Shigesada , K. , Hirano , M. & Imai , M . Autogenous regulation of the gene for transcription termination factor rho in Escherichia coli: localization and function of its attenuators . J. Bacteriol . 166 , 945 – 958 ( 1986 ). OpenUrl Abstract / FREE Full Text 31. ↵ Silva , I. J. et al. SraL sRNA interaction regulates the terminator by preventing premature transcription termination of rho mRNA . Proc. Natl. Acad. Sci . 116 , 3042 – 3051 ( 2019 ). OpenUrl Abstract / FREE Full Text 32. ↵ Wang , B. et al. Transcription termination factor ρ polymerizes under stress . bioRxiv 2023.08.18.553922 ( 2023 ) doi: 10.1101/2023.08.18.553922 . OpenUrl Abstract / FREE Full Text 33. ↵ Pérez-Varela , M. et al. Evidence for Rho-dependent control of a virulence switch in Acinetobacter baumannii . mBio 15 , e02708 – 23 ( 2024 ). OpenUrl 34. ↵ Ingham , C. J. , Dennis , J. & Furneaux , P. A . Autogenous regulation of transcription termination factor Rho and the requirement for Nus factors in Bacillus subtilis . Mol. Microbiol . 31 , 651 – 663 ( 1999 ). OpenUrl CrossRef PubMed Web of Science 35. ↵ Le , K. Y. & Otto , M . Quorum-sensing regulation in staphylococci—an overview . Front. Microbiol . 6 , ( 2015 ). 36. ↵ Novick , R . p., et al. Synthesis of staphylococcal virulence factors is controlled by a regulatory RNA molecule . EMBO J . 12 , 3967 – 3975 ( 1993 ). OpenUrl CrossRef PubMed Web of Science 37. ↵ Huntzinger , E. et al. Staphylococcus aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression . EMBO J . 24 , 824 – 835 ( 2005 ). OpenUrl Abstract / FREE Full Text 38. ↵ Gupta , R. Kr ., Luong , T. T. & Lee , C. Y . RNAIII of the Staphylococcus aureus agr system activates global regulator MgrA by stabilizing mRNA . Proc. Natl. Acad. Sci. U. S. A . 112 , 14036 – 14041 ( 2015 ). OpenUrl Abstract / FREE Full Text 39. ↵ Chevalier , C. et al. Staphylococcus aureus RNAIII Binds to Two Distant Regions of coa mRNA to Arrest Translation and Promote mRNA Degradation . PLoS Pathog . 6 , e1000809 ( 2010 ). OpenUrl CrossRef PubMed 40. ↵ Morfeldt , E. , Taylor , D. , von Gabain , A. & Arvidson , S . Activation of alpha-toxin translation in Staphylococcus aureus by the trans-encoded antisense RNA, RNAIII . EMBO J . 14 , 4569 – 4577 ( 1995 ). OpenUrl CrossRef PubMed Web of Science 41. ↵ Boisset , S. et al. Staphylococcus aureus RNAIII coordinately represses the synthesis of virulence factors and the transcription regulator Rot by an antisense mechanism . Genes Dev . 21 , 1353 – 1366 ( 2007 ). OpenUrl Abstract / FREE Full Text 42. ↵ Villanueva , M. et al. Sensory deprivation in Staphylococcus aureus . Nat. Commun . 9 , 523 ( 2018 ). 43. ↵ Benito , Y. et al. Probing the structure of RNAIII, the Staphylococcus aureus agr regulatory RNA, and identification of the RNA domain involved in repression of protein A expression . RNA 6 , 668 – 679 ( 2000 ). OpenUrl Abstract 44. ↵ Desgranges , E. et al. The 3′UTR-derived sRNA RsaG coordinates redox homeostasis and metabolism adaptation in response to glucose-6-phosphate uptake in Staphylococcus aureus . Mol. Microbiol . 117 , 193 – 214 ( 2022 ). OpenUrl CrossRef PubMed 45. ↵ Bronesky , D. et al. A multifaceted small RNA modulates gene expression upon glucose limitation in Staphylococcus aureus . EMBO J . 38 , e99363 ( 2019 ). OpenUrl Abstract / FREE Full Text 46. ↵ Tomasini , A. et al. The RNA targetome of Staphylococcus aureus non-coding RNA RsaA: impact on cell surface properties and defense mechanisms . Nucleic Acids Res . 45 , 6746 – 6760 ( 2017 ). OpenUrl CrossRef PubMed 47. ↵ Mann , M. , Wright , P. R. & Backofen , R . IntaRNA 2.0: enhanced and customizable prediction of RNA-RNA interactions . Nucleic Acids Res . 45 , W435 – W439 ( 2017 ). OpenUrl CrossRef PubMed 48. Wright , P. R. et al. CopraRNA and IntaRNA: predicting small RNA targets, networks and interaction domains . Nucleic Acids Res . 42 , W119 – W123 ( 2014 ). OpenUrl CrossRef PubMed Web of Science 49. Busch , A. , Richter , A. S. & Backofen , R . IntaRNA: efficient prediction of bacterial sRNA targets incorporating target site accessibility and seed regions . Bioinforma. Oxf. Engl . 24 , 2849 – 2856 ( 2008 ). OpenUrl 50. ↵ Raden , M. et al. Freiburg RNA tools: a central online resource for RNA-focused research and teaching . Nucleic Acids Res . 46 , W25 – W29 ( 2018 ). OpenUrl CrossRef PubMed 51. ↵ Boisset , S. , et al. Staphylococcus aureus RNAIII coordinately represses the synthesis of virulence factors and the transcription regulator Rot by an antisense mechanism . Genes Dev . 21 , 1353 – 1366 ( 2007 ). OpenUrl Abstract / FREE Full Text 52. ↵ Yeo , W.-S. et al. A Membrane-Bound Transcription Factor is Proteolytically Regulated by the AAA+ Protease FtsH in Staphylococcus aureus . J. Bacteriol . 202 , e00019 – 20 ( 2020 ). OpenUrl PubMed 53. ↵ Kinkel , T. L. , Roux , C. M. , Dunman , P. M. & Fang , F. C . The Staphylococcus aureus SrrAB Two-Component System Promotes Resistance to Nitrosative Stress and Hypoxia . mBio 4 , e00696 – 13 ( 2013 ). OpenUrl CrossRef PubMed 54. ↵ de Haas , C. J. C. et al. Chemotaxis Inhibitory Protein of Staphylococcus aureus, a Bacterial Antiinflammatory Agent . J. Exp. Med . 199 , 687 – 695 ( 2004 ). OpenUrl Abstract / FREE Full Text 55. ↵ Smith , E. J. , Visai , L. , Kerrigan , S. W. , Speziale , P. & Foster , T. J . The Sbi Protein Is a Multifunctional Immune Evasion Factor of Staphylococcus aureus ▿ . Infect. Immun . 79 , 3801 – 3809 ( 2011 ). OpenUrl Abstract / FREE Full Text 56. ↵ Guan , Z. et al. Staphylococcus aureus β-Hemolysin Up-Regulates the Expression of IFN-γ by Human CD56bright NK Cells . Front. Cell. Infect. Microbiol . 11 , 658141 ( 2021 ). 57. ↵ Chabelskaya , S. , Bordeau , V. & Felden , B . Dual RNA regulatory control of a Staphylococcus aureus virulence factor . Nucleic Acids Res . 42 , 4847 – 4858 ( 2014 ). OpenUrl CrossRef PubMed 58. ↵ Chabelskaya , S. , Gaillot , O. & Felden , B . A Staphylococcus aureus small RNA is required for bacterial virulence and regulates the expression of an immune-evasion molecule . PLoS Pathog . 6 , e1000927 ( 2010 ). OpenUrl CrossRef PubMed 59. ↵ Charpentier , E. et al. Novel Cassette-Based Shuttle Vector System for Gram-Positive Bacteria . Appl. Environ. Microbiol . 70 , 6076 – 6085 ( 2004 ). OpenUrl Abstract / FREE Full Text 60. ↵ Tejada-Arranz , A. , De Crecy-Lagard , V. & De Reuse , H . Bacterial RNA degradosomes . Trends Biochem. Sci . 45 , 42 – 57 ( 2020 ). OpenUrl CrossRef PubMed 61. ↵ Romilly , C. et al. Loop-loop interactions involved in antisense regulation are processed by the endoribonuclease III in Staphylococcus aureus . RNA Biol . 9 , 1461 – 1472 ( 2012 ). OpenUrl CrossRef PubMed 62. ↵ Li , Y. et al. Nitrate Reductase NarGHJI Modulates Virulence via Regulation of agr Expression in Methicillin-Resistant Staphylococcus aureus Strain USA300 LAC . Microbiol. Spectr . 11 , e0359622 ( 2023 ). OpenUrl 63. ↵ Pagels , M. et al. Redox sensing by a Rex-family repressor is involved in the regulation of anaerobic gene expression in Staphylococcus aureus . Mol. Microbiol . 76 , 1142 – 1161 ( 2010 ). OpenUrl CrossRef PubMed 64. ↵ Fuchs , S. , Pané-Farré , J. , Kohler , C. , Hecker , M. & Engelmann , S . Anaerobic gene expression in Staphylococcus aureus . J. Bacteriol . 189 , 4275 – 4289 ( 2007 ). OpenUrl Abstract / FREE Full Text 65. ↵ Fischer , M. & Bacher , A. Biosynthesis of flavocoenzymes . Nat. Prod. Rep . 22 , 324 – 350 ( 2005 ). OpenUrl CrossRef PubMed 66. ↵ Winkler , W. C. , Cohen-Chalamish , S. & Breaker , R. R . An mRNA structure that controls gene expression by binding FMN . Proc. Natl. Acad. Sci. U. S. A . 99 , 15908 – 15913 ( 2002 ). OpenUrl Abstract / FREE Full Text 67. ↵ Wang , H. et al. Dual-Targeting Small-Molecule Inhibitors of the Staphylococcus aureus FMN Riboswitch Disrupt Riboflavin Homeostasis in an Infectious Setting . Cell Chem. Biol . 24 , 576 – 588.e6 ( 2017 ). OpenUrl CrossRef PubMed 68. ↵ Hollands , K. et al. Riboswitch control of Rho-dependent transcription termination . Proc. Natl. Acad. Sci . 109 , 5376 – 5381 ( 2012 ). OpenUrl Abstract / FREE Full Text 69. ↵ Takemoto , N. , Tanaka , Y. & Inui , M . Rho and RNase play a central role in FMN riboswitch regulation in Corynebacterium glutamicum . Nucleic Acids Res . 43 , 520 – 529 ( 2015 ). OpenUrl CrossRef PubMed 70. ↵ Geiger , T. , Goerke , C. , Mainiero , M. , Kraus , D. & Wolz , C . The virulence regulator Sae of Staphylococcus aureus: promoter activities and response to phagocytosis-related signals . J. Bacteriol . 190 , 3419 – 3428 ( 2008 ). OpenUrl Abstract / FREE Full Text 71. ↵ Marincola , G. & Wolz , C . Downstream element determines RNase Y cleavage of the saePQRS operon in Staphylococcus aureus . Nucleic Acids Res . 45 , 5980 – 5994 ( 2017 ). OpenUrl CrossRef PubMed 72. ↵ Numata , S. , Nagata , M. , Mao , H. , Sekimizu , K. & Kaito , C . CvfA Protein and Polynucleotide Phosphorylase Act in an Opposing Manner to Regulate Staphylococcus aureus Virulence . J. Biol. Chem . 289 , 8420 – 8431 ( 2014 ). OpenUrl Abstract / FREE Full Text 73. ↵ Jeong , D.-W. et al. Identification of the P3 Promoter and Distinct Roles of the Two Promoters of the SaeRS Two-Component System in Staphylococcus aureus . J. Bacteriol . 193 , 4672 – 4684 ( 2011 ). OpenUrl Abstract / FREE Full Text 74. ↵ Collins , M. M. et al. The Accessory Gene saeP of the SaeR/S Two-Component Gene Regulatory System Impacts Staphylococcus aureus Virulence During Neutrophil Interaction . Front. Microbiol . 11 , 561 ( 2020 ). 75. ↵ Jeong , D.-W. et al. The auxiliary protein complex SaePQ activates the phosphatase activity of sensor kinase SaeS in the SaeRS two-component system of Staphylococcus aureus . Mol. Microbiol . 86 , 331 – 348 ( 2012 ). OpenUrl CrossRef PubMed 76. ↵ Li , D. & Cheung , A . Repression of hla by rot is dependent on sae in Staphylococcus aureus . Infect. Immun . 76 , 1068 – 1075 ( 2008 ). OpenUrl Abstract / FREE Full Text 77. ↵ Podkowik , M. et al. Quorum-sensing agr system of Staphylococcus aureus primes gene expression for protection from lethal oxidative stress . eLife 12 , RP89098 ( 2024 ). 78. ↵ Nitzan , M. et al. A defense-offense multi-layered regulatory switch in a pathogenic bacterium . Nucleic Acids Res . 43 , 1357 – 1369 ( 2015 ). OpenUrl CrossRef PubMed 79. ↵ Bidnenko , V. , et al. Complex Sporulation-Specific Expression of Transcription Termination Factor Rho Highlights Its Involvement in Bacillus Subtilis Cell Differentiation . http://biorxiv.org/lookup/doi/10.1101/2023.12.01.569620 ( 2023 ) doi: 10.1101/2023.12.01.569620 . OpenUrl Abstract / FREE Full Text 80. ↵ Yuan , A. H. & Hochschild , A . A bacterial global regulator forms a prion . Science 355 , 198 – 201 ( 2017 ). OpenUrl Abstract / FREE Full Text 81. ↵ Arnaud , M. , Chastanet , A. & Débarbouillé , M . New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, gram-positive bacteria . Appl. Environ. Microbiol . 70 , 6887 – 6891 ( 2004 ). OpenUrl Abstract / FREE Full Text 82. ↵ Mercier , N. et al. MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria . J. Vis. Exp . 61731 ( 2021 ) doi: 10.3791/61731-v . OpenUrl CrossRef 83. ↵ Bolger , A. M. , Lohse , M. & Usadel , B . Trimmomatic: a flexible trimmer for Illumina sequence data . Bioinforma. Oxf. Engl . 30 , 2114 – 2120 ( 2014 ). OpenUrl 84. ↵ Blankenberg , D. et al. Manipulation of FASTQ data with Galaxy . Bioinformatics 26 , 1783 – 1785 ( 2010 ). OpenUrl CrossRef PubMed Web of Science 85. ↵ Caldelari , I. et al. Complete Genome Sequence and Annotation of the Staphylococcus aureus Strain HG001 . Genome Announc . 5 , e00783 – 17 ( 2017 ). OpenUrl 86. ↵ Langmead , B. , Trapnell , C. , Pop , M. & Salzberg , S. L . Ultrafast and memory-efficient alignment of short DNA sequences to the human genome . Genome Biol . 10 , R25 ( 2009 ). 87. ↵ Anders , S. , Pyl , P. T. & Huber , W . HTSeq--a Python framework to work with high-throughput sequencing data . Bioinforma. Oxf. Engl . 31 , 166 – 169 ( 2015 ). OpenUrl 88. ↵ Varet , H. , Brillet-Guéguen , L. , Coppée , J.-Y. & Dillies , M.-A . SARTools: A DESeq2- and EdgeR-Based R Pipeline for Comprehensive Differential Analysis of RNA-Seq Data . PloS One 11 , e0157022 ( 2016 ). OpenUrl CrossRef PubMed 89. ↵ Afgan , E. et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update . Nucleic Acids Res . 44 , W3 – W10 ( 2016 ). OpenUrl CrossRef PubMed 90. ↵ Khusainov , I. et al. Structure of the 70S ribosome from human pathogen Staphylococcus aureus . Nucleic Acids Res . 45 , 1026 ( 2017 ). OpenUrl CrossRef PubMed 91. ↵ Pivard , M. et al. Complex Regulation of Gamma-Hemolysin Expression Impacts Staphylococcus aureus Virulence . Microbiol. Spectr . 11 , e01073 – 23 . 92. ↵ Spaan , A. N. et al. The staphylococcal toxins γ-haemolysin AB and CB differentially target phagocytes by employing specific chemokine receptors . Nat. Commun . 5 , 5438 ( 2014 ). OpenUrl CrossRef PubMed 93. ↵ Hartford , O. , Francois , P. , Vaudaux , P. & Foster , T. J . The dipeptide repeat region of the fibrinogen-binding protein (clumping factor) is required for functional expression of the fibrinogen-binding domain on the Staphylococcus aureus cell surface . Mol. Microbiol . 25 , 1065 – 1076 ( 1997 ). OpenUrl CrossRef PubMed Web of Science View the discussion thread. Back to top Previous Next Posted July 24, 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. 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Share Quorum sensing dependent repression of the Staphylococcus aureus transcription termination factor Rho is necessary for pathogenicity Laura Barrientos , Marion Blum , Florence Couzon , François Vandenesch , Karen Moreau , Pascale Romby , Isabelle Caldelari bioRxiv 2025.07.24.666493; doi: https://doi.org/10.1101/2025.07.24.666493 Share This Article: Copy Citation Tools Quorum sensing dependent repression of the Staphylococcus aureus transcription termination factor Rho is necessary for pathogenicity Laura Barrientos , Marion Blum , Florence Couzon , François Vandenesch , Karen Moreau , Pascale Romby , Isabelle Caldelari bioRxiv 2025.07.24.666493; doi: https://doi.org/10.1101/2025.07.24.666493 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7637) Biochemistry (17705) Bioengineering (13899) Bioinformatics (41970) Biophysics (21463) Cancer Biology (18605) Cell Biology (25526) Clinical Trials (138) Developmental Biology (13385) Ecology (19911) Epidemiology (2067) Evolutionary Biology (24329) Genetics (15615) Genomics (22514) Immunology (17743) Microbiology (40424) Molecular Biology (17194) Neuroscience (88650) Paleontology (667) Pathology (2835) Pharmacology and Toxicology (4827) Physiology (7648) Plant Biology (15160) Scientific Communication and Education (2046) Synthetic Biology (4302) Systems Biology (9825) Zoology (2271)