Subtle variations in a client protein determine bacterial Hsp90 dependence

Subtle variations in a client protein determine bacterial Hsp90 dependence | 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 Subtle variations in a client protein determine bacterial Hsp90 dependence Marie Corteggiani , Amine Ali Chaouche , Miha Bahun , Flora A. Honoré , Deborah Byrne , Sébastien Dementin , View ORCID Profile Mathieu E. Rebeaud , View ORCID Profile Olivier Genest doi: https://doi.org/10.1101/2025.07.01.662558 Marie Corteggiani a Aix-Marseille Univ, CNRS, BIP UMR 7281, IMM , 31 Chemin Joseph Aiguier, 13402 Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Amine Ali Chaouche a Aix-Marseille Univ, CNRS, BIP UMR 7281, IMM , 31 Chemin Joseph Aiguier, 13402 Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Miha Bahun b Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana , Jamnikarjeva 101, SI-1000, Ljubljana, Slovenia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Flora A. Honoré a Aix-Marseille Univ, CNRS, BIP UMR 7281, IMM , 31 Chemin Joseph Aiguier, 13402 Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Deborah Byrne c Aix-Marseille Univ, CNRS, IMM-FR3479 , 31 Chemin Joseph Aiguier, 13402, Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sébastien Dementin a Aix-Marseille Univ, CNRS, BIP UMR 7281, IMM , 31 Chemin Joseph Aiguier, 13402 Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mathieu E. Rebeaud d Institute of Physics, School of Basic Sciences , École Polytechnique Fédérale de Lausanne - EPFL, Lausanne, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mathieu E. Rebeaud Olivier Genest a Aix-Marseille Univ, CNRS, BIP UMR 7281, IMM , 31 Chemin Joseph Aiguier, 13402 Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Olivier Genest For correspondence: ogenest{at}imm.cnrs.fr Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Chaperones ensure protein homeostasis and are conserved across species. The ATP-dependent chaperone Hsp90 is present from bacteria to eukaryotes, where it stabilizes and activates a wide range of substrate proteins called clients. However, what determines whether a protein depends on Hsp90 remains an open question. Here, we focused on the bacterial chaperone Hsp90 and its obligate client TilS (referred to as TilS So ) in the bacterium Shewanella oneidensis . Although Hsp90 is indispensable in S. oneidensis under heat stress by protecting the essential protein TilS So from degradation by the protease HslUV, Hsp90 is dispensable in Escherichia coli , suggesting that E. coli TilS (TilS Ec ) is Hsp90 independent. We therefore compared the TilS orthologs with respect to in vitro stability, in vivo degradation, and interaction with Hsp90 to identify the determinants of the Hsp90 dependence. We found that in contrast to TilS So , TilS Ec was more stable, was not degraded by protease in the absence of Hsp90, and did not interact with Hsp90, indicating that TilS Ec is not a client of Hsp90. Chimeras between TilS So and TilS Ec as well as directed mutagenesis revealed a region of TilS So that is key for protease degradation and Hsp90 protection. Consistent with these results, the growth of S. oneidensis producing TilS Ec was no longer dependent on Hsp90 under heat stress. Conversely, Hsp90 became essential for the growth of E. coli that produced TilS So instead of TilS Ec . Taken together, these results provide new insights into the mechanism of client protection by Hsp90 and the interplay between chaperones and proteases. Introduction Chaperone proteins are present in all organisms to assist other proteins in folding, unfolding, preventing aggregation, disaggregation, and in some cases, degradation [ 1 – 3 ]. How chaperones identify and interact with these proteins (also called clients) remains an open question. Some chaperones interact with hydrophobic regions of the clients that are exposed during the folding process before being buried in the native conformation, thereby preventing protein aggregation, and promoting folding towards the native conformation. One of the most studied chaperones, Hsp70 (DnaK in prokaryotes) interacts with a stretch of hydrophobic residues exposed in virtually all unfolded proteins, although it can also target proteins later in the folding process [ 4 – 7 ]. For other chaperones such as Hsp90, no consensus sequence in clients is known. However, client features, such as hydrophobicity or intrinsically disordered regions, could direct Hsp90 recognition, and Hsp90 is believed to act late in the folding process [ 8 – 11 ]. Hsp90 is a highly conserved ATP-dependent chaperone in bacteria and eukaryotes [ 9 , 12 – 15 ]. It is a dimeric three-domain protein whose activity is regulated by large conformational changes induced by ATP binding and hydrolysis [ 13 ]. The N-terminal domain interacts with nucleotides, the middle domain mainly binds most of the clients, and the C-terminal domain allows dimerization. Several cochaperones, found only in eukaryotes, interact with Hsp90 to control and modulate its functional cycle depending on the client [ 2 , 16 ]. In both eukaryotes and in bacteria, Hsp90 functions in concert with the DnaK/Hsp70 chaperone system through direct interactions between Hsp70/DnaK and Hsp90 [ 12 ]. Although many clients of eukaryotic Hsp90 have been identified, the determinants that render a protein Hsp90-dependent are not fully understood and additional studies with other clients are needed. Shewanella oneidensis is an aquatic gram-negative bacterium with a strong ability to adapt to environmental changes and stresses [ 17 ]. For example, it can support variations in a wide range of temperatures. Interestingly, we have shown that the Hsp90 chaperone is required in this bacterium to support growth under heat stress, making S. oneidensis a powerful model to study the Hsp90 chaperone [ 18 ]. Using a genetic selection, we have identified the client of Hsp90, TilS, which is responsible for Hsp90 essentiality at high temperature [ 18 ]. We found that Hsp90 protects TilS from degradation by HslUV, a conserved chaperone-protease machinery ( Figure 1A ) [ 19 ]. We demonstrated that direct binding between DnaK and Hsp90 is required to enable TilS protection and activation, suggesting a transfer of TilS from DnaK to Hsp90 and showing the interplay between chaperones and protease for post-translational TilS control [ 19 , 20 ]. Download figure Open in new tab Figure 1: Generalities on the TilS protein. (A) Simplified hypothetical model of TilS So requirement for Hsp90. In S. oneidensis , Hsp90 in collaboration with Hsp70/DnaK, is essential to promote TilS So folding and thereby prevent its degradation. (B) Structure of TilS Ec (PDB: 1NI5). Four domains are identified: the N-terminal (N) domain in green, the Helical (H) domain in gold, the C-terminal 1 (C1) in light blue, and the C-terminal 2 (C2) in orange. (C) Taxonomic tree of bacterial taxa based on TilS sequences. This circular taxonomic tree represents the taxonomic relationships between the different bacterial groups that possess TilS. Colors highlight the main bacterial clades, including Bacillati (blue), Betaproteobacteria (orange), Gammaproteobacteria (red), Pseudomonadales (cyan), Enterobacterales (purple), Pseudomonadota (light red) as well as other minor bacterial groups. (D) Determination of melting temperatures of TilS So and TilS Ec by Thermal Shift Assay. TilS So or TilS Ec were labeled with SYPRO Orange and incubated with increasing temperatures. The protein unfolding curves of three replicates from three independent experiments are shown as mean ± SD of derivatives of the fluorescence. The table indicates the melting temperatures as mean ± SD. TilS is a conserved enzyme that modifies a tRNA to allow translation of the rare AUA codon into isoleucine, making it essential in bacteria [ 21 – 24 ]. TilS consists of an N-terminal catalytic domain, a long α-helix linker, and two C-terminal subdomains ( Figure 1B ). The mechanism of TilS activity has been elegantly demonstrated and involves large conformational changes: the two C-terminal subdomains hold and lock the tRNA while the N-terminal domain specifically catalyzes the addition of a lysine at cytidine 34 of the anticodon loop, resulting in a lysidine and switching the tRNA from methionine-specific to isoleucine-specific [ 24 ]. Surprisingly, although (i) TilS is essential in Escherichia coli [ 22 ], and (ii) Hsp90 is required for the protection and activation of TilS in S. oneidensis under heat stress [ 18 ], an E. coli strain that does not produce Hsp90 Ec is only slightly affected by the absence of Hsp90 under heat stress [ 25 ]. One hypothesis to explain these observations is that TilS from E. coli can reach its native conformation without the action of Hsp90, and therefore is not a client of Hsp90. If this hypothesis is correct, comparing the homologous TilS proteins from E. coli and S. oneidensis with respect to Hsp90 dependence could provide valuable insight into client recognition and specificity by Hsp90, as well as the interplay between Hsp90 and the HslUV protease. Here, by comparing TilS orthologs from E. coli, S. oneidensis and other bacteria, we provide insight into the determinants that render TilS dependent or independent of Hsp90. Using in vitro approaches, chimera constructions, and in vivo phenotypes, we found that the degradation-sensitive TilS proteins, including TilS from S. oneidensis (TilS So ), require Hsp90 for their stabilization. In contrast, E. coli TilS (TilSE c ) did not require Hsp90 for protection, and therefore did not interact with Hsp90. In addition, we mapped a region of S. oneidensis TilS (TilS So ) that is responsible for protein degradation by protease and protection by Hsp90. Taken together, these results provide a better understanding of the mechanism of client recognition by chaperones and proteases. Results Comparison of the TilS proteins from E. coli and S. oneidensis A taxonomic tree based on 10500 TilS proteins distributed over more than 6000 different bacteria indicated that TilS is present in most bacteria and that its evolution follows bacterial phylogeny ( Figure 1C ). We focused on the TilS proteins from E. coli (TilS Ec ) and from S. oneidensis (TilS So ). The two TilS proteins share 35% sequence identity, TilS So (464 amino acids, 52.1 kDa, Uniprot ID: Q8EGF9) is larger than TilS Ec (432 amino acids, 48.2 kDa, Uniprot ID: P52097), and the key catalytic residues (including D130, E133 and R203, S. oneidensis numbering) are conserved [ 24 , 26 ] ( Figure S1A ). The N-terminal domain and the helical linker present a higher level of conservation (46 % identity) than the C1 and C2 domains (28 % identity). Additional amino acids are found at the N-terminal extremity (10 amino acids) and in the two C-terminal domains of TilS So ( Figure S1A ). A comparison of the X-ray crystal structure of TilS Ec (PDB 1NI5) with the AlphaFold model of TilS So (AF-Q8EGF9) is shown in Fig. S1B [ 27 ]. To experimentally compare the two proteins, TilS So and TilS Ec were produced in E. coli and were purified. A small shift in size exclusion chromatography elution profiles was observed, in agreement with the low molecular mass difference of the two proteins ( Figure S1C ). Interestingly, thermal shift assays revealed that while the melting temperature (Tm) of TilS Ec was 46.4°C, it was only 38.2°C for TilS So ( Figure 1D ). This large difference in Tm values indicates a higher stability of TilS Ec compared to TilS So , consistent with the hypothesis of a stronger requirement of chaperones for TilS So compared to TilS Ec under heat stress. In the same line of thought, we found that TilS So was more susceptible to trypsin degradation than TilS Ec , again suggesting that TilS So is less stable than TilS Ec ( Figure S1D ). TilS Ec is Hsp90-independent in contrast to TilS So Since we have previously shown that TilS So is a client of Hsp90 and that it interacts with Hsp90 [ 18 ], we wondered whether TilS Ec behaves similarly as TilS So or not. We first conducted bacterial two-hybrid experiments [ 28 ]. In these assays, the Hsp90 proteins from E. coli (Hsp90 Ec ) and from S. oneidensis (Hsp90 So ), as well as the TilS proteins from E. coli (TilS Ec ) and from S. oneidensis (TilS So ) were produced from plasmids as fusion proteins with the T18 or T25 catalytic domains of the adenylate cyclase from Bordetella pertussis . Combinations of plasmids were introduced in an E. coli strain deleted of the gene encoding the adenylate cyclase. If Hsp90 and TilS interact, the two catalytic domains are in proximity, reconstituting the enzymatic activity of the adenylate cyclase, leading to cAMP production and in turn to β-galactosidase activity. As already shown, we observed that TilS So interacted with Hsp90 So ( Figure 2A ). TilS So also interacted with Hsp90 Ec , indicating that TilS So is recognized by both Hsp90 Ec and Hsp90 So . In contrast, background levels of β-galactosidase activity were measured when TilS Ec was produced with Hsp90 Ec or Hsp90 So , similarly to the levels measured when T18-TilS Ec was produced with the T25 domain alone. These results indicate that Hsp90 does not interact with TilS Ec . As positive controls, we found that the Hsp90 fusion proteins retained their ability to dimerize since production of the T18 and T25 fusion proteins containing Hsp90 So or Hsp90 Ec led to a strong level of β -galactosidase activity. Finally, Western blot analysis indicated that the T18-TilS Ec and T18-TilS So fusion proteins were produced ( Figure S2A ). Download figure Open in new tab Figure 2: TilS Ec is not a client of Hsp90. (A) Bacterial two-hybrid experiments showing the interaction between TilS and Hsp90. Hsp90 So and Hsp90 Ec were fused to the T25 domain of B. pertussis adenylate cyclase, and TilS So and TilS Ec were fused to the T18 domain. Bth101 Δhsp90 E. coli strains were transformed with two plasmids allowing the production of T18 and T25 fusions, respectively. The interaction between TilS and Hsp90 was monitored indirectly by following β-galactosidase activity. In negative control (−), the T25 domain alone was used, and in positive control the T18 domain was fused to Hsp90 Ec or Hsp90 So to monitor the interaction of Hsp90 dimer. Data from at least three replicates are shown as mean ± SD. (B) Microscale thermophoresis experiment to determine the interaction between Hsp90 and TilS So or TilS Ec . 85 nM of TilS labeled with RED-tris-NTA 2nd generation dye (NanoTemper) was added to serial 2-times dilutions of Hsp90 So up to 50 µM. Fluorescence at 670 nm was measured, and values at 2.5 s were used to determine K d . The curves shown are representative of three independent experiments for TilS So and two for TilS Ec . The mean of the Kd with standard deviation from three independent experiments is indicated. (C) Western blot showing the amount of TilS So or TilS Ec in S. oneidensis . The strains WT or Δhsp90 with plasmids allowing the production of TilS So or TilS Ec with hexahistidine tag were grown at 34°C (heat stress conditions) and 0.02% arabinose was added. Two hours later, samples were analyzed by Western blot using anti-His antibody. Anti-Hsp90 So antibody was used as a control, and a contaminating band revealed by the anti-AtcJ antibody was used as a loading control. (D) Quantification of TilS from the Western blots shown in C . The peaks corresponding to the pixels of each band were quantified using ImageJ software. The amount of TilS So or TilS Ec in WT strain was set to 1, and each WT is compared to Δhsp90 producing the corresponding TilS So or TilS Ec respectively. Data from six replicates are shown as mean ± SD. (E) Western blot showing the amount of TilS So or TilS Ec in E. coli in the same conditions as in C . Anti-Hsp90 Ec was used as control. The band above Hsp90 Ec is a contaminant detected by the anti-Hsp90 Ec antibody. In C and E , the Western blots shown are representative of at least four independent experiments. (F) Quantification as explained in D of the anti-His Western blot shown in E . Data from four replicates are shown as mean ± SD. In A, D and F , results of one-way ANOVA indicate whether the differences are significant (*** P ≤ 0.001, * P ≤ 0.05) or not (ns, P > 0.05). To confirm the results observed by two-hybrid and obtain binding parameters between the purified TilS proteins and Hsp90, we performed microscale thermophoresis (MST) experiments. This approach quantifies interactions by measuring the intensity variations of the fluorescently labeled proteins along a microscopic temperature gradient in the presence of increasing concentrations of an unlabeled partner protein. TilS Ec and TilS So were first labeled with RED-tris-NTA fluorescent dye that specifically interacts with the 6-His tag of the two proteins. In the MST experiments, the concentration of the RED-tris-NTA labeled TilS so or TilS Ec was kept constant (85 nM), while the concentration of the non-labeled binding partner Hsp90 So was varied between 0 µM-50 µM. For TilS So , the signal was fitted as a one-site binding curve indicating that the two proteins interact with a K d of 1.09 µM ( Figure 2B and S2B ). As also shown by bacterial two-hybrid experiments, TilS Ec did not interact with Hsp90 since no variation in fluorescence between the two proteins was observed by MST under these conditions ( Figure 2B and S2B ). We have previously shown that the amount of TilS So is dramatically reduced under heat stress in the absence of Hsp90 in S. oneidensis due to the activity of the HslUV protease [ 19 ]. To test whether TilS Ec is also degraded in the absence of Hsp90, TilS So or TilS Ec with a 6-His tag at the N-terminal extremity was produced from an inducible promoter in the WT and Δ hsp90 S. oneidensis strains grown under heat stress (34°C), and the amount of the TilS protein was quantified by Western blot. Although the amount of TilS So was reduced to about 30% in the Δ hsp90 strain as expected, no reduction in the amount of TilS Ec was observed in the absence of Hsp90 ( Figure 2C and D ). Interestingly, similar results were observed in E. coli WT or Δ hsp90 strains ( Figure 2E and F ). These results show that in contrast to TilS So at 34°C, TilS Ec is not susceptible to degradation in the absence of Hsp90 both in S. oneidensis and in E. coli . When grown at 28°C, the amount of TilS So and TilS Ec did not significantly vary in E. coli and S. oneidensis WT or Δ hsp90 strains ( Figure S2C-F ). Altogether, these experiments show that in contrast to TilS So , TilS Ec does not interact with Hsp90 in our experimental conditions, and its amount is not reduced in the absence of Hsp90, strongly suggesting that TilS Ec is not an Hsp90 client, whereas TilS So is. TilS identity determines the Hsp90-dependent growth of bacteria As proposed above, TilS Ec does not require the activity of the Hsp90 chaperone to be functional, unlike TilS So . It would imply that the growth defect of the S. oneidensis Δ hsp90 strain under heat stress would be rescued by the production of TilS Ec instead of TilS So . To test this hypothesis, tilS So was replaced by tilS Ec on the chromosome of the S. oneidensis MR1 WT and Δ hsp90 strains using homologous recombination, leading to the strains Δ tilS So tilS Ec+ and Δ hsp90 So Δ tilS So tilS Ec + , respectively. We measured the growth of these strains as well as the WT and Δ hsp90 So strains that were incubated at either 28°C (the non-stress temperature at which Hsp90 So is dispensable) or 35°C (the heat stress temperature at which Hsp90 So is required). We first observed that TilS Ec can functionally substitute for TilS So to support growth of S. oneidensis since the four strains grew similarly at 28°C ( Figure 3A and S3A ), and under heat stress (35°C) the strain producing TilS Ec instead of TilS So in the presence of Hsp90 (Δ tilS So tilS Ec + ) grew as the WT strain ( Figure 3B ). As expected, the absence of Hsp90 led to a strong growth defect at 35°C (Δ hsp90 So vs WT), but not at 28°C ( Figure 3A and B ) [ 18 ]. Interestingly, replacing tilS So by tilS Ec in the S. oneidensis hsp90 -knockout strain (Δ hsp90 So Δ tilS So tilS Ec + ) dramatically improved growth of the Δ hsp90 So strain at 35°C ( Figure 3B ). Similarly, the growth defect under heat stress of the Δ hsp90 So strain was rescued by the production of TilS Ec instead of TilS So when bacterial growth was performed on LB-agar plates ( Figure S3B and C ). Additional growth temperatures (34°C and 36°C) were also tested. Although at 34°C, the presence of TilS Ec almost totally rescued the growth defect of the Δ hsp90 So strain, it only slightly rescued growth at 36°C ( Figure S3C and D ). This could suggest that additional proteins, besides TilS, that are important for bacterial growth are impacted by the absence of Hsp90 at temperatures above 35°C. In agreement with this hypothesis, the slight growth defect of the Δ hsp90 So Δ tilS So tilS Ec+ at 35°C was fully rescued by the production of Hsp90 from a plasmid ( Figure S3E ). Download figure Open in new tab Figure 3: The identity of TilS dictates the dependence on Hsp90 in S. oneidensis and in E. coli . (A) Bacterial growth of S. oneidensis strains carrying tilS from E. coli . The strains S. oneidensis MR-1 wild-type (WT), deleted of hsp90 ( Δhsp90 ) with natural TilS or replaced by TilS from E. coli (Δ tilS So tilS Ec + ) were grown in microplates in LB medium at 28°C with shaking for 24 hours. (B) Bacterial growth of the strains used in A at 35°C. (C) Bacterial growth of E. coli strains carrying tilS from S. oneidensis . The strains E. coli MG1655 wild-type (WT), deleted of hsp90 ( Δhsp90 ) with natural TilS or replaced by TilS from S. oneidensis (Δ tilS Ec tilS So + ) were grown in microplates in LB medium at 28°C with shaking for 24 hours. (D) Bacterial growth of the strains used in C at 35°C. In A, B, C and D , data from three replicates are shown as mean ± SD. In contrast to S. oneidensis , the absence of hsp90 in E. coli does not lead to significant growth defects even under heat stress [ 25 ]. Since we know that Hsp90 is essential for TilS So stabilization at temperatures above 35°C, we wondered whether we could transform E. coli from an Hsp90-independent strain to an Hsp90-dependent strain only by exchanging its TilS ( i . e . TilS Ec ) for TilS So . The tilS gene in E. coli MG1655 WT and Δ hsp90 Ec was replaced by tilS So by homologous recombination leading to strains Δ tilS Ec tilS So + and Δ hsp90 Ec Δ tilS Ec tilS So + , respectively. At 28°C, the four strains grew similarly ( Figure 3C ). However, at 35°C, the growth of Δ tilS Ec tilS So + was delayed compared to WT, indicating that TilS So cannot fully compensate for the absence of TilS Ec . Nevertheless, we found that deleting hsp90 dramatically reduced the growth of the E. coli strain that produces TilS So instead of TilS Ec ( Figure 3D ). Therefore, by exchanging the TilS proteins, E. coli was successfully converted to a strain whose growth depends on the Hsp90 chaperone. Similar results were observed when growth was performed on LB-agar plates ( Figure S3F and G ). Altogether, these experiments demonstrate that the origin of the TilS protein (i.e. from S. oneidensis or from E. coli ) dictates the dependence of a strain on the Hsp90 chaperone. Indeed, the growth defect of a S. oneidensis strain devoid of Hsp90 can be rescued by TilS Ec , a protein that does not need Hsp90 to be functional. Conversely, an E. coli strain that produces TilS So requires Hsp90 for growth at 35°C, a temperature at which the TilS So protein needs Hsp90 to be protected. The C-terminal domain of TilS So is responsible for the dependence on Hsp90 We next wanted to identify the region of TilS So that confers dependence on Hsp90. To this end, we constructed chimeras by swapping domains between TilS So and TilS Ec ( Figure 4A ). As described above, TilS is constituted by an N-terminal domain (N), a long alpha helix (H), and a C-terminal domain divided in the two subdomains C1 and C2 ( Figures 1B , S1A-B ). The chimeras were named according to the origin of each of their four domains, with “S” for S. oneidensis and “E” for E. coli . For example, “SEEE” stands for the chimera with the N-terminal domain of TilS So and the three other domains of TilS Ec . The chimeras were produced from a pBad inducible promoter in the S. oneidensis WT and Δ hsp90 strains grown at 28°C and 34°C. To quantify the amount of the TilS chimeras in the two strains, total proteins were separated on SDS-PAGE and the chimeras were detected by Western blot ( Figures 4B and C , and S4A and B ). Download figure Open in new tab Figure 4: The C-terminal domain of TilS dictates Hsp90 dependence. (A) Schematic showing the organization in domains of TilS and how they were mixed to give 6 TilS Ec – TilS So chimeras. « E » corresponds to TilS domain from E. coli , and « S » to domain from S. oneidensis . (B) Western blot showing the abundance of TilS chimeras in S. oneidensis WT or Δ hsp90 . The strains WT or Δ hsp90 with plasmid pBad33 allowing the production of 6His-TilS chimeras were grown at 34°C, and 0.02% arabinose was added. After 2 hours, samples were analyzed by Western blot with anti-His antibody. Anti-Hsp90 was used as a control and Anti-AtcJ as neutral loading control. This Western blot is representative of at least three independent experiments. (C) Quantification of the Western blot shown in B . The peaks corresponding to the pixels of each band were quantified using ImageJ software. The amount of each chimera in WT strain was set to 1, and each WT is compared to Δhsp90 producing the corresponding chimera respectively. Data from at least three replicates are shown as mean ± SD. Results of one-way ANOVA indicate whether the differences are significant (**** P ≤ 0.0001, *** P ≤ 0.001) or not (ns, P > 0.05). Although, as indicated above, the amount of TilS So is considerably reduced in the absence of Hsp90 ( Figures 2C, 2D , and 4C ), we found that replacing the single C2 domain of TilS So with that of TilS Ec (“SSSE” chimera) was sufficient to stabilize TilS So in the absence of Hsp90. Exchanging the C1 and C2 domains (chimera “SSEE”) or H, C1, and C2 domains (chimera “SEEE”) of TilS So by the domains of TilS Ec also led to stabilization of the chimeras in the absence of Hsp90 ( Figure 4B and C ). Contrarily, although the amount of TilS Ec was not reduced in the absence of Hsp90 compared to WT ( Figures 2C, 2D , and 4C ), substituting the C2 domain of TilS Ec by the one of TilS So (chimera “EEES”) was sufficient to lead to a lower level of chimera in the absence of Hsp90, therefore indicating a dependence on the Hsp90 chaperone ( Figure 4B and C ). Similar results, i.e., a lower amount of chimeras in the absence of Hsp90, were obtained with the chimeras “EESS” and “ESSS”. These results strongly suggest that the C2 domain of TilS So is responsible for the dependence on the Hsp90 chaperone. When Hsp90 does not protect it, this domain most likely directs TilS So to degradation. Indeed, we found that Hsp90, which does not interact with TilS Ec ( Figure 2A and B ) protects the “EEES” chimera, possessing only the C2 domain of TilS So , from degradation ( Figure 4 ). Interestingly, the AlphaFold3 prediction of the binding between Hsp90 So and TilS So positions the two proteins in interaction via the C2 domain of TilS So , while no binding was predicted with TilS Ec ( Figure S5 ). Identification of point mutations in the C2 domain of TilS So that modify Hsp90 dependence Based on our observations that TilS So is an obligate client of Hsp90, while TilS Ec is not, we aimed to predict Hsp90 essentiality in a given strain by analyzing its TilS protein. To this end, we hypothesized that TilS proteins evolutionary close to TilS Ec would not require Hsp90 for stability, while those that are close to TilS So would. To test this, a phylogenetic tree was built based on TilS C2 domains ( Figure S6 ), and several TilS orthologs were selected and tested for their stability in vivo in S. oneidensis . However, we could not validate this hypothesis because some TilS proteins close to TilS Ec , such as TilS from Salmonella Typhymurium, were dependent on Hsp90 as shown with our in vivo degradation assay ( SI results, and Figure S7 ). Interestingly, we were surprised to observe that TilS from Shewanella xiamenensis (TilS Sx ), a protein with over 90% identity to TilS So , did not behave like TilS So with regard to Hsp90 dependence. Indeed, we found that the amount of TilS Sx was not reduced in the absence of Hsp90 So , as observed with TilS So ( Figures 5A , S7A, and S7C ). Comparison of the primary sequences between the two proteins revealed a few point mutations in the C2 domain, and in particular the L340 residue in TilS So that is replaced by a proline residue in TilS Sx ( Figure 5B ). We constructed the single mutant Hsp90 So L340P to test whether this proline residue could be responsible for the stabilization of TilS Sx in the absence of Hsp90. Interestingly, we found that, in contrast to the wild-type protein, the amount of the mutant protein TilS So L340P was only slightly reduced in the absence of Hsp90 ( Figures 5A and S8A ). Replacing L340 of TilS So by residues other than proline (serine or glycine) ( Figure S8B ), or replacing the last 7 residues of TilS So by the 8 last ones of TilS Sx ( Figure S8C ) did not lead to stabilization of TilS So in the absence of Hsp90. These results indicate that mutating the leucine 340 of TilS So into a proline is crucial to stabilize the protein in the absence of Hsp90, as naturally found in TilS Sx . Download figure Open in new tab Figure 5: A single point mutation in TilS So decreases Hsp90 dependence. (A) Graph showing the amount of TilS Sx , and TilS So WT or mutants from the Western blot in Fig. S8A. The S. oneidensis WT or Δhsp90 strains with plasmid allowing the production of TilS So or TilS Sx with hexahistidine tag were grown at 34°C (heat stress conditions) and 0.02% arabinose was added. Two hours later, samples were analyzed by Western blot using anti-His antibody. This graph shows the quantification of three independent Western blots. The peaks corresponding to the pixels of each band were quantified using ImageJ software. The amount of TilS So or TilS Sx in WT strain was set to 1, and each WT is compared to Δhsp90 producing the corresponding TilS So or TilS Sx respectively. Data from three replicates are shown as mean ± SD. The results of one-way ANOVA indicate whether the differences are significant (**** P ≤ 0.0001) or not (ns, P > 0.05). (B) Partial alignment of the sequences of TilS So and TilS from S. xiamenensis (TilS Sx ) using the multiple sequence alignment tool «Multalin» ( http://multalin.toulouse.inra.fr/multalin/ ). TheESPript program ( https://espript.ibcp.fr/ESPript/ESPript/ ) was used to display the alignment. Numbering of TilS So and TilS Sx . The red arrows indicate the residues that were mutated. (C) Bacterial growth in liquid media of the S. oneidensis strains MR-1 wild-type (WT) or deleted of hsp90 ( Δhsp90 ) with the tilS gene WT or coding for the mutation L340P. The cells were grown in microplates in LB medium at 35°C with shaking for 24 hours. Data from six replicates are shown as mean ± SD. (D) Determination of the melting temperatures of TilS So WT or L340P, TilS Ec , or TilS Sx by Thermal Shift Assay. The TilS proteins were labeled with SYPRO Orange and incubated with increasing temperatures. The protein unfolding curves of at least three replicates are shown as mean ± SD of derivatives of the fluorescence. The table indicates the melting temperatures as mean ± SD. (E) Bacterial two-hybrid experiments showing interaction of TilS So WT or mutants, or TilS Sx with Hsp90 So . Hsp90 So was fused to the T25 domain of B. pertussis adenylate cyclase and the five TilS proteins were fused to the T18 domain. Bth101 Δhsp90 strains were transformed with two plasmids allowing the production of T18 and T25 fusions, respectively. The interaction between TilS and Hsp90 was monitored indirectly by following β-galactosidase activity. In the negative control (−), T25 or T18 domains alone were used, and in the positive control T25 and T18 domain were both fused to Hsp90 So to monitor the interaction of Hsp90 dimer. Data from at least three replicates are shown as mean ± SD. The results of one-way ANOVA indicate whether the differences are significant (* P ≤ 0.05) or not (ns, P > 0.05). We then wondered whether the stabilization of TilS So by the mutation L340P would allow bacteria to grow with a lower dependence on Hsp90 under heat stress. The corresponding mutation in tilS So was introduced in the chromosome of S. oneidensis WT and Δ hsp90 . In the WT background, the strain producing TilS So L340P instead of TilS So grew similarly to the WT strain at 28°C and 35°C ( Figures 5C and S8D-F ). More importantly, production of TilS So L340P did partially rescue the growth defect of the S. oneidensis Δ hsp90 strain ( Figure 5C ), confirming that TilS So L340P can be functional without the assistance of Hsp90. We further characterized TilS Sx and TilS So L340P to investigate why they can work independently of Hsp90, as was observed with TilS Ec . The proteins were purified and size exclusion chromatography elution profiles of the two proteins were similar to TilS So ( Figure S9A ). Surprisingly, we found that TilS Sx and TilS So L340P behaved more like TilS So than TilS Ec with regard to intrinsic protein stability and interaction with Hsp90. Indeed, thermal shift assays indicated that the Tm of TilS Sx (37.0°C) and TilS So L340P (38.5°C) were closer to TilS So (38.2°C) than TilS Ec (46.4°C) ( Figure 5D ). Similarly, the trypsin digestion profiles of TilS Sx and TilS So L340P resembled those obtained with TilS So ( Figure S9B ). Finally, two-hybrid and MST experiments revealed that TilS Sx and TilS So L340P interacted with Hsp90, as observed with TilS So , but in contrast to TilS Ec ( Figure 5E and S9C ). Altogether, based on the TilS ortholog from S. xiamenensis , we identified a single mutation, L340P, in TilS So that switches the TilS protein from Hsp90-dependent (TilS So WT) to Hsp90-independent (TilS So L340P). Since TilS So L340P still interacts with Hsp90, this suggests that the mutation prevents the degradation by the HslUV protease. Discussion In this study, we aimed to explore the determinants that govern whether a protein is dependent on the bacterial Hsp90 chaperone. Our client model was the TilS protein, an essential enzyme whose function is to modify a tRNA. We have previously found that TilS is an obligate Hsp90 client under heat stress in the bacterium S. oneidensis [ 18 ]. Indeed, we have shown that Hsp90 protects TilS So from degradation by the HslUV protease [ 18 , 19 ]. Since TilS is also essential in E. coli , but Hsp90 is dispensable for growth in this bacterium, we hypothesized that TilS So and TilS Ec do not have the same requirement for Hsp90. Therefore, we compared these TilS proteins. We found that although the two TilS orthologs share 35 % sequence identity and a common global fold ( Figure S1 A-B ), they do not behave similarly with respect to Hsp90 dependence. Indeed, in contrast to TilS So , whose level was dramatically reduced under heat stress in the absence of Hsp90, the level of TilS Ec did not differ significantly between WT and Δ hsp90 strains ( Fig 2C-F ). In addition, TilS Ec did not interact with Hsp90, whereas TilS So did ( Figure 2A-B ), suggesting that TilS Ec , unlike TilS So , is not a client of Hsp90. To more precisely define the regions of TilS that dictate Hsp90 dependence, chimeras between the two TilS proteins were constructed, allowing the identification of the C2 domain of TilS So that is responsible for TilS degradation and Hsp90 dependence ( Figure 4 ). Indeed, the amount of the chimera consisting of TilS Ec with only the C2 domain of TilS So (named “EEES”) was strongly reduced in the Δ hsp90 strain, whereas TilS So was stabilized by exchanging its C2 domain with that of TilS Ec (named “SSSE”). In addition, we found that replacement of residue L340 of TilS So with a proline was sufficient to partially abolish its degradation in the absence of Hsp90 ( Figure 5 ). The mutation of this leucine residue to serine or glycine did not stabilize TilS So . This suggests that the local reorientation of the region near L340, or the increased rigidity of the protein induced by proline, is sufficient to prevent TilS So degradation, rather than the nature of the leucine residue per se. Taken together, these experiments indicate that the C2 domain of TilS So is key to triggering TilS So degradation in the absence of Hsp90, and to allowing TilS to be protected by Hsp90. Based on our results, we can propose a model in which Hsp90 interacts with the C2 domain of TilS So , thereby limiting the accessibility of the HslUV protease to an as yet unidentified degron probably located in the C2 domain of TilS So . After Hsp90-assisted folding of TilS So , the region recognized by HslUV could be buried and no longer accessible to the protease. This model is also supported by AlphaFold 3 predictions ( Figure S5 ), which propose an interaction between a dimer of Hsp90 So and the C2 domain of TilS So . In these predictions, binding to Hsp90 occurs through the cleft located between the two monomers of Hsp90 So , which is known to be important for binding to multiple clients [ 29 ]. Interestingly, mutations in this region of Hsp90 (W476R and L563A, Hsp90 So numbering) have been shown to prevent binding to clients, including TilS So [ 18 , 29 ]. Alternative models obtained with AlphaFold 3 always position the C2 domain of TilS So in contact with Hsp90 So , but the other domains of TilS So adopt different orientations without interacting with Hsp90 ( Figure S5B ). How Hsp90 interacts with its clients has long been of interest [ 8 – 11 ]. Studies performed with eukaryotic Hsp90 clients, including for example the Tau protein [ 30 ] and α-synuclein [ 31 ], suggest that hydrophobic patches lead to Hsp90-client stabilization. Recently, the laboratory of Brian Freeman identified over 1000 proteins that associated with Hsp90 in yeast using a cross-linking approach followed by mass spectrometry [ 11 ]. Mapping the chaperone binding sites in the clients revealed that Hsp90 preferentially interacts with intrinsically disordered regions of proteins. We used predictor tools including AIUPred algorithm to search for putative intrinsically disordered regions in the TilS So sequence; however, no such regions were identified ( Figure S10A ) [ 32 ]. Interestingly, AlphaFold 3 indicates the presence of an unstructured loop in the C2 domain of TilS So , in the vicinity of residue L340, but this loop is not shown in the X-ray crystal structure of TilS Ec (PDB 1NI5) ( Figure S10B ). While this may seem appealing, it must be treated with high caution since the structures of TilS So and TilS Sx shown here are only predictions and this loop could appear as an artefact. The conclusions regarding the stability of TilS and its dependence on Hsp90 were supported by in vivo phenotypes. E. coli could be transformed from an Hsp90-dispensable strain to an Hsp90-essential strain by producing TilS So instead of TilS Ec ( Figure 3D ) [ 25 ]. Conversely, although S. oneidensis requires Hsp90 for growth under heat stress, we generated a S. oneidensis strain whose growth became Hsp90-independent only by replacing TilS So with TilS Ec ( Figure 3B ), or by introducing the single mutation L340P in TilS So ( Figure 5C ). However, the restoration of the growth phenotype was almost lost when the temperature increased by only 1°C, although we know that TilS Ec is functional at this temperature in E. coli (regular E. coli growth temperature is 37°C) ( Figure S3D ). Therefore, we can hypothesize that, although TilS So is the only essential protein whose level dramatically decreases in the absence of Hsp90 at 35°C, the absence of Hsp90 at 36°C would impact several other proteins. In that case, compensating for the loss of TilS So by producing TilS Ec would not be sufficient to support growth of S. oneidensis because several other proteins or another essential protein would be inactivated by the increased temperature in the absence of Hsp90 (36°C). The interplay between the Hsp90 chaperone and the HslUV protease has previously been documented [ 19 , 33 , 34 ]. Fauvet et al . proposed that aggregation-prone proteins are degraded by HslUV in an Hsp90-dependent process [ 34 ]. We have shown that TilS So is degraded by the protease HslUV when it is not protected by Hsp90, and the severe growth defect of the S. oneidensis Δ hsp90 strain was suppressed by deletion of hslUV [ 19 ]. Similarly, defects in colibactin toxin production in E. coli hsp90 knockout strain were rescued by deletion of the gene encoding the HslV protease [ 33 ]. These observations suggest that the main function of Hsp90 is to protect its clients from protease degradation, most likely by facilitating their folding. The present study highlights differences in TilS orthologs regarding chaperone dependence and protease degradation. Based on protein intrinsic stability, we found that TilS Ec and the TilS proteins from the Shewanella genus (TilS So and TilS Sx ) fell into two different categories. Indeed, thermal shift assays on purified proteins indicate that TilS So and TilS Sx are less stable than TilS Ec , with a shift higher than 8°C in the melting temperatures of the proteins from the two categories ( Figure 1D and 5D ). This result correlates with the fact that Hsp90 interacts with TilS So and TilS Sx , but not with TilS Ec ( Figure 2A-B , 5E , and S9C ). These observations are in agreement with a global study in yeast demonstrating that the intrinsically unstable state of kinases leads to recognition by Hsp90 [ 35 ]. An example is provided by the oncogenic v-Src kinase, an Hsp90-dependent protein, and the Hsp90-independent c-Src kinase, that share 98 % sequence identity [ 36 ]. c-Src was more stable than v-Src in thermal unfolding measurements, with a 4°C difference in the melting temperature of the two proteins, and v-Src was more prone to aggregation than c-Src. Interestingly, additional differences were found in the in vivo stability of the TilS orthologs belonging to the Shewanella genus. Indeed, TilS So was degraded by HslUV in the absence of Hsp90 [ 19 ], whereas TilS Sx was not degraded ( Figure 5A ). Thus, despite conserving their ability to interact with Hsp90, these two proteins have evolved to be either protease sensitive (TilS So ) or resistant (TilS Sx ). This difference may be due to a single mutation, since mutating L340 to P in TilS So is sufficient to stabilize it in the absence of Hsp90. Therefore, TilS So L340P and TilS Sx are interesting proteins for studying the interplay between chaperones and proteases, as their behavior uncouples Hsp90 protective activity from HslUV proteolytic activity. The physiological relevance of TilS Sx still interacting with Hsp90, despite not being degraded, remains unclear. It is possible that under certain stress conditions, this TilS protein requires the chaperone activity of Hsp90 to be functional. These observations raise the question of the evolution of TilS in relation to its dependence on Hsp90. A taxonomic tree was constructed based on full-length TilS ( Figure 1C ) to assess the distribution of TilS, and a phylogenetic tree of the C2 domain of TilS ( Figure S6 ). Although the evolution of TilS follows the bacterial phylogeny, we could not correlate the evolution of the C2 domain of TilS with a dependence on Hsp90 ( SI Results and Figure S7 ). Indeed, as explained above, although the TilS proteins from the bacteria S. oneidensis and S. xiamenensis have more than 90 % sequence identity, TilS So required Hsp90 for protection against protease degradation, while TilS Sx did not. Interestingly, mutations may arise in the essential TilS protein because of the protection provided by the Hsp90 chaperone. Consistent with this, Hsp90 has been shown to play an important role in evolution in both eukaryotes and bacteria, and Hsp90 belongs to the family of “global modifiers” [ 37 – 41 ]. Hsp90 buffers the phenotypic effects of mutations that occur in proteins. Indeed, by stabilizing and allowing the folding of a mutated client, Hsp90 prevents the phenotype associated with that mutation from being expressed. These phenotypes can be revealed under conditions in which Hsp90 is titrated, for instance, by other clients. The physiological relevance of the dependence on Hsp90 and/or degradation by protease regarding the cellular availability of the essential TilS protein is unclear, though it may play a significant role in regulating bacterial fitness in response to environmental stressors. For example, we can hypothesize that a less stable TilS may confer a fitness advantage to certain bacterial species. With the assistance of Hsp90, TilS So may be better adapted than TilS Ec to the likely more diverse environmental conditions of the ecological niche inhabited by S. oneidensis , compared to the relatively stable environment of the intestinal tract where E. coli resides [ 17 ]. In conclusion, our study on the TilS So protein and some of its close orthologs highlights the variety of mechanisms implemented in bacteria to achieve the native functional protein, including chaperone requirement and protease susceptibility. Future work will aim to better understand the interplay between clients, chaperones, and proteases to gain a broader view of the mechanisms of protein homeostasis in bacteria. Material and methods Strains, Plasmids and Growth conditions Strains used in this study are listed in Table S1 . The reference S. oneidensis strain used is MR1-R [ 42 ] and the reference strain for E. coli is MG1655 (K12) [ 43 ]. The strains S. oneidensis Δ hsp90 and E. coli Δ hsp90 were constructed previously [ 18 , 29 ]. S. oneidensis and E. coli WT or Δ hsp90 strains with natural tilS So or tilS Ec replaced by tilS Ec or tilS So , respectively, were constructed for this study as described in Supplementary Materials and Methods. Plasmids used in this study are listed in Table S2 . Plasmid construction is described in Supplementary Materials and Methods. Plasmids were introduced by conjugation in S. oneidensis and by transformation in E. coli . Site-directed mutagenesis was performed with the Quickchange mutagenesis kit (Agilent) according to manufacturer’s instructions. Mutations were checked by sequencing. Sequences of the TilS So -TilS Ec chimeras are indicated in Table S3 . When necessary, chloramphenicol (25 µg/mL), kanamycin (50 µg/mL), ampicillin (50 µg/mL) or streptomycin (100 µg/mL) was added. Computational analysis of TilS orthologs Taxonomic tree To construct a taxonomic tree based on the TilS protein, we searched in InterPro IPR015262, which identifies the substrate-binding domain of TilS [ 44 ]. A total of approximately 18,500 proteins were retrieved, with the majority exhibiting two predominant domain architectures: IPR011063 -IPR015262 - IPR012796 (59%) and IPR011063 - IPR015262 (39%). Focusing on proteins possessing the IPR011063 - IPR015262 - IPR012796 architecture, we identified ∼10,500 proteins distributed across more than 6,000 bacterial species. Using the NCBI taxonomy [ 45 ], a taxonomic tree of these species was generated, demonstrating the widespread presence of TilS across bacteria. Pseudomonadota (Proteobacteria) were the most represented group, accounting for 72% of the sequences, followed by the Bacillati (Terrabacteria) group at 18%. The taxonomic analysis delves into the evolutionary distribution of TilS and its conservation across bacterial lineages. To further characterize TilS close to S. oneidensis and based on the phylogenetic analysis and the structure of TilS organization, we reduced the distribution of proteins to those best defined on Uniprot [ 26 ]. The reviewed TilS were recovered (with the addition of the TilS of Shewanella xiamenensis , due to its proximity to S. oneidensis , see Table S4 for the sequences) and the C2 domains of these proteins were aligned with MUSCLE v3 [ 46 ]. Evolutionary history was inferred by using the Maximum Likelihood method and JTT matrix-based model with MEGA X [ 47 ]. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site ( Table S5 for the Newick file). Based on this tree, we then selected the subtree presented ( Figure S6 ), with the 73 sequences revolving around E. coli and S. oneidensis to select several sequences to perform experiments in vivo . TilS sequence alignment TilS So and TilS Sx sequences were aligned using “Multalin” tool ( http://multalin.toulouse.inra.fr/multalin/ ). The ESPript program ( https://espript.ibcp.fr/ESPript/ESPript/ ) was used to display the alignment. Thermal Shift Assays to determine TilS melting temperature TilS proteins were purified as described in Supplementary Materials and Methods. 5 µg of proteins in 25 mM Tris-HCl pH 7.5, 50 mM KCl labeled with 10X SYPRO Orange (Sigma Life Science) were exposed to increasing temperatures from 15 to 90°C at a scan rate of 0.5°C per 30 s using BioRad CFX96 Touch RealTime PCR instrument. The protein unfolding curves were monitored by detecting changes in SYPRO Orange fluorescence. Melting temperatures were determined using the first derivative values of raw fluorescence data using Bio-Rad CFX Manager 3.1 software. Microscale Thermophoresis 90 µL of TilS (340 nM) in PBS-Tween 0.05 %, pH 7.3, was labeled with 90 µL of 100 nM RED-tris-NTA 2 nd generation dye (NanoTemper). According to the manufacturer protocol, after 30 minutes incubation at room temperature, sample was centrifuged 10 minutes at 15 000 g and 10 µL of labeled TilS at 170 nM was transferred into 16 tubes containing 10 µL of serial 2-fold dilutions of Hsp90 So in PBS-Tween 0.05 %, pH 7.3. Final concentrations in capillary are 85 nM TilS and 50 µM Hsp90 So at highest concentration to 1.5 nM at the lowest. The resulting solutions were loaded onto 16 Monolith capillaries (NanoTemper). Fluorescence was measured at 670 nm at an ambient temperature of 25°C using Monolith NT.115 according to the manufacturer instructions. Instrument parameters were adjusted to 50% LED power and medium MST power. Data of two independently pipetted measurements were analyzed (MO.Affinity Analysis 3 software version 2.6.3, NanoTemper Technologies) using the signal from an MST on-time of 2.5 s. Bacterial two-hybrid assays Bacterial two-hybrid assays were performed as described by Battesti and Bouveret with modifications [ 28 ]. The Bth101 Δ hsp90 strains lacking adenylate cyclase and Hsp90 corresponding genes were transformed with two plasmids. The first encoded the fusion protein between the T18 domain of B. pertussis adenylate cyclase at the N-terminal extremity with TilS from E. coli, S. oneidensis or S. xiamenensis , at the C-terminal extremity. The second encoded the fusion protein between the T25 domain of B. pertussis adenylate cyclase at the N-terminal extremity with Hsp90 from S. oneidensis or E. coli at the C-terminal extremity. As negative controls, vectors producing the T25 or T18 domains were used. Transformants were incubated at 28°C. After 3 days, colonies were inoculated in LB medium supplemented with ampicillin, kanamycin and isopropylthio-β-galactoside. After overnight culture at 28°C, cells were lysed with 1mg/mL lysozyme and Popculture Reagent solution (Millipore) for 15 minutes. Then, Z buffer was added (100 mM phosphate buffer pH=7, 10 mM KCl, 1 mM MgSO 4 , 50 mM β-mercaptoethanol). Before measurement, 2.2 mM orthonitrophenyl-β-galactoside was added. β-galactosidase activity was measured via a modified Miller assay adapted for use in a Tecan Spark microplate reader as described previously [ 48 ]. Western blot analysis to evaluate TilS amount TilS amount was determined by Western blots. The p tilS -6his plasmids carrying tilS genes from several species were introduced into S. oneidensis or E. coli WT or Δ hsp90 strains. The strains were grown at 28°C overnight with chloramphenicol, diluted to OD 600 = 0.1, and incubated at 28°C or 34°C. After 3 h, 0.02% arabinose was added and 2 h later the same amount of cells was collected. Pellets were resuspended in denaturing loading buffer and heat treated at 95°C. Proteins were separated by SDS-PAGE and transferred by Western blot. TilS was detected using anti-6his antibody (Thermo). Hsp90 was detected with anti-Hsp90 So or anti-Hsp90 Ec antibody. As neutral loading control, a non-specific band detected by the anti-AtcJ antibody was used [ 48 ]. Bacterial growth of S. oneidensis or E. coli These experiments were performed as previously described with modifications [ 20 ]. After overnight precultures at 28°C in LB medium, the S. oneidensis or E. coli strains were inoculated to OD 600 = 0.1 in LB and incubated at 28°C until late exponential phase. For growth on liquid media, cells were diluted to OD 600 = 0.0005 in LB. Growth was measured in a microplate reader at indicated temperatures. CRediT authorship contribution statement Marie Corteggiani: Conceptualization, Investigation, Validation, Visualization, Writing – original draft, Writing – review and editing. Amine Ali-Chaouche: Investigation, Validation, Writing – review and editing. Miha Bahun: Investigation, Validation, Writing – review and editing. Flora Honoré: Investigation, Validation, Writing – review and editing. Deborah Byrne: Resources, Validation, Writing – review and editing. Sébastien Dementin: Investigation, Validation, Writing – review and editing. Mathieu E. Rebeaud: Conceptualization, Investigation, Validation, Visualization, Writing – original draft, Writing – review and editing. Olivier Genest: Conceptualization, Investigation, Validation, Visualization, Writing – original draft, Writing – review and editing, Supervision, Project administration, Funding acquisition. Declaration of competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement We thank members of our groups for help and fruitful discussions, and Yann Denis from the transcriptomic platform of the IMM (CNRS) for assistance with thermal shift assay. M.E.R. would like to thank Paolo De Los Rios for helpful discussions and the opportunity to work on several interesting side projects. This work was supported by the Centre National de la Recherche Scientifique, Aix Marseille Université, and the Agence Nationale de la Recherche (ANR-16-CE11-0002-01 and ANR-20-CE44-0017). 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Share Subtle variations in a client protein determine bacterial Hsp90 dependence Marie Corteggiani , Amine Ali Chaouche , Miha Bahun , Flora A. Honoré , Deborah Byrne , Sébastien Dementin , Mathieu E. Rebeaud , Olivier Genest bioRxiv 2025.07.01.662558; doi: https://doi.org/10.1101/2025.07.01.662558 Share This Article: Copy Citation Tools Subtle variations in a client protein determine bacterial Hsp90 dependence Marie Corteggiani , Amine Ali Chaouche , Miha Bahun , Flora A. Honoré , Deborah Byrne , Sébastien Dementin , Mathieu E. 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