# 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

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Marie Corteggiani, Amine Ali Chaouche, Miha Bahun, Flora A Honoré, Deborah Byrne, et al.. Subtle variations in a client protein determine bacterial Hsp90 dependence. Journal of Molecular Biology, 2025, 437 (21), pp.169378. 10.1016/j.jmb.2025.169378. hal-05248273

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Subtle variations in a client protein determine bacterial Hsp90 dependence

Marie Corteggianiª, Amine Ali Chaoucheª, Miha Bahunᵇ, Flora A. Honoréª, Deborah Byrneᶜ, Sébastien Dementinª, Mathieu E. Rebeaudᵈ, Olivier Genestª, *

ªAix-Marseille Univ, CNRS, BIP UMR 7281, IMM, 31 Chemin Joseph Aiguier, 13402 Marseille, France.

ᵇDepartment of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1000, Ljubljana, Slovenia.

ᶜAix-Marseille Univ, CNRS, IMM-FR3479, 31 Chemin Joseph Aiguier, 13402, Marseille, France.

ᵈInstitute of Physics, School of Basic Sciences, École Polytechnique Fédérale de Lausanne - EPFL, Lausanne, Switzerland.

*Corresponding author:

Olivier Genest, CNRS, BIP UMR 7281, 31 Chemin Joseph Aiguier, 13402 Marseille, Cedex 20, France.

Email: [ogenest@imm.cnrs.fr](mailto:ogenest@imm.cnrs.fr)

Phone: +33 4 91 16 46 53

**Keywords:**

Chaperone; Protease; Hsp90; Client specificity; Bacteria; HtpG; HslU HslV
**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<sub>SO</sub>) in the bacterium *Shewanella oneidensis*. Although Hsp90 is indispensable in *S. oneidensis* under heat stress by protecting the essential protein TilS<sub>SO</sub> from degradation by the protease HsIUV, Hsp90 is dispensable in *Escherichia coli*, suggesting that *E. coli* TilS (TilS<sub>Ec</sub>) is Hsp90 independent. We therefore compared the TilS orthologs with respect to *in vitro* stability, *in vivo* degradation, and interaction with Hsp90 to identify determinants of Hsp90 dependence. We found that in contrast to TilS<sub>SO</sub>, TilS<sub>Ec</sub> was more stable, was not degraded by protease in the absence of Hsp90, and did not interact with Hsp90, indicating that TilS<sub>Ec</sub> is not a client of Hsp90. Chimeras between TilS<sub>SO</sub> and TilS<sub>Ec</sub> as well as directed mutagenesis revealed a region of TilS<sub>SO</sub> that is key for protease degradation and Hsp90 protection. Consistent with these results, the growth of *S. oneidensis* producing TilS<sub>Ec</sub> was no longer dependent on Hsp90 under heat stress. Conversely, Hsp90 became essential for the growth of *E. coli* that produced TilS<sub>SO</sub> instead of TilS<sub>Ec</sub>. Altogether, our work reveals that protein-specific features, such as stability and degradation sensitivity, can determine whether orthologous proteins require the bacterial Hsp90 chaperone in vivo.
Introduction

Chaperone proteins are present in all organisms and assist other proteins in folding, unfolding,
preventing aggregation, disaggregating, and in some cases, targeting for 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 into 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].

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 Hsp90EC 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*<sub>So</sub>), require Hsp90 for their stabilization. In contrast, *E. coli* TilS (*TilSE*<sub>c</sub>) did not require Hsp90 for protection, and therefore did not interact with Hsp90. In addition, we mapped a region of *S. oneidensis* TilS (*TilS*<sub>So</sub>) that is responsible for protein degradation by protease and protection by Hsp90. Taken together, these findings illustrate the interplay between Hsp90 and proteases, whose involvement may vary in the stabilization or degradation of orthologous proteins.

## 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*<sub>Ec</sub>) and from *S. oneidensis* (*TilS*<sub>So</sub>). The two TilS proteins share 35% sequence identity, *TilS*<sub>So</sub> (464 amino acids, 52.1 kDa, Uniprot ID: Q8EGF9) is larger than *TilS*<sub>Ec</sub> (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*<sub>So</sub> (Figure S1A). A comparison of the X-ray crystal structure of *TilS*<sub>Ec</sub> (PDB 1NI5) with the AlphaFold model of *TilS*<sub>So</sub> (AF-Q8EGF9) is shown in Fig. S1B [27].

To experimentally compare the two proteins, *TilS*<sub>So</sub> and *TilS*<sub>Ec</sub> 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*<sub>Ec</sub> was 46.4°C, it was only 38.2°C for *TilS*<sub>So</sub> (Figure 1D). This large difference in Tm values indicates a higher stability of *TilS*<sub>Ec</sub> compared to
TilS<sub>So</sub>, consistent with the hypothesis of a stronger requirement of chaperones for TilS<sub>So</sub> compared to TilS<sub>Ec</sub> under heat stress. In the same line of thought, we found that TilS<sub>So</sub> was more susceptible to trypsin degradation than TilS<sub>Ec</sub>, again suggesting that TilS<sub>So</sub> is less stable than TilS<sub>Ec</sub> (Figure S1D).

TilS<sub>Ec</sub> is Hsp90-independent in contrast to TilS<sub>So</sub>.

Since we have previously shown that TilS<sub>So</sub> is a client of Hsp90 and that it interacts with Hsp90 [18], we wondered whether TilS<sub>Ec</sub> behaves similarly as TilS<sub>So</sub> or not. We first conducted bacterial two-hybrid experiments [28]. In these assays, the Hsp90 proteins from *E. coli* (Hsp90<sub>Ec</sub>) and from *S. oneidensis* (Hsp90<sub>So</sub>), as well as the TilS proteins from *E. coli* (TilS<sub>Ec</sub>) and from *S. oneidensis* (TilS<sub>So</sub>) 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<sub>So</sub> interacted with Hsp90<sub>So</sub> (Figure 2A) [18]. TilS<sub>So</sub> also interacted with Hsp90<sub>Ec</sub>, indicating that TilS<sub>So</sub> is recognized by both Hsp90<sub>Ec</sub> and Hsp90<sub>So</sub>. In contrast, background levels of β-galactosidase activity were measured when TilS<sub>Ec</sub> was produced with Hsp90<sub>Ec</sub> or Hsp90<sub>So</sub>, similarly to the levels measured when T18-TilS<sub>Ec</sub> was produced with the T25 domain alone (Figure 2A). These results indicate that Hsp90 does not interact with TilS<sub>Ec</sub>. 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<sub>So</sub> or Hsp90<sub>Ec</sub> led to a strong level of β-galactosidase activity. Finally, Western blot analysis indicated that the T18-TilS<sub>Ec</sub> and T18-TilS<sub>So</sub> fusion proteins were produced (Figure S2A).

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<sub>Ec</sub> and TilS<sub>So</sub> 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<sub>So</sub> or TilS<sub>Ec</sub> was kept constant (85 nM), while the
concentration of the non-labeled binding partner Hsp90<sub>So</sub> was varied between 0 µM and 50 µM. For
TilS<sub>So</sub>, the signal was fitted as a one-site binding curve indicating that the two proteins interact with a
K<sub>d</sub> of 1.09 µM (Figure 2B and S2B). As also shown by bacterial two-hybrid experiments, TilS<sub>Ec</sub> 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<sub>So</sub> 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<sub>Ec</sub>
is also degraded in the absence of Hsp90, TilS<sub>So</sub> or TilS<sub>Ec</sub> with a 6-His tag at the N-terminal extremity
was produced from an inducible promoter in the WT and Δhsp90 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<sub>So</sub> was reduced to about 25% in the Δhsp90 strain as expected, no reduction in the
amount of TilS<sub>Ec</sub> was observed in the absence of Hsp90 (Figure 2C and D). Interestingly, similar results
were observed in E. coli WT or Δhsp90 strains (Figure 2E and F). These results show that in contrast to
TilS<sub>So</sub> at 34°C, TilS<sub>Ec</sub> 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<sub>So</sub> and TilS<sub>Ec</sub> did not significantly vary in S.
oneidensis and E. coli WT or Δhsp90 strains (Figure S2C-F). Note that in some experiments, TilS<sub>Ec</sub>
levels were increased in the absence of Hsp90, for reasons that remain unclear (Figure 2D, S2D and
S2F).

Altogether, these experiments show that in contrast to TilS<sub>So</sub>, TilS<sub>Ec</sub> does not interact with Hsp90 in
our experimental conditions, and its amount is not reduced in the absence of Hsp90, strongly
suggesting that TilS<sub>Ec</sub> is not an Hsp90 client, whereas TilS<sub>So</sub> is.
TilS identity determines the Hsp90-dependent growth of bacteria.

As proposed above, TilS<sub>Ec</sub> does not require the activity of the Hsp90 chaperone to be functional, unlike TilS<sub>So</sub>. 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<sub>Ec</sub> instead of TilS<sub>So</sub>. To test this hypothesis, *tilS*<sub>So</sub> was replaced by *tilS*<sub>Ec</sub> on the chromosome of the *S. oneidensis* MR1 WT and Δ*hsp90* strains using homologous recombination, leading to the strains Δ*tilS*<sub>So</sub> *tilS*<sub>Ec<sup>+</sup></sub> and Δ*hsp90*<sub>So</sub> Δ*tilS*<sub>So</sub> *tilS*<sub>Ec<sup>+</sup></sub>, respectively. We measured the growth of these strains as well as the WT and Δ*hsp90*<sub>So</sub> strains that were incubated at either 28°C (the non-stress temperature at which Hsp90<sub>So</sub> is dispensable) or 35°C (the heat stress temperature at which Hsp90<sub>So</sub> is required). We first observed that TilS<sub>Ec</sub> can functionally substitute for TilS<sub>So</sub> 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<sub>Ec</sub> instead of TilS<sub>So</sub> in the presence of Hsp90 (Δ*tilS*<sub>So</sub> *tilS*<sub>Ec<sup>+</sup></sub>) grew as the WT strain (**Figure 3B**). As expected, the absence of Hsp90 led to a strong growth defect at 35°C (Δ*hsp90*<sub>So</sub> vs WT), but not at 28°C (**Figure 3A and B**) [18]. Interestingly, replacing *tilS*<sub>So</sub> by *tilS*<sub>Ec</sub> in the *S. oneidensis* hsp90-knockout strain (Δ*hsp90*<sub>So</sub> Δ*tilS*<sub>So</sub> *tilS*<sub>Ec<sup>+</sup></sub>) dramatically improved growth of the Δ*hsp90*<sub>So</sub> strain at 35°C (**Figure 3B**). Similarly, the growth defect under heat stress of the Δ*hsp90*<sub>So</sub> strain was rescued by the production of TilS<sub>Ec</sub> instead of TilS<sub>So</sub> when bacterial growth was performed on LB-agar plates (**Figure S3B**). Additional growth temperatures (34°C and 36°C) were also tested. Although at 34°C, the presence of TilS<sub>Ec</sub> almost totally rescued the growth defect of the Δ*hsp90*<sub>So</sub> 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*<sub>So</sub>Δ*tilS*<sub>So</sub> *tilS*<sub>Ec<sup>+</sup></sub> at 35°C was fully rescued by the production of Hsp90 from a plasmid (**Figure S3E**).
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<sub>So</sub>* 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<sub>Ec</sub>* ) for *TilS<sub>So</sub>*. The *tilS* gene in *E. coli* MG1655 WT and *Δhsp90<sub>Ec</sub>* was replaced by *tilS<sub>So</sub>* by homologous recombination leading to strains *ΔtilS<sub>Ec</sub> tilS<sub>So</sub><sup>+</sup>* and *Δhsp90<sub>Ec</sub> ΔtilS<sub>Ec</sub> tilS<sub>So</sub><sup>+</sup>* , respectively. At 28°C, the four strains grew similarly (**Figure 3C**). However, at 35°C, the growth of *ΔtilS<sub>Ec</sub> tilS<sub>So</sub><sup>+</sup>* was delayed compared to WT, indicating that *TilS<sub>So</sub>* cannot fully compensate for the absence of *TilS<sub>Ec</sub>. Nevertheless, we found that deleting *hsp90* dramatically reduced the growth of the *E. coli* strain that produces *TilS<sub>So</sub>* instead of *TilS<sub>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*<sub>Ec*, a protein that does not need
*Hsp90* to be functional. Conversely, an *E. coli* strain that produces *TilS*<sub>So* requires *Hsp90* for growth at
35°C, a temperature at which the *TilS*<sub>So</sub> protein needs *Hsp90* to be protected.

The C-terminal domain of *TilS*<sub>So</sub> is responsible for the dependence on *Hsp90*.

We next wanted to identify the region of *TilS*<sub>So</sub> that confers dependence on *Hsp90*. To this end, we
constructed chimeras by swapping domains between *TilS*<sub>So</sub> and *TilS*<sub>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*<sub>So</sub> and the three other domains of
TilS<sub>Ec</sub>. The chimeras were produced from a pBad inducible promoter in the *S. oneidensis* WT and Δhsp90 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).

Although, as indicated above, the amount of TilS<sub>So</sub> is considerably reduced in the absence of Hsp90 (Figures 2C and 2D), we found that replacing the single C2 domain of TilS<sub>So</sub> with that of TilS<sub>Ec</sub> ("SSSE" chimera) was sufficient to stabilize TilS<sub>So</sub> in the absence of Hsp90. Exchanging the C1 and C2 domains (chimera "SSEE") or H, C1, and C2 domains (chimera "SEEE") of TilS<sub>So</sub> by the domains of TilS<sub>Ec</sub> also led to stabilization of the chimeras in the absence of Hsp90 (Figure 4B and C). Contrarily, although the amount of TilS<sub>Ec</sub> was not reduced in the absence of Hsp90 compared to WT (Figures 2C and 2D), substituting the C2 domain of TilS<sub>Ec</sub> by the one of TilS<sub>So</sub> (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<sub>So</sub> is responsible for the dependence on the Hsp90 chaperone. When Hsp90 does not protect it, this domain most likely directs TilS<sub>So</sub> to degradation. Indeed, we found that Hsp90, which does not interact with TilS<sub>Ec</sub> (Figure 2A and B) protects the "EEES" chimera, possessing only the C2 domain of TilS<sub>So</sub>, from degradation (Figure 4). Interestingly, the AlphaFold3 prediction of the binding between Hsp90<sub>S0</sub> and TilS<sub>So</sub> positions the two proteins in interaction via the C2 domain of TilS<sub>So</sub>, while no binding was predicted with TilS<sub>Ec</sub> (Figure S5).

## Identification of point mutations in the C2 domain of TilS<sub>So</sub> that modify Hsp90 dependence.

Based on our observations that TilS<sub>So</sub> is an obligate client of Hsp90, while TilS<sub>Ec</sub> 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<sub>Ec</sub> would not require Hsp90 for stability, while those that
are close to TilS<sub>So</sub> 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<sub>Ec</sub>, such as TilS
from *Salmonella Typhimurium*, 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<sub>Sx</sub>), a protein with over 90% identity to TilS<sub>So</sub>, did not behave like TilS<sub>So</sub>
with regard to Hsp90 dependence. Indeed, we found that the amount of TilS<sub>Sx</sub> was not reduced in the
absence of Hsp90<sub>So</sub>, as observed with TilS<sub>So</sub> (Figures 5A, 5B, 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<sub>So</sub> that is replaced by a proline residue in TilS<sub>Sx</sub> (Figure 5C). We
produced TilS<sub>So</sub>L340P variant to test whether this proline residue could be responsible for the
stabilization of TilS<sub>Sx</sub> in the absence of Hsp90. Interestingly, we found that, in contrast to the wild-type
protein, the amount of the mutant protein TilS<sub>So</sub>L340P was only slightly reduced in the absence of
Hsp90 (Figures 5A and 5B). Replacing L340 of TilS<sub>So</sub> by residues other than proline (serine or glycine)
(Figure S8A), or replacing the last 7 residues of TilS<sub>So</sub> by the 8 last ones of TilS<sub>Sx</sub> (Figure S8B) did not
lead to stabilization of TilS<sub>So</sub> in the absence of Hsp90. These results indicate that mutating the leucine
340 of TilS<sub>So</sub> into a proline is crucial to stabilize the protein in the absence of Hsp90, as naturally
found in TilS<sub>Sx</sub>.

We then wondered whether the stabilization of TilS<sub>So</sub> by the mutation L340P would allow bacteria to
grow with a lower dependence on Hsp90 under heat stress. The corresponding mutation in *tilS<sub>So</sub>* was
introduced in the chromosome of *S. oneidensis* WT and Δ*hsp90*. In the WT background, the strain
producing TilS<sub>So</sub>L340P instead of TilS<sub>So</sub> grew similarly to the WT strain at 28°C and 35°C (Figures 5D
and S8C-E). More importantly, production of TilS<sub>So</sub>L340P did partially rescue the growth defect of the
*S. oneidensis* Δ*hsp90* strain (Figure 5D and S8E), confirming that TilS<sub>So</sub>L340P can be functional without
the assistance of Hsp90.
We further characterized TilS<sub>Sx</sub> and TilS<sub>So</sub>L340P to investigate why they can work independently of Hsp90, as was observed with TilS<sub>Ec</sub>. The proteins were purified and size exclusion chromatography elution profiles of the two proteins were similar to TilS<sub>So</sub> (Figure S9A). Surprisingly, we found that TilS<sub>Sx</sub> and TilS<sub>So</sub>L340P behaved more like TilS<sub>So</sub> than TilS<sub>Ec</sub> with regard to intrinsic protein stability and interaction with Hsp90. Indeed, thermal shift assays indicated that the Tm of TilS<sub>Sx</sub> (37.0°C) and TilS<sub>So</sub>L340P (38.5°C) were closer to TilS<sub>So</sub> (38.2°C) than TilS<sub>Ec</sub> (46.4°C) (Figure 5E). Similarly, the trypsin digestion profiles of TilS<sub>Sx</sub> and TilS<sub>So</sub>L340P resembled those obtained with TilS<sub>So</sub> (Figure S9B compared to S1D). Finally, two-hybrid experiments showed that TilS<sub>Sx</sub> and TilS<sub>So</sub>L340P interact with Hsp90, similar to TilS<sub>So</sub>, but in contrast to TilS<sub>Ec</sub> (compare Figures 5F and 2A). This interaction was further confirmed by MST experiments, which indicated that both TilS<sub>Sx</sub> and TilS<sub>So</sub>L340P bind to Hsp90 (Figure S9C). However, the L340P mutation slightly reduced binding affinity, with the dissociation constant increasing from Kd=1.09 µM for TilS<sub>So</sub> (Fig. 2B) to Kd=20.35 µM for TilS<sub>So</sub>L340P (Fig. S9C).

Altogether, based on the TilS ortholog from *S. xiamenensis*, we identified a single mutation, L340P, in TilS<sub>So</sub> that switches the TilS protein from Hsp90-dependent (TilS<sub>So</sub> WT) to Hsp90-independent (TilS<sub>So</sub>L340P). Since TilS<sub>So</sub>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 *in vivo*. Our client model was the TilS protein, an essential enzyme responsible for modifying a specific 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<sub>So</sub> 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<sub>So</sub> and TilS<sub>Ec</sub> 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<sub>so</sub>, whose level was dramatically reduced under heat stress in the absence of Hsp90, the level of TilS<sub>Ec</sub> did not differ significantly between WT and Δ*hsp90* strains (Fig 2C-F). In addition, TilS<sub>Ec</sub> did not interact with Hsp90, whereas TilS<sub>so</sub> did (Figure 2A-B), suggesting that TilS<sub>Ec</sub>, unlike TilS<sub>so</sub>, 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<sub>so</sub> that is responsible for TilS degradation and Hsp90 dependence (Figure 4). Indeed, the amount of the chimera consisting of TilS<sub>Ec</sub> with only the C2 domain of TilS<sub>so</sub> (named “EEES”) was strongly reduced in the Δ*hsp90* strain, whereas TilS<sub>so</sub> was stabilized by exchanging its C2 domain with that of TilS<sub>Ec</sub> (named “SSSE”). In addition, we found that replacement of residue L340 of TilS<sub>so</sub> 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<sub>so</sub>. 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<sub>so</sub> degradation, rather than the nature of the leucine residue per se. Taken together, these experiments indicate that the C2 domain of TilS<sub>so</sub> is key to triggering TilS<sub>so</sub> 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<sub>so</sub>, thereby limiting the accessibility of the HslUV protease to an as yet unidentified degron probably located in the C2 domain of TilS<sub>so</sub>. After Hsp90-assisted folding of TilS<sub>so</sub>, 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<sub>so</sub> and the C2 domain of TilS<sub>so</sub>. In these predictions, binding to Hsp90 occurs through the cleft located between the two monomers of Hsp90<sub>so</sub>, which is known to be important for binding to multiple clients [29]. Interestingly, mutations in this region of Hsp90 (W476R and L563A, Hsp90<sub>so</sub> numbering) have been shown to prevent binding to clients, including TilS<sub>so</sub> [18,29]. Alternative models obtained
with AlphaFold 3 always position the C2 domain of TilS<sub>SO</sub> in contact with Hsp90<sub>SO</sub>, but the other domains of TilS<sub>SO</sub> 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<sub>SO</sub> 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<sub>SO</sub>, in the vicinity of residue L340, but this loop is shorter in the X-ray crystal structure of TilS<sub>EC</sub> (PDB 1NI5) (Figure S10B). While this may seem appealing, it must be treated with high caution since the structures of TilS<sub>SO</sub> and TilS<sub>EX</sub> 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<sub>SO</sub> instead of TilS<sub>EC</sub> (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<sub>SO</sub> with TilS<sub>EC</sub> (Figure 3B), or by introducing the single mutation L340P in TilS<sub>SO</sub> (Figure 5D). However, the restoration of the growth phenotype was almost lost when the temperature increased by only 1°C, although we know that TilS<sub>EC</sub> 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<sub>SO</sub> 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<sub>SO</sub> by producing TilS<sub>EC</sub> 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<sub>so</sub> 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<sub>Ec</sub> and the TilS proteins from the *Shewanella* genus (TilS<sub>so</sub> and TilS<sub>sx</sub>) fell
into two different categories. Indeed, thermal shift assays on purified proteins indicate that TilS<sub>so</sub> and
TilS<sub>sx</sub> are less stable than TilS<sub>Ec</sub>, with a shift higher than 8°C in the melting temperatures of the
proteins from the two categories (**Figure 1D and 5E**). This result correlates with the fact that Hsp90
interacts with TilS<sub>so</sub> and TilS<sub>sx</sub>, but not with TilS<sub>Ec</sub> (**Figure 2A-B, 5F, and S9C**). These observations are in
agreement with a global study demonstrating that the intrinsically unstable state of eukaryotic
kinases leads to their 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<sub>so</sub> was degraded by HslUV in the absence of Hsp90 [19], whereas TilS<sub>sx</sub> was not degraded (**Figure 5A, 5B**). Thus, despite conserving their ability to interact with Hsp90,
these two proteins have evolved to be either protease sensitive (TilS<sub>SO</sub>) or resistant (TilS<sub>SX</sub>). This
difference may be due to a single mutation, since mutating L340 to P in TilS<sub>SO</sub> is sufficient to stabilize it
in the absence of Hsp90. Therefore, TilS<sub>SO</sub>L340P and TilS<sub>SX</sub> 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<sub>SX</sub> 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.
To assess the distribution and evolutionary relationships of TilS, we constructed a taxonomic tree
based on full-length TilS (Figure 1C) and a phylogenetic tree focusing on its C2 domain (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<sub>SO</sub> required Hsp90 for protection against protease degradation, while TilS<sub>SX</sub>
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. This has also been interestingly shown in
humans, where disease-associated mutant proteins with impaired folding interact with Hsp90, which
in turn buffers the phenotypic impact of these mutations [42].

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*<sub>So</sub> may be better adapted than *TilS*<sub>Ec*</sub> 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*<sub>So</sub> 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 [43] and the reference strain for *E. coli* is MG1655 (K12) [44]. 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*<sub>So</sub> or *tilS*<sub>Ec*</sub> replaced by *tilS*<sub>Ec</sub> or *tilS*<sub>So</sub>, 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*<sub>So</sub>-*TilS*<sub>Ec*</sub> 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 [45]. 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 [46], 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 [47]. Evolutionary history was inferred by using the Maximum Likelihood method and JTT matrix-based model with MEGA X [48]. 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<sub>S0</sub> and TilS<sub>Sx</sub> sequences were aligned using "Multalin" tool (http://multalin.toulouse.inra.fr/multalin/). The ESPript program

(https://escript.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<sub>so</sub> in PBS-Tween 0.05 %, pH 7.3. Final concentrations in capillary are 85 nM TilS and 50 µM Hsp90<sub>so</sub> 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 MgSO4, 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 [49].

**Western blot analysis to evaluate TilS amount.** TilS amount was determined by Western blots. The *ptilS*-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 OD₆₀₀ = 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<sub>So</sub> or anti-Hsp90<sub>Ec</sub> antibody. As neutral loading control, a non-specific band detected by the anti-AtcJ antibody was used [49].

**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 OD₆₀₀ = 0.1 in LB and incubated at 28°C until late exponential phase. For growth on liquid media, cells were diluted to OD₆₀₀ = 0.0005 in LB. Growth was measured in a microplate reader at indicated temperatures.

**Statistical analysis.** Statistical analysis were performed with GraphPad Prism software (version 8) as indicated using t-tests or one way ANOVA tests. Student t-tests were performed assuming Gaussian distribution of residuals and equal SDs. One-way ANOVA tests were performed with Tukey's multiple
comparisons test assuming Gaussian distribution of residuals. Results were shown as significant
(****P ≤ 0.0001, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05) or not significant (ns, P > 0.05).

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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## Figure Legends

**Figure 1: Generalities on the TilS protein.** (A) Simplified hypothetical model of TilS<sub>so</sub> requirement for Hsp90. In *S. oneidensis*, Hsp90 in collaboration with Hsp70/DnaK, is essential to promote TilS<sub>so</sub> folding and thereby prevent its degradation. (B) Structure of TilS<sub>Ec</sub> (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<sub>so</sub> and TilS<sub>Ec</sub> by Thermal Shift Assay. TilS<sub>so</sub> or TilS<sub>Ec</sub> were labeled with SYPRO Orange and incubated with increasing temperatures. The protein unfolding curves of nine replicates from three independent experiments are shown as mean ± SEM of derivatives of the fluorescence. The table indicates the melting temperatures as mean ± SEM.

**Figure 2: TilS<sub>Ec</sub> is not a client of Hsp90.** (A) Bacterial two-hybrid experiments showing the interaction between TilS and Hsp90. Hsp90<sub>so</sub> and Hsp90<sub>Ec</sub> were fused to the T25 domain of *B. pertussis* adenylate cyclase, and TilS<sub>so</sub> and TilS<sub>Ec</sub> 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<sub>Ec</sub> or Hsp90<sub>so</sub> to monitor the interaction of Hsp90 dimer. Data from at least three replicates are shown as mean ± SEM. Results of one-way ANOVA indicate whether the differences (versus T18-TilS<sub>so</sub> – T25) are significant (****P ≤ 0.0001, ***P ≤ 0.001) or not (ns, P > 0.05). (B) Microscale thermophoresis experiment to determine the interaction between Hsp90 and TilS<sub>so</sub> or
TilS<sub>Ec</sub>, 85 nM of TilS labeled with RED-tris-NTA 2nd generation dye (NanoTemper) was added to serial 2-times dilutions of Hsp90<sub>So</sub> up to 50 µM. Fluorescence at 670 nm was measured, and values at 2.5 s were used to determine K<sub>d</sub>. The curves shown are representative of three independent experiments for TilS<sub>So</sub> and two for TilS<sub>Ec</sub>. The mean of the K<sub>d</sub> with SEM from three independent experiments is indicated. (C) Western blot showing the amount of TilS<sub>So</sub> or TilS<sub>Ec</sub> in *S. oneidensis*. The strains WT or Δhsp90 with plasmids allowing the production of TilS<sub>So</sub> or TilS<sub>Ec</sub> 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<sub>So</sub> 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 average of the amount of TilS<sub>So</sub> or TilS<sub>Ec</sub> in WT strain was set to 1, and each WT is compared to Δhsp90 producing the corresponding TilS<sub>So</sub> or TilS<sub>Ec</sub>, respectively. Data from five replicates are shown as mean ± SEM. Distinct symbols are used to represent data from each replicate. (E) Western blot showing the amount of TilS<sub>So</sub> or TilS<sub>Ec</sub> in *E. coli* in the same conditions as in C. Anti-Hsp90<sub>Ec</sub> was used as control. The band above Hsp90<sub>Ec</sub> is a contaminant detected by the anti-Hsp90<sub>Ec</sub> antibody. In C and E, the Western blots shown are representative of at least five independent experiments. (F) Quantification as explained in D of the anti-His Western blot shown in E. Data from seven replicates are shown as mean ± SEM. Distinct symbols are used to represent data from each replicate. In D and F, results of t-tests indicate whether the differences are significant (*P* ≤ 0.05, **P* ≤ 0.01) or not (ns, *P* > 0.05).

<caption>**Figure 3: The identity of TilS dictates the dependence on Hsp90 in S. oneidensis and in E. coli.** (A)</caption>

Bacterial growth of *S. oneidensis* strains carrying *tilS* from *E. coli*. The strains *S. oneidensis* MR-1 wild-type (WT), deleted of *hsp90* (Δ*hsp90*) with natural *TilS* or replaced by *TilS* from *E. coli* (Δ*TilS*<sub>So</sub> *tilS*<sub>Ec</sub><sup>+</sup>) 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* (Δ*hsp90*) with natural *TilS* or replaced by *TilS* from *S. oneidensis* (Δ*tilS*<sub>Ec</sub> *tilS*<sub>So</sub><sup>+</sup>) 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 independent experiments are shown as mean ± SEM.

**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<sub>Ec</sub>-TilS<sub>So</sub> 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 five 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 average of the amount of each chimera in WT strain was set to 1, and each WT is compared to Δ*hsp90* producing the corresponding chimera, respectively. Data from at least five replicates are shown as mean ± SEM. Distinct symbols are used to represent data from each replicate. Results of t-tests indicate whether the differences are significant (*P* ≤ 0.05, ***P* ≤ 0.001, ****P* ≤ 0.0001) or not (ns, *P* > 0.05).

**Figure 5: A single point mutation in TilS<sub>So</sub> decreases Hsp90 dependence.**

(A) Western blot showing the amount of TilS<sub>So</sub> WT or mutants, or TilS<sub>Sx</sub> in *S. oneidensis* WT or Δ*hsp90* strains. The strains WT or Δ*hsp90* with plasmid allowing the production of TilS<sub>So</sub> WT or mutants or TilS<sub>Sx</sub> with hexahistidine tag were grown at 34°C and 0.02% arabinose was added. Two hours later,
samples were analyzed by Western blot using anti-His antibody. As a control, anti-Hsp90 antibody was used. The Western blot is representative of six independent experiments. (B) Graph showing the amount of TilS<sub>Sx</sub>, and TilS<sub>So</sub> WT or mutants from the Western blot in A. This graph shows the quantification of six independent Western blots. The peaks corresponding to the pixels of each band were quantified using ImageJ software. The average of the amount of TilS<sub>So</sub> or TilS<sub>Sx</sub> in WT strain was set to 1, and each WT is compared to Δ*hsp90* producing the corresponding TilS<sub>So</sub> or TilS<sub>Sx</sub> respectively. Data from six replicates are shown as mean ± SEM. Distinct symbols are used to represent data from each replicate. The results of t-tests indicate whether the differences are significant (***P ≤ 0.001) or not (ns, *P* > 0.05). (C) Alignment of the sequences of TilS<sub>So</sub> and TilS from *S. xiamenensis* (TilS<sub>Sx</sub>), from residue 300 (TilS<sub>So</sub> numbering), using the multiple sequence alignment tool «Multalin» (http://multalin.toulouse.inra.fr/multalin/). TheESPrigt program

(https://escript Ibcp.fr/ESPrigt/ESPrigt/) was used to display the alignment. The red arrows indicate
the residues that were mutated. (D) Bacterial growth in liquid media of the *S. oneidensis* strains MR-1
wild-type (WT) or deleted of *hsp90* (Δ*hsp90*) with the *tiIS* gene WT or coding the L340P mutant. 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 ± SEM. (E) Determination of the melting temperatures of TilS<sub>So</sub> L340P
and TilS<sub>Sx</sub> 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 ± SEM of derivatives of the fluorescence. The table indicates the melting temperatures as
mean ± SEM. Unfolding curves of TilS<sub>So</sub> WT and TilS<sub>Ec</sub> presented in Figure 1D are shown for
comparison. (F) Bacterial two-hybrid experiments showing interaction of TilS<sub>So</sub> WT or mutants, or
TilS<sub>Sx</sub> with Hsp90<sub>So</sub>. Hsp90<sub>So</sub> was fused to the T25 domain of *B. pertussis* adenylate cyclase and the
five TilS proteins were fused to the T18 domain. Bth101 Δ*hsp90* 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<sub>so</sub> to monitor the interaction of Hsp90 dimer. Data from at least three replicates are
shown as mean ± SEM. The results of one-way ANOVA indicate whether the differences are
significant (*P* ≤ 0.05) or not (ns, *P* > 0.05).
<figure><img src="image_2.png" /><figcaption>Figure 1</figcaption></figure>

<table><thead><tr><td>Protein</td><td>Tm (°C)</td><td>SEM</td></tr></thead><tbody><tr><td>TilS<sub>So</sub></td><td>38.2</td><td>± 0.1</td></tr><tr><td>TilS<sub>Ec</sub></td><td>46.4</td><td>± 0.1</td></tr></tbody></table>
<figure><img src="image_3.png" /><figcaption>Figure 2</figcaption></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>
<figure><img src="image_9.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>Figure 4</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>Figure 5</figcaption></figure>
Subtle variations in a client protein determine bacterial Hsp90 dependence

Marie Corteggiani<sup>a</sup>, Amine Ali Chaouche<sup>a</sup>, Miha Bahun<sup>b</sup>, Flora A. Honoré<sup>a</sup>, Deborah Byrne<sup>c</sup>, Sébastien Dementin<sup>a</sup>, Mathieu E. Rebeaud<sup>d</sup>, Olivier Genest<sup>a, *</sup>

<sup>a</sup>Aix-Marseille Univ, CNRS, BIP UMR 7281, IMM, 31 Chemin Joseph Aiguier, 13402 Marseille, France.

<sup>b</sup>Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1000, Ljubljana, Slovenia.

<sup>c</sup>Aix-Marseille Univ, CNRS, IMM-FR3479, 31 Chemin Joseph Aiguier, 13402, Marseille, France.

<sup>d</sup>Institute of Physics, School of Basic Sciences, École Polytechnique Fédérale de Lausanne - EPFL, Lausanne, Switzerland.

This supplementary document includes:

- Supplementary Results

- Supplementary Materials and Methods

- Supplementary Tables S1 to S3

- Supplementary References

- Supplementary Figures 1 to 10, and Legends
**<u>Supplementary Results</u>**

Hsp90 dependence for TilS protection is not an evolutionary conserved process.

Since TilS<sub>Ec</sub> is not dependent on Hsp90, but TilS<sub>So</sub> is, we hypothesized that we could link the evolution
of TilS to its dependence on Hsp90. Based on TilS sequences, we built a phylogenetic tree with the
sequence of the C2 domains of several TilS orthologs, including TilS<sub>Ec</sub> and TilS<sub>So</sub> (<strong>Figure S6</strong>). We
expected that the TilS with C2 domains close to TilS<sub>So</sub> would be degraded in <em>S. oneidensis</em> Δhsp90,
whereas TilS with C2 sequences close to TilS<sub>Ec</sub> would be stable even in the absence of Hsp90. The C2
of TilS<sub>So</sub> is in the same subgroup as two other <em>Shewanella</em> species: <em>S. xiamenensis</em> and <em>S. woodyi</em>. The
C2 of TilS<sub>Ec</sub> and TilS from <em>Yersinia pestis</em>, <em>Salmonella enterica</em> serovar Typhimurium, and <em>Vibrio cholera</em>
also forms a subgroup.

We cloned several *tilS* genes representative of the different groups and subgroups of orthologous TilS
proteins classified by their C2 including *Shewanella woodyi*, *Shewanella xiamenensis*, *Vibrio cholera*,
and *Salmonella enterica* serovar Typhimurium. The resulting plasmids were introduced into *S. oneidensis* WT or Δ*hsp90* strains, and the resulting strains were grown under heat stress (34°C) or permissive (28°C) temperatures. The amount of TilS was determined by Western blot (Figure S7 A-B) and quantification of the bands (Figure S7 C-D).

As expected, the TilS of *V. cholerae* and *Y. pestis* belonging to the TilS<sub>Ec</sub> subgroup were not degraded
in the Δ*hsp90* strain at both temperatures suggesting that these two proteins are not dependent on
the chaperone Hsp90. Surprisingly, TilS of *S. enterica* is less abundant in the absence of the chaperone
both at 34°C and 28°C.

In contrast to our expectations, TilS of *S. xiamenensis* did not seem to be degraded in the absence of Hsp90. For *S. woodyi*, the variability between replicates did not allow us to draw a clear conclusion on the dependence of its TilS on Hsp90.

Altogether these results suggest that it is not possible to draw a correlation between the amino acid
sequences of the C2 domain of TilS and the dependence on the chaperone Hsp90. Surprisingly, TilS of
*S. xiamenensis* which is not degraded in the absence of the chaperone, shares 92% identity with TilS<sub>So</sub>.
**Strain constructions.** The *S. oneidensis* WT or *Δhsp90* strains with natural *tilS*<sub>So</sub> replaced by *tilS*<sub>Ec* or *tilS*<sub>So</sub>(L340P), were constructed using homologous recombination as previously described with some modifications [1]. For the replacement of *tilS*<sub>So</sub> by *tilS*<sub>Ec* gene, *tilS*<sub>Ec* was amplified by PCR from *E. coli* MG1655 chromosome and 500 bp long flanking sequences upstream and downstream *tilS*<sub>So</sub> locus were amplified from *S. oneidensis* MR1-R chromosome. For the *tilS*<sub>So</sub>(L340P) mutation, two flanking sequences of 500 bp upstream and downstream of the codon of interest were amplified. The resulting constructions were cloned in the pKNG101 suicide vector using NEBuilder® HiFi DNA Assembly kit (NEB) according to the manufacturer protocol, and subsequently introduced in *E. coli* CC118λpir strain. The resulting plasmids were introduced in *S. oneidensis* by conjugation. After integration into the chromosome, plasmids were removed using sucrose. For mutations on the chromosome of *E. coli*, the *tilS*<sub>So</sub> gene with 600 bp flanking sequences from upstream and downstream *tilS*<sub>Ec* was amplified by PCR and cloned in the pKO3 suicide vector using NEBuilder® HiFi DNA Assembly kit (NEB) according to the manufacturer protocol. The resulting plasmid was transformed in *E. coli* MG1655 strain. After integration into the chromosome at 42°C because of thermosensitive replication of the pKO3, the plasmid was removed using sucrose. All the constructions were first checked by PCR with specific primers and further confirmed by sequencing.

**Plasmid constructions.** The pBad33 plasmids carrying *tilS* genes were obtained by PCR amplification of the *tilS*<sub>Ec* and/ or *tilS*<sub>So</sub> genes from the chromosomes of *E. coli* MG1655 and *S. oneidensis* MR1-R, respectively. For the chimeric constructions, the sequences amplified from *tilS*<sub>Ec* or *tilS*<sub>So</sub> are indicated in Table S3. Orthologous *tilS* other than *tilS*<sub>Ec* or *tilS*<sub>So</sub> were amplified from genes synthesized by the Genecust company. The resulting fragments were cloned into the pBad33 vector digested with Xmal and Xbal restriction enzymes using NEBuilder Assembly kit (NEB).
The pT18 plasmids carrying *tilS* genes were obtained as described for the pBad33 plasmids above. The pT18 plasmid was digested with EcoRI and Xhol enzymes. The pET24b plasmids carrying *tilS* were obtained as described above. The pET24b plasmids were digested with Ndel and Sacl enzymes.

The pBad33 plasmids carrying *hsp90<sub>So</sub>* or *hsp90<sub>Ec</sub>* under the control of their own promoter were obtained by the amplification of *hsp90<sub>So</sub>* or *hsp90<sub>Ec</sub>* including their respective 344 bp upstream the ATG codon. *hsp90<sub>So</sub>* was inserted into pBad33 digested with Xmal and Xbal enzymes and *hsp90<sub>Ec</sub>* was inserted into pBad33 digested with KpnI using NEBuilder Assembly kit (NEB). All plasmid constructions were verified by sequencing.

**Growth of S. oneidensis or E. coli.** These experiments were performed as previously described with modifications [2]. After overnight precultures at 28°C in LB medium, the *S. oneidensis* or *E. coli* strains were inoculated to OD<sub>600</sub> = 0.1 in LB and incubated at 28°C until late exponential phase. For strains carrying pBad33 plasmid, chloramphenicol was added. For growth on liquid media, cells were diluted to OD<sub>600</sub> = 0.0005 in LB. Growth was measured in a microplate reader at indicated temperatures. For growth on solid media, cells were diluted to OD<sub>600</sub> = 1 and 3 µL of 10x serial dilutions were spotted on LB agar plates. Plates were incubated at 28°C overnight and at 35°C for 24 h.

**Proteins.** To purify *TilS<sub>So</sub>*, *TilS<sub>Ec</sub>*, *TilS<sub>Sx</sub>* and *TilS<sub>So</sub>*L340P, the corresponding pET24b-*tilS* plasmids were introduced into the BL21 (DE3) strain by transformation. The resulting strains were grown in LB at 37°C. At OD<sub>600</sub> = 0.8, 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added, and the strains were grown overnight at 16°C, cells were collected by centrifugation, lysed by French Press and centrifuged at 13 000 rpm, 4°C, 15 min. After a second centrifugation step at 45,000 rpm, 4°C, 45 min, the supernatant was loaded onto a HisTrap HP 1mL column (Cytiva), and purification was performed as described by the manufacturer. To improve purification, *TilS* extract from HisTrap column were loaded onto Gel Filtration Superdex® 16 600 Hiload S200 (Cytiva). *Hsp90<sub>So</sub>* purification was performed as
described previously [2,3]. Purified proteins were stored at -80°C in 50 mM Tris-HCl pH=7.5, 100 mM KCl, 1 mM DTT, and 10% glycerol. Concentrations are given as monomeric proteins.

**Gel Filtration analysis.** 8 µM of proteins in 300 µL of 25mM Tris HCl pH 7.5, 50 mM KCl, 1 mM DTT and 10% glycerol were loaded onto gel filtration Superdex® 200 Increase 10 300 GL column (Cytiva).

**Limited proteolysis experiments.** TilsS<sub>So</sub>, TilsS<sub>Ec</sub>, TilsS<sub>Sx</sub> or TilsS<sub>So</sub>L340P (15 µg) was mixed (50 µL final volume) with trypsin (0.25 µg) in 50 mM Tris-HCl buffer pH 7.5 containing 100 mM KCl, 10% glycerol and 1 mM DTT and incubated at 37°C. At the indicated times, 10 µL was removed, mixed with 5 µL of loading buffer, denatured for 5 min at 95°C and analyzed by SDS-PAGE. Trypsin was purchased from Sigma.
## Supplementary Tables

<table><caption>Table S1: Strains used in this study</caption><thead><tr><th>STRAINS</th><th>CHARACTERISTICS</th><th>SOURCE</th></tr></thead><tbody><tr><td><strong><em>S. oneidensis</em> strains</strong></td><td></td><td></td></tr><tr><td>MR1-R</td><td><em>S. oneidensis</em> MR1 strain ATCC 700550, Rifampicine resistance.</td><td>[5]</td></tr><tr><td>MR1-R Δ<i>hsp90</i><sub>So</sub></td><td>MR1-R with deletion of <i>hsp90</i> gene.</td><td>[3]</td></tr><tr><td>MR1-R Δ<i>tilS</i><sub>So</sub> <i>tilS</i><sub>Ec</sub><sup>+</sup></td><td>MR1-R with substitution of the whole <i>tilS</i><sub>So</sub> gene by <i>tilS</i><sub>Ec</sub>.</td><td>This study</td></tr><tr><td>MR1-R Δ<i>hsp90</i><sub>So</sub> Δ<i>tilS</i><sub>So</sub> <i>tilS</i><sub>Ec</sub><sup>+</sup></td><td>MR1-R with substitution of the whole <i>tilS</i><sub>So</sub> gene by <i>tilS</i><sub>Ec</sub> in Δ<i>hsp90</i><sub>So</sub> background.</td><td>This study</td></tr><tr><td>MR1-R <i>tilS</i><sub>So</sub>(L340P)</td><td>MR1-R with mutations in <i>tilS</i> gene leading to L340P substitutions in <i>TilS</i><sub>So</sub>.</td><td>This study</td></tr><tr><td>MR1-R Δ<i>hsp90</i><sub>So</sub> <i>tilS</i><sub>So</sub>(L340P)</td><td>MR1-R with mutations in <i>tilS</i> gene leading to L340P substitutions in <i>TilS</i><sub>So</sub>, in Δ<i>hsp90</i><sub>So</sub> background.</td><td>This study</td></tr><tr><td><strong><em>E. coli</em> strains</strong></td><td></td><td></td></tr><tr><td>Bth101 Δ<i>hsp90</i><sub>Ec</sub></td><td><i>cya</i>-, deleted of <i>hsp90</i>.</td><td>[6]</td></tr><tr><td>MG1655</td><td><em>E. coli</em> MG1655 strain.</td><td>[7]</td></tr><tr><td>MG1655 Δ<i>hsp90</i><sub>Ec</sub></td><td>MG1655 strain with deletion of <i>hsp90</i> gene.</td><td>[8]</td></tr><tr><td>MG1655 Δ<i>tilS</i><sub>Ec</sub> <i>tilS</i><sub>So</sub><sup>+</sup></td><td>MG1655 with substitution of the whole <i>tilS</i><sub>Ec</sub> gene by <i>tilS</i><sub>So</sub>.</td><td>This study</td></tr><tr><td>MG1655 Δ<i>hsp90</i><sub>Ec</sub> Δ<i>tilS</i><sub>Ec</sub> <i>tilS</i><sub>So</sub><sup>+</sup></td><td>MG1655 with substitution of the whole <i>tilS</i><sub>Ec</sub> gene by <i>tilS</i><sub>So</sub> in Δ<i>hsp90</i><sub>Ec</sub> background.</td><td>This study</td></tr><tr><td>BI21(DE3)</td><td>Used for protein overproduction. Codes the T7 RNA polymerase.</td><td>Novagen</td></tr><tr><td>1047 /pRK2013</td><td><em>E. coli</em> "helper" strain for conjugation.</td><td>[9]</td></tr><tr><td>CC118λ<i>pir</i></td><td><i>λpir</i> lysogens of <em>E. coli</em> CC118. Used for pKNG101 plasmid replication.</td><td>[10]</td></tr></tbody></table>
<table><caption>Table S2: Plasmids used in this study</caption><thead><tr><th>PLASMIDS</th><th>CHARACTERISTICS</th><th>SOURCE</th></tr></thead><tbody><tr><td>Plasmids used for measuring TilS amount</td><td></td><td></td></tr><tr><td>pBad33</td><td>pBad33 plasmid. Arabinose inducible. Chloramphenicol resistance.</td><td>[11]</td></tr><tr><td>ptilS<sub>So</sub></td><td>pBad33 plasmid carrying tilS<sub>So</sub> gene and a sequence coding six histidines (6His) tag. Production of a fusion TilS protein with 6His tag in N-terminus.</td><td>[3]</td></tr><tr><td>ptilS<sub>Ec</sub></td><td>pBad33 plasmid carrying tilS<sub>Ec</sub> gene and a sequence coding six histidines (6His) tag. Production of a fusion TilS protein with 6His tag in N-terminus.</td><td>This study</td></tr><tr><td>ptilS<sub>EEES</sub></td><td>pBad33 plasmid carrying a chimeric tilS gene and a sequence coding six histidines (6His) tag. Production of the TilS chimera EEES protein fused with 6His tag in N-terminus.</td><td>This study</td></tr><tr><td>ptilS<sub>EESS</sub></td><td>pBad33 plasmid carrying a chimeric tilS gene and a sequence coding six histidines (6His) tag. Production of the TilS chimera EESS protein fused with 6His tag in N-terminus.</td><td>This study</td></tr><tr><td>ptilS<sub>ESSS</sub></td><td>pBad33 plasmid carrying a chimeric tilS gene and a sequence coding six histidines (6His) tag. Production of the TilS chimera ESSS protein fused with 6His tag in N-terminus.</td><td>This study</td></tr><tr><td>ptilS<sub>SSSE</sub></td><td>pBad33 plasmid carrying a chimeric tilS gene and a sequence coding six histidines (6His) tag. Production of the TilS chimera SSSE protein fused with 6His tag in N-terminus.</td><td>This study</td></tr><tr><td>ptilS<sub>SSEE</sub></td><td>pBad33 plasmid carrying a chimeric tilS gene and a sequence coding six histidines (6His) tag. Production of the TilS chimera SSEE protein fused with 6His tag in N-terminus.</td><td>This study</td></tr><tr><td>ptilS<sub>SEEE</sub></td><td>pBad33 plasmid carrying a chimeric tilS gene and a sequence coding six histidines (6His) tag. Production of the TilS chimera SEEE protein fused with 6His tag in N-terminus.</td><td>This study</td></tr><tr><td>ptilS<sub>St</sub></td><td>pBad33 plasmid carrying tilS<sub>St</sub> gene of Salmonella enterica serovar Typhimurium and a sequence coding six histidines (6His) tag. Production of a fusion TilS protein with 6His tag in N-terminus.</td><td>This study</td></tr><tr><td>ptilS<sub>Sw</sub></td><td>pBad33 plasmid carrying tilS<sub>Sw</sub> gene of Shewanella woodyi and a sequence coding six histidines (6His) tag. Production of a fusion TilS protein with 6His tag in N-terminus.</td><td>This study</td></tr><tr><td>ptilS<sub>Sx</sub></td><td>pBad33 plasmid carrying tilS<sub>Sx</sub> gene of Shewanella xiamenensis and a sequence coding six histidines (6His) tag. Production of a fusion TilS protein with 6His tag in N-terminus.</td><td>This study</td></tr></tbody></table>
<table><tr><td>ptilS<sub>Yp</sub></td><td>pBad33 plasmid carrying <i>tilS<sub>Yp</sub></i> gene of <i>Yersinia pestis</i> and a sequence coding six histidines (6His) tag. Production of a fusion TilS protein with 6His tag in N-terminus.</td><td>This study</td></tr><tr><td>ptilS<sub>Vc</sub></td><td>pBad33 plasmid carrying <i>tilS<sub>Vc</sub></i> gene of <i>Vibrio cholerae</i> and a sequence coding six histidines (6His) tag. Production of a fusion TilS protein with 6His tag in N-terminus.</td><td>This study</td></tr><tr><td>ptilS<sub>So</sub>(L340P)</td><td>pBad33 plasmid carrying <i>tilS<sub>So</sub></i> mutant leading to L340P substitution in <i>TilS<sub>So</sub></i> and a sequence coding six histidines (6His) tag. Production of a fusion TilS protein with 6His tag in N-terminus.</td><td>This study</td></tr><tr><td>ptilS<sub>So</sub>(L340P-A344E)</td><td>pBad33 plasmid carrying <i>tilS<sub>So</sub></i> mutant leading to L340P-A344E substitutions in <i>TilS<sub>So</sub></i> and a sequence coding six histidines (6His) tag. Production of a fusion TilS protein with 6His tag in N-terminus.</td><td>This study</td></tr><tr><td>ptilS<sub>So</sub>(L340S)</td><td>pBad33 plasmid carrying <i>tilS<sub>So</sub></i> mutant leading to L340S substitution in <i>TilS<sub>So</sub></i> and a sequence coding six histidines (6His) tag. Production of a fusion TilS protein with 6His tag in N-terminus.</td><td>This study</td></tr><tr><td>ptilS<sub>So</sub>(L340G)</td><td>pBad33 plasmid carrying <i>tilS<sub>So</sub></i> mutant leading to L340G substitution in <i>TilS<sub>So</sub></i> and a sequence coding six histidines (6His) tag. Production of a fusion TilS protein with 6His tag in N-terminus.</td><td>This study</td></tr><tr><td>ptilS<sub>So</sub> (8-Ct<sub>Sx</sub>)</td><td>pBad33 plasmid carrying <i>tilS<sub>So</sub></i> mutant leading to replacement of 7 C-terminus last residues of <i>TilS<sub>So</sub></i> by 8 C-terminus last residues of <i>TilS<sub>Sx</sub></i> and a sequence coding six histidines (6His) tag. Production of a fusion TilS protein with 6His tag in N-terminus.</td><td>This study</td></tr></table>

<table><thead><tr><th colspan="3">Plasmids used for two-hybrid experiments</th></tr></thead><tbody><tr><td>pT18</td><td>pUT18C-linker vector. Carries the gene coding T18 adenylate cyclase domain of <i>B. pertussis</i>. IPTG inducible. Ampicillin resistance.</td><td>[12]</td></tr><tr><td>pT18-hsp90<sub>So</sub></td><td>pUT18C-linker plasmid carrying hsp90So gene.</td><td>[3]</td></tr><tr><td>pT18-tilS<sub>So</sub></td><td>pUT18C-linker plasmid carrying <i>tilS<sub>So</sub></i> gene.</td><td>[3]</td></tr><tr><td>pT18-tilS<sub>Ec</sub></td><td>pUT18C-linker plasmid carrying <i>tilS<sub>Ec</sub></i> gene.</td><td>This study</td></tr><tr><td>pT18-tilS<sub>Sx</sub></td><td>pUT18C-linker plasmid carrying <i>tilS<sub>Sx</sub></i> gene.</td><td>This study</td></tr><tr><td>pT18-tilS<sub>So</sub>(L340P)</td><td>pUT18C-linker plasmid carrying <i>tilS<sub>So</sub></i> mutant leading to L340P substitution in <i>TilS<sub>So</sub></i>.</td><td>This study</td></tr><tr><td>pT18-tilS<sub>So</sub>(L340P-A344E)</td><td>pUT18C-linker plasmid carrying <i>tilS<sub>So</sub></i> mutant leading to L340P-A344E substitutions in <i>TilS<sub>So</sub></i>.</td><td>This study</td></tr><tr><td>pT18-tilS<sub>So</sub>(L340S)</td><td>pUT18C-linker plasmid carrying <i>tilS<sub>So</sub></i> mutant leading to L340S substitution in <i>TilS<sub>So</sub></i>.</td><td>This study</td></tr><tr><td>pT25</td><td>pKT25-linker vector. Carries the gene coding T25 adenylate cyclase domain of <i>B. pertussis</i>. IPTG inducible. Kanamycin resistance.</td><td>[12]</td></tr><tr><td>pT25-hsp90<sub>So</sub></td><td>pKT25-linker plasmid carrying <i>hsp90<sub>So</sub></i> gene.</td><td>[3]</td></tr><tr><td>pT25-hsp90<sub>Ec</sub></td><td>pKT25-linker plasmid carrying <i>hsp90<sub>Ec</sub></i> gene.</td><td>[13]</td></tr></tbody></table>
<table><thead><tr><th colspan="3">Plasmids for complementation experiments</th></tr></thead><tbody><tr><td>phsp90<sub>So</sub> (own promoter)</td><td>pBad33 plasmid allowing the expression of hsp90<sub>So</sub> under the control of its own promoter.</td><td>This study</td></tr><tr><td>phsp90<sub>Ec</sub> (own promoter)</td><td>pBad33 plasmid allowing the expression of hsp90<sub>Ec</sub> under the control of its own promoter.</td><td>This study</td></tr></tbody><thead><tr><th colspan="3">Plasmids for protein purification</th></tr></thead><tbody><tr><td>pET24b-tilS<sub>So</sub></td><td>Plasmid allowing the production of TilS<sub>So</sub> and a sequence coding six histidines (6His) tag. Production of a fusion TilS protein with 6His tag in C-terminus.</td><td>[3]</td></tr><tr><td>pET24b-tilS<sub>Ec</sub></td><td>Plasmid allowing the production of TilS<sub>Ec</sub> and a sequence coding six histidines (6His) tag. Production of a fusion TilS protein with 6His tag in C-terminus.</td><td>This study</td></tr><tr><td>pET24b-tilS<sub>Sx</sub></td><td>Plasmid allowing the production of TilS<sub>Sx</sub> and a sequence coding six histidines (6His) tag. Production of a fusion TilS protein with 6His tag in C-terminus.</td><td>This study</td></tr><tr><td>pET24b-tilS<sub>So</sub>(L340P)</td><td>Plasmid allowing the production of TilS<sub>So</sub> L340P and a sequence coding six histidines (6His) tag. Production of a fusion TilS protein with 6His tag in C-terminus.</td><td>This study</td></tr><tr><td>pET24b-hsp90<sub>So</sub></td><td>Plasmid allowing the production of Hsp90<sub>So</sub> wild-type.</td><td>[3]</td></tr></tbody><thead><tr><th colspan="3">Suicide vectors</th></tr></thead><tbody><tr><td>pKNG101</td><td>Suicide vector used for gene substitution and mutagenesis in S. oneidensis. Carries the sacB cassette which causes sensitivity to sucrose, and a λ-pir-dependent origin of replication. Streptomycin resistance.</td><td>[14]</td></tr><tr><td>pKO3</td><td>Suicide vector used for gene substitution and mutagenesis in E. coli. Carries the sacB cassette which causes sensitivity to sucrose, and a thermosensitive origin of replication. Chloramphenicol resistance.</td><td>[15]</td></tr></tbody></table>
<table><caption>Table S3: Sequences of the chimeras</caption><tr><td><b>&gt;TilS_Ecoli_Uniprot_P52097</b><br>MTLTLNRQLLTSRQILVAFSGGLDSTVLLHQLVQWRTENPGVALRAIHVHHGLSANADAWVTHCENVCQQWQVPLVVERVQLAQEGLGIEAQARQARYQAFARTLLPGEVLVTAQHLDDQCETFLLALKRGSGPAGLSAMAEVSEFAGTRLIRPLLARTRGELVQWARQYDLRWIEDES NQDDSYDRNFLRLVVPLLQQRWP HFAEATARSAALCAEQESLLDELLADLAHCQSPQGTLQIVPMLAMSDARRAAIIRRWLAGQNAPMPSRDALVRIWQEVALAREDASPCLRLGAFEIRRYQSQLWWIKSVTGQSENIVPWQTWLQPLELPAGLGSVQLNAGGDIRPPRADEAVSVRFKAPGLLHIVGRNGGRKLKKIWQELGVPPWLRDTTPLLFYGETLIAAAGVFVTQEGVAEGENGVSFVWQKTLS</td></tr><tr><td><b>&gt;TilS_Soneidensis_Uniprot_Q8EGF9</b><br>MTAQDLSVHIARWLDSLPLQAGSKLVLAYSGGVDSEVLAYGLSEYAKQRPDLRYQLIYVHHGLSPNADNWAKHCQARAAIYGLPVTVERVQLILGPRVSVEAEARKARYQAILPHLNPQDILLTAHHEDDQLETILLALKRGGPKGLAAMGQIQPLSLADKGSLQVRPLLDISREMIETFAQTRQLVHIEDESNNQDDKYDRNFLRLEIIPRLKARWPSIATTASRSAQLCAEQQAIVETEVSERLPKLLVKAPVTEQTVLKSELAAQPIEWQGILLLRGFIESQEFSLPSYVQLQQMLQQLIHIKEDAKVHIRINDCVLRRFAGMLYLDSEGETLSTALHITARDLHQEILTLLTQASAMVEDKIVPFALVTTGPRLRVLTTGPRLRLPKADEVVS LGYGLPGQFRCQPHFRDKGRELKKLWQECAVPPWLRAEVGFLFYNDKLVMAFGLWVEKAFCAQGDEIGLSYLIANP</td></tr><tr><td><b>Chimera 1: EEES</b><br>MTLTLNRQLLTSRQILVAFSGGLDSTVLLHQLVQWRTENPGVALRAIHVHHGLSANADAWVTHCENVCQQWQVPLVVERVQLAQEGLGIEAQARQARYQAFARTLLPGEVLVTAQHLDDQCETFILALKRGSGPAGLSAMAEVSEFAGTRLIRPLLARTRGELVQWARQYDLRWIEDES NQDDSYDRNFLRLVVPLLQQRWPHFAEATARSAALCAEQESLLDELLADLAHCQSPQGTLQIVPMLAMSDARRAAIIRRWLAGQNAPMPSRDALVRIWQEVALAREDASPCLRLGAFEIRRYQSQLWWIKSVGETLSTALHITARDLHQEILTLLTQASAMVEDKIVPFALVTTGPRLRPLPKADEVVS LGYGLPGQFRCQPHFRDKGRELKKLWQECAVPPWLRAEVGFLFYNDKLVMAFGLWVEKAFCAQGDEIGLSYLIANP</td></tr><tr><td><b>Chimera 2: EEES</b><br>MTLTLNRQLLTSRQILVAFSGGLDSTVLLHQLVQWRTENPGVALRAIHVHHGLSANADAWVTHCENVCQQWQVPLVVERVQLAQEGLGIEAQARQARYQAFARTLLPGEVLVTAQHLDDQCETFLLALKRGSGPAGLSAMAEVSEFAGTRLIRPLLARTRGELVQWARQYDLRWIEDES NQDDSYDRNFLRLVVPLLQQRWPSIATTASRSAQLCAEQQAIVETEVSERLPKLLVKAPVTEQTVLKSELAAQPIEWQGILLLRGFIESQEFSLPSYVQLQQMLQQLIHIKEDAKVHIRINDCVLRRFAGMLYLDSEGETLSTALHITARDLHQEILTLLTQASAMVEDKIVPFALVTTGPRLRPLPKADEVVS LGYGLPGQFRCQPHFRDKGRELKKLWQECAVPPWLRAEVGFLFYNDKLVMAFGLWVEKAFCAQGDEIGLSYLIANP</td></tr><tr><td><b>Chimera 3: ESSS</b><br>MTLTLNRQLLTSRQILVAFSGGLDSTVLLHQLVQWRTENPGVALRAIHVHHGLSANADAWVTHCENVCQQWQVPLVVERVQLAQEGLGIEAQARQARYQAFARTLLPGEVLVTAQHLDDQCETFLLALKRGSGPAGLSAMAEVSEFAGTRLIRPLLARTRGELVQWARQYDLRWIEDES NQDDSYDRNFLRLVVPLLQQRWPSIATTASRSAQLCAEQQAIVETEVSERLPKLLVKAPVTEQTVLKSELAAQPIEWQGILLLRGFIESQEFSLPSYVQLQQMLQQLIHIKEDAKVHIRINDCVLRRFAGMLYLDSEGETLSTALHITARDLHQEILTLLTQASAMVEDKIVPFALVTTGPRLRPLPKADEVVS LGYGLPGQFRCQPHFRDKGRELKKLWQECAVPPWLRAEVGFLFYNDKLVMAFGLWVEKAFCAQGDEIGLSYLIANP</td></tr><tr><td><b>Chimera 4: SSSE</b><br>MTAQDLSVHIARWLDSLPLQAGSKLVLAYSGGVDSEVLAYGLSEYAKQRPDLRYQLIYVHHGLSPNADNWAKHCQARAAIYGLPVTVERVQLILGPRVSVEAEARKARYQAILPHLNPQDILLTAHHEDDQLETILLALKRGGPKGLAAMGQIQPLSLADKGSLQVRPLLDISREMIETFAQTRQLVHIEDESNNQDDKYDRNFLRLEIIPRLKARWPSIATTASRSAQLCAEQQAIVETEVSERLPKLLVKAPVTEQTVLKSELAAQPIEWQGILLLRGFIESQEFSLPSYVQLQQMLQQLIHIKEDAKVHIRINDCVLRRFAGMLYLDSTGQSENIVPWQTWLQPLELPAGLGSVQLNAGGDIRP PRADEAVSVRFKAPGLLHIVGRNGGRKLKKIWQELGVPPWLRDTTPLLFYGETLIAAAGVFVTQEGVAEGENGVSFVWQKTLS</td></tr><tr><td><b>Chimera 5: SSEE</b><br>MTAQDLSVHIARWLDSLPLQAGSKLVLAYSGGVDSEVLAYGLSEYAKQRPDLRYQLIYVHHGLSPNADNWAKHCQARAAIYGLPVTVERVQLILGPRVSVEAEARKARYQAILPHLNPQDILLTAHHEDDQLETILLALKRGGPKGLAAMGQIQPLSLADKGSLQVRPLLDISREMIETFAQTRQLVHIEDESNNQDDKYDRNFLRLEIIPRLKARWPSIATTASRSAQLCAEQQAIVETEVSERLPKLPQGTLQIVPMLAMSDARRAAIIRRWLAGNAPMPSRDALVRIWQEVALAREDASPCLRLGAFEIRRYQSQLWWIKSVTGQSENIVPWQTWLQPLELPAGLGSVQLNAGGDIRPPRADEAVSVRFKAPGLLHIVGRNGGRKLKKIWQELGVPPWLRDTTPLLFYGETLIAAAGVFVTQEGVAEGENGVSFVWQKTLS</td></tr><tr><td><b>Chimera 6: SEEES</b><br>MTAQDLSVHIARWLDSLPLQAGSKLVLAYSGGVDSEVLAYGLSEYAKQRPDLRYQLIYVHHGLSPNADNWAKHCQARAAIYGLPVTVERVQLILGPRVSVEAEARKARYQAILPHLNPQDILLTAHHEDDQLETILLALKRGGPKGLAAMGQIQPLSLADKGSLQVRPLLDISREMIETFAQTRQLVHIEDESNNQDDKYDRNFLRLEIIPRLKARWPHFAEATARSAALCAEQESLLDELLADLAHCQSPQGTLQIVPMLAMSDARRAAIIRRWLRWLAGQNAPMPSRDALVRIWQEVALAREDASPCLRLGAFEIRRYQSQLWWIKSVTGQSENIVPWQTWLQPLELPAGLGSVQLNAGGDIRPPRADEAVSVRFKAPGLLHIVGRNGGRKLKKIWQELGVPPWLRDTTPLLFYGETLIAAAGVFVTQEGVAEGENGVSFVWQKTLS</td></tr></table>
**Supplementary References**

[1] C. Baraquet, L. Théraulaz, C. Iobbi-Nivol, V. Méjean, C. Jourlin-Castelli, Unexpected chemoreceptors mediate energy taxis towards electron acceptors in Shewanella oneidensis, Mol. Microbiol. 73 (2009) 278-290. https://doi.org/10.1111/j.1365-2958.2009.06770.x.

[2] M. Corteggiani, N. Bossuet-Greif, J.-P. Nougayrède, D. Byrne, M. Ilbert, S. Dementin, M.-T. Giudici-Orticoni, V. Méjean, E. Oswald, O. Genest, Uncoupling the Hsp90 and DnaK chaperone activities revealed the in vivo relevance of their collaboration in bacteria, Proc. Natl. Acad. Sci. U. S. A. 119 (2022) e2201779119. https://doi.org/10.1073/pnas.2201779119.

[3] F.A. Honoré, V. Méjean, O. Genest, Hsp90 Is Essential under Heat Stress in the Bacterium Shewanella oneidensis, Cell Rep. 19 (2017) 680-687. https://doi.org/10.1016/j.celrep.2017.03.082.

[4] N.J. Maillot, F.A. Honoré, D. Byrne, V. Méjean, O. Genest, Cold adaptation in the environmental bacterium Shewanella oneidensis is controlled by a J-domain co-chaperone protein network, Commun. Biol. 2 (2019) 323. https://doi.org/10.1038/s42003-019-0567-3.

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[6] O. Genest, J.R. Hoskins, A.N. Kravats, S.M. Doyle, S. Wickner, Hsp70 and Hsp90 of E. coli Directly Interact for Collaboration in Protein Remodeling, J. Mol. Biol. 427 (2015) 3877-3889. https://doi.org/10.1016/j.jmb.2015.10.010.

[7] F.R. Blattner, G. Plunkett, C.A. Bloch, N.T. Perna, V. Burland, M. Riley, J. Collado-Vides, J.D. Glasner, C.K. Rode, G.F. Mayhew, J. Gregor, N.W. Davis, H.A. Kirkpatrick, M.A. Goeden, D.J. Rose, B. Mau, Y. Shao, The complete genome sequence of Escherichia coli K-12, Science 277 (1997) 1453-1462. https://doi.org/10.1126/science.277.5331.1453.

[8] O. Genest, M. Reidy, T.O. Street, J.R. Hoskins, J.L. Camberg, D.A. Agard, D.C. Masison, S. Wickner, Uncovering a region of heat shock protein 90 important for client binding in E. coli and chaperone function in yeast, Mol. Cell 49 (2013) 464-473. https://doi.org/10.1016/j.molcel.2012.11.017.

[9] V. de Lorenzo, M. Herrero, U. Jakubzik, K.N. Timmis, Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria, J. Bacteriol. 172 (1990) 6568-6572. https://doi.org/10.1128/jb.172.11.6568-6572.1990.

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[11] L.M. Guzman, D. Belin, M.J. Carson, J. Beckwith, Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter, J. Bacteriol. 177 (1995) 4121-4130.

[12] D. Gully, E. Bouveret, A protein network for phospholipid synthesis uncovered by a variant of the tandem affinity purification method in Escherichia coli, Proteomics 6 (2006) 282-293. https://doi.org/10.1002/pmic.200500115.

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**Supplementary Figures**

<figure><img src="image_12.png" /><figcaption>**Figure S1:** A comparison between *TilS* of *E. coli* (*TilS*<sub>Ec</sub>) and *TilS* of *S. oneidensis* (*TilS*<sub>So</sub>). (A) Sequence alignments of *TilS*<sub>So</sub> and *TilS*<sub>Ec</sub> using the multiple sequence alignment tool «Multalin» (http://multalin.toulouse.inra.fr/multalin/). The ESPrigt program (https://esprigt.ibcp.fr/ESPrigt/ESPrigt/) was used to display the alignment. The four domains of *TilS*<sub>So</sub> are represented: the N-terminal domain (green), the Helical domain (wheat), the C-terminal 1 domain (blue) and the C-terminal 2 domain (orange). The key catalytic residues are indicated by red asterisk. (B) Structure of *TilS*<sub>Ec</sub> (PDB 1NI5) and model of *TilS*<sub>So</sub> generated with AlphaFold 3.0 Software, pTM = 0.81. Each *TilS* domain is colored as in A. (C) Gel filtration analysis. Purified *TilS*<sub>So</sub> or *TilS*<sub>Ec</sub> (8 µM, 100 µL) were analyzed on a Superdex 200 Increase 10/300 GL size exclusion chromatography column. Normalized values of absorbance at 280 nm are shown. (D) Limited proteolysis profiles of purified *TilS*<sub>Ec</sub> and *TilS*<sub>So</sub> (15 µg) digested with trypsin (0.25 µg) for various times at 37°C in Tris-HCl buffer (50 mM, pH 7.5) containing 100 mM KCl, 10% glycerol and 1 mM DTT in a final reaction volume of 50 µL. At the indicated times, 10 µL was removed and analyzed by SDS-PAGE.</figcaption></figure>
<figure><img src="image_13.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>C</figcaption></figure>

S. oneidensis strains

<figure><img src="image_15.png" /><figcaption>D</figcaption></figure>

S. oneidensis strains

<figure><img src="image_16.png" /><figcaption>E</figcaption></figure>

E. coli strains

<figure><img src="image_17.png" /><figcaption>F</figcaption></figure>

E. coli strains

<figure><img src="image_18.png" /></figure>

**Figure S2: Controls related to Figure 2.** (A) Western blots showing the production of T18-TilS<sub>Ec</sub> and T18-TilS<sub>So</sub> used in bacterial two-hybrid experiments. Anti-RecA antibody was used as neutral loading control. *E. coli* strains with plasmid allowing the production of T18-TilS<sub>Ec</sub> or TilS<sub>So</sub> were grown at 28°C overnight with 1mM IPTG to allow the expression of the corresponding genes. After 12 hours, samples were analyzed by Western blot using anti-Adenylate cyclase (T18) antibody. (B) Replicates of microscale thermophoresis experiment to determine the interaction between Hsp90 and TilS<sub>So</sub> or TilS<sub>Ec</sub>. 85 nM of TilS labeled with RED-tris-NTA 2<sup>nd</sup> generation dye (NanoTemper) was added to serial 2-times dilutions of Hsp90<sub>So</sub> up to 50 µM. Fluorescence at 670 nm was measured, and values at 2.5 s were used to determine K<sub>d</sub>. (C) Western blots showing the amount of TilS<sub>So</sub> or TilS<sub>Ec</sub> in *S. oneidensis*. The strains WT or Δhsp90 with plasmid allowing the production of TilS<sub>So</sub> or TilS<sub>Ec</sub> with hexahistidine tag were grown at 28°C and 0.02% arabinose was added. Two hours later, samples were analyzed by Western blot using anti-His antibody. Anti-Hsp90<sub>So</sub> antibody was used as a control and a contaminant band revealed by the anti-AtcJ antibody was used as neutral sample control. (D) Quantification of anti-His Western blot shown in C. The peaks corresponding to the pixels of each band were quantified using ImageJ software. The average of the amount of TilS<sub>So</sub> or TilS<sub>Ec</sub> in WT strain
was set to 1, and each WT is compared to Δhsp90 producing the corresponding TilS<sub>So</sub> or TilS<sub>Ec</sub>, respectively. Data from three replicates are shown as mean ± SEM. Distinct symbols are used to represent data from each replicate. (E) Western blots showing the amount of TilS<sub>So</sub> or TilS<sub>Ec</sub> in *E. coli*. The protocol is similar to what was done in C. Anti-Hsp90<sub>Ec</sub> was used as control and a contaminant band revealed by the anti-AtcJ antibody was used as neutral loading control. (F) Quantification of anti-His Western blot shown in *E*. The method used is similar to *D*. In *A*, *C*, and *E*, the Western blots shown are representative of three independent experiments. In *D* and *F*, results of t-tests indicate that the differences are not significant (ns, *P* > 0.05).
<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

**Figure S3:** Growth of the strains used in Fig. 3. (A) Bacterial growth on solid media 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* (*ΔtilS*<sub>So</sub> *tilS*<sub>Ec</sub><sup>+</sup>) were grown at 28°C in liquid media and 10-fold serial diluted to be spotted on LB plates. The plates were incubated for 24 hours at 28°C. (B) Bacterial growth on solid media of the strains used in A, at 35°C. (C) 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* (*ΔtilS*<sub>So</sub> *tilS*<sub>Ec</sub><sup>+</sup>) were grown in microplates in LB medium at 34°C with shaking for 24 hours. (D) Bacterial growth of *S. oneidensis* strains presented in C. The experiments is the same as in C except that strains were grown at 36°C. (E) Complementation of 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* (*ΔtilS*<sub>So</sub> *tilS*<sub>Ec</sub><sup>+</sup>) with empty vector or allowing the production of Hsp90 from *E. coli* (*hsp90*<sub>Ec</sub>) or from *S. oneidensis* (*hsp90*<sub>So</sub>) were grown in microplates in LB medium at 35°C with shaking for 24 hours. (F) Bacterial growth on solid media 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* (*ΔtilS*<sub>Ec</sub> *tilS*<sub>So</sub><sup>+</sup>) were grown at 28°C in liquid media and 10-fold serial diluted to be spotted on LB plates. The plates were incubated for 24 hours at 28°C. (G) Bacterial growth on solid media of the strains used in *F*, at 35°C. In *A*, *B*, *F* and *G*, the plates are representative of three independent experiments. In *C*, *D*, and *E*, data from at least three replicates are shown as mean ± SEM.
A

<figure><img src="image_26.png" /></figure>

B

<figure><img src="image_27.png" /></figure>

**Figure S4:** TilS chimeras are not dependent on Hsp90 at 28°C. (A) Western blot showing the abundance of TilS chimera in S. oneidensis WT or Δhsp90 at 28°C. The strains WT or Δhsp90 with plasmid pBad33 allowing the production of 6His-TilS chimera were grown at 28°C, and 0.02% arabinose was added. After 2 hours, samples were analyzed by Western blot with anti-His antibody. Anti-Hsp90<sub>so</sub> was used as a control and Anti-AtcJ as neutral loading control. This Western blot is representative of at least five independent experiments. (B) Quantification of the Western blot shown in A. The peaks corresponding to the pixels of each band were quantified using ImageJ software. The average of 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 five replicates are shown as mean ± SEM. Distinct symbols are used to represent data from each replicate. Results of t-tests indicate that the differences are not significant (ns, *P* > 0.05).
<figure><img src="image_28.png" /><figcaption>**Figure S5:** AlphaFold 3 prediction models of the interaction of Hsp90 with *TilS*<sub>So</sub> or *TilS*<sub>Ec</sub>. (A-B) Prediction model of a monomer of *TilS*<sub>So</sub> (Uniprot: Q8EGF9) with a dimer of *Hsp90*<sub>So</sub> (Uniprot: Q8EFF7) by AlphaFold 3.0 software, ipTM = 0.38 pTM = 0.47. *TilS*<sub>So</sub> is colored by domains: the N-terminal domain (green), the Helical domain (wheat), the C-terminal 1 domain (blue) and the C-terminal 2 domain (orange). The W476 (purple) and L563 (pink) residues of Hsp90 important for the interaction with clients are shown. Among the five models presented by the software, four are similar to the model presented in A, and one shows *TilS*<sub>So</sub> as in B. In A right panel, *TilS*<sub>So</sub> structure was truncated to better visualize the insertion of the C-</figcaption></figure>
terminal 2 domain into the cleft formed by Hsp90<sub>So</sub>. (C) Prediction model of a monomer of TilS<sub>Ec</sub> (Uniprot: P52097) with a dimer of Hsp90<sub>So</sub> (Uniprot: Q8EFF7) by AlphaFold 3.0 software, ipTM = 0.39, pTM = 0.5. TilS<sub>Ec</sub> is colored by domains as in A. (D-E) Heatmaps showing the predicted interaction confidence between TilS<sub>So</sub> (D) or TilS<sub>Ec</sub> (E) and the Hsp90<sub>So</sub> dimer, based on residue-residue contact probabilities.
<figure><img src="image_29.png" /><figcaption>**Figure S6:** Phylogenetic tree of the TiIS C2 domain in selected bacterial species. This maximum-likelihood phylogenetic tree illustrates the evolutionary relationships of the TiIS C2 domain among bacterial taxa. Branches are labelled with UniProt ID and species names. Selected taxa are *Shewanella oneidensis* MR-1 (red) and *Escherichia coli* K12 (blue). Branches length indicates the substitution rate per site. Sequences of interest are indicated by asterisks.</figcaption></figure>
<figure><img src="image_30.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_31.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_32.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_33.png" /></figure>

**Figure S7: Controls related to TilS orthologues.** (A) Western blot showing the amount of TilS orthologues. The strains of *S. oneidensis* WT or Δhsp90 with plasmid allowing the production of TilS from *Shewanella woodyi*, *Shewanella xiamenensis*, *Vibrio cholerae*, *Yersinia pestis* or *Salmonella enterica* serovar Typhimurium 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<sub>so</sub> antibody was used as a control and anti-AtcJ antibody was used as neutral sample control. (B) Same experiment as in *B*, except that the strains were grown at 28°C. (C-D) Quantification of the Western blot shown in *A* and *B*. The peaks corresponding to the pixels of each band were quantified using ImageJ software. The average of the amount of each TilS protein in WT strain was set to 1, and each WT is compared to Δ*hsp90* producing the corresponding chimera respectively. Data from four replicates are shown as mean ± SEM. Distinct symbols are used to represent data from each replicate. Results of t-tests indicate whether the differences are significant (*P* ≤ 0.05) or not (ns, *P* > 0.05).
<figure><img src="image_34.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_35.png" /><figcaption>C</figcaption></figure>

S. oneidensis strains

<figure><img src="image_36.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_37.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_38.png" /></figure>

**Figure S8: Controls related to Figure 5.** (A) Western blot showing the amount of TilS<sub>So</sub> L340S and L340G mutants in S. oneidensis WT or Δ*hsp90*. The strains WT or *Δhsp90* with plasmid allowing the production of TilS mutants with hexahistidine tag were grown at 34°C and 0.02% arabinose was added. Two hours later, samples were analyzed by Western blot using anti-His antibody. As a control, anti-Hsp90 antibody was used. (B) Western blot showing the amount of TilS<sub>So</sub> with the 7 last residues replaced by the 8 last residues of TilS<sub>Sx</sub> (TilS<sub>So</sub> 8-Ct<sub>Sx</sub>) in S. oneidensis WT or Δ*hsp90*. The strains WT or Δ*hsp90* with plasmid allowing the production of TilS<sub>So</sub> 8-Ct<sub>Sx</sub> with hexahistidine tag were grown at 28°C or 34°C and 0.02% arabinose was added. Two hours later, samples were analyzed by Western blot using anti-His antibody. As a control, anti-Hsp90 antibody was used. As control, WT or Δ*hsp90* with plasmid allowing the production of TilS<sub>So</sub> L340P is shown. In A and B, Western blots shown are representative of two independent experiments. (C) Bacterial growth on liquid media at 28°C of the S. oneidensis strains MR-1 wild-type (WT) or deleted of *hsp90* (Δ*hsp90*) with the *tilS* gene WT or coding for the mutation L340P. The cells were grown in microplates in LB medium at 28°C with shaking during 24 hours. Data from six replicates are shown as mean ± SEM. (D) Bacterial growth on solid media of the strains described in C. Cells were grown at 28°C in liquid media and 10-fold serial diluted to be spotted on LB plates. The plates were incubated for 24 hours at 28°C. (E) Bacterial growth on solid media of the strains described in C. Cells were grown at 28°C in liquid media and 10-fold serial diluted to be spotted on LB plates. The plates were incubated for 24 hours at 35°C. In D and E, the plates shown are representative of three independent experiments.
A

<figure><img src="image_39.png" /></figure>

B

<figure><img src="image_40.png" /></figure>

C

<figure><img src="image_41.png" /></figure>

<table><thead><tr><td>Protein</td><td>K<sub>d</sub> (µM)</td><td>SEM</td></tr></thead><tbody><tr><td>TiIS<sub>So</sub> L340P</td><td>20.35</td><td>± 8.85</td></tr><tr><td>TiIS<sub>Sx</sub></td><td>0.76</td><td>± 0.16</td></tr></tbody></table>

<figure><img src="image_42.png" /></figure>

**Figure S9: In vitro characterization of the TiIS<sub>So</sub> single mutant.** (A) Gel filtration analysis of TiIS proteins. Purified TiIS<sub>So</sub> WT or L340P, TiIS<sub>Ec</sub>, or TiIS<sub>Sx</sub> (8 µM, 100 µL) were analyzed on a Superdex 200 Increase 10/300 GL size exclusion chromatography column. Normalized values of absorbance at 280 nm are shown. (B) Limited proteolysis profiles of purified TiIS<sub>So</sub> L340P or TiIS<sub>Sx</sub> (15 µg) digested with trypsin (0.25 µg) for various times at 37°C in Tris-HCl buffer (50 mM, pH 7.5) containing 100 mM KCl, 10% glycerol and 1 mM DTT in a final reaction volume of 50 µL. At the indicated times, 10 µL was removed and analyzed by SDS-PAGE. (C) Microscale thermophoresis experiment to determine the interaction between Hsp90 and TiIS<sub>So</sub> L340P or TiIS<sub>Sx</sub>, 85 nM of TiIS labeled with RED-tris-NTA 2nd generation dye (NanoTemper) was added to serial 2-times dilutions of Hsp90<sub>So</sub> up to 50 µM. Fluorescence at 670 nm was measured, and values at 2.5 s were used to determine K<sub>d</sub>. K<sub>d</sub> is represented as mean ± SEM based on two independent experiments shown on the two panels.
A

<figure><img src="image_43.png" /></figure>

B

<figure><img src="image_44.png" /></figure>

**Figure S10: In silico characterization of the TilS<sub>So</sub> single mutant.** (A) Disorder analysis of TilS<sub>So</sub> and TilS<sub>Ec</sub> using AIUPred algorithm (https://iupred.elte.hu/) with the options "AIUPred – Only disorder" and "Default smoothing". The graph shows disorder prediction score by aminoacid. A residue or region showing a score superior to 0.5 can be considered as disordered. (B) Comparison of the structures of TilS<sub>So</sub> (AlphaFold3 prediction, pTM = 0.81) and TilS<sub>Ec</sub> (PDB: 1NI5). The green box delineates the region in the vicinity of the leucine residue in position 340 of TilS<sub>So</sub> (shown in yellow).