Genome-scale co-evolutionary inference identifies functions and clients of bacterial Hsp90

Abstract

The molecular chaperone Hsp90 is essential in eukaryotes, in which it facilitates the folding of developmental regulators and signal transduction proteins known as Hsp90 clients. In contrast, Hsp90 is not essential in bacteria, and a broad characterization of its molecular and organismal function is lacking. To enable such characterization, we used a genome-scale phylogenetic analysis to identify genes that co-evolve with bacterial Hsp90. We find that genes whose gain and loss were coordinated with Hsp90 throughout bacterial evolution tended to function in flagellar assembly, chemotaxis, and bacterial secretion, suggesting that Hsp90 may aid assembly of protein complexes. To add to the limited set of known bacterial Hsp90 clients, we further developed a statistical method to predict putative clients. We validated our predictions by demonstrating that the flagellar protein FliN and the chemotaxis kinase CheA behaved as Hsp90 clients in Escherichia coli, confirming the predicted role of Hsp90 in chemotaxis and flagellar assembly. Furthermore, normal Hsp90 function is important for wild-type motility and/or chemotaxis in E. coli. This novel function of bacterial Hsp90 agreed with our subsequent finding that Hsp90 is associated with a preference for multiple habitats and may therefore face a complex selection regime. Taken together, our results reveal previously unknown functions of bacterial Hsp90 and open avenues for future experimental exploration by implicating Hsp90 in the assembly of membrane protein complexes and adaptation to novel environments.

Figure 1. The distribution of hsp90A across a bacterial phylogeny.

Branches are colored according to phyla. Large taxonomic groups are labeled. Branch lengths are ignored for ease of display. The phylogeny constructed by Ciccarelli et al. is used (see Methods). For distribution of other bacterial Hsp90 paralogs, see Figure S1C. hsp90B and hsp90C are not displayed, and are ignored throughout the analysis.

Figure 2. Flagellar genes and secretion system genes show distinct signatures of co-evolution with hsp90A.

Schematic diagrams of the models describing the co-evolution of hsp90A with the flagellar gene fliI (A) and the non-flagellar Type III secretion gene yscN (B). The four boxes represent the four possible states of presence and absence in each model, and arrows represent transitions between them (gain or loss events). Arrow widths in each diagram are scaled to represent the rate of each transition. The average transition rate and standard deviation across multiple BayesTraits runs are displayed (see Methods). Box plots of the rates of gain and loss of all hsp90A-associated flagellar genes (n = 27; C) and all hsp90A-associated Type III secretion genes (n = 10; D) further demonstrate consistent co-evolutionary dynamics of genes in these categories. A box plot of all hsp90A-associated secretion genes (including all types) is provided as Figure S4.

Figure 3. The distribution of the Putative Client Index, PCI, among hsp90A-associated genes.

Lower values indicate behavior closer to that expected of a client. The 18 genes most likely to be clients are listed in Table 2. Prominent functional groups are highlighted, as well as two chaperone-encoding genes.

Figure 4. ΔhtpG E. coli cells spread less efficiently on soft-agar plates.

Upon equal mixing, WT and ΔhtpG cells were competed for 8 hours at 34° on the same soft-agar plates, where bacteria spread in a motility- and chemotaxis-dependent fashion. Samples from the outer edge of the plate are thus enriched in cells with optimal chemotaxis and motility, whereas cells from the center are less chemotactic and/or motile. (A) A representative image of assay plate. (B) Quantitation of different genotypes as determined by percentage of the YFP-labeled WT vs. CFP-labeled ΔhtpG cells at the indicated locations. YFP and CFP expression was induced by 1 µM IPTG. An essentially identical result was obtained for the CFP-labeled WT vs. YFP-labeled ΔhtpG cells (data not shown), confirming that it is label-independent. Error bars indicate standard errors from four replicates. Results were similar at 42°C (Table S3).

Figure 5. Growth-stage-dependent interaction of HtpG with FliN and CheA.

(A) Efficiency of FRET between HtpG-YFP or HtpG(E34A)-YFP and FliN-CFP or CFP-CheA as a function of growth stage (indicated by OD₆₀₀ value), measured by acceptor photobleaching in wild-type cells (Methods). Error bars indicate standard errors from three replicates. For these assays, a truncated form of CheA lacking the first 97 amino acids (CheA_s) was used because this fusion was more stable against spontaneous proteolysis than the fusion to full-length CheA, but showed similar interaction with HtpG (Table S4) (B) Growth-stage dependence of motility in cultures used for FRET measurements in (A), assayed as a percentage of motile cells The onset of cell motility is substantially delayed in cells expressing HtpG(E34A). Error bars indicate standard errors from three replicates.

Figure 6. Habitat preference affects the gain and loss of hsp90A in bacteria.

Rates of gain and loss of hsp90A throughout bacterial evolution with relation to multiple habitat preference. Standard deviation across 100 runs was smaller than 0.001 in all cases.