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. 2016 Oct 5;8(9):2979-2991.
doi: 10.1093/gbe/evw176.

The Molecular Chaperone DnaK Is a Source of Mutational Robustness

José Aguilar-Rodríguez  1 , Beatriz Sabater-Muñoz  2 , Roser Montagud-Martínez  3 , Víctor Berlanga  3 , David Alvarez-Ponce  4 , Andreas Wagner  5 , Mario A Fares  6
Affiliations

The Molecular Chaperone DnaK Is a Source of Mutational Robustness

José Aguilar-Rodríguez et al. Genome Biol Evol. .

Abstract

Molecular chaperones, also known as heat-shock proteins, refold misfolded proteins and help other proteins reach their native conformation. Thanks to these abilities, some chaperones, such as the Hsp90 protein or the chaperonin GroEL, can buffer the deleterious phenotypic effects of mutations that alter protein structure and function. Hsp70 chaperones use a chaperoning mechanism different from that of Hsp90 and GroEL, and it is not known whether they can also buffer mutations. Here, we show that they can. To this end, we performed a mutation accumulation experiment in Escherichia coli, followed by whole-genome resequencing. Overexpression of the Hsp70 chaperone DnaK helps cells cope with mutational load and completely avoid the extinctions we observe in lineages evolving without chaperone overproduction. Additionally, our sequence data show that DnaK overexpression increases mutational robustness, the tolerance of its clients to nonsynonymous nucleotide substitutions. We also show that this elevated mutational buffering translates into differences in evolutionary rates on intermediate and long evolutionary time scales. Specifically, we studied the evolutionary rates of DnaK clients using the genomes of E. coli, Salmonella enterica, and 83 other gamma-proteobacteria. We find that clients that interact strongly with DnaK evolve faster than weakly interacting clients. Our results imply that all three major chaperone classes can buffer mutations and affect protein evolution. They illustrate how an individual protein like a chaperone can have a disproportionate effect on the evolution of a proteome.

Keywords: DnaK; Escherichia coli; experimental evolution; molecular chaperones; mutational robustness; protein evolution.

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Figures

<sc>Fig</sc>. 1.—
Fig. 1.—
Mutation accumulation experiment. Evolutionary history of the populations evolved in this study from the first daily transfer or single-cell bottleneck (T0) until the end of the evolution experiment (T85). We constructed two strains derived from an ancestral Escherichia coli K-12 MG1655 strain lacking the mismatch repair gene mutS. The DnaK+ strain harbours the ∼15-copy plasmid pKJE7 that contains the DnaK/DnaJ/GrpE chaperone system under the control of the promoter PBAD inducible by L-arabinose (Nishihara et al. 1998). The DnaK− strain contains a control pKJE7-derived plasmid where the operon dnaK–dnaJ–grpE has been deleted. We evolved in parallel multiple independent populations of both strains through single-cell bottlenecks under the effect of strong genetic drift at two different temperatures (37 °C and 42 °C). At each temperature we evolved some populations in the presence of L-arabinose (L-ara+), and some in the absence of this expression inducer (L-ara–). (A) During the evolution of 68 DnaK+ populations, five out of eight lines evolving in the absence of inducer went extinct (indicated by a cross). None of the 60 lines evolving under DnaK overexpression experienced any extinction. (B) Of the 16 independent DnaK− populations, 12 populations went extinct. We finished the evolution experiment after 85 single-cell bottlenecks (T85), or ∼1,870 generations.
<sc>Fig</sc>. 2.—
Fig. 2.—
DnaK abundance at the beginning and the end of the mutation accumulation experiment. We measured the abundance of the chaperone DnaK for the 8 sequenced lines evolved trough 85 single-cell bottlenecks (∼1,870 generations) at 37 °C or 42 °C. For comparison, we also measured the abundance of the chaperone in the ancestral DnaK+ and DnaK− strains at both temperatures. We determined DnaK levels in the presence and absence of the inducer L-arabinose (L-ara+ and L-ara–, respectively), as described in Materials and Methods (“Verification of DnaK overexpression”), via the intensity of the DnaK band in a Western blot. The evolved lines did not lose the ability to overexpress DnaK in the presence of the inducer L-arabinose except for a DnaK+ line evolved at 42 °C (line #2), which explains the decrease in the average DnaK abundance at the end of the evolution experiment. However, this loss of overexpression occurred late in the evolution experiment, and it is not even complete for most of the daily growth cycle of this line (supplementary fig. S2, Supplementary Material online). The height of the bars indicates mean DnaK abundance across two experimental replicates per strain and condition. Error bars represent 1 SD of the mean.
<sc>Fig</sc>. 3.—
Fig. 3.—
Nonsynonymous mutations accumulated in DnaK clients. (A) The proportion of nonsynonymous mutations that affect DnaK clients is significantly higher in DnaK+ lines that overexpress DnaK than in the control DnaK− lines that do not express the chaperone at such high levels. This is observed both for lines evolved at 37 °C and 42 °C. We combined mutations across DnaK+ lines evolved at the same temperature. The significance of the difference in the proportions was evaluated using a binomial test. (B) At both temperatures, strong clients have accumulated significantly more nonsynonymous substitutions than weak clients in DnaK+ lines. Strong clients include those clients with the highest DnaK dependency, whereas weak clients include clients with the lowest chaperone dependency. Statistical significance was evaluated using Fisher’s test.
<sc>Fig</sc>. 4.—
Fig. 4.—
DnaK accelerates protein evolution on intermediate and long evolutionary time scales. Scatter-plots showing the relationship between DnaK dependency (calculated as a relative enrichment factor that indicates the fraction of cellular protein bound to DnaK at 37 °C, horizontal axis) and the degree of divergence over (A) intermediate time scales, measured as nonsynonymous divergence (Spearman rank correlation coefficient, ρ = 0.367, N = 627, P < 2.2 × 10 − 16), and (B) long time scales, measured as protein (amino acid) distance (ρ = 0.257, N = 311, P = 4.4 × 10 − 6) (vertical axes). Solid lines represent the best fit to the points. Note the logarithmic scale on both axes.
<sc>Fig</sc>. 5.—
Fig. 5.—
Strong clients evolve faster than weak clients. (A) We find that strong clients evolve faster than weak clients on intermediate evolutionary time scales, measured as the rate of nonsynonymous substitutions (Wilcoxon rank-sum test, P < 2.2 × 10 − 16). (B) On long evolutionary time scales, we also find that strong clients evolve faster than weak clients (Wilcoxon rank-sum test, P = 2.3 × 10 − 3). The thick horizontal line in the middle of each box represents the median of the data, whereas the bottom and top of each box represent the 25th and 75th percentiles, respectively. Note the logarithmic scale on the y-axis in (A).
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