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Nature Ecology & Evolution, 4, ISSN:2397-334X

Linking the molecular effects of mutations to fitness is central to understanding evolutionary dynamics. Here we establish a quantitative relation between the global effect of mutations on the E. coli proteome and bacterial fitness. We created E. coli strains with specific destabilizing mutations in the chromosomal folA gene encoding dihydrofolate reductase (DHFR) and quantified the ensuing changes in the abundances of 2000+ E. coli proteins in mutant strains using tandem mass tags with subsequent LC-MS/MS. mRNA abundances in the same E. coli strains were also quantified. The proteomic effects of mutations in DHFR are quantitatively linked to phenotype: the standard deviations of the distributions of logarithms of relative (to wild-type) protein abundances anti-correlate with bacterial growth rates. Proteomes hierarchically cluster first by media conditions, and within each condition, by the severity of the perturbation to DHFR function. These results highlight the importance of a systems-level layer in the genotype-phenotype relationship.

Several genes exhibit gene dosage toxicity yet its molecular underpinnings remain unknown. Here we demonstrate that overexpression of DHFR inE. colicauses toxic metabolic imbalance triggered by interactions with several enzymes involved in 1-carbon metabolism, in particular GlyA and PurH. DHFR overexpression partially inhibits activity of these enzymes, but at physiological concentrations, PurH-DHFR interaction enhances catalytic efficiency of DHFR, implying a functional interactionin vivo. Surprisingly, overexpression of orthologous DHFRs from other bacterial species caused minimal metabolic and fitness perturbations, despite pulling out more interacting partners than overexpressed endogenous DHFR. Orthologous DHFRs were less potent in inhibitingE. coliGlyA and PurH, or gaining a catalytic improvement upon interaction with PurH, indicating a partial loss of interaction specificity due to evolutionary divergence. This study shows how protein overexpression perturbs a dynamic network of weak yet potentially functional PPI with consequences for the metabolic state of cells and their fitness.

Fitness landscapes of drug resistance constitute powerful tools to elucidate mutational pathways of antibiotic escape. Here, we developed a predictive biophysics-based fitness landscape of trimethoprim (TMP) resistance for Escherichia coli dihydrofolate reductase (DHFR). We investigated the activity, binding, folding stability, and intracellular abundance for a complete set of combinatorial DHFR mutants made out of three key resistance mutations and extended this analysis to DHFR originated from Chlamydia muridarum and Listeria grayi. We found that the acquisition of TMP resistance via decreased drug affinity is limited by a trade-off in catalytic efficiency. Protein stability is concurrently affected by the resistant mutants, which precludes a precise description of fitness from a single molecular trait. Application of the kinetic flux theory provided an accurate model to predict resistance phenotypes (IC50) quantitatively from a unique combination of the in vitro protein molecular properties. Further, we found that a controlled modulation of the GroEL/ES chaperonins and Lon protease levels affects the intracellular steady-state concentration of DHFR in a mutation-specific manner, whereas IC50 is changed proportionally, as indeed predicted by the model. This unveils a molecular rationale for the pleiotropic role of the protein quality control machinery on the evolution of antibiotic resistance, which, as we illustrate here, may drastically confound the evolutionary outcome. These results provide a comprehensive quantitative genotype–phenotype map for the essential enzyme that serves as an important target of antibiotic and anticancer therapies.

Horizontal gene transfer (HGT) plays a central role in bacterial evolution, yet the molecular and cellular constraints on functional integration of the foreign genes are poorly understood. Here we performed inter-species replacement of the chromosomal folA gene, encoding an essential metabolic enzyme dihydrofolate reductase (DHFR), with orthologs from 35 other mesophilic bacteria. The orthologous inter-species replacements caused a marked drop (in the range 10–90%) in bacterial growth rate despite the fact that most orthologous DHFRs are as stable as E.coli DHFR at 37°C and are more catalytically active than E. coli DHFR. Although phylogenetic distance between E. coli and orthologous DHFRs as well as their individual molecular properties correlate poorly with growth rates, the product of the intracellular DHFR abundance and catalytic activity (k cat/KM), correlates strongly with growth rates, indicating that the drop in DHFR abundance constitutes the major fitness barrier to HGT. Serial propagation of the orthologous strains for ~600 generations dramatically improved growth rates by largely alleviating the fitness barriers. Whole genome sequencing and global proteome quantification revealed that the evolved strains with the largest fitness improvements have accumulated mutations that inactivated the ATP-dependent Lon protease, causing an increase in the intracellular DHFR abundance. In one case DHFR abundance increased further due to mutations accumulated in folA promoter, but only after the lon inactivating mutations were fixed in the population. Thus, by apparently distinguishing between self and non-self proteins, protein homeostasis imposes an immediate and global barrier to the functional integration of foreign genes by decreasing the intracellular abundance of their products. Once this barrier is alleviated, more fine-tuned evolution occurs to adjust the function/expression of the transferred proteins to the constraints imposed by the intracellular environment of the host organism., Author Summary Horizontal gene transfer (HGT) is central to bacterial evolution. The outcome of an HGT event (fixation in a population, elimination, or separation as a subdominant clone) depends not only on the availability of a new gene but crucially on the fitness cost or benefit of the genomic incorporation of the foreign gene and its expression in recipient bacteria. Here we studied the fitness landscape for inter-species chromosomal replacement of an essential protein, dihydrofolate reductase (DHFR) encoded by the folA gene, by its orthologs from other mesophilic bacteria. We purified and biochemically characterized 33 out of 35 orthologous DHFRs and found that most of them are stable and more catalytically active than E. coli DHFR. However, the inter-species replacement of DHFR caused significant fitness loss for most transgenic strains due to low abundance of orthologous DHFRs in E. coli cytoplasm. Laboratory evolution resulted in an increase in orthologous DHFR abundance leading to a dramatic fitness improvement. Genomic and proteomic analyses of “naive” and evolved strains suggest a new function of protein homeostasis to discriminate between “self” and “non-self” proteins, thus creating fitness barriers to HGT.

This protocol is used to assay the effect of protein over-expression on fitness of E. coli. It is based on a plasmid expression of a protein of interest from an arabinose-regulated pBAD promoter followed by the measurement of the intracellular protein abundance by Western blot along with the measurement of growth parameters of E. coli cell expressing this protein.

What are the molecular properties of proteins that fall on the radar of protein quality control (PQC)? Here we mutate the E. coli’s gene encoding dihydrofolate reductase (DHFR), and replace it with bacterial orthologous genes to determine how components of PQC modulate fitness effects of these genetic changes. We find that chaperonins GroEL/ES and protease Lon compete for binding to molten globule intermediate of DHFR, resulting in a peculiar symmetry in their action: Over-expression of GroEL/ES and deletion of Lon both restore growth of deleterious DHFR mutants and most of the slow-growing orthologous DHFR strains. Kinetic steady-state modeling predicts and experimentation verifies that mutations affect fitness by shifting the flux balance in cellular milieu between protein production, folding and degradation orchestrated by PQC through the interaction with folding intermediates.

Mutations create the genetic diversity on which selective pressures can act, yet also create structural instability in proteins. How, then, is it possible for organisms to ameliorate mutation-induced perturbations of protein stability while maintaining biological fitness and gaining a selective advantage? Here we used site-specific chromosomal mutagenesis to introduce a selected set of mostly destabilizing mutations into folA —an essential chromosomal gene of Escherichia coli encoding dihydrofolate reductase (DHFR)—to determine how changes in protein stability, activity, and abundance affect fitness. In total, 27 E. coli strains carrying mutant DHFR were created. We found no significant correlation between protein stability and its catalytic activity nor between catalytic activity and fitness in a limited range of variation of catalytic activity observed in mutants. The stability of these mutants is strongly correlated with their intracellular abundance, suggesting that protein homeostatic machinery plays an active role in maintaining intracellular concentrations of proteins. Fitness also shows a significant correlation with intracellular abundance of soluble DHFR in cells growing at 30 °C. At 42 °C, the picture was mixed, yet remarkable: A few strains carrying mutant DHFR proteins aggregated, rendering them nonviable, but, intriguingly, the majority exhibited fitness higher than wild type. We found that mutational destabilization of DHFR proteins in E. coli is counterbalanced at 42 °C by their soluble oligomerization, thereby restoring structural stability and protecting against aggregation.

Antibiotic resistance is an evolving threat to public health. Understanding the evolution of antibiotic resistance at the genetic level is critical to develop novel strategies to diagnose and treat antibiotic resistant infections. We recently developed an automated microbial selection device, the “morbidostat”, which is used to study the evolution of antibiotic resistance in dynamically sustained drug selection. The morbidostat adjusts drug concentrations to maintain nearly constant inhibition of bacterial growth even as evolving bacterial populations acquire higher resistance. using the morbidostat and next generation sequencing, we identified striking features in the evolution of trimethoprim resistance in five E. coli populations evolving in parallel. We found that resistance was acquired in a stepwise manner, through multiple mutations almost exclusively restricted to the gene encoding trimethoprim's target, dihydrofolate reductase (DHFR). Multiple distinct genotypes produced very similar trimethoprim resistant phenotypes, with each highly evolved strain each containing four mutations from a set of six possibilities, that were acquired in a non-random order. Never were more than four mutations acquired, despite sustained selection for further increases in drug resistance, indicating that these genotypes were local adaptive peaks.In order to understand how the adaptive landscape of drug resistance contained multiple peaks, all combinatorial sets of adaptive mutations in the DHFR gene (96 strains) were constructed and characterized. These measurements showed that resistance evolves through an almost maximally rugged adaptive landscape with direct and indirect trajectories leading to distinct peaks. The ruggedness was not explained by pairwise incompatibilities between mutations, instead indicating ‘high-order' genetic interactions between mutations. These high-order interactions were responsible for the existence of multiple adaptive peaks. One mutation was seen to have the power to control the adaptive landscape: its presence or absence largely defined the ruggedness or smoothness of the adaptive landscape.