RESUMEN
The rampant variability in codon bias existing between bacterial genomes is expected to interfere with horizontal gene transfer (HGT), a phenomenon that drives bacterial adaptation. However, delineating the constraints imposed by codon bias on functional integration of the transferred genes is complicated by multiple genomic and functional barriers controlling HGT, and by the dependence of the evolutionary outcomes of HGT on the host's environment. Here, we designed an experimental system in which codon composition of the transferred genes is the only variable triggering fitness change of the host. We replaced Escherichia coli's chromosomal folA gene encoding dihydrofolate reductase, an essential enzyme that constitutes a target for trimethoprim, with combinatorial libraries of synonymous codons of folA genes from trimethoprim-sensitive Listeria grayi and trimethoprim-resistant Neisseria sicca. The resulting populations underwent selection at a range of trimethoprim concentrations, and the ensuing changes in variant frequencies were used to infer the fitness effects of the individual combinations of codons. We found that when HGT causes overstabilization of the 5'-end mRNA, the fitness contribution of mRNA folding stability dominates over that of codon optimality. The 5'-end overstabilization can also lead to mRNA accumulation outside of the polysome, thus preventing the decay of the foreign transcripts despite the codon composition-driven reduction in translation efficiency. Importantly, the fitness effects of mRNA stability or codon optimality become apparent only at sub-lethal levels of trimethoprim individually tailored for each library, emphasizing the central role of the host's environment in shaping the codon bias compatibility of horizontally transferred genes.
Asunto(s)
Antibacterianos , Trimetoprim , Antibacterianos/farmacología , Codón , ARN Mensajero , Farmacorresistencia Microbiana/genética , Trimetoprim/farmacologíaRESUMEN
Natural product methyltransferases (NPMTs) represent an emerging class of enzymes that can be of great use for the structural and functional diversification of bioactive compounds, such as the strategic modification of C-, N-, O- and S-moieties. To assess the activity and the substrate scope of the ever-expanding repertoire of NPMTs, a simple, fast, and robust assay is needed. Here, we report a continuous spectroscopic assay, in which S-adenosyl-L-methionine-dependent methylation is linked to NADH oxidation through the coupled activities of S-adenosyl-L-homocysteine (SAH) deaminase and glutamate dehydrogenase. The assay is highly suitable for a high-throughput evaluation of small molecule methylation and for determining the catalytic parameters of NPMTs under conditions that remove the potent inhibition by SAH. Through the modular design, the assay can be extended to match the needs of different aspects of methyltransferase cascade reactions and respective applications.
Asunto(s)
Productos Biológicos , Metiltransferasas , Ensayos Analíticos de Alto Rendimiento , Metilación , Metiltransferasas/metabolismo , S-Adenosilhomocisteína/metabolismo , S-Adenosilmetionina/metabolismoRESUMEN
The relationship between sequence variation and phenotype is poorly understood. Here, we use metabolomic analysis to elucidate the molecular mechanism underlying the filamentous phenotype of E. coli strains that carry destabilizing mutations in dihydrofolate reductase (DHFR). We find that partial loss of DHFR activity causes reversible filamentation despite SOS response indicative of DNA damage, in contrast to thymineless death (TLD) achieved by complete inhibition of DHFR activity by high concentrations of antibiotic trimethoprim. This phenotype is triggered by a disproportionate drop in intracellular dTTP, which could not be explained by drop in dTMP based on the Michaelis-Menten-like in vitro activity curve of thymidylate kinase (Tmk), a downstream enzyme that phosphorylates dTMP to dTDP. Instead, we show that a highly cooperative (Hill coefficient 2.5) in vivo activity of Tmk is the cause of suboptimal dTTP levels. dTMP supplementation rescues filamentation and restores in vivo Tmk kinetics to Michaelis-Menten. Overall, this study highlights the important role of cellular environment in sculpting enzymatic kinetics with system-level implications for bacterial phenotype.
Asunto(s)
Escherichia coli , Mutación Puntual , Escherichia coli/genética , FenotipoRESUMEN
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: overexpression 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.
Asunto(s)
Escherichia coli/genética , Mutación Missense , Pliegue de Proteína , Tetrahidrofolato Deshidrogenasa/metabolismo , Algoritmos , Secuencias de Aminoácidos , Secuencia de Bases , Chaperonina 10/genética , Chaperonina 10/metabolismo , Chaperonina 60/genética , Chaperonina 60/metabolismo , Escherichia coli/enzimología , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Técnicas de Inactivación de Genes , Aptitud Genética , Homeostasis , Cinética , Viabilidad Microbiana , Modelos Biológicos , Datos de Secuencia Molecular , Proteasa La/genética , Proteasa La/metabolismo , Biosíntesis de Proteínas , Proteolisis , Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/genéticaRESUMEN
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.
Asunto(s)
Farmacorresistencia Bacteriana/genética , Escherichia coli/efectos de los fármacos , Tetrahidrofolato Deshidrogenasa/genética , Trimetoprim/farmacología , Secuencia de Aminoácidos , Biofisica/métodos , Chlamydia muridarum/genética , Evolución Molecular Dirigida , Estabilidad de Enzimas/genética , Epistasis Genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Concentración 50 Inhibidora , Listeria/genética , Datos de Secuencia Molecular , Mutación , Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/metabolismo , Trimetoprim/metabolismoRESUMEN
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 (kcat/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.
Asunto(s)
Evolución Molecular , Transferencia de Gen Horizontal/genética , Filogenia , Tetrahidrofolato Deshidrogenasa/genética , Secuencia de Aminoácidos/genética , Escherichia coli/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Homeostasis/genética , Mutación , Especificidad de la EspecieRESUMEN
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.
Asunto(s)
Proteínas de Escherichia coli/genética , Escherichia coli/enzimología , Escherichia coli/genética , Aptitud Genética , Mutación/genética , Tetrahidrofolato Deshidrogenasa/genética , Cromosomas Bacterianos/genética , Estabilidad de Enzimas , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/metabolismo , Estructura Cuaternaria de Proteína , Solubilidad , TemperaturaRESUMEN
Enzymes of the central metabolism tend to assemble into transient supramolecular complexes. However, the functional significance of the interactions, particularly between enzymes catalyzing non-consecutive reactions, remains unclear. Here, by co-localizing two non-consecutive enzymes of the TCA cycle from Bacillus subtilis, malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICD), in phase separated droplets we show that MDH-ICD interaction leads to enzyme agglomeration with a concomitant enhancement of ICD catalytic rate and an apparent sequestration of its reaction product, 2-oxoglutarate. Theory demonstrates that MDH-mediated clustering of ICD molecules explains the observed phenomena. In vivo analyses reveal that MDH overexpression leads to accumulation of 2-oxoglutarate and reduction of fluxes flowing through both the catabolic and anabolic branches of the carbon-nitrogen intersection occupied by 2-oxoglutarate, resulting in impeded ammonium assimilation and reduced biomass production. Our findings suggest that the MDH-ICD interaction is an important coordinator of carbon-nitrogen metabolism.
Asunto(s)
Bacillus subtilis , Carbono , Ciclo del Ácido Cítrico , Isocitrato Deshidrogenasa , Ácidos Cetoglutáricos , Malato Deshidrogenasa , Nitrógeno , Nitrógeno/metabolismo , Carbono/metabolismo , Malato Deshidrogenasa/metabolismo , Malato Deshidrogenasa/genética , Bacillus subtilis/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/enzimología , Isocitrato Deshidrogenasa/metabolismo , Isocitrato Deshidrogenasa/genética , Ácidos Cetoglutáricos/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Compuestos de Amonio/metabolismoRESUMEN
The distribution of fitness effects of protein mutations is still unknown. Of particular interest is whether accumulating deleterious mutations interact, and how the resulting epistatic effects shape the protein's fitness landscape. Here we apply a model system in which bacterial fitness correlates with the enzymatic activity of TEM-1 beta-lactamase (antibiotic degradation). Subjecting TEM-1 to random mutational drift and purifying selection (to purge deleterious mutations) produced changes in its fitness landscape indicative of negative epistasis; that is, the combined deleterious effects of mutations were, on average, larger than expected from the multiplication of their individual effects. As observed in computational systems, negative epistasis was tightly associated with higher tolerance to mutations (robustness). Thus, under a low selection pressure, a large fraction of mutations was initially tolerated (high robustness), but as mutations accumulated, their fitness toll increased, resulting in the observed negative epistasis. These findings, supported by FoldX stability computations of the mutational effects, prompt a new model in which the mutational robustness (or neutrality) observed in proteins, and other biological systems, is due primarily to a stability margin, or threshold, that buffers the deleterious physico-chemical effects of mutations on fitness. Threshold robustness is inherently epistatic-once the stability threshold is exhausted, the deleterious effects of mutations become fully pronounced, thereby making proteins far less robust than generally assumed.
Asunto(s)
Epistasis Genética , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Flujo Genético , Mutación/genética , Selección Genética , beta-Lactamasas/químicaRESUMEN
Homomers are prevalent in bacterial proteomes, particularly among core metabolic enzymes. Homomerization is often key to function and regulation, and interfaces that facilitate the formation of homomeric enzymes are subject to intense evolutionary change. However, our understanding of the molecular mechanisms that drive evolutionary variation in homomeric complexes is still lacking. How is the diversification of protein interfaces linked to variation in functional regulation and structural integrity of homomeric complexes? To address this question, we studied quaternary structure evolution of bacterial methionine S-adenosyltransferases (MATs)-dihedral homotetramers formed along a large and conserved dimeric interface harboring two active sites, and a small, recently evolved, interdimeric interface. Here, we show that diversity in the physicochemical properties of small interfaces is directly linked to variability in the kinetic stability of MAT quaternary complexes and in modes of their functional regulation. Specifically, hydrophobic interactions within the small interface of Escherichia coli MAT render the functional homotetramer kinetically stable yet impose severe aggregation constraints on complex assembly. These constraints are alleviated by electrostatic interactions that accelerate dimer-dimer assembly. In contrast, Neisseria gonorrhoeae MAT adopts a nonfunctional dimeric state due to the low hydrophobicity of its small interface and the high flexibility of its active site loops, which perturbs small interface integrity. Remarkably, in the presence of methionine and ATP, N. gonorrhoeae MAT undergoes substrate-induced assembly into a functional tetrameric state. We suggest that evolution acts on the interdimeric interfaces of MATs to tailor the regulation of their activity and stability to unique organismal needs.
Asunto(s)
Metionina Adenosiltransferasa , Proteínas , Dominio Catalítico , Escherichia coli/metabolismo , Metionina , Metionina Adenosiltransferasa/química , Metionina Adenosiltransferasa/genética , Metionina Adenosiltransferasa/metabolismo , Modelos Moleculares , Proteínas/química , Relación Estructura-ActividadRESUMEN
S-Adenosylmethionine lyase (SAMase) of bacteriophage T3 degrades the intracellular SAM pools of the host Escherichia coli cells, thereby inactivating a crucial metabolite involved in a plethora of cellular functions, including DNA methylation. SAMase is the first viral protein expressed upon infection, and its activity prevents methylation of the T3 genome. Maintenance of the phage genome in a fully unmethylated state has a profound effect on the infection strategy. It allows T3 to shift from a lytic infection under normal growth conditions to a transient lysogenic infection under glucose starvation. Using single-particle cryoelectron microscopy (cryo-EM) and biochemical assays, we demonstrate that SAMase performs its function by not only degrading SAM but also by interacting with and efficiently inhibiting the host's methionine S-adenosyltransferase (MAT), the enzyme that produces SAM. Specifically, SAMase triggers open-ended head-to-tail assembly of E. coli MAT into an unusual linear filamentous structure in which adjacent MAT tetramers are joined by two SAMase dimers. Molecular dynamics simulations together with normal mode analyses suggest that the entrapment of MAT tetramers within filaments leads to an allosteric inhibition of MAT activity due to a shift to low-frequency, high-amplitude active-site-deforming modes. The amplification of uncorrelated motions between active-site residues weakens MAT's substrate binding affinity, providing a possible explanation for the observed loss of function. We propose that the dual function of SAMase as an enzyme that degrades SAM and as an inhibitor of MAT activity has emerged to achieve an efficient depletion of the intracellular SAM pools. IMPORTANCE Self-assembly of enzymes into filamentous structures in response to specific metabolic cues has recently emerged as a widespread strategy of metabolic regulation. In many instances, filamentation of metabolic enzymes occurs in response to starvation and leads to functional inactivation. Here, we report that bacteriophage T3 modulates the metabolism of the host E. coli cells by recruiting a similar strategy: silencing a central metabolic enzyme by subjecting it to phage-mediated polymerization. This observation points to an intriguing possibility that virus-induced polymerization of the host metabolic enzymes is a common mechanism implemented by viruses to metabolically reprogram and subdue infected cells.
Asunto(s)
Bacteriófago T3/enzimología , Escherichia coli/enzimología , Interacciones Microbiota-Huesped , Metionina Adenosiltransferasa/antagonistas & inhibidores , Polímeros/metabolismo , Proteínas Virales/metabolismo , Bacteriófago T3/genética , Microscopía por Crioelectrón , Escherichia coli/genética , Hidrolasas/metabolismo , Lisogenia , Metionina Adenosiltransferasa/genética , Metionina Adenosiltransferasa/metabolismo , Polimerizacion , Polímeros/química , Proteínas Virales/genéticaRESUMEN
Evolutionary dynamics in large asexual populations is strongly influenced by multiple competing beneficial lineages, most of which segregate at very low frequencies. However, technical barriers to tracking a large number of these rare lineages in bacterial populations have so far prevented a detailed elucidation of evolutionary dynamics. Here, we overcome this hurdle by developing a chromosomal-barcoding technique that allows simultaneous tracking of approximately 450,000 distinct lineages in Escherichia coli, which we use to test the effect of sub-inhibitory concentrations of common antibiotics on the evolutionary dynamics of low-frequency lineages. We find that populations lose lineage diversity at distinct rates that correspond to their antibiotic regimen. We also determine that some lineages have similar fates across independent experiments. By analysing the trajectory dynamics, we attribute the reproducible fates of these lineages to the presence of pre-existing beneficial mutations, and we demonstrate how the relative contribution of pre-existing and de novo mutations varies across drug regimens. Finally, we reproduce the observed lineage dynamics by simulations. Altogether, our results provide a valuable methodology for studying bacterial evolution as well as insights into evolution under sub-inhibitory antibiotic levels.
Asunto(s)
Escherichia coli , Evolución Molecular , Antibacterianos , MutaciónRESUMEN
The divergence of new gene functions is described by various scenarios that involve gene duplication, albeit, at fundamentally different stages. We performed experimental measurements and developed a subsequent model, aimed at predicting the rate of divergence under different scenarios. We used gene libraries of TEM-1 beta-lactamase that were drifted under purifying selection toward the original penicillinase activity or under no selection at all. The frequency of genes conferring a new function (degradation of a cephalosporin antibiotic) was measured at various stages of the drift, and a model that accounts for the differences in the observed adaptation dynamics of the drifting TEM-1 populations was derived. The results indicate that rapid nonfunctionalization in the population relieved from selection (Ohno's model) afforded only a narrow window of adaptation to cefotaxime (neofunctionalization). The trade-off between TEM-1's original function and the new evolving function also disfavored the "gene sharing" model. The rate of adaptation was maximal when selection for the original function was partially relieved to enable the accumulation of potentially adaptive mutations, while still purging a large fraction of otherwise deleterious mutations. Altogether, scenarios of subfunctionalization seem more feasible, whereby sustaining the original function by two copies facilitates the accumulation potentially adaptive mutations while purging nonfunctionalization mutations.
Asunto(s)
Adaptación Fisiológica/genética , Modelos Genéticos , Mutación , beta-Lactamasas/genética , Antibacterianos/farmacología , Cefotaxima/farmacología , Farmacorresistencia Bacteriana/genética , Escherichia coli , Duplicación de Gen , Frecuencia de los Genes , Flujo Genético , Supresión GenéticaRESUMEN
We address recent developments in the area of laboratory, or directed evolution, with a focus on enzymes and on new methodologies of generic potential. We survey three main areas: (i) library making techniques, including the application of computational and rational methods for library design; (ii) screening and selection techniques, including recent applications of enzyme screening by FACS (fluorescence activated cell sorter); (iii) new approaches for performing directed evolution, and in particular, the application of 'neutral drifts' (libraries generated by rounds of mutation and selection for the enzyme's original function) and of consensus mutations to generate highly evolvable starting points for directed evolution.
Asunto(s)
Evolución Molecular Dirigida/métodos , Enzimas/genética , Enzimas/metabolismo , Citometría de Flujo , Biblioteca de Genes , MutaciónRESUMEN
Methionine S-adenosyltransferases (MATs) are predominantly homotetramers, comprised of dimers of dimers. The larger, highly conserved intradimeric interface harbors two active sites, making the dimer the obligatory functional unit. However, functionality of the smaller, more diverged, and recently evolved interdimeric interface is largely unknown. Here, we show that the interdimeric interface of Ureaplasmaurealiticum MAT has evolved to control the catalytic activity and structural integrity of the homotetramer in response to product accumulation. When all four active sites are occupied with the product, S-adenosylmethionine (SAM), binding of four additional SAM molecules to the interdimeric interface prompts a â¼45° shift in the dimer orientation and a concomitant â¼60% increase in the interface area. This rearrangement inhibits the enzymatic activity by locking the flexible active site loops in a closed state and renders the tetramer resistant to proteolytic degradation. Our findings suggest that the interdimeric interface of MATs is subject to rapid evolutionary changes that tailor the molecular properties of the entire homotetramer to the specific needs of the organism.
Asunto(s)
Metionina Adenosiltransferasa/química , Multimerización de Proteína , Ureaplasma/enzimología , Sitios de Unión , Espectrometría de Masas , Metionina Adenosiltransferasa/metabolismo , Modelos Moleculares , Conformación Proteica , Estabilidad Proteica , Proteolisis , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Relación Estructura-ActividadRESUMEN
Bridging the gap between the molecular properties of proteins and organismal/population fitness is essential for understanding evolutionary processes. This task requires the integration of the several physical scales of biological organization, each defined by a distinct set of mechanisms and constraints, into a single unifying model. The molecular scale is dominated by the constraints imposed by the physico-chemical properties of proteins and their substrates, which give rise to trade-offs and epistatic (non-additive) effects of mutations. At the systems scale, biological networks modulate protein expression and can either buffer or enhance the fitness effects of mutations. The population scale is influenced by the mutational input, selection regimes, and stochastic changes affecting the size and structure of populations, which eventually determine the evolutionary fate of mutations. Here, we summarize the recent advances in theory, computer simulations, and experiments that advance our understanding of the links between various physical scales in biology.
Asunto(s)
Biofisica/métodos , Evolución Molecular , Proteínas/química , Proteínas/metabolismo , Secuencia de Aminoácidos , Genética de Población , Proteínas/genéticaRESUMEN
Gene dosage toxicity (GDT) is an important factor that determines optimal levels of protein abundances, yet its molecular underpinnings remain unknown. Here, we demonstrate that overexpression of DHFR in E. coli causes a toxic metabolic imbalance triggered by interactions with several functionally related enzymes. Though deleterious in the overexpression regime, surprisingly, these interactions are beneficial at physiological concentrations, implying their functional significance in vivo. Moreover, we found that overexpression of orthologous DHFR proteins had minimal effect on all levels of cellular organization - molecular, systems, and phenotypic, in sharp contrast to E. coli DHFR. Dramatic difference of GDT between 'E. coli's self' and 'foreign' proteins suggests the crucial role of evolutionary selection in shaping protein-protein interaction (PPI) networks at the whole proteome level. This study shows how protein overexpression perturbs a dynamic metabolon of weak yet potentially functional PPI, with consequences for the metabolic state of cells and their fitness.
Asunto(s)
Proteínas de Escherichia coli/toxicidad , Escherichia coli/metabolismo , Dosificación de Gen , Proteínas Recombinantes/toxicidad , Tetrahidrofolato Deshidrogenasa/toxicidad , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Metaboloma , Unión Proteica , Mapas de Interacción de Proteínas , Proteínas Recombinantes/metabolismo , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
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 2,000+ 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 SDs of the distributions of logarithms of relative (to WT) protein abundances anticorrelate 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.