RESUMEN
CRISPR-Cas-based genome editing is widely used in bacteria at scales ranging from construction of individual mutants to massively parallel libraries. This procedure relies on guide RNA-directed cleavage of the genome followed by repair with a template that introduces a desired mutation along with synonymous "immunizing" mutations to prevent re-cleavage of the genome after editing. Because the immunizing mutations do not change the protein sequence, they are often assumed to be neutral. However, synonymous mutations can change mRNA structures in ways that alter levels of the encoded proteins. We have tested the assumption that immunizing mutations are neutral by constructing a library of over 50,000 edits that consist of only synonymous mutations in Escherichia coli. Thousands of edits had substantial effects on fitness during growth of E. coli on acetate, a poor carbon source that is toxic at high concentrations. The percentage of high-impact edits varied considerably between genes and at different positions within genes. We reconstructed clones with high-impact edits and found that 69% indeed had significant effects on growth in acetate. Interestingly, fewer edits affected fitness during growth in glucose, a preferred carbon source, suggesting that changes in protein expression caused by synonymous mutations may be most important when an organism encounters challenging conditions. Finally, we showed that synonymous edits can have widespread effects; a synonymous edit at the 5' end of ptsI altered expression of hundreds of genes. Our results suggest that the synonymous immunizing edits introduced during CRISPR-Cas-based genome editing should not be assumed to be innocuous.
Asunto(s)
Escherichia coli , ARN Guía de Sistemas CRISPR-Cas , Escherichia coli/genética , Biblioteca de Genes , Carbono , AcetatosRESUMEN
New enzymes often evolve by duplication and divergence of genes encoding enzymes with promiscuous activities that have become important in the face of environmental opportunities or challenges. Amplifications that increase the copy number of the gene under selection commonly amplify many surrounding genes. Extra copies of these coamplified genes must be removed, either during or after evolution of a new enzyme. Here we report that amplicon remodeling can begin even before mutations occur in the gene under selection. Amplicon remodeling and mutations elsewhere in the genome that indirectly increase fitness result in complex population dynamics, leading to emergence of clones that have improved fitness by different mechanisms. In this work, one of the two most successful clones had undergone two episodes of amplicon remodeling, leaving only four coamplified genes surrounding the gene under selection. Amplicon remodeling in the other clone resulted in removal of 111 genes from the genome, an acceptable solution under these selection conditions, but one that would certainly impair fitness under other environmental conditions.
Asunto(s)
Amplificación de Genes , Genómica , Mutación , Dinámica PoblacionalRESUMEN
PdxB (erythronate 4-phosphate dehydrogenase) is expected to be required for synthesis of the essential cofactor pyridoxal 5'-phosphate (PLP) in Escherichia coli Surprisingly, incubation of the ∆pdxB strain in medium containing glucose as a sole carbon source for 10 d resulted in visible turbidity, suggesting that PLP is being produced by some alternative pathway. Continued evolution of parallel lineages for 110 to 150 generations produced several strains that grow robustly in glucose. We identified a 4-step bypass pathway patched together from promiscuous enzymes that restores PLP synthesis in strain JK1. None of the mutations in JK1 occurs in a gene encoding an enzyme in the new pathway. Two mutations indirectly enhance the ability of SerA (3-phosphoglycerate dehydrogenase) to perform a new function in the bypass pathway. Another disrupts a gene encoding a PLP phosphatase, thus preserving PLP levels. These results demonstrate that a functional pathway can be patched together from promiscuous enzymes in the proteome, even without mutations in the genes encoding those enzymes.
Asunto(s)
Deshidrogenasas de Carbohidratos/genética , Proteínas de Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/genética , Genoma Bacteriano , Fosfato de Piridoxal/biosíntesis , Deshidrogenasas de Carbohidratos/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Evolución Molecular Dirigida/métodos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Esenciales , Glucosa/metabolismo , Redes y Vías Metabólicas/genética , Microorganismos Modificados Genéticamente , Mutación , Fosfato de Piridoxal/genéticaRESUMEN
Synonymous mutations do not alter the specified amino acid but may alter the structure or function of an mRNA in ways that impact fitness. There are few examples in the literature, however, in which the effects of synonymous mutations on microbial growth rates have been measured, and even fewer for which the underlying mechanism is understood. We evolved four populations of a strain of Salmonella enterica in which a promiscuous enzyme has been recruited to replace an essential enzyme. A previously identified point mutation increases the enzyme's ability to catalyze the newly needed reaction (required for arginine biosynthesis) but decreases its ability to catalyze its native reaction (required for proline biosynthesis). The poor performance of this enzyme limits growth rate on glucose. After 260 generations, we identified two synonymous mutations in the first six codons of the gene encoding the weak-link enzyme that increase growth rate by 41 and 67%. We introduced all possible synonymous mutations into the first six codons and found substantial effects on growth rate; one doubles growth rate, and another completely abolishes growth. Computational analyses suggest that these mutations affect either the stability of a stem-loop structure that sequesters the start codon or the accessibility of the region between the Shine-Dalgarno sequence and the start codon. Thus, these mutations would be predicted to affect translational efficiency and thereby indirectly affect mRNA stability because translating ribosomes protect mRNA from degradation. Experimental data support these hypotheses. We conclude that the effects of the synonymous mutations are due to a combination of effects on mRNA stability and translation efficiency that alter levels of the weak-link enzyme. These findings suggest that synonymous mutations can have profound effects on fitness under strong selection and that their importance in evolution may be under-appreciated.
Asunto(s)
Proteínas Bacterianas/genética , Aptitud Genética , ARN Mensajero/genética , Salmonella enterica/crecimiento & desarrollo , Mutación Silenciosa , Codón , Variaciones en el Número de Copia de ADN , Evolución Molecular , Conformación de Ácido Nucleico , Operón , Fosfotransferasas (aceptor de Grupo Carboxilo)/genética , Fosfotransferasas (aceptor de Grupo Carboxilo)/metabolismo , Mutación Puntual , Proteómica , Estabilidad del ARN , Ribosomas/genética , Salmonella enterica/genética , Secuenciación Completa del GenomaRESUMEN
UNLABELLED: When microbes are faced with an environmental challenge or opportunity, preexisting enzymes with promiscuous secondary activities can be recruited to provide newly important functions. Mutations that increase the efficiency of a new activity often compromise the original activity, resulting in an inefficient bifunctional enzyme. We have investigated the mechanisms by which growth of Escherichia coli can be improved when fitness is limited by such an enzyme, E383A ProA (ProA*). ProA* can serve the functions of both ProA (required for synthesis of proline) and ArgC (required for synthesis of arginine), albeit poorly. We identified four genetic changes that improve the growth rate by up to 6.2-fold. Two point mutations in the promoter of the proBA* operon increase expression of the entire operon. Massive amplification of a genomic segment around the proBA* operon also increases expression of the entire operon. Finally, a synonymous point mutation in the coding region of proB creates a new promoter for proA* This synonymous mutation increases the level of ProA* by 2-fold but increases the growth rate by 5-fold, an ultrasensitive response likely arising from competition between two substrates for the active site of the inefficient bifunctional ProA*. IMPORTANCE: The high-impact synonymous mutation we discovered in proB is remarkable for two reasons. First, most polar effects documented in the literature are detrimental. This finding demonstrates that polar effect mutations can have strongly beneficial effects, especially when an organism is facing a difficult environmental challenge for which it is poorly adapted. Furthermore, the consequence of the synonymous mutation in proB is a 2-fold increase in the level of ProA* but a disproportionately large 5.1-fold increase in growth rate. While ultrasensitive responses are often found in signaling networks and genetic circuits, an ultrasensitive response to an adaptive mutation has not been previously reported.
Asunto(s)
Proteínas de Escherichia coli/genética , Escherichia coli/enzimología , Escherichia coli/crecimiento & desarrollo , Glutamato-5-Semialdehído Deshidrogenasa/genética , Fosfotransferasas (aceptor de Grupo Carboxilo)/genética , Secuencia de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Glutamato-5-Semialdehído Deshidrogenasa/metabolismo , Cinética , Datos de Secuencia Molecular , Mutación Missense , Operón , Fosfotransferasas (aceptor de Grupo Carboxilo)/metabolismo , Mutación Puntual , Regiones Promotoras GenéticasRESUMEN
In E. coli, editing efficiency with Cas9-mediated recombineering varies across targets due to differences in the level of Cas9:gRNA-mediated DNA double-strand break (DSB)-induced cell death. We found that editing efficiency with the same gRNA and repair template can also change with target position, cas9 promoter strength, and growth conditions. Incomplete editing, off-target activity, nontargeted mutations, and failure to cleave target DNA even if Cas9 is bound also compromise editing efficiency. These effects on editing efficiency were gRNA-specific. We propose that differences in the efficiency of Cas9:gRNA-mediated DNA DSBs, as well as possible differences in binding of Cas9:gRNA complexes to their target sites, account for the observed variations in editing efficiency between gRNAs. We show that editing behavior using the same gRNA can be modified by mutating the gRNA spacer, which changes the DNA DSB activity. Finally, we discuss how variable editing with different gRNAs could limit high-throughput applications and provide strategies to overcome these limitations.
Asunto(s)
Sistemas CRISPR-Cas/genética , Escherichia coli/genética , Edición Génica/métodos , Roturas del ADN de Doble Cadena , Escherichia coli/metabolismo , Galactoquinasa/genética , Mutación , Regiones Promotoras Genéticas , ARN Guía de Kinetoplastida/metabolismoRESUMEN
New enzymes often evolve by gene amplification and divergence. Previous experimental studies have followed the evolutionary trajectory of an amplified gene, but have not considered mutations elsewhere in the genome when fitness is limited by an evolving gene. We have evolved a strain of Escherichia coli in which a secondary promiscuous activity has been recruited to serve an essential function. The gene encoding the 'weak-link' enzyme amplified in all eight populations, but mutations improving the newly needed activity occurred in only one. Most adaptive mutations occurred elsewhere in the genome. Some mutations increase expression of the enzyme upstream of the weak-link enzyme, pushing material through the dysfunctional metabolic pathway. Others enhance production of a co-substrate for a downstream enzyme, thereby pulling material through the pathway. Most of these latter mutations are detrimental in wild-type E. coli, and thus would require reversion or compensation once a sufficient new activity has evolved.