RESUMO
The degeneracy of the genetic code allows nucleic acids to encode amino acid identity as well as noncoding information for gene regulation and genome maintenance. The rare arginine codons AGA and AGG (AGR) present a case study in codon choice, with AGRs encoding important transcriptional and translational properties distinct from the other synonymous alternatives (CGN). We created a strain of Escherichia coli with all 123 instances of AGR codons removed from all essential genes. We readily replaced 110 AGR codons with the synonymous CGU codons, but the remaining 13 "recalcitrant" AGRs required diversification to identify viable alternatives. Successful replacement codons tended to conserve local ribosomal binding site-like motifs and local mRNA secondary structure, sometimes at the expense of amino acid identity. Based on these observations, we empirically defined metrics for a multidimensional "safe replacement zone" (SRZ) within which alternative codons are more likely to be viable. To evaluate synonymous and nonsynonymous alternatives to essential AGRs further, we implemented a CRISPR/Cas9-based method to deplete a diversified population of a wild-type allele, allowing us to evaluate exhaustively the fitness impact of all 64 codon alternatives. Using this method, we confirmed the relevance of the SRZ by tracking codon fitness over time in 14 different genes, finding that codons that fall outside the SRZ are rapidly depleted from a growing population. Our unbiased and systematic strategy for identifying unpredicted design flaws in synthetic genomes and for elucidating rules governing codon choice will be crucial for designing genomes exhibiting radically altered genetic codes.
Assuntos
Arginina/genética , Escherichia coli/genética , RNA Mensageiro/genética , Aminoácidos/genética , Códon/genética , Genes Essenciais/genética , Código Genético , Genoma Bacteriano , Biossíntese de Proteínas/genética , RNA Mensageiro/biossínteseRESUMO
Selection has been invaluable for genetic manipulation, although counter-selection has historically exhibited limited robustness and convenience. TolC, an outer membrane pore involved in transmembrane transport in E. coli, has been implemented as a selectable/counter-selectable marker, but counter-selection escape frequency using colicin E1 precludes using tolC for inefficient genetic manipulations and/or with large libraries. Here, we leveraged unbiased deep sequencing of 96 independent lineages exhibiting counter-selection escape to identify loss-of-function mutations, which offered mechanistic insight and guided strain engineering to reduce counter-selection escape frequency by â¼40-fold. We fundamentally improved the tolC counter-selection by supplementing a second agent, vancomycin, which reduces counter-selection escape by 425-fold, compared colicin E1 alone. Combining these improvements in a mismatch repair proficient strain reduced counter-selection escape frequency by 1.3E6-fold in total, making tolC counter-selection as effective as most selectable markers, and adding a valuable tool to the genome editing toolbox. These improvements permitted us to perform stable and continuous rounds of selection/counter-selection using tolC, enabling replacement of 10 alleles without requiring genotypic screening for the first time. Finally, we combined these advances to create an optimized E. coli strain for genome engineering that is â¼10-fold more efficient at achieving allelic diversity than previous best practices.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Engenharia Genética/métodos , Proteínas de Membrana Transportadoras/genética , Alelos , Biomarcadores , Escherichia coli/genética , Deleção de Genes , Duplicação Gênica , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , FenótipoRESUMO
Previous studies showed that deletion of genes c3405 to c3410 from PAI-metV, a genomic island from Escherichia coli CFT073, results in a strain that fails to compete with wild-type CFT073 after a transurethral cochallenge in mice and is deficient in the ability to independently colonize the mouse kidney. Our analysis of c3405 to c3410 suggests that these genes constitute an operon with a role in the internalization and utilization of an unknown carbohydrate. This operon is not found in E. coli K-12 but is present in a small number of pathogenic E. coli and Shigella boydii strains. One of the genes, c3406, encodes a protein with significant homology to the sugar isomerase domain of arabinose 5-phosphate isomerases but lacking the tandem cystathionine beta-synthase domains found in the other arabinose 5-phosphate isomerases of E. coli. We prepared recombinant c3406 protein, found it to possess arabinose 5-phosphate isomerase activity, and characterized this activity in detail. We also constructed a c3406 deletion mutant of E. coli CFT073 and demonstrated that this deletion mutant was still able to compete with wild-type CFT073 in a transurethral cochallenge in mice and could colonize the mouse kidney. These results demonstrate that the presence of c3406 is not essential for a pathogenic phenotype.
Assuntos
Aldose-Cetose Isomerases/metabolismo , Escherichia coli/enzimologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Ilhas Genômicas/genética , Escherichia coli Uropatogênica/enzimologia , Aldose-Cetose Isomerases/genética , Sequência de Aminoácidos , Animais , Cistite/microbiologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Nefropatias/microbiologia , Camundongos , Dados de Sequência Molecular , Mutação , Escherichia coli Uropatogênica/genéticaRESUMO
We describe the construction and characterization of a genomically recoded organism (GRO). We replaced all known UAG stop codons in Escherichia coli MG1655 with synonymous UAA codons, which permitted the deletion of release factor 1 and reassignment of UAG translation function. This GRO exhibited improved properties for incorporation of nonstandard amino acids that expand the chemical diversity of proteins in vivo. The GRO also exhibited increased resistance to T7 bacteriophage, demonstrating that new genetic codes could enable increased viral resistance.
Assuntos
Aminoácidos/genética , Bacteriófago T7/fisiologia , Códon de Terminação/genética , Escherichia coli/genética , Escherichia coli/virologia , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/virologia , Substituição de Aminoácidos/genética , Proteínas de Escherichia coli/genética , Engenharia Genética , Genoma Bacteriano , Terminação Traducional da Cadeia Peptídica/genética , Fatores de Terminação de Peptídeos/genéticaRESUMO
Lambda Red recombineering is a powerful technique for making targeted genetic changes in bacteria. However, many applications are limited by the frequency of recombination. Previous studies have suggested that endogenous nucleases may hinder recombination by degrading the exogenous DNA used for recombineering. In this work, we identify ExoVII as a nuclease which degrades the ends of single-stranded DNA (ssDNA) oligonucleotides and double-stranded DNA (dsDNA) cassettes. Removing this nuclease improves both recombination frequency and the inheritance of mutations at the 3' ends of ssDNA and dsDNA. Extending this approach, we show that removing a set of five exonucleases (RecJ, ExoI, ExoVII, ExoX, and Lambda Exo) substantially improves the performance of co-selection multiplex automatable genome engineering (CoS-MAGE). In a given round of CoS-MAGE with ten ssDNA oligonucleotides, the five nuclease knockout strain has on average 46% more alleles converted per clone, 200% more clones with five or more allele conversions, and 35% fewer clones without any allele conversions. Finally, we use these nuclease knockout strains to investigate and clarify the effects of oligonucleotide phosphorothioation on recombination frequency. The results described in this work provide further mechanistic insight into recombineering, and substantially improve recombineering performance.
Assuntos
Bacteriófago lambda/genética , Escherichia coli/genética , Engenharia Genética/métodos , Recombinação Genética , Alelos , Segregação de Cromossomos/genética , Primers do DNA/genética , DNA de Cadeia Simples/genética , Escherichia coli/virologia , Exonucleases/metabolismo , Técnicas Genéticas , Genótipo , Oligonucleotídeos/genética , Oligonucleotídeos Fosforotioatos/genéticaRESUMO
Contaminated drinking water is responsible for causing diarrheal diseases that kill millions of people a year. Additionally, toxin-producing blue-green algae associated with diarrhea and neurologic effects continues to be an issue for many drinking water supplies. Disinfection has been used to reduce these risks. A novel gravity-fed household drinking water system with canisters containing N-halamine bromine or chlorine media was challenged with MS2 bacteriophage and microcystin. Chlorine and bromine systems were effective against this virus, with an mean +/- SE reduction of 2.98 +/- 0.26 log(10) and 5.02 +/- 0.19 log(10), respectively. Microcystin toxin was reduced by 27.5% and 88.5% to overall mean +/- SE concentrations of 1,600 +/- 98 ng/L and 259 +/- 50 ng/L for the chlorine and bromine canisters, respectively. Only the bromine units consistently produced microcystin effluent < 1,000 ng/L (the World Health Organization recommended level) when challenged with 2,500 ng/L and consistently surpassed the U.S. Environmental Protection Agency virus reduction goal of 99.99%.