Mutations are central to evolution, providing the genetic variation upon which selection acts. A mutation’s effect on the suitability of a gene to perform a particular function (gene fitness) can be positive, negative, or neutral. Knowledge of the distribution of fitness effects (DFE) of mutations is fundamental for understanding evolutionary dynamics, molecular-level genetic variation, complex genetic disease, the accumulation of deleterious mutations, and the molecular clock. We present comprehensive DFEs for point and codon mutants of the Escherichia coli TEM-1 β-lactamase gene and missense mutations in the TEM-1 protein. These DFEs provide insight into the inherent benefits of the genetic code’s architecture, support for the hypothesis that mRNA stability dictates codon usage at the beginning of genes, an extensive framework for understanding protein mutational tolerance, and evidence that mutational effects on protein thermodynamic stability shape the DFE. Contrary to prevailing expectations, we find that deleterious effects of mutation primarily arise from a decrease in specific protein activity and not cellular protein levels.

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We introduce PFunkel, a versatile method for extensive, researcher-defined DNA mutagenesis using a ssDNA or dsDNA template. Once the template DNA is prepared, the method can be completed in a single day in a single tube, and requires no intermediate DNA purification or sub-cloning. PFunkel can be used for site-directed mutagenesis at an efficiency approaching 100%. More importantly, PFunkel allows researchers the unparalleled ability to efficiently construct user-defined libraries. We demonstrate the creation of a library with site-saturation at four distal sites simultaneously at 70% efficiency. We also employ PFunkel to create a comprehensive codon mutagenesis library of the TEM-1 ß-lactamase gene. We designed this library to contain 18,081 members, one for each possible codon substitution in the gene (287 positions in TEM-1 x 63 possible codon substitutions). Deep sequencing revealed that ∼97% of the designed single codon substitutions are present in the library. From such a library we identified 18 previously unreported adaptive mutations that each confer resistance to the ß-lactamase inhibitor tazobactam. Three of these mutations confer resistance equal to or higher than that of the most resistant reported TEM-1 allele and have the potential to emerge clinically.

An important goal of evolutionary biology is to understand the constraints that shape the dynamics and outcomes of evolution. Here, we address the extent to which the structure of the standard genetic code constrains evolution by analyzing adaptive mutations of the antibiotic resistance gene TEM-1 β-lactamase and the fitness distribution of codon substitutions in two influenza hemagglutinin inhibitor genes. We find that the architecture of the genetic code significantly constrains the adaptive exploration of sequence space. However, the constraints endow the code with two advantages: the ability to restrict access to amino acid mutations with a strong negative effect and, most remarkably, the ability to enrich for adaptive mutations. Our findings support the hypothesis that the standard genetic code was shaped by selective pressure to minimize the deleterious effects of mutation yet facilitate the evolution of proteins through imposing an adaptive mutation bias.

Background Recurrent Clostridioides diffícile infection (RCDI) is associated with major bacterial dysbiosis and colitis. Fecal microbiota transplantation (FMT) is a highly effective therapeutic modality for RCDI. While several studies have identified bacterial species associated with resolution of symptoms in patients, characterization of the fecal microbiome at the bacterial strain level in RCDI patients before and after FMT and healthy donors, has been lacking. The aim of this study was to examine the ability of bacterial strains from healthy donors to engraft in the gastrointestinal tract of patients with RCDI following FMT. Methods Fecal samples were collected from 22 patients with RCDI before and after FMT and their corresponding healthy donors. Total DNA was extracted from each sample and analyzed by shotgun metagenomic sequencing. The Cosmos-ID analysis platform was used for taxonomic assignment of sequences and calculation of the relative abundance (RA) of bacterial species and strains. From these data, the total number of bacterial strains (BSI), Shannon diversity index, dysbiosis index (DI), and bacterial engraftment factor, were calculated for each strain. Findings A marked reduction (p<0·0001) in the RA of total and specific bacterial strains, especially from phylum Firmicutes, was observed in RCDI patients prior to FMT. This change was associated with an increase in the DI (p<0·0001) and in pathobiont bacterial strains from phylum Proteobacteria, such as Escherichia coli O157:H7 and Klebsiella pneumoniae UCI 34. BSI was significantly lower in this group of patients as compared to healthy donors and correlated with the Shannon Index. (p<0·0001). Identification and engraftment of bacterial strains from healthy donors revealed a greater diversity and higher relative abundance of short-chain fatty acid (SCFA)-producing bacterial strains, including Lachnospiraceae bacterium 5_1_63FAA_u_t, Dorea formicigenerans ATCC 27755, Anaerostipes hadrusand others, in RCDI patients after FMT. Interpretation These observations identify a group of SCFA-producing bacterial strains from healthy donors that engraft well in patients with RCDI following FMT and are associated with complete resolution of clinical symptoms and bacterial dysbiosis.