Biology subjects

Copley, S. D.

Publications and source records attributed to Copley, S. D..

4 recordsLinked to original sources

bioRxiv2025

A cheater founds the winning lineages during evolution of a novel metabolic pathway

Underground metabolic pathways--leaks in the metabolic network caused by promiscuous enzyme activities and non-enzymatic transformations--can provide the starting point for emergence of novel protopathways if a mutation or environmental change increases flux to a physiologically significant level. This early stage in the evolution of metabolic pathways is typically hidden from our view. We have evolved a novel protopathway in {Delta}pdxB E. coli, which lacks an enzyme required for synthesis of the essential cofactor pyridoxal 5'-phosphate (PLP). This protopathway is comprised of four steps catalyzed by promiscuous enzymes that are still serving their native functions. Complex population dynamics occurred during the evolution experiment. The dominant strain after 150 population doublings, JK1, had acquired four mutations. We constructed every intermediate between the {Delta}pdxB strain and JK1 and identified the order in which mutations arose in JK1 and the physiological effect of each. Three of the mutations together increased the PLP accumulation rate by 32-fold. The second mutation created a cheater that was less fit on its own but thrived in the population by scavenging nutrients released from the fragile parental cells. Notably, the dominant lineages at the end of the experiment all derived from this cheater strain.

evolutionary biology↗

bioRxiv2024

CRISPR-Cas9-assisted genome editing in E. coli elevates the frequency of unintended mutations

Cas-assisted lambda Red recombineering techniques have rapidly become a mainstay of bacterial genome editing. Such techniques have been used to construct both individual mutants and massive libraries to assess the effects of genomic changes. We have found that a commonly used Cas9-assisted editing method results in unintended mutations elsewhere in the genome in 26% of edited clones. The unintended mutations are frequently found over 200 kb from the intended edit site and even over 10 kb from potential off-target sites. We attribute the high frequency of unintended mutations to error-prone polymerases expressed in response to dsDNA breaks introduced at the edit site. Most unintended mutations occur in regulatory or coding regions and thus may have phenotypic effects. Our findings highlight the risks associated with genome editing techniques involving dsDNA breaks in E. coli and likely other bacteria and emphasize the importance of sequencing the genomes of edited cells to ensure the absence of unintended mutations. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/584922v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@476263org.highwire.dtl.DTLVardef@8c891eorg.highwire.dtl.DTLVardef@7e32b4org.highwire.dtl.DTLVardef@132d3fc_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

The transcriptomic and proteomic ramifications of segmental amplification

Gene amplification can drive adaptation by rapidly increasing the cellular dosage of critical gene products. Segmental amplifications often encompass large genomic regions surrounding the gene(s) under selection for higher dosage. Overexpression of co-amplified neighboring genes imposes a substantial metabolic burden. While compensatory mutations can decrease inappropriate overexpression of co-amplified genes, it takes time for such mutations to arise. The extent to which intrinsic regulatory mechanisms modulate expression of co-amplified genes in the immediate aftermath of segmental amplification is largely unknown. To address the collateral effects of segmental amplification, we evolved replicate cultures of an Escherichia coli mutant under conditions that select for higher dosage of an inefficient enzyme whose weak activity limits growth rate. Segmental amplifications encompassing the gene encoding the weak-link enzyme arose in all populations. Amplicons ranged in size (9 to 125 kb) and copy number (2 to 12 copies). We performed RNA-seq and label-free proteomics to quantify expression of amplified genes present at 2, 6, and 12 copies. mRNA expression generally scales with gene copy number, but protein expression scales less well with both gene copy number and mRNA expression. We characterize the molecular mechanisms underlying discrepancies between gene copy number and expression for several cases. We also show that segmental amplifications can have system-wide consequences by indirectly altering expression of non-amplified genes. Our findings indicate that the fitness benefit derived from segmental amplification depends on the combined effects of amplification size, gene content, and copy number as well as collateral effects on non-amplified genes. Significance StatementGene amplification frequently drives rapid adaptation when organisms are challenged by harsh conditions. However, gene amplification rarely amplifies only the gene(s) under selection for higher dosage. Segmental amplifications often include many co-amplified neighbor genes. Because segmental amplifications can reach very high copy number, overexpression of co-amplified genes can impose an energetic burden and perturb physiology, yet this aspect of gene amplification has received little attention. Here, we performed transcriptomic and proteomic analysis of laboratory-evolved Escherichia coli strains containing large, high-copy-number amplifications. We found that mRNA levels, but often not protein levels, scale well with gene copy number. We identified amplified genes that exhibit discrepancies between gene copy number and mRNA/protein expression and examine the molecular mechanisms underlying these discrepancies.

evolutionary biology↗

bioRxiv2019

Mutations that improve the efficiency of a weak-link enzyme are rare compared to adaptive mutations elsewhere in the genome

New enzymes often evolve by amplification and divergence of genes encoding enzymes with a weak ability to provide a new function. Experimental studies to date have followed the evolutionary trajectory of an amplified gene, but have not addressed other mutations in the genome when fitness is limited by an evolving gene. We have adapted Escherichia coli in which an enzymes weak secondary activity has been recruited to serve an essential function. While the gene encoding the \"weak-link\" enzyme amplified in all eight populations, mutations improving the new activity occurred in only one. This beneficial allele quickly swept the amplified array, displacing the parental allele. Most adaptive mutations, however, occurred elsewhere in the genome. We have identified the mechanisms by which three of the classes of mutations increase fitness. These mutations may be detrimental once a new enzyme has evolved, and require reversion or compensation, leading to permanent changes in the genome.

evolutionary biology↗