The evolutionary significance of stress-induced mutagenesis was evaluated by studying mutagenesis in aging colonies (MAC) of Escherichia coli natural isolates. A large fraction of isolates exhibited a strong MAC, and the high MAC variability reflected the diversity of selective pressures in ecological niches. MAC depends on starvation, oxygen, and RpoS and adenosine 3,5-monophosphate regulons; thus it may be a by-product of genetic strategies for improving survival under stress. MAC could also be selected through beneficial mutations that it generates, as shown by computer modeling and the patterns of stressinducible and constitutive mutagenesis. We suggest that irrespective of the causes of their emergence, stress-induced mutations participate in adaptive

http://fire.biol.wwu.edu/cmoyer/zztemp_fire/biol405_F09/papers/Bjedov_stress_muts_sci03.pdf

Stress-Induced Mutagenesis in Bacteria

Knowledge of the rate and nature of spontaneous mutation is fundamental to understanding evolutionary and molecular processes. In this report, we analyze spontaneous mutations accumulated over thousands of generations by wild-type Escherichia coli and a derivative defective in mismatch repair (MMR), the primary pathway for correcting replication errors. The major conclusions are (i) the mutation rate of a wild-type E. coli strain is ∼1 × 10−3 per genome per generation; (ii) mutations in the wild-type strain have the expected mutational bias for G:C > A:T mutations, but the bias changes to A:T > G:C mutations in the absence of MMR; (iii) during replication, A:T > G:C transitions preferentially occur with A templating the lagging strand and T templating the leading strand, whereas G:C > A:T transitions preferentially occur with C templating the lagging strand and G templating the leading strand; (iv) there is a strong bias for transition mutations to occur at 5′ApC3′/3′TpG5′ sites (where bases 5′A and 3′T are mutated) and, to a lesser extent, at 5′GpC3′/3′CpG5′ sites (where bases 5′G and 3′C are mutated); (v) although the rate of small (≤4 nt) insertions and deletions is high at repeat sequences, these events occur at only 1/10th the genomic rate of base-pair substitutions. MMR activity is genetically regulated, and bacteria isolated from nature often lack MMR capacity, suggesting that modulation of MMR can be adaptive. Thus, comparing results from the wild-type and MMR-defective strains may lead to a deeper understanding of factors that determine mutation rates and spectra, how these factors may differ among organisms, and how they may be shaped by environmental conditions.

https://www.pnas.org/content/pnas/109/41/E2774.full.pdf

Rate and molecular spectrum of spontaneous mutations in the bacterium Escherichia coli as determined by whole-genome sequencing

The variable lymphocyte receptors (VLRs) of jawless vertebrates such as lamprey and hagfish are composed of highly diverse modular leucine-rich repeats. Each lymphocyte assembles a unique VLR by rearrangement of the germline gene. In the lamprey genome, we identify here about 850 distinct cassettes encoding leucine-rich repeat modules that serve as sequence templates for the hypervariable VLR repertoires. The data indicate a gene conversion-like process in VLR diversification. Genomic analysis suggested a link between the VLR and platelet glycoprotein receptors. Lamprey lymphocytes express two putative deaminases of the AID-APOBEC family that may be involved in VLR diversification, as indicated by in vitro mutagenesis and recombination assays. Vertebrate acquired immunity could have therefore originated from lymphocyte receptor diversification by an ancestral AID-like DNA cytosine deaminase.

Evolution and Diversification of Lamprey Antigen Receptors: Evidence for Involvement of an AID-APOBEC Family Cytosine Deaminase

https://www.cell.com/molecular-cell/pdf/S1097-2765(05)01522-4.pdf

A Switch from High-Fidelity to Error-Prone DNA Double-Strand Break Repair Underlies Stress-Induced Mutation

Cytosine base editors (CBEs) enable targeted C•G-to-T•A conversions in genomic DNA. Recent studies report that BE3, the original CBE, induces a low frequency of genome-wide Cas9-independent off-target C•G-to-T•A mutation in mouse embryos and in rice. Here we develop multiple rapid, cost-effective methods to screen the propensity of different CBEs to induce Cas9-independent deamination in Escherichia coli and in human cells. We use these assays to identify CBEs with reduced Cas9-independent deamination and validate via whole-genome sequencing that YE1, a narrowed-window CBE variant, displays background levels of Cas9-independent off-target editing. We engineered YE1 variants that retain the substrate-targeting scope of high-activity CBEs while maintaining minimal Cas9-independent off-target editing. The suite of CBEs characterized and engineered in this study collectively offer ~10-100-fold lower average Cas9-independent off-target DNA editing while maintaining robust on-target editing at most positions targetable by canonical CBEs, and thus are especially promising for applications in which off-target editing must be minimized.

Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors

Use of the rpoB gene to determine the specificity of base substitution mutations on the Escherichia coli chromosome.

We searched for genes that create mutator phenotypes when put on to a multicopy plasmid in Escherichia coli. In many cases, this will result in overexpression of the gene in question. We constructed a random shotgun library with E. coli genomic fragments between 3 and 5 kbp in length on a multicopy plasmid vector that was transformed into E. coli to screen for frameshift mutators. We identified a total of 115 independent genomic fragments that covered 17 regions on the E. coli chromosome. Further studies identified 12 genes not previously known as causing mutator phenotypes when overproduced. A striking finding is that overproduction of the multidrug resistance transcription regulator, EmrR, results in a large increase in frameshift and base substitution mutagenesis. This suggests a link between multidrug resistance and mutagenesis. Other identified genes include those encoding DNA helicases (UvrD, RecG, RecQ), truncated forms of the DNA mismatch repair protein (MutS) and a primosomal component (DnaT), a negative modulator of initiation of replication/GATC-binding protein (SeqA), a stationary phase regulator AppY, a transcriptional regulator PaaX and three putative open reading frames, ycgW, yfjY and yjiD, encoding hypothetical proteins. In addition, we found three genes encoding proteins that were previously known to cause mutator effects under overexpression conditions: error-prone polymerase IV (DinB), DNA methylase (Dam) and sigma S factor (RpoS). This genomic strategy offers an approach to identify novel mutator effects resulting from the multicopy cloning (MCC) of specific genes and therefore complementing the conventional gene inactivation approach to finding mutators.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2958.2004.04125.x

Amy Diep

Papers

Use of the rpoB gene to determine the specificity of base substitution mutations on the Escherichia coli chromosome.

Identification of mutator genes and mutational pathways in Escherichia coli using a multicopy cloning approach