Selection-Enhanced Mutagenesis of lac Genes Is Due to Their Coamplification with dinB Encoding an Error-Prone DNA Polymerase

Abstract

To test whether growth limitation induces mutations, Cairns and Foster constructed an Escherichia coli strain whose mutant lac allele provides 1–2% of normal ability to use lactose. This strain cannot grow on lactose, but produces ∼50 Lac+ revertant colonies per 108 plated cells over 5 days. About 80% of revertants carry a stable lac+ mutation made by the error-prone DinB polymerase, which may be induced during growth limitation; 10% of Lac+ revertants are stable but form without DinB; and the remaining 10% grow by amplifying their mutant lac allele and are unstably Lac+. Induced DinB mutagenesis has been explained in two ways: (1) upregulation of dinB expression in nongrowing cells (“stress-induced mutagenesis”) or (2) selected local overreplication of the lac and dinB+ genes on lactose medium (selected amplification) in cells that are not dividing. Transcription of dinB is necessary but not sufficient for mutagenesis. Evidence is presented that DinB enhances reversion only when encoded somewhere on the F’lac plasmid that carries the mutant lac gene. A new model will propose that rare preexisting cells (1 in a 1000) have ∼10 copies of the F’lac plasmid, providing them with enough energy to divide, mate, and overreplicate their F’lac plasmid under selective conditions. In these clones, repeated replication of F’lac in nondividing cells directs opportunities for lac reversion and increases the copy number of the dinB+ gene. Amplification of dinB+ increases the error rate of replication and increases the number of lac+ revertants. Thus, reversion is enhanced in nondividing cells not by stress-induced mutagenesis, but by selected coamplification of the dinB and lac genes, both of which happen to lie on the F’lac plasmid.

Selection and Plasmid Transfer Underlie Adaptive Mutation in Escherichia coli

“…Third, formation of stable Lac + revertants under selection requires that the F9lac plasmid carrying the lac allele must also carry a functional copy of the dinB + gene, which encodes an error-prone DNA repair polymerase (PolIV). A chromosomal copy of the dinB + gene is not sufficient to support a high level of reversion (Slechta et al 2003;Yamayoshi et al 2018). Selection favors increases in the copy number of a plasmid that includes the lac and dinB + genes (Yamayoshi et al 2018).…”

“…A chromosomal copy of the dinB + gene is not sufficient to support a high level of reversion (Slechta et al 2003;Yamayoshi et al 2018). Selection favors increases in the copy number of a plasmid that includes the lac and dinB + genes (Yamayoshi et al 2018). Induced transcription of the dinB + gene by global regulatory systems is not sufficient for mutagenesis, but provides a level of DinB that can become mutagenic if amplified by plasmid copy number increases.…”

“…In strains lacking the error-prone DNA polymerase DinB, the yield of stable revertants drops 10-fold but is not eliminated. Unstable Lac + revertants form at the same rate with or without DinB (McKenzie et al 2001;Yamayoshi et al 2018). The above results have been interpreted either as support for stress-induced mutagenesis (Rosenberg et al 2012) or as evidence that selection favors improved lactose degradation by acting through standard functions involved in plasmid transfer and DNA repair Roth 2015, 2016).…”

“…Because of their extra copies of the leaky lac allele, the preexisting initiator cells can divide on selective medium and develop into lac + revertants. Selection acts on the plasmid population within initiator cells by a multistep process that involves very few divisions of the plated cell population (Roth et al 2006;Wrande et al 2008;Yamayoshi et al 2018). The problem is to understand the process by which selection acts on the plasmid population within an initiator cell Roth 2015, 2016).…”

Transposable element insertions in fission yeast drive adaptation to environmental stress

“…One potential solution is the hypothesis that cells possess a mechanism that responds to environmental changes by increasing errors in DNA replication (Cairns et al 1988;Cairns and Foster 1991). However, this possibility remains unresolved because model reporters suggest stressinduced growth limitation may not influence replication errors (Roth et al 2006;Maisnier-Patin and Roth 2015;Yamayoshi et al 2018). Thus, while point mutations in regulatory or coding sequences have the potential to allow survival following environmental shock, they may often be inadequate.…”