Plasmid Copy Number Underlies Adaptive Mutability in Bacteria

Abstract

The origin of mutations under selection has been intensively studied using the Cairns-Foster system, in which cells of an Escherichia coli lac mutant are plated on lactose and give rise to 100 Lac+ revertants over several days. These revertants have been attributed variously to stress-induced mutagenesis of nongrowing cells or to selective improvement of preexisting weakly Lac+ cells with no mutagenesis. Most revertant colonies (90%) contain stably Lac+ cells, while others (10%) contain cells with an unstable amplification of the leaky mutant lac allele. Evidence is presented that both stable and unstable Lac+ revertant colonies are initiated by preexisting cells with multiple copies of the F′lac plasmid, which carries the mutant lac allele. The tetracycline analog anhydrotetracycline (AnTc) inhibits growth of cells with multiple copies of the tetA gene. Populations with tetA on their F′lac plasmid include rare cells with an elevated plasmid copy number and multiple copies of both the tetA and lac genes. Pregrowth of such populations with AnTc reduces the number of cells with multiple F′lac copies and consequently the number of Lac+ colonies appearing under selection. Revertant yield is restored rapidly by a few generations of growth without AnTc. We suggest that preexisting cells with multiple F′lac copies divide very little under selection but have enough energy to replicate their F′lac plasmids repeatedly until reversion initiates a stable Lac+ colony. Preexisting cells whose high-copy plasmid includes an internal lac duplication grow under selection and produce an unstable Lac+ colony. In this model, all revertant colonies are initiated by preexisting cells and cannot be stress induced.

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

“…In this model, pre-existing initiator cells have a 10-fold increased lac copy number. These cells form during non-selective pre-growth and are held at a high steady state frequency (about 1 in a 1000) dictated by their rates of copy number increase and decrease (REAMS et al 2010;SANO et al 2014). After plating, selection enhances reversion by allowing initiator cells to replicate their F'lac plasmid more than their chromosome, which provides more opportunities for reversion.…”

“…This observed distribution was like that seen for revertants accumulation in the Cairns-Foster system. The Poisson distribution suggested that revertants either form on the selection plate (CAIRNS AND FOSTER 1991) or are initiated by pre-existing copy number variants that are not subject to LuriaDelbrück fluctuation (SANO et al 2014) . In this E. coli position effect test, revertant number was increased by a lexA(Def) mutation which causes constitutive expression of dinB by SOS derepression and was reduced by removal of the dinB + from the F'lac plasmid (KIM et al 2001).…”

“…Later evidence demonstrated in a different way that Lac + revertants are initiated by cells that form prior to plating -even though these cells show no Luria-Delbruck fluctuation (SANO et al 2014).…”

“…These cells are able to replicate their lac genes more than they replicate their chromosomes (SANO et al 2014; MAISNIER-PATIN AND ROTH 2015; MAISNIER-PATIN AND ROTH 2016;…”

Natural selection underlies apparent stress-induced mutagenesis in a bacteriophage infection model

“…Furthermore, fluctuation tests carried out under these conditions showed a Poisson distribution of the number of mutants, as opposed to the stochastic fluctuating number of mutants obtained by Luria and Delbruck in their experiments, suggesting that the revertants arose after plating on selective medium 5 . Proponents of the 'stress-induced mutagenesis' (SIM) model proposed that the higher mutation rate occurs in response to the growth limitation 5,7,8 , whereas opponents explained the rates in terms of slow DNA replication/cell division (hereafter termed 'cryptic growth') shaped by natural selection [9][10][11][12][13] . The renewed debate centred around whether the stressed cells induce mechanisms to cause increased DNA replication in the absence of growth, resulting in increased mutation rates, or whether cryptic growth occurs, allowing natural selection of randomly generated beneficial mutations.…”