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Selection and Plasmid Transfer Underlie Adaptive Mutation in Escherichia coli.

ABSTRACT: In the Cairns-Foster adaptive mutation system, a +1 lac frameshift mutant of Escherichia coli is plated on lactose medium, where the nondividing population gives rise to Lac+ revertant colonies during a week under selection. Reversion requires the mutant lac allele to be located on a conjugative F'lac plasmid that also encodes the error-prone DNA polymerase, DinB. Rare plated cells with multiple copies of the mutant F'lac plasmid initiate the clones that develop into revertants under selection. These initiator cells arise before plating, and their extra lac copies allow them to divide on lactose and produce identical F'lac-bearing daughter cells that can mate with each other. DNA breaks can form during plasmid transfer and their recombinational repair can initiate rolling-circle replication of the recipient plasmid. This replication is mutagenic because the amplified plasmid encodes the error-prone DinB polymerase. A new model proposes that Lac+ revertants arise during mutagenic over-replication of the F'lac plasmid under selection. This mutagenesis is focused on the plasmid because the cell chromosome replicates very little. The outer membrane protein OmpA is essential for reversion under selection. OmpA helps cells conserve energy and may stabilize the long-term mating pairs that produce revertants.

SUBMITTER: Maisnier-Patin S

PROVIDER: S-EPMC6218229 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Selection and Plasmid Transfer Underlie Adaptive Mutation in Escherichia coli.

Maisnier-Patin Sophie S Roth John R JR

Genetics 20180907 3

In the Cairns-Foster adaptive mutation system, a +1 lac frameshift mutant of Escherichia coli is plated on lactose medium, where the nondividing population gives rise to Lac+ revertant colonies during a week under selection. Reversion requires the mutant lac allele to be located on a conjugative F'lac plasmid that also encodes the error-prone DNA polymerase, DinB. Rare plated cells with multiple copies of the mutant F'lac plasmid initiate the clones ...[more]

PMID: 30194073

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Project description:Spontaneous plasmid transformation of Escherichia coli is independent of the DNA uptake machinery for single-stranded DNA (ssDNA) entry. The one-hit kinetic pattern of plasmid transformation indicates that double-stranded DNA (dsDNA) enters E. coli cells on agar plates. However, DNA uptake and transformation regulation remain unclear in this new type of plasmid transformation. In this study, we developed our previous plasmid transformation system and induced competence at early stationary phase. Despite of inoculum size, the development of competence was determined by optical cell density. DNase I interruption experiment showed that DNA was taken up exponentially within the initial 2 minutes and most transforming DNA entered E. coli cells within 10 minutes on LB-agar plates. A half-order kinetics between recipient cells and transformants was identified when cell density was high on plates. To determine whether the stationary phase master regulator RpoS plays roles in plasmid transformation, we investigated the effects of inactivating and over-expressing its encoding gene rpoS on plasmid transformation. The inactivation of rpoS systematically reduced transformation frequency, while over-expressing rpoS increased plasmid transformation. Normally, RpoS recognizes promoters by its lysine 173 (K173). We found that the K173E mutation caused RpoS unable to promote plasmid transformation, further confirming a role of RpoS in regulating plasmid transformation. In classical transformation, DNA was transferred across membranes by DNA uptake proteins and integrated by DNA processing proteins. At stationary growth phase, RpoS regulates some genes encoding membrane/periplasmic proteins and DNA processing proteins. We quantified transcription of 22 of them and found that transcription of only 4 genes (osmC, yqjC, ygiW and ugpC) encoding membrane/periplasmic proteins showed significant differential expression when wildtype RpoS and RpoS(K173E) mutant were expressed. Further investigation showed that inactivation of any one of these genes did not significantly reduce transformation, suggesting that RpoS may regulate plasmid transformation through other/multiple target genes.

Project description:Adaptive mutation refers to the continuous outgrowth of new mutants from a non-dividing cell population during selection, in apparent violation of the neo-Darwinian principle that mutation precedes selection. One explanation is that of retromutagenesis, in which a DNA lesion causes a transcriptional mutation that yields a mutant protein, allowing escape from selection. This enables a round of DNA replication that establishes heritability. Because the model requires that gene expression precedes DNA replication, it predicts that during selection, new mutants will arise from damage only to the transcribed DNA strand. As a test, we used a lacZ amber mutant of Escherichia coli that can revert by nitrous acid-induced deamination of adenine residues on either strand of the TAG stop codon, each causing different DNA mutations. When stationary-phase, mutagenized cells were grown in rich broth before being plated on lactose-selective media, only non-transcribed strand mutations appeared in the revertants. This result was consistent with the known high sensitivity to deamination of the single-stranded DNA in a transcription bubble, and it provided an important control because it demonstrated that the genetic system we would use to detect transcribed-strand mutations could also detect a bias toward the non-transcribed strand. When residual lacZ transcription was blocked beforehand by catabolite repression, both strands were mutated about equally, but if revertants were selected immediately after nitrous acid exposure, transcribed-strand mutations predominated among the revertants, implicating retromutagenesis as the mechanism. This result was not affected by gene orientation. Retromutagenesis is apt to be a universal method of evolutionary adaptation, which enables the emergence of new mutants from mutations acquired during counterselection rather than beforehand, and it may have roles in processes as diverse as the development of antibiotic resistance and neoplasia.

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Selection and Plasmid Transfer Underlie Adaptive Mutation in Escherichia coli.

Publications

Selection and Plasmid Transfer Underlie Adaptive Mutation in <i>Escherichia coli</i>.

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