Mutability and importance of a hypermutable cell subpopulation that produces stress-induced mutants in Escherichia coli

Abstract

In bacterial, yeast, and human cells, stress-induced mutation mechanisms are induced in growth-limiting environments and produce non-adaptive and adaptive mutations. These mechanisms may accelerate evolution specifically when cells are maladapted to their environments, i.e., when they are are stressed. One mechanism of stress-induced mutagenesis in Escherichia coli occurs by error-prone DNA double-strand break (DSB) repair. This mechanism was linked previously to a differentiated subpopulation of cells with a transiently elevated mutation rate, a hypermutable cell subpopulation (HMS). The HMS could be important, producing essentially all stress-induced mutants. Alternatively, the HMS was proposed to produce only a minority of stress-induced mutants, i.e., it was proposed to be peripheral. We characterize three aspects of the HMS. First, using improved mutation-detection methods, we estimate the number of mutations per genome of HMS-derived cells and find that it is compatible with fitness after the HMS state. This implies that these mutants are not necessarily an evolutionary dead end, and could contribute to adaptive evolution. Second, we show that stress-induced Lac(+) mutants, with and without evidence of descent from the HMS, have similar Lac(+) mutation sequences. This provides evidence that HMS-descended and most stress-induced mutants form via a common mechanism. Third, mutation-stimulating DSBs introduced via I-SceI endonuclease in vivo do not promote Lac(+) mutation independently of the HMS. This and the previous finding support the hypothesis that the HMS underlies most stress-induced mutants, not just a minority of them, i.e., it is important. We consider a model in which HMS differentiation is controlled by stress responses. Differentiation of an HMS potentially limits the risks of mutagenesis in cell clones.

Figure 1. Lac⁺ Mutation Sequences in HMS-Descended Cells.

The sequences of stress-induced Lac⁺ frameshift-reversion mutations are nearly all -1 deletions in small mononucleotide repeats at the positions shown. Those from cells carrying chromosomal “secondary” mutations, detected in our screens, (•, this study) are indistinguishable from stress-induced Lac⁺ frameshift reversions from cells without detected secondary mutations (X, data from [12],[13]). The 30 new mutants sequenced (•) were identified in a previous screen for Lac⁺ mutants with chromosomal loss-of-function mutations conferring the following phenotypes: Mal⁻ (15 mutants); Xyl⁻ (10 mutants); minimal temperature sensitive (TS), which grow on minimal medium at 37° but not at 42° (1 mutant); Mal⁻ Xyl⁻ double mutants (3 mutants); and Mal⁻ minimal TS (1 mutant).

Figure 2. Different Models for the Role of the HMS in Mutagenesis: Predictions for How Mutagenesis Is Enhanced by I-SceI Endonuclease.

(A) Model 1: the HMS generates few stress-induced Lac⁺ mutants and does so via mechanism(s) not relevant to most stress-induced mutagenesis. These models predict that when the main DSB-repair-dependent mechanism of stress-induced mutagenesis (open bars) is stimulated by I-SceI-mediated DSBs made near lac in vivo , Lac⁺ mutagenesis will increase from cells not undergoing genome-wide mutagenesis (open bars). This would cause a decrease in the frequency of genome-wide secondary mutations (present only in the red-dotted fraction) per total Lac⁺ mutant (open and red-dotted total). (B) Model 2: the HMS generates most/all stress-induced Lac⁺ mutants. Models in which genome-wide mutagenesis necessarily accompanies most/all stress-induced Lac reversion predict that the proportion of Lac⁺ mutants with additional chromosomal mutations (red dotted) will not decrease when mutation is stimulated by I-SceI-induced DSBs.

Figure 3. Lac⁺ Mutations and Genome-Wide Mutagenesis Remain Coupled during I-SceI-Mediated Stimulation of Stress-Induced Mutagenesis.

(A) I-SceI-mediated DNA cleavage near the lac gene stimulates stress-induced Lac reversion. Representative experiment. Strains: SMR6280; I-SceI DSBs (enzyme+cutsite) (♦), SMR6276; No I-SceI DSBs (enzyme only) (▪), SMR6281; No I-SceI DSBs (cutsite only) (▴). (B) Data from (A) displayed with the y axis expanded. (C) Viable cell measurements of the Lac⁻ cells during the experiment shown in A and B show no significant growth or death of the strains during the experiment. Because it takes two days for a Lac⁺ cell to form a colony on lactose minimal medium, these viable cell measurements on days 1, 2 and 3 pertain to Lac⁺ colonies visible on days 3, 4 and 5, respectively. (D) Stress-induced mutation rates are increased by I-SceI action near lac. Data from two independent experiments, mean±range (error bars). Lac⁺ mutations accumulated over five days of selection in a strain without I-SceI-induced DSBs (No I-SceI DSBs, SMR6276), and in an I-SceI-mediated-DSB-inducible strain (I-SceI DSBs, SMR6280), showing a ∼70-fold increase in mutation rate when both I-SceI enzyme and its cutsite near lac are present. (E) Frequencies of secondary chromosomal mutations (auxotrophic mutants plus Mal⁻, Xyl⁻, and mucoid from Table 3) per Lac⁺ point mutant are not decreased by I-SceI-mediated DSB stimulation of mutagenesis. The slight increase in the frequency of secondary mutations in the I-SceI-cut-induced strain (I-SceI DSBs, SMR6280) relative to the non- I-SceI-cut-inducible strain (No I-SceI DSBs, SMR6276) is significant: p = 0.001 (z-test with Yates correction). Error bars show 95% confidence limits for binomial populations.

Figure 4. Model for the Differentiation of the HMS.

(A) We suggest that differentiation of the HMS results from the convergence of three events: acquisition of a DNA double-strand break (DSB) or double-strand end (DSE, one end of a DSB); induction of the SOS DNA-damage response; and induction of the RpoS general stress-response (modified from Figure 5 in [2]). Spontaneous SOS induction occurs in about 1% (steady-state levels) of growing cells, about 60% of which were induced because of a DSB or DSE . Individual cells may cycle in and out of the steady-state SOS-induced population, obtaining DNA damage, inducing SOS, then repairing the damage, and turning off SOS induction (rising and falling blue lines). Because repair of a DSB with SOS induction is not sufficient to cause mutagenesis—either stationary phase or induction of the RpoS response is also required —we suggest that when the SOS-induced subpopulation is additionally induced for the RpoS stress response (yellow field), for example upon starvation, it becomes hypermutable: the HMS (green box). (B) Expectation for the HMS in experiments in which I-SceI-induced DSBs increased Lac⁺ mutagenesis. In these experiments (Tables 3,4 and Figure 3), I-SceI is induced from the P_BAD promoter when the cells run out of glucose (stationary phase) and are plated onto lactose medium on which leaky expression from P_BAD promotes I-SceI induction, DNA cleavage, and mutagenesis . With stimulation of mutagenesis by I-SceI, Lac⁺ mutations remained coupled with chromosomal secondary mutations (Table 3, Figure 3E). This can be understood as depicted here: upon I-SceI induction, the fraction of cells with a DSB and an SOS response increases, causing an increase in the fraction of cells that will become the HMS when the RpoS response is induced upon starvation, and thus no decrease in the proportion of secondary mutations per Lac⁺ mutant (Table 3, Figure 3E). However, not all of the starved cells become HMS cells, in that most (Lac⁻ stressed cells) do not show the high genome-wide mutagenesis seen among Lac⁺ point mutants (Table 4), the descendents of the HMS. This might be because many cells receive no DSB, or because DSBs induced during starvation might induce SOS inefficiently.