Competition of Escherichia coli DNA Polymerases I, II and III with DNA Pol IV in Stressed Cells

Escherichia coli has five DNA polymerases, one of which, the low-fidelity Pol IV or DinB, is required for stress-induced mutagenesis in the well-studied Lac frameshift-reversion assay. Although normally present at ∼200 molecules per cell, Pol IV is recruited to acts of DNA double-strand-break repair, and causes mutagenesis, only when at least two cellular stress responses are activated: the SOS DNA-damage response, which upregulates DinB ∼10-fold, and the RpoS-controlled general-stress response, which upregulates Pol IV about 2-fold. DNA Pol III was also implicated but its role in mutagenesis was unclear. We sought in vivo evidence on the presence and interactions of multiple DNA polymerases during stress-induced mutagenesis. Using multiply mutant strains, we provide evidence of competition of DNA Pols I, II and III with Pol IV, implying that they are all present at sites of stress-induced mutagenesis. Previous data indicate that Pol V is also present. We show that the interactions of Pols I, II and III with Pol IV result neither from, first, induction of the SOS response when particular DNA polymerases are removed, nor second, from proofreading of DNA Pol IV errors by the editing functions of Pol I or Pol III. Third, we provide evidence that Pol III itself does not assist with but rather inhibits Pol IV-dependent mutagenesis. The data support the remaining hypothesis that during the acts of DNA double-strand-break (DSB) repair, shown previously to underlie stress-induced mutagenesis in the Lac system, there is competition of DNA polymerases I, II and III with DNA Pol IV for action at the primer terminus. Up-regulation of Pol IV, and possibly other stress-response-controlled factor(s), tilt the competition in favor of error-prone Pol IV at the expense of more accurate polymerases, thus producing stress-induced mutations. This mutagenesis assay reveals the DNA polymerases operating in DSB repair during stress and also provides a sensitive indicator for DNA polymerase competition and choice in vivo

Atypical Role for PhoU in Mutagenic Break Repair under Stress in Escherichia coli.

Mechanisms of mutagenesis activated by stress responses drive pathogen/host adaptation, antibiotic and anti-fungal-drug resistance, and perhaps much of evolution generally. In Escherichia coli, repair of double-strand breaks (DSBs) by homologous recombination is high fidelity in unstressed cells, but switches to a mutagenic mode using error-prone DNA polymerases when the both the SOS and general (σS) stress responses are activated. Additionally, the σE response promotes spontaneous DNA breakage that leads to mutagenic break repair (MBR). We identified the regulatory protein PhoU in a genetic screen for functions required for MBR. PhoU negatively regulates the phosphate-transport and utilization (Pho) regulon when phosphate is in excess, including the PstB and PstC subunits of the phosphate-specific ABC transporter PstSCAB. Here, we characterize the PhoU mutation-promoting role. First, some mutations that affect phosphate transport and Pho transcriptional regulation decrease mutagenesis. Second, the mutagenesis and regulon-expression phenotypes do not correspond, revealing an apparent new function(s) for PhoU. Third, the PhoU mutagenic role is not via activation of the σS, SOS or σE responses, because mutations (or DSBs) that restore mutagenesis to cells defective in these stress responses do not restore mutagenesis to phoU cells. Fourth, the mutagenesis defect in phoU-mutant cells is partially restored by deletion of arcA, a gene normally repressed by PhoU, implying that a gene(s) repressed by ArcA promotes mutagenic break repair. The data show a new role for PhoU in regulation, and a new regulatory branch of the stress-response signaling web that activates mutagenic break repair in E. coli

Pho-regulon-repression defect without <i>pst/phoBR</i> suppressor mutations in <i>phoU83</i>::<i>Tn</i>10dCam strains.

<p>Colony size was observed on M9 B1 glycerol medium. <i>pstSCAB</i> suppressor mutations or their absence were identified either by whole-genome sequencing (strains SMR4562, SMR4953, SMR20344, SMR21643, SMR21644) or by targeted sequencing of those genes (SMR13353).</p><p>Pho-regulon-repression defect without <i>pst/phoBR</i> suppressor mutations in <i>phoU83</i>::<i>Tn</i>10dCam strains.</p

<i>pho</i> mutations do not strongly affect generation-dependent Lac⁺ reversion rates.

<p>^aMutation rates were calculated as described in Materials and Methods. Exp. 1 and 2 consisted of 19 and 14–15 independent cultures of each strain respectively. Four to six Lac⁺ derivatives of each strain were plated in parallel as controls as described in Materials and Methods.</p><p><i>pho</i> mutations do not strongly affect generation-dependent Lac⁺ reversion rates.</p

<i>pho</i> mutation effects on speed and efficiency of Lac⁺-colony formation do not account for MBR-deficiency.

<p>^aValues are means ± one standard deviation (SD). In each case, four-six independent day-5 or day-6 Lac⁺ mutants were used as controls for the time of colony formation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123315#sec002" target="_blank">Materials and Methods</a>), with the exception of SMR4562 for which day-2 mutants were used (they behave similarly [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123315#pone.0123315.ref041" target="_blank">41</a>]). Exp. 1 was carried out to day 5, and Exp. 2 to day 6.</p><p>^b Only two Lac⁺ control strains were used in this case and so a range, rather than SD is given.</p><p>^c We note that the time to Lac⁺ colony formation for different isolates of Δ<i>pstS</i> Δ<i>phoBR</i> strain SMR6760 varies from two to five days. They all form normal-size colonies and are not detectably amplified (amplified Lac⁺ take 3–5 days to form, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123315#pone.0123315.ref027" target="_blank">27</a>]).</p><p><i>pho</i> mutation effects on speed and efficiency of Lac⁺-colony formation do not account for MBR-deficiency.</p

<b>PhoU is not substituted by SOS-induced levels of DinB (the SOS response) or by DSBs, the role of the σ^E response</b>, indicating that PhoU promotes MBR other than or in addition to by formation of DSBs, activation of the σ^E or SOS responses.

<p>(A, B) Representative experiments. (C, D) Multiple experiments. Lac⁺ mutation rates are Lac⁺ colonies/10⁸ cells /day from days 3–5 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123315#pone.0123315.ref046" target="_blank">46</a>] (mean of 2–3 experiments ± range or SEM, respectively). The first set of isogenic strains carry a <i>dinB</i> operator-constitutive allele <i>dinB</i>(o^c) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123315#pone.0123315.ref045" target="_blank">45</a>], which produces SOS-induced levels of DinB protein at all times, and completely substitutes for a functional SOS response in MBR [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123315#pone.0123315.ref045" target="_blank">45</a>]. <i>dinB</i>(o^c) does not substitute for functional PhoU, indicating that PhoU promotes mutagenesis other than or in addition to by promoting the SOS response. The isogenic strains in the right panel (and right side of the left panel) carry either inducible I-<i>Sce</i>I endonuclease and a cutsite near <i>lac</i> (DSB), or the cutsite-only (“No-DSB”), which has spontaneous DSBs but not additional DSBs induced by I-SceI. I-SceI-induced DSBs substitute for all components that contribute to spontaneous DSBs in the <i>lac</i> region: σ^E [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123315#pone.0123315.ref017" target="_blank">17</a>], TraI [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123315#pone.0123315.ref015" target="_blank">15</a>]; Mfd and RNA-DNA hybrids [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123315#pone.0123315.ref036" target="_blank">36</a>], but do not substitute for PhoU. Strains: WT, SMR4562; <i>phoU</i>, SMR4953; DSB, SMR6280; “No-DSB”, SMR6281; <i>phoU</i> DSB, SMR19235; <i>dinB</i>(o^c), SMR17049; <i>phoU dinB</i>(o^c), SMR20214. Rates were calculated from 3 separate experiments for <i>phoU</i>, wild-type and DSB strains, and error bars represent one SEM. For <i>dinB</i>(o^c), error bars represent range calculated from two independent experiments.</p

Model of regulation of the Pho regulon.

<p>Figure based on conclusions, models and interpretations of Hsieh and Wanner [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123315#pone.0123315.ref019" target="_blank">19</a>].</p

<i>Escherichia coli</i> strains and plasmids used.

<p>^aThis strain is derived from a Lac⁺ colony isolated from a stress-induced mutagenic break-repair experiment and so may carry additional mutations.</p><p>^bThese are independent Lac⁺ stress-induced point mutants. See [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123315#pone.0123315.ref061" target="_blank">61</a>], for the sequence to which the nt positions correspond.</p><p><i>Escherichia coli</i> strains and plasmids used.</p

Mutations affecting the Pho regulon can decrease stress-induced Lac⁺ reversion.

<p>(A) Representative experiment. Strains (top to bottom in legend): SMR4562, SMR4061, SMR4604, SMR4059, SMR4047, SMR4953, and SMR5235. Values are means ± one SEM for eight independent cultures per strain in one representative experiment (where not visible, error bars are smaller than the symbol). (B) Mean of multiple experiments. Complex effects of double and triple mutations affecting the Pho regulon on MBR in the Lac assay. Strains (top to bottom in legend): SMR5235, SMR4953, SMR4059, SMR7351, SMR6762, SMR6760, SMR6759, SMR4061, SMR5860, SMR4047, and SMR4604. Fold decrease in the change in Lac⁺ from day 4 to day 5 relative to the <i>pho</i>⁺ strain SMR4562 was calculated for each genotype in several experiments of multiple cultures (like that shown in A). The values (shown next to the bars) are the mean fold decreases in mutagenesis from multiple experiments ± SEM (error bars, n ≥ 3). (C) A different <i>phoU</i>::Tn<i>10</i> transposon insertion (SMR4954) also depresses MBR, indicating that the <i>phoU</i> mutagenesis-deficiency is not the result of a specific truncation/fusion protein. Representative experiment.</p

PhoU is required for MBR in the <i>E</i>. <i>coli</i> chromosome.

<p>(A) Diagram of relevant genetic elements in the <i>E</i>. <i>coli</i> chromosome. Experimental design of [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123315#pone.0123315.ref016" target="_blank">16</a>]. Cells expressing a chromosomal regulatable I-<i>Sce</i>I endonuclease gene and carrying a chromosomal cutsite near a <i>tet</i> +1bp frameshift allele are starved in liquid for 84 hours (with no tetracycline), rescued to rich medium then plated on rich tetracycline and no-drug plates to score tetracycline-resistant (TetR) mutant colonies. (B) PhoU is required for I-SceI-induced MBR under stress, and DSBs do not substitute for PhoU in mutagenesis. DSB strains have I-<i>Sce</i>I enzyme and cutsite and control “No-DSB” strains have I-<i>Sce</i>I cutsite only. Strains: “No-DSB”, SMR10865; DSB, SMR10866; <i>phoU</i> DSB, SMR20344. The DSB mutant frequency is 14.5 Tet^R mutants /10⁸ cells (1.5 x 10^-7 TetR mutants per cell). Mean ± range of two independent experiments.</p