Error-prone DNA polymerase IV is controlled by the stress-response sigma factor, RpoS, in Escherichia coli

Abstract

An insertion in rpoS, which encodes the general stress response sigma factor sigma 38, was isolated as an antimutator for 'stationary-phase' or 'adaptive' mutation. In the rpoS mutant strain the levels of error-prone DNA polymerase Pol IV were reduced. Pol IV is encoded by the dinB gene, and the amount of its transcript was also reduced in rpoS mutant cells. In wild-type cells, the levels of Pol IV increased in late stationary phase and stayed elevated for several days of continuous incubation, whereas in rpoS defective cells Pol IV was not induced and declined during prolonged incubation. Even in cells missing LexA, the repressor of dinB, maximum Pol IV expression required RpoS. These results suggest that induction of Pol IV is part of a cellular response to starvation and other stresses.

Fig. 1

A recG mutation enhances the Pol IV-dependent component of adaptive mutation. The accumulation of Lac⁺ mutations in Lac⁻ cells incubated on lactose minimal medium. Wild type = FC40; ΔdinB = FC1354; recG = FC438; ΔdinB recG = FC1355. Two experiments are shown, one with FC40 and FC1354, and one with FC438 and FC1355. Data are the mean number of Lac⁺ colonies appearing each day from days 3–5; each point is the mean of four cultures ± SEM.

Fig. 2

Levels of Pol IV are increased in a recG mutant strain but are not affected by ruv mutations. Western blot showing levels of Pol IV in (from left to right) wild type = FC40; recG = FC465; Δ (ruvAC) = FC836; recG Δ (ruvAC) = FC897; ΔdinB = FC1240. Samples consisting of 10 μg of total protein were loaded in each lane of a precast SDS-PAGE gel (Bio-Rad Laboratories). The intensities of the bands for each strain relative to the wild-type strain are given below each lane; the intensity of only the upper band relative to that of the wild-type strain is in parenthesis.

Fig. 3

A rpoS mutation decreases adaptive mutation. A. The accumulation of Lac⁺ mutations in Lac⁻ cells incubated on lactose minimal medium. Circles, recG = FC526; triangles, recG rpoS = PFG36. Data are the cumulative number of Lac⁺ colonies appearing from days 3–5; each point is the mean of four cultures ± SEM (some error bars are smaller than the symbols). B. The survival of Lac⁻ cells while incubating on lactose minimal medium. Circles, recG = FC526; triangles, recG rpoS::Cm = PFG36. Because 10-fold more PFG36 cells were plated than FC526 cells, the numbers of FC526 cells on each day were multiplied by 10; then the results for both strains were normalized to the value for FC526 on day 0. Because it takes two days for a Lac⁺ revertant to make a visible colony, the values shown in B correspond to the points two days later in the Lac⁺ curves in A.

Fig. 4

An rpoS mutation reduces the cellular levels of Pol IV protein. A. Western blot showing levels of Pol IV in (from left to right) ΔdinB = FC1240; recG rpoS = PFG36; recG = FC526; wild type = FC40. B. A different Western blot showing levels of Pol IV in (from left to right) wild type = FC40; rpoS = PFG72. Samples consisting of 40 μg of total protein were loaded in each lane of each blot; the blot in B was developed long enough to enhance the band from the rpoS mutant strain. The intensities of the bands for each strain relative to the wild type strain are given below each lane; the intensity of only the upper band relative to that of the wild type strain is in parenthesis.

Fig. 5

A rpoS mutation reduces the cellular levels of Pol IV transcript. A. Ethidium stained agarose gel showing the dinB RT-PCR products from 250 ng of total endogenous RNA extracted from the wild type strain FC40 (upper band) and the RT-PCR products from the truncated dinB competitor RNA added to the same reactions at 1, 2, 4, 8, and 16 pg (lower band). M = 500 bp molecular weight marker. B. Ethidium stained agarose gel showing the dinB RT-PCR products from 250 ng of total endogenous RNA extracted from the rpoS::Cm strain PFG72 (upper band) and the RT-PCR products from the truncated dinB competitor RNA added to the same reactions at 0.5, 1, 2, 4, and 8 pg (lower band). M = 500 bp molecular weight marker. C. Plot and the least-squares fit of the log of the ratios of endogenous to competitor band intensities vs. the log of the amount of competitor RNA added. (To give a better fit, the 4 pg value for FC40, which appears to be an outlier, has been excluded; if it is included the line crosses y = 0 at x = 0.56 or 4.2 pg instead of at × = 0.69 or 4.9 pg).

Fig. 6

Pol IV levels increase in late stationary phase. A. Western blot showing the amount of Pol IV protein in wild type (= FC40) and rpoS::Cm (= PFG72) cells at the time points indicated in B. Two blots are shown: on the left are samples taken during exponential growth through stationary phase; on the right are samples taken one and two days later. Samples c and d were reloaded for the blot on the right. Samples consisting of 40 μg of total protein were loaded (only half of the ΔdinB control lane on the left is visible). B. The growth curve of the wild type (= FC40) and rpoS::Cm (= PFG72) mutant strain in minimal glycerol medium. Samples were taken at the times indicated by arrows. C. The relative amounts of Pol IV protein in the wild type (= FC40) cells and rpoS::Cm (= PFG72) cells at the time points indicated in B. Bands on the two blots were normalized by first setting the most intense band for each strain on each blot equal; these were the band at d for the wild type strain, and the band at c for the rpoS::Cm strain. The intensity of each band was then recalculated relative to these. These values were then normalized to the wild-type strain sample a.

Fig. 7

A rpoS mutation reduces adaptive mutation in various backgrounds. The accumulation of Lac⁺ mutations in Lac⁻ cells incubated on lactose minimal medium. From left to right: Wild type = FC40; rpoS = PFG72; sulA = FC1417; sulA rpoS = PFG185; LexA(Ind⁻) = FC1418; LexA(Ind⁻) rpoS = PFG184; sulA LexA(Def) = FC1419, sulA LexA(Def) rpoS = PFG186; recG = FC526; recG rpoS = PFG36. Data are the mean number of Lac⁺ colonies appearing each day from days 3–5; each point is the mean of four cultures ± SEM. Three experiments are shown: the first was with the wild type and rpoS mutant strain; the second was with all the derivatives of the sulA mutant strain; and, for comparison, the data for recG and recG rpoS from the experiment shown in Fig. 3 were recalculated in the same format.

Fig. 8

A rpoS mutation reduces the cellular levels of Pol IV protein even in the absence of LexA. Western blot showing levels of Pol IV in (from left to right); sulA LexA(Def) = FC1413; sulA LexA(Def) rpoS

Fig. 9

The dinB promoter region. The DNA sequence 5′ to the dinB gene. Relevant features are in bold. The minimum 3-base inverted repeats that define the LexA box are boxed. A possible promoter element is underlined. SD = possible Shine-Delgarno sequence. Start = the dinB translational start. Stop = stop codon of the preceding gene, mbhA.