Error-prone DNA polymerase IV is regulated by the heat shock chaperone GroE in Escherichia coli

Abstract

An insertion in the promoter of the operon that encodes the molecular chaperone GroE was isolated as an antimutator for stationary-phase or adaptive mutation. The groE operon consists of two genes, groES and groEL; point mutations in either gene conferred the same phenotype, reducing Lac+ adaptive mutation 10- to 20-fold. groE mutant strains had 1/10 the amount of error-prone DNA polymerase IV (Pol IV). In recG+ strains, the reduction in Pol IV was sufficient to account for their low rate of adaptive mutation, but in recG mutant strains, a deficiency of GroE had some additional effect on adaptive mutation. Pol IV is induced as part of the SOS response, but the effect of GroE on Pol IV was independent of LexA. We were unable to show that GroE interacts directly with Pol IV, suggesting that GroE may act indirectly. Together with previous results, these findings indicate that Pol IV is a component of several cellular stress responses.

FIG. 1.

Mutant alleles of groE decrease adaptive mutation in E. coli. The accumulation of Lac⁺ revertants during incubation on lactose-minimal medium. Four to six independent cultures were plated for each strain; data are the mean numbers of Lac⁺ colonies appearing each day from days 3 to 5 ± standard errors of the means (SEM). The results of four experiments are shown. (A) Wild type = FC40; groES::Cm = PFG361; groE⁺ = PFG60 and PFG64; groEL100 = PFG61; groES30 = PFG65. (B) Wild type = FC526; groES::Cm = PFG351; groE⁺ = PFG62 and PFG66; groEL100 = G63; groES30 = PFG67. PFG60 through PFG67 are also purA::Tn10.

FIG. 2.

The reduction in the level of adaptive mutation due to deficiency of GroE is not due to loss of viability. (A and B) Accumulation of Lac⁺ revertants of groE⁺ and groEL100 mutant strains during incubation on lactose-minimal medium. Data are the cumulative number of Lac⁺ colonies divided by the number of viable Lac⁻ cells on the plate 2 days earlier; each point is the mean ± SEM of results for 20 independent cultures (some error bars are smaller than the symbols). Because it takes 2 days for a Lac⁺ revertant to make a visible colony, the values are displaced 2 days earlier to correspond to the points in the survival curves in panels C and D. (A) Circles, PFG60 (groE⁺); triangles, PFG61 (groEL100). (B) Circles, PFG62 (ΔrecG263 groE⁺); triangles, PFG63 (ΔrecG263 groEL100). All strains are also purA::Tn10. (C and D) Survival of Lac⁻ cells during incubation on lactose-minimal medium. Each point is the mean ± SEM of results for three independent cultures. (C) Circles, PFG60 (groE⁺); triangles, PFG61 (groEL100). Numbers have been normalized to the value for PFG60 on day 0. (D) Circles, PFG62 (ΔrecG263 groE⁺); triangles, PFG63 (ΔrecG263 groEL100). Because about 100-fold-more PFG63 cells than PFG62 cells were plated, the numbers of PFG62 cells were multiplied by 100; then the results for both strains were normalized to this value for PFG62 on day 0.

FIG. 3.

A mutant allele of groE reduces the cellular amount of Pol IV protein. Shown is a Western blot indicating the amounts of Pol IV in (from left to right) PFG67 (recG groES30), PFG65 (recG⁺ groES30), FC1230 (dinB⁺⁺), FC1240 (ΔdinB), FC526 (recG), and FC40 (wild type). Only one blot is shown, but lanes with irrelevant samples have been removed; the rightmost three lanes have been previously published (34). Samples consisting of 40 μg of total protein were loaded in each lane. The intensities of the bands for each strain relative to those of 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 parentheses.

FIG. 4.

A mutant allele of groE reduces the cellular amount of Pol IV protein in the absence of LexA. Shown is a Western blot indicating the levels of Pol IV in (from left to right): PFG324 [sulA lexA(Def)], PFG325 [sulA lexA(Def) groEL100], PFG326 (sulA), FC526 (dinB⁺⁺), and FC1240 [Δ(dinB)]. PFG324, PFG325, and PFG326 are F⁻ strains, so their only dinB alleles are chromosomal; these strains are also sulA11 [which prevents lethal filamentation due to the lexA(Def) allele]. Only one blot is shown, but lanes with irrelevant samples have been removed. Samples consisting of 40 μg of total protein were loaded. The intensities of the bands for each strain relative to those of the wild-type strain are given below each lane.

FIG. 5.

A groE mutant allele reduces adaptive mutation in the absence of LexA. The accumulation of Lac⁺ revertants during incubation on lactose-minimal medium is shown. Three independent cultures were plated for each strain; data are the mean numbers of Lac⁺ colonies appearing each day from days 3 to 5 ± SEM. lexA(Def) groE⁺ = PFG328: lexA(Def) groEL100 = PFG329; lexA⁺ = PFG340; lexA⁺ groEL100 = PFG341. All strains are sulA11 and purA::Tn10.

FIG. 6.

The groEL100 and ΔdinB::Zeo mutant alleles are epistatic in recG⁺ but not in recG mutant strains. Shown is the accumulation of Lac⁺ revertants of groE⁺, ΔdinB::Zeo, groEL100, and groEL100 ΔdinB::Zeo strains during incubation on lactose-minimal medium. Data are the cumulative number of Lac⁺ colonies divided by the number of viable Lac⁻ cells on the plate 2 days earlier. Because it takes 2 days for a Lac⁺ revertant to make a visible colony, the values shown are displaced 2 days earlier. Each point is the mean ± 95% confidence limits of results for six cultures (some error bars are smaller than the symbols). (A) Closed circles, PFG60 (groE⁺); open circles, PFG250 (ΔdinB::Zeo); closed triangles, PFG61 (groEL100); open triangles, PFG251 (ΔdinB::Zeo groEL100). (B) Closed circles, PFG62 (groE⁺); open circles, PFG305 (ΔdinB::Zeo); closed triangles, PFG63 (groEL100); open triangles, PFG307 (ΔdinB::Zeo groEL100). For clarity, the results for PF62 after day 1 are not shown; see Fig. 2 for comparable data for this strain. All strains are also purA::Tn10.