A role for the umuDC gene products of Escherichia coli in increasing resistance to DNA damage in stationary phase by inhibiting the transition to exponential growth

Abstract

The umuDC gene products, whose expression is induced by DNA-damaging treatments, have been extensively characterized for their role in SOS mutagenesis. We have recently presented evidence that supports a role for the umuDC gene products in the regulation of growth after DNA damage in exponentially growing cells, analogous to a prokaryotic DNA damage checkpoint. Our further characterization of the growth inhibition at 30 degrees C associated with constitutive expression of the umuDC gene products from a multicopy plasmid has shown that the umuDC gene products specifically inhibit the transition from stationary phase to exponential growth at the restrictive temperature of 30 degrees C and that this is correlated with a rapid inhibition of DNA synthesis. These observations led to the finding that physiologically relevant levels of the umuDC gene products, expressed from a single, SOS-regulated chromosomal copy of the operon, modulate the transition to rapid growth in E. coli cells that have experienced DNA damage while in stationary phase. This activity of the umuDC gene products is correlated with an increase in survival after UV irradiation. In a distinction from SOS mutagenesis, uncleaved UmuD together with UmuC is responsible for this activity. The umuDC-dependent increase in resistance in UV-irradiated stationary-phase cells appears to involve, at least in part, counteracting a Fis-dependent activity and thereby regulating the transition to rapid growth in cells that have experienced DNA damage. Thus, the umuDC gene products appear to increase DNA damage tolerance at least partially by regulating growth after DNA damage in both exponentially growing and stationary-phase cells.

FIG. 1

umuDC-dependent inhibition of growth at 30°C is growth phase specific. (A) A stationary-phase culture of a lexA(Def) strain carrying a multicopy umuD⁺C⁺ plasmid [GW8025(pSE117)] was diluted 1:125 into fresh LB medium, and growth at 42°C was monitored by measuring OD₆₀₀ (□). A portion of this culture was shifted to 30°C (time of shift indicated by arrows) during lag phase (■) and exponential growth (●), and subsequent growth was monitored by measuring OD₆₀₀. (B) Stationary-phase cultures of strains GW8025(pBR322kan) and GW8025(pSE117) were diluted into fresh LB medium, grown at 42°C for 1 h, and shifted to 30°C. Growth at 30°C was monitored by measuring OD₆₀₀. □, GW8025(pBR322kan), 1:125 dilution; ■, GW8025(pSE117), 1:20 dilution; ●, GW8025(pSE117), 1:125 dilution; ▴, GW8025(pSE117), 1:400 dilution.

FIG. 2

UmuDC-mediated inhibition of DNA synthesis at 30°C. Stationary-phase cultures of GW8025(pBR322kan) (□) and GW8025(pSE117) (▴) were diluted 1:125 in fresh LB medium and grown at 42°C. A portion of these cultures was shifted to 30°C during lag phase and exponential growth. At various times subsequently, the rate of DNA synthesis at 42 and 30°C was determined. Incorporation of [methyl-³H]thymidine was normalized at each time point to the OD₆₀₀ of the culture. The ratios of the rates of DNA synthesis of the ΔumuDC to umuDC⁺ cultures are indicated in the following in parentheses (A) 42°C, lag phase (1.0); (B) 42°C, exponential growth (1.0); (C) 30°C for 10 min after shift in lag phase (2.7); (D) 30°C for 10 min after shift in exponential growth (0.8); (E) 30°C for 2 h after shift in lag phase (5.0); (F) 30°C for 2 h after shift in exponential growth (2.0).

FIG. 3

Effect of prolonged starvation on umuDC-mediated growth inhibition at 30°C. (A) The following strains were grown in LB medium at 42°C: GW8025(pBR322kan) (□), GW8025(pSE115) (●), and GW8025(pSE117) (▴). At various times during growth (as indicated by arrows), a portion of each culture was diluted 1:125 in fresh LB medium, grown at 42°C for 1 h, and shifted to 30°C. Subsequent growth at 30°C was monitored by measuring OD₆₀₀. (B to D) Results are shown for cultures 8 (B), 16 (C) and 24 (D) h old at the time of nutrient upshift.

FIG. 4

Effect of the umuDC gene products on survival and growth of UV-irradiated stationary-phase cultures after nutrient upshift. Cultures were grown at 37°C. GW2771 (umuD⁺C⁺) and GW8023 (ΔumuDC) were grown in M9 medium to stationary phase. One milliliter of each culture was exposed to a UV dose of 50 J/m². UV-irradiated and unirradiated cultures were diluted 1:100 in fresh M9 medium and grown in the dark. At various times during growth, serial dilutions were plated to quantify CFU per milliliter. Symbols: ●, GW2771 (umuD⁺C⁺) without UV irradiation; □, GW8023 (ΔumuDC) without UV irradiation; ○, GW2771 (umuD⁺C⁺) with UV irradiation; ■, GW8023 (ΔumuDC) with UV irradiation.

FIG. 5

Effect of uncleaved UmuD and UmuC on survival and growth of UV-irradiated stationary-phase cultures after nutrient upshift. Cultures were grown at 37°C. GW8027 [recA430 lexA(Def) umuD⁺C⁺] and GW8040 [recA430 lexA(Def) ΔumuDC] were grown in M9 medium to stationary phase. One milliliter of each culture was exposed to a UV dose of 50 J/m². UV-irradiated and unirradiated cultures were diluted 1:100 in fresh M9 medium and grown in the dark. At various times during growth, serial dilutions were plated to quantify CFU per milliliter. Symbols: ●, GW8027 (recA430 umuD⁺C⁺) without UV irradiation; □, GW8040 (recA430 ΔumuDC) without UV irradiation; ○, GW8027 (recA430 umuD⁺C⁺) with UV irradiation; ■, GW8040 (recA430 ΔumuDC) with UV irradiation.

FIG. 6

Effect of fis::767 on survival after UV irradiation in stationary phase. Cultures were grown at 37°C. Symbols: ■, GW2771 (fis⁺ umuD⁺C⁺); ●, GW8023 (fis⁺ ΔumuDC); ▵, GW8037 (fis::767 umuD⁺C⁺); ○, GW8038 (fis::767 ΔumuDC). Survival of cells UV irradiated after 11 h in stationary phase is shown.

FIG. 7

Effect of fis::767 on survival after UV irradiation of exponentially growing cultures. Cultures were grown at 37°C. Symbols: ■, GW2771 (fis⁺ umuD⁺C⁺); ●, GW8023 (fis⁺ ΔumuDC); ▵, GW8037 (fis::767 umuD⁺C⁺); ○, GW8038 (fis::767 ΔumuDC). Survival of cells UV irradiated in exponential growth is shown.