DdrA, DdrD, and PprA: components of UV and mitomycin C resistance in Deinococcus radiodurans R1

Abstract

Mutants created by deleting the ddrA, ddrB, ddrC, ddrD, and pprA loci of Deinococcus radiodurans R1alone and in all possible combinations of pairs revealed that the encoded gene products contribute to this species' resistance to UV light and/or mitomycin C. Deleting pprA from an otherwise wild type cell sensitizes the resulting strain to UV irradiation, reducing viability by as much as eight fold relative to R1. If this deletion is introduced into a ΔddrA or ΔddrD background, the resulting strains become profoundly sensitive to the lethal effects of UV light. At a fluence of 1000 Jm⁻², the ΔddrA ΔpprA and ΔddrD ΔpprA strains are 100- and 1000-fold more sensitive to UV relative to the strain that has only lost pprA. Deletion of ddrA results in a 100 fold increase in strain sensitivity to mitomycin C, but in backgrounds that combine a deletion of ddrA with deletions of either ddrC or ddrD, mitomycin resistance is restored to wild type levels. Inactivation of ddrB also increases D. radiodurans sensitivity to mitomycin, but unlike the ddrA mutant deleting ddrC or ddrD from a ΔddrB background further increases that sensitivity. Despite the effect that loss of these gene products has on DNA damage resistance, none appear to directly affect either excision repair or homologous recombination suggesting that they participate in novel processes that facilitate tolerance to UV light and interstrand crosslinks in this species.

Figure 1. Survival curves for ΔddrA, ΔddrB , ΔddrC, ΔddrD, and ΔpprA derivatives of D. radiodurans R1 strains exposed to UV light.

Exponential phase cultures of D. radiodurans R1, TNK101 ΔddrC, TNK102 ΔddrB, TNK103 ΔddrD, TNK104 ΔddrA, TNK105 ΔpprA, TNK106 ΔrecA, and LSU2000 uvrA1 uvs::TnDrCat were exposed to UV light at 25 J m^-2s^-1. Values are the means ± standard deviations of three independent experiments (n = 9).

Figure 2. Survival curves for ΔddrA derivatives of D. radiodurans R1 exposed to UV light.

The UV resistances of TNK114 ΔddrA ΔddrC, TNK117 ΔddrA ΔddrB, TNK119 ΔddrA ΔddrD, TNK121 ΔddrA ΔpprA are compared with TNK104 ΔddrA, TNK105 ΔpprA, and D. radiodurans R1. Values are the means ± standard deviations of four independent experiments (n = 12).

Figure 3. Survival curves for ΔddrB and ΔddrC derivatives of D. radiodurans R1 exposed to UV light.

The UV resistances of TNK112 ΔddrB ΔddrC, TNK113 ΔddrC ΔddrD, TNK115 ΔddrC ΔpprA, TNK116 ΔddrB ΔddrD, and TNK118 ΔddrB ΔpprA are compared with TNK101 ΔddrC, and TNK102 ΔddrB. Values are the means ± standard deviations of six independent experiments (n = 18).

Figure 4. The survival curve of TNK120 exposed to UV light.

The UV resistances of TNK120 ΔddrD ΔpprA compared with the survival curves of TNK103 ΔddrD, TNK104 ΔddrA, and TNK105 ΔpprA. Values are the means ± standard deviations of four independent experiments (n = 12).

Figure 5. Survival curves for ΔpprA derivatives D. radiodurans R1 exposed to mitomycin C.

TNK120 ΔddrD ΔpprA and TNK121 ΔddrA ΔpprA are compared with D. radiodurans R1, TNK103 ΔddrD, and TNK105 ΔpprA. Values are the means ± standard deviations of three independent experiments (n = 9).

Figure 6. Survival curves for ΔddrC and ΔddrD derivatives of TNK104 ΔddrA exposed to mitomycin C.

TNK114 ΔddrA ΔddrC, TNK117 ΔddrA ΔddrB, and TNK119 ΔddrA ΔddrD are compared with D. radiodurans R1 and TNK104 ΔddrA. Values are the means ± standard deviations of three independent experiments (n = 9).

Figure 7. Survival curves for ΔddrC and ΔddrD, and ΔpprA derivatives D. radiodurans R1 exposed to mitomycin C.

TNK101 ΔddrC, TNK103 ΔddrD, and TNK113 ΔddrC ΔddrD are compared with D. radiodurans R1. Values are the means ± standard deviations of eight independent experiments (n = 24).

Figure 8. Survival curves for ΔddrC and ΔddrD derivatives of TNK104 ΔddrB exposed to mitomycin C.

TNK102 ΔddrB, TNK112 ΔddrB ΔddrC, and TNK116 ΔddrB ΔddrD are compared with D. radiodurans R1. Values are the means ± standard deviations of eight independent experiments (n = 24).