Indirect Ultraviolet - Reactivation of Phage x ( mutagenesis / repair of DNA / bacterial conjugation / Escherichia coli )

When an Frecipient Escherichia coli K12 bacterium receives Hfr or F-lac+ DNA from an ultraviolet-irradiated donor, its capacity to promote DNA repair and mutagenesis of ultraviolet-damaged phage X is substantially increased. We call this phenomenon indirect ultraviolet-reactivation, since its features are essentially the same as those of ultraviolet-reactivation; this repair process occurs in pyrimidine dimer excision-deficient strains and produces clear plaque mutations of the restored phage. Moreover, this process is similar to indirect ultraviolet-induction of prophage X, since it is promoted by conjugation. However, contrarily to indirect induction, it is produced by Hfr donors and occurs in recipients restricting the incoming ultraviolet-damaged donor DNA. The occurrence of indirect ultraviolet-reactivation provides evidence for the existence in E. coli of an inducible error-prone mechanism for the repair of DNA. The survival of ultraviolet (UV)-irradiated phage X is increased when the host cell has been exposed to a low UV dose before infection. This phenomenon, known as UV-reactivation (1), is accompanied by a high rate of mutation (UV-mutagenesis) of the reactivated phage (2). UV-reactivation as well as UV-mutagenesis of phage X require for their occurrence the bacterial recA and lex gene functions (3, 4). This requirement is also shared by UV-induction of prophage X (5-8) as well as by UV-mutagenesis of Escherichia coli K12 (9, 10). This has led Defais et al. (4) to suggest that common pathways might be involved in the abovementioned phenomena. Since lysogenic induction in a lysogenic recipient cell can be obtained "indirectly" by conjugation with a UV-irradiated F+ (11, 12) or F' donor (13), if the above hypothesis is correct, one should be able to produce UV-reactivation of phage X as well as its mutagenesis in a recipient host cell mated with a UV-damaged F' donor. This paper shows that this is indeed the case. Therefore, we call the repair process described here indirect UV-reactivation. Its conditions of occurrence have been compared to those of indirect UV-induction. The occurrence of indirect UV-reactivation provides evidence for the existence in E. coli of an inducible error-prone DNA repair mechanism. MATERIALS AND METHODS Bacteria and Bacteriophages. Bacteria used are described in Table 1. Symbols conform to the recommendations of Demerec Abbreviation: UV, ultraviolet. * Present address: Unite de Genetique Cellulaire, Institut de Biologie Moleculaire, 2 Place Jussieu, 75005, Paris. t Present address: Laboratoire de Byophysique et Radiobiologie, Universite Libre de Bruxelles, Rhode-St-Genese. et al. (14). All Fstrains were derived from GY 158: Fthr-4 leu-8 thi-1 pyrF49 dra-34 thyA34 lacYl tonA101 supE, except AB 2480. F2-lac+ was that from Jacob and Adelberg. Bacteria were rendered resistant to streptomycin (StrR) or spectinomycin (SpcR) by transduction with phage P1 grown on GY 2049 str or on GY 2339 spc. Bacteria resistant to phage X (LamR) were selected against Xvir as white colonies on eosinmethylene blue-maltose plates. Bacteria resistant to phage P1 (PonR) were obtained as resistant to phage 62c (this paper). Phage Xpapa was used throughout for UV-reactivation experiments. In restricting crosses (see Table 2), it was modified by previous growth on P1CM (15) lysogens. Indirect UV-Reactivation Experiments were performed as follows: logarithmic phase cultures of donor and recipient bacteria at 2 X 108 cells per ml were centrifuged at 40, and the pellets were resuspended in cold 0.01 M MgSO4 solution. Aliquots of the suspensions were exposed to UV light; when only one UV dose was given to the donor it was 1600 ergs/ mm2. Irradiated and nonirradiated samples were centrifuged and the pellets resuspended in broth, donor bacteria being concentrated 5-fold. The survival of donor and recipient bacteria was then measured. Bacterial conjugation was carried out by mixing equal volumes of donor and recipient cultures, which were immediately distributed into small tubes and incubated in the dark for 25 min at 370 with gentle agitation. Then, dilutions of UV-irradiated (or not) phage X were added at multiplicities of infection of less than 10-2 to the small tubes in which phage adsorption took place for 25 min at 37°. The suspensions were then plated with AB 2480 indicator bacteria. In the mating mixture, the strain that was not host for phage X was always LamR. Plaques were counted after overnight incubation at 37°. The efficiency of conjugation was determined at 25 min, before the addition of phage to the mixture, by measuring the number of recipients having acquired the lac+ gene from the nonirradiated donor. Direct UV-Reactivation Experiments were performed under conditions similar to those described above, except that donors were not added, since their addition gave identical results in control experiments. Host bacteria were exposed to 800 or 50 ergs/mm2, according to whether or not they were able to excise pyrimidine dimers (Uvr+ or Uvr-). UV-Reactivation Factor. UV-reactivation of X was always determined at about 10-2 survival of the phage plated either on Uvr+ or Uvrbacteria; exposure doses of the phage to UV light were 1500 and 450 ergs/mm2, respectively. The reactivation factor was estimated as the ratio of X survival on a host carrying IJV-damaged bacterial DNA over that on a host without UV-damage to the bacterial DNA. For the sake of