Construction and analysis of photolyase mutants of Pseudomonas aeruginosa and Pseudomonas syringae: Contribution of photoreactivation, nucleotide excision repair, and mutagenic DNA repair to cell survival and mutability following exposure to UV-B radiation

Based on nucleotide sequence homology with the Escherichia coli photolyase gene (phr), the phr sequence of Pseudomonas aeruginosa PAO1 was identified from the genome sequence, amplified by PCR, cloned, and shown to complement a known phr mutation following expression in Escherichia coli SY2, Stable, insertional phr mutants containing a tetracycline resistance gene cassette were constructed in P. aeruginosa PAO1 and P. syringae pv. syringae FF5 by homologous recombination and sucrose-mediated counterselection. These muta nts showed a decrease in survival compared to the wild type of as much as 1 9-fold after irradiation at UV-B doses of 1,000 to 1,550 J m(-2) followed b y a recovery period under photoreactivating conditions. A phr uvrA mutant o f P. aeruginosa PAO1 was markedly sensitive to UV-B irradiation exhibiting a decrease in survival of 6 orders of magnitude following a UV-B dose of 25 0 J m-2. Complementation of the phr mutations in P. aeruginosa PAO1 and P. syringae pv, syringae FF5 using the cloned phr gene from strain PAO1 result ed in a restoration of survival following UV-B irradiation and recovery und er photoreactivating conditions. The UV-B survival of the phr mutants could also be complemented by the P. syringae mutagenic DNA repair determinant r ulAB. Assays for increases in the frequency of spontaneous rifampin-resista nt mutants in UV-B-irradiated strains containing rulAB indicated that signi ficant UV-B mutability (up to a 51-fold increase compared to a nonirradiate d control strain) occurred even in the wild-type PAO1 background in which r ulAB only enhanced the UV-B survival by 2-fold under photoreactivating cond itions. The frequency of occurrence of spontaneous nalidixic acid-resistant mutants in the PAO1 uvrA and uvrA phr backgrounds complemented with rulAB were 3.8 x 10(-5) and 2.1 x 10(-3), respectively, following a UV-B dose of 1,550 J m(-2). The construction and characterization of phr mutants in the present study will facilitate the determination of the roles of light and d ark repair systems in organisms exposed to solar radiation in their natural habitats.