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.

Natural sunlight has a profound effect on much of the life on earth, both from the positive standpoint of the utilization of radiant energy for photochemical and photobiological processes and from the negative standpoint of the harmful effects of UV radiation (UVR) on organisms. The UV wavelengths which reach the earth's surface are classified as UV-A (320 to 400 nm) and UV-B (290 to 320 nm). The UV-A or far-UV wavelengths typically cause only indirect damage to cellular DNA through catalyzing the formation of chemical intermediates such as reactive oxygen species (10). In contrast, UV-B or near-UV radiation can cause direct DNA damage by inducing the formation of DNA photoproducts, of which the cyclobutane pyrimidine dimer (CPD) and the pyrimidine(6-4)pyrimidinone (6-4PP) are the most common (29). The accumulation of DNA photoproducts can be lethal to cells through the blockage of DNA replication and RNA transcription. The effect of UV-B radiation on the ecology of microorganisms has been studied in aquatic systems and in the habitat of the plant leaf surface or phyllosphere. In marine environments, bacterioplankton are highly susceptible to UV-B, with results from field studies indicating that exposure to natural solar UVR can result in decreases in total cell density, reduction in amino acid uptake, increases in measurable CPDs, and significant inhibition of protein and DNA synthesis (3, 16, 26, 27). Solar UVRs also affect the population levels and species composition of bacteria recovered from the peanut phyllosphere at different times of day (39), and increased UV-B irradiation above ambient levels results in alterations of the relative abundance of certain fungal species in the oak phyllosphere (28). Examinations of the effects of UVR on individual bacterial isolates from marine or phyllosphere environments have shown a range of sensitivity levels among different species and among isolates within a species (2, 18, 39, 41). In one large-scale analysis, the majority of culturable isolates recovered from the peanut phyllosphere community were tolerant to relatively large doses of UV-C (254 nm) radiation in vitro (39). Bacteria are particularly vulnerable to UVR damage because their small size limits effective cellular shading (13) and their unicellular nature places a paramount importance on the successful replication of the genome for growth to continue. Thus, the possession of mechanisms to repair UVR-induced DNA damage is an essential contributor to the ecological fitness of organisms that are regularly exposed to solar UVR. Bacteria have evolved four main mechanisms in the repair or damage tolerance of UVR-damaged DNA, including photoreactivation, nucleotide excision repair (NER), mutagenic DNA repair (MDR), and recombinational DNA repair (12). Photoreactivation in bacteria involves a single enzyme called photolyase which binds CPDs and, in a light-dependent step, monomerizes the CPD and dissociates from the repaired lesion (47). The photolyase enzyme contains two distinct chromophores, of which the first one (either 5,10-methenyl tetrahydrofolate or 8-hydroxy-5-deazaflavin) harvests light energy and transfers it to a reduced flavin chromophore that acts as the reaction center in CPD monomerization (22). CPD photolyases are widely but also sporadically distributed among eubacteria, archaea, and eukaryotes comprising two distinct classes: class I CPD photolyases are found in bacteria and lower eukaryotes, and class II CPD photolyases, with one exception, are found in higher eukaryotes (47). Few ecological studies have delineated the importance of individual DNA repair or damage tolerance mechanisms in organisms for survival following exposure to solar UVR in their native habitats. An MDR system, encoded by the rulAB determinant of Pseudomonas syringae, confers a UVR tolerance phenotype that enables strains to maintain significantly larger populations in the bean phyllosphere following UV-B irradiation (40, 41). Spores of Bacillus subtilus were shown to require NER, spore photoproduct lyase (a photolyase), and an additional unknown mechanism for survival following exposure to solar radiation (45). The role of photoreactivation and dark repair has been inferred from field studies of CPD kinetics in bacterioplankton samples from the marine water column (16, 17). These studies showed that CPD levels followed a diel pattern with maximum damage present at late-afternoon samplings and subsequent reductions in CPDs during night-time hours (17). An additional study using artificial UV-B wavelengths and solar radiation also suggested the importance of exposure to photoreactivating wavelengths in bacterioplankton recovery following irradiation (19). While these studies suggest that both photoreactivation and dark repair are important processes in bacterioplankton, they have not resolved the importance of individual repair mechanisms nor have they accounted for the possibility of artifacts caused by differences in the relative abundance of particular organisms at each sampling time. Our long-term goal is to perform ecological studies in which the survival of and relative rates of DNA repair of organism derivatives with alterations in specific DNA repair pathways can be examined in microcosms and in the field. In the present study, we isolated the gene encoding photolyase (phr) from P. aeruginosa, a ubiquitous soil and aquatic organism, and utilized the gene to construct stable insertional phr mutants of P. aeruginosa and P. syringae. We then assessed the contribution of photoreactivation, NER, and MDR to survival and mutability following UV-B irradiation.