Mutations in the Escherichia coli katF gene (hydroperoxidase II) result in sensitivity to inactivation by H202 and broad-spectrum near-UV (NUV; 300 to 400 nm) radiation. Another mutation, nur, originally described as conferring sensitivity to inactivation by broad-spectrum and monochromatic NUV, also confers sensitivity to inactivation by H202. Genetic analysis via transduction suggests that the nur mutation is a mutant allele of the katF locus. As previously reported for broad-spectrum and monochromatic NUV wavelengths, the sensitivity of a particular strain to H202 inactivation is also independent of the recA and uvrA alleles. Extracts of nur and katF strains lack catalase (hydroperoxidase II) as revealed by polyacrylamide gels stained for such activity, which is consistent with the genetic results.The mutagenic and inactivating effects of both monochromatic and broad-spectrum near-UV (NUV) wavelengths (300 to 400 nm) have been the subject of numerous investigations, which have been extensively reviewed (10,13,17,18,40).A mutation in an Escherichia coli gene (nur) has been described which sensitizes cells to inactivation by NUV without affecting sensitivity to far-UV (FUV) inactivation (35-37). Specifically, it was shown that the recA13, recAl, and uvrA6 mutations did not affect the sensitivity of stationary-phase cells to NUV inactivation. However, the polAl mutation did influence the sensitivity of E. coli cells to inactivation by NUV in an nur+ genetic background (36). The fact that the polAl mutation sensitizes E. coli to NUV inactivation and that E. coli xthA mutants (exonuclease III deficient) are sensitive to inactivation by H202 (8) and NUV (31) might mean that repair of or protection against NUVand H202-induced damage is based on a complex oxidative defense system (4).Further evidence that H202 is involved in NUV inactivation comes from the observation that incorporation of bovine catalase into the plating medium or the irradiated cell suspension protects E. coli cells from both inactivation and mutagenesis by broad-spectrum NUV (32). Hartman (14) has also presented evidence that H202 is involved with NUV inactivating events in stationary-phase E. coli cells.Pretreatment of E. coli or Salmonella typhimurium cells with a sublethal concentration of H202 results in protection against inactivation by a lethal concentration of H202 (4, 7) as well as by broad-spectrum NUV (33, 39). Tyrrell (39) has shown that pretreatment of growing E. coli cells with low fluences of NUV protects against inactivation by H202. If H202 were one product of NUV irradiation in cells, it would be expected that cells lacking catalase should be sensitive to inactivation by NUV. Recently, Leowen and his colleagues have described mutants which are defective in catalase activity (21)(22)(23). In this paper, we present evidence that lesions in the katF gene, but not the katE or katG gene, result in sensitivity to broad-spectrum NUV as well as to * Corresponding author.

—Catalase incorporated into plating medium protects against inactivation and mutagenesis by broad‐spectrum near‐ultraviolet wavelength (300‐400 nm) (NUV) radiation in strains of Escherichia coll. Plating medium containing catalase does not provide protection against inactivation by wavelengths in the FUV region. Catalase added to the cell suspension during or added immediately after NUV exposure also protects against inactivation. The protection provided by catalase suggests a possible role for hydrogen peroxide in the processes of inactivation and mutagenesis by broad‐spectrum NUV.

Abstract— Strains of Escherichia coli carrying the four possible combinations of the alleles nur, nur+, uvrAb, and uvrA+ were either untreated or pretreated with a sublethal dose of H202 prior to inactivation with NUV radiation. Pretreated cells exhibited a greater resistance to NUV than did untreated cells. Pretreatment with H2O2 did not induce resistance to FUV radiation. The induction of resistance to NUV inactivation by H2O2 pretreatment apparently leads to protection against the damage caused by NUV radiation. Although pretreatment of cells with H202 leads to resistance of such cells to inactivation by H2O2 and NUV, survival of H2O2 treated bacteriophage PI cml clr100 is not enhanced when assayed on H2O2 pretreated E. coli host cells.

Abstract— Four strains carrying all four possible combinations of the alleles nur, nur+, uvr A6 and uvr A+ were transduced to hemA8. The hemA8 mutation blocks the synthesis of δ‐aminolevulinic acid (δ‐ALA), one of the first steps in the synthesis of porphyrin and, ultimately, cytochromes essential for aerobic respiration. The cells were grown either with or without δ‐ALA and treated with broad‐spectrum near‐ultraviolet light (NUV; 300–400 nm). hemA8 defective cells grown without δ‐ALA were resistant to inactivation by NUV while hemA8 cells were sensitive to such inactivation when supplemented with δ‐ALA. The sensitivity to NUV inactivation conferred by the nur gene was retained in the hemA8 derivatives. The sensitivity of such cells to NUV inactivation can be controlled by varying the level of δ‐ALA supplementation. The level of δ‐ALA supplementation did not influence the sensitivity of the cells to inactivation by far‐UV light (FUV; 200–300 nm). The near‐UV sensitivity of hemA+ cells was not significantly altered when grown with δ‐ALA suppiementation suggesting that endogenously formed δ‐ALA supports the normal, regulated level of porphyrin synthesis. These results can be interpreted to mean that porphyrin components of the respiratory chain in E. coli represent chromophores involved specifically in broad‐spectrum NUV inactivating events.

To determine if membrane-bound cytochromes function as endogenous near-UV photosensitizers, strains containing the cloned cydA and cydB genes were tested for near-UV sensitivity. A strain contaiping both cloned genes overproduced cytochromes biss, b595, and d. Another strain containing only cloned cyd4 overproduced cytochrome b558. Both cytochrome-overproducing strains were hypersensitive to broad-spectrum near-UV inactivation. The presence of excess cytochromes did not affect sensitivity to far-UV radiation and provided protection against H202 inactivation.The pronounced oxygen dependence of near-UV (320-to 400-nm) inactivation has led to the conclusion that near-UV inactivation is based on a photodynamic process. The chromophores involved in near-UV inactivation events have not been identified. Jagger (11) genes into a cydA background to obtain strains which overexpress the cytochromes of the Cyd complex. In these strains, cytochromes are overexpressed at levels three-to fourfold higher than levels in wild-type cells, while the cydA recipient produces none of the cytochromes of this complex (7, 9).The bacterial strains and plasmids used in this study are listed in Table 1. Cells were grown to stationary phase in minimal A medium (14) supplemented with 0.3% lactic acid (Sigma Chemical Co.), 1 mM MgSO4, 0.1 mM CaCl2, 0.15% Casamino Acids (Difco Laboratories), and 5 ,uM nicotinic acid (Sigma) at 37°C with moderate aeration to achieve maximum cytochrome expression (6). Near-UV and far-UV fluence survival responses were assessed as previously described (23), except that the plating medium was minimal A medium supplemented with lactic acid and nicotinic acid solidified with 1.2% agar (Difco).The H202-generating system used was a glucose-glucose oxidase system modified from one described previously (21). The original description of the system is incorrect and should be 10 mM glucose in 0.85% saline, to which was added approximately 1,000 U of glucose oxidase per ml, resulting in a reaction mixture generating 0.1 mM H202 per min. Hemin solution was prepared by dissolving 10 mg of hemin chloride (Sigma) in 9.6 ml of H20 and 0.4 ml of 100% triethanolamine with vigorous shaking followed by heating at 60°C for 10 min. The solution was filter sterilized before use. Appropriate volumes of the hemin stock solution were added to 10 ml of the glucose-glucose oxidase reaction mixture to get a hemin concentration of 10 or 100 ,ug/ml. When a hemin concentration of 100 ,ug/ml was required, 1.0 ml of the hemin stock solution had to be used. To compensate for any dilution of glucose in the reaction mixture, an appropriate volume of glucose was added from a sterile glucose stock solution. This adjustment was not done for experiments utilizing hemin at a concentration of 10 ,ug/ml.The UV sources were the same as those used in our previous experiments. The broad-spectrum near-UV source 5304