Bacterial Genes Involved in Response to Near-Ultraviolet Radiation

BACTERIAL GENES INVOLVED IN RESPONSE TO N EAR-ULTRAVIOLET RADIATION A. Eisenstork Division of Biological Sciences and Department of Microbiology, University of Missouri, Columbia, Missouri 6521 1

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Significance , . . . . . . . . B. Model for NUV Action . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . s (Photoreceptors) of NUV

. . . . . . . . . . . . . .. . . . . . . . . . . . ,

.

......

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tRNA . . . . . . , C. Porphyrins and Other M ........... D. Amino Acids and Polypeptides.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Photoprotective Chromophores 111. Oxidative Photoproducts of NUV. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Hydrogen Peroxide . . . . . . . . . . . . . .

C. Hydroxyl Radic

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A. Catalase . . . ... . . .. . . . . . . . . . . . . . . . . . .. .. . . . . . . . . . . . . . . . . . . .. .. . ... B. Superoxide Dismutase C. Exonuclease I11 and Endonuclease IV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C. Membrane Effects . . . . . . . . .

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100 100 101 103 103 105 107 108 110 111 112 113 114 115 115 119 119 122 123 124 126 126 126 128 129 129 131 132 132 132 134 136

99 ADVANCES IN GENETICS, Vol 26

Copyright Q 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

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I. Introduction

A. SIGNIFICANCE Solar near-ultraviolet radiation (NUV; 290-400 nm), because of its abundance, is perhaps the most mutagenic agent to which organisms are exposed. In addition to its mutagenic effect, NUV influences biological systems in unique and often subtle ways. Many microbial, plant, and animal biochemical reactions may be induced, cued, and modulated by NUV in the organism’s developmental, growth and behavioral activities. Solar NUV may have deleterious effects on bacteria; therefore, bacteria must constantly cope with fluctuating solar NUV conditions (Calkins, 1982; Gameson and Gould, 1975).This is particularly true of Escherichia coli, which lives in both aerobic and anaerobic environments, as well as in both sunlight and darkness as the organism cycles between hosts. The genetic mechanisms that have evolved in bacteria to cope with solar NUV stress are the focus of this article. There is increasing concern that additional NUV (particularly UV-B; 290-320 nm) may impinge on the earth’s surface as a result of depletion of the ozone filter in the stratosphere; this depletion may result from use of fluorocarbon aerosols, nitrogen oxide pollution, and supersonic flights, as well as from other products and procedures of modern technology. Recently, even greater focus on this problem has resulted from the finding that the stratospheric ozone screen may be lost over the Antarctic; the effect of this excess NUV on zooplankton and other organisms in the food chain is not yet known (Calkins, 1982; Helene et al., 1982). Faced with the possibility of this altered global environment, it is appropriate that we seriously assess the mutagenic and toxic nature of NUV. In addition to natural radiation, many humans receive excessive NUV from artificial illumination, such as sun lamps and therapeutic lamps. Also, photosensitizing molecules, with 290- to 400-nm absorbance, abound in natural substances and may affect human health (Ames, 1983; Blum, 1941; Straight and Spikes, 1985). One of the effects of NUV may be t o generate excess active oxygen species; these have been implicated in a wide variety of environmental and health effects (Adelman et al., 1988; Halliwell, 1987; Marx, 19871, including premature aging (Bissett et al., 19871, circulatory diseases, rheumatoid arthritis, and induction of cancers and cataracts (Adelman et al., 1988; Ames, 1983; Petkau, 1986; Touati, 1988a). Critical microbial experiments may be traced to the late Alexander

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Hollaender (1943), whose pioneering paper stimulated UV research. This was followed by numerous NUV research and review papers (Eisenstark, 1971, 1985, 1987; Helene et al., 1982; Kubitschek et al., 1986; Webb, 19771, plus a book by Jagger (1985). Answers to major questions have emerged recently, particularly as to the identification of NUV chromophores, targets, toxic photoproducts and their subsequent quenching action, the specific nature of DNA lesions (mutagenic and nonmutagenic), induced synthesis of protective molecules, and repair of DNA damage following NUV radiation. This review will focus on NUV lethal and mutagenic effects on bacteria and phages, and will attempt to identify some decisive experiments that might resolve unanswered questions. Effects on higher organisms have been reviewed (Calkins, 1982; Helene et al., 1982; Jagger, 1985). Also, the chemistry of the generation of toxic oxygen species, which are photoproducts of NUV, both with and without added photosensitizers, may be found in excellent reviews (Jagger, 1985; Sies, 1985; Sohal and Allen, 1986).

B. MODELFOR NUV ACTION The complex mechanism for coping with NUV may be divided into two parts: (1) detoxification of reactive molecules that result from photooxidation (e.g., H202, 0 2 - , OH., and singlet oxygen) and (2) repair of DNA damage and resynthesis of damaged tRNA. Because there are multiple responses to NUV, it is difficult to follow a single set of events; much depends on the initial photoreceptor and subsequent reactions. Some of these reactions occur simultaneously. 1. By analogy with far-ultraviolet radiation (FUV) effects, the simplest reaction would be that NUV photons directly alter DNA and produce mutations or other deleterious effects, and that the cell copes with the changes to return to a “nonstressed” state. Although the frequency of such lesions may be considerably less than for lesions produced by indirect photo action, they still may be significant. 2. Instead of (or in addition to) direct photo action, the DNA damage could be indirect, via a n endogenous photosensitizer (photodynamic action) (Straight and Spikes, 1985). There are abundant heme, flavin, and other cellular molecules that could serve as photoreceptors, with energy transfer causing damage to DNA. Neither the specific DNA lesion nor the recovery from indirect action need be the same as from direct photo action.

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3. H202 and other reactive oxygen species generated by NUV may (a) peroxidize lipids and other molecules, (b) damage DNA, and (c) signal a cascade of coping events, including the induction of several gene products, some under control of the oxyR regulon. The oxidative damage to the DNA could produce a lesion that might differ from the lesions of 1and 2, above, and could perhaps induce a different regulon. 4. Even at low fluences, there are profound biological consequences, such as when thiolated tRNA molecules are inactivated by NUV (Favre et al., 1985; Kramer et al., 1988); these include abrupt cellular growth delay and reduction of cell size. This effect on biomolecules could be independent of H202. The triggering of guanosine tetraphosphate (ppGpp) synthesis or other nucleotides (alarmones) is a n intriguing aspect of the recovery process, since mutants lacking thiolated tRNA (nuu) or mutants defective in the stringent response (relA)are sensitive to low fluence of NUV (Favre et al., 1985; Kramer et al., 1988). Also, dihydroxy acid dehydratase (DHAD), a key enzyme in the amino acid biosynthetic pathway, is sensitive t o NUV (Wilke, 1988) and could trigger a protective response. 5. One of the earliest biological effects a t very low fluences occurs a t the membrane level and is detected by sharp exclusion of exogenous molecules. One of the intriguing aspects here is that the presence of catalase hydroperoxidase I (HPI) overcomes this exclusion. 6. Initially, there is a derepression of a regulatory gene(& followed by derepression of appropriate stress genes and synthesis of proteins necessary to cope with the stress. These inductions include detoxifying molecules (e.g., catalase HPI and Mn superoxide dismutase), ppGppinducible proteins, and DNA repair enzymes (xthA, dam, and polA genes are involved in repairing DNA damage, but they are not inducible by NUV). Along with synthesis of some new proteins, NUV shuts off synthesis of many other proteins. Also, other gene products, not yet identified, may be involved. 7. One of the effects of NUV is nitrogenous base destruction. After removal of the base, the AP site is recognized by exonuclease I11 (exoIII) (xthA gene product), producing nicks 5’ to the base-free deoxyribose. The polA gene product, polymerase I (polI), could remove free sugar (5’ to 3’). The product of the d a m gene may also be involved in the recovery process (Yallaly, 1988). The precise role of ligase in the final step is obscure (Zig mutants are not NUV sensitive). 8. Following DNA damage, several pathways might be available for repair or bypass, depending on the specific nature of the lesion. The SOS pathway may be involved indirectly, but other enzymatic actions also occur. Among SOS gene products, the recombination function of

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the product of recA may be important for repair or bypass of NUV lesions. Glucosylase is probably not involved in recovery (ung-I mutants are not NUV sensitive). 9. Whether NUV mutagenesis is the same as 02-mutagenesis remains unresolved. However, in both cases, excess catalase HPI and absence of ex0111 are antimutagenic. 10. The product of the katF gene has a role in protection against NUV. It is a regulatory gene that is necessary for the production of catalase HPII and ex0111 (Sak et al., 1989). 11. There may be two distinct types of pathways of damage and recovery, one type occurring a t low NUV fluence rates (Kramer and Ames, 1987; Lang et al., 1986) or H202doses (Linn and Imlay, 1987) and other pathways occurring at higher fluence rates and doses. The lower fluences and doses may involve iron (Fenton chemistry). 12. The model must also account for the need of a delicate quantitative balance of some of the enzymes (excess may have a deleterious effect), as well as quantitative and changing ratios between different enzymes and radical scavengers a t different steps in a pathway.

II. Chromophores (Photoreceptors) of NUV

A. DNA Studies on effects of ultraviolet radiation are usually performed with germicidal lamps that emit radiation maximally at 254 nm (farultraviolet radiation). At this wavelength, DNA (A,, = 260 nm) is the major chromophore, and the mechanisms of DNA mutagenic and lethal events have been described in some detail. Thymine dimers, as well as other DNA photoproducts, are formed and the cell responds accordingly. However, studies of NUV effects show that these differ from FUV, and they are much more complex (Ferron et al., 1972); this would be expected since cells have both DNA and a number of non-DNA photoreceptors with A,, in the range 290-400 nm. Although DNA may still absorb a small quantity of radiation (e.g., -0.1%, at 320 nm, of that which is absorbed at 260 nm) (Jagger, 19851, this may not be sufficient t o account for all NUV-induced mutations (Cabrera-Juarez, 1981; Favre et al., 1985; Kubitschek et al., 1986; Moody and Hassan, 1982; Turner and Webb, 1981). However, even a t low absorbance, long exposures to NUV could account for accumulated DNA damage. Therefore, despite several decades of study, the role of

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DNA as a critical NUV chromophore still needs to be sorted out from the effects on non-DNA chromophores. While DNA may not be the critical chromophore for NUV, it is a main target of NUV, resulting in death, mutation inhibition of DNA synthesis (Parsons and Hayward, 19851, and biochemical alteration. “Target” is defined as the damaged molecule that results in death, mutation, or other physiological alteration. As noted, DNA may be both a chromophore and a target for NUV (1)via direct photon action on DNA similar to FUV, although the lesions differ; (2) photodynamically by striking an absorbing chromophore (e.g., hematin) that transfers the energy to adjacent DNA; (3)via photooxidation to generate toxic oxygen species (e.g., H202, Oa-, OH., and singlet oxygen); (4) via destruction of critical enzymes that assist in DNA lesion formation (e.g., catalase, ribonuclease reductase, and dihydroxy acid dehydratase); and (5)via destruction of thiolated tRNA and 2-thiouracil photosensitization (Peak et al., 1987b, 1988). Thus DNA alterations may occur via several distinctly different routes; step 1 assumes that DNA is the photoreceptor, but steps 2-5 assume that DNA is the target, with energy transferred from another molecule. In addition to physical measurements that show 290- to 320-nm NUV is absorbed by DNA, there is biological evidence of direct DNA alteration. Perhaps the most supportive evidence comes from studies of photoreactivation (see Section II,E), in which DNA damaged by FUV is partially repaired by NUV directly, even without the photoreactivating enzyme. This indicates that the DNA bases, although altered by FUV, can absorb NUV directly. Nonenzymatic photoreactivation and other photoreversal effects occur in cells devoid of photoreactivating enzymes ( p h r mutants) and under stringent anoxic conditions; this should preclude effects via intermediate reactive oxygen species, and indicates that the NUV action is directly on DNA. Another direct way to determine whether DNA is a n effective NUV chromophore is to irradiate transforming DNA under anoxic conditions and to assay for mutations and other destructive effects (Cadet et al., 1986; Cabrera-Juarez, 1964, 1981; Cabrera-Juarez and Setlow, 1976). Despite some debate about whether other molecules might tenaciously remain as part of a DNA preparation and act as photosensitizers, it is clear that DNA may directly absorb NUV photons, resulting in genetic damage [Cabrera-Juarez, 1964, 1981; CabreraJuarez and Setlow, 1976; see Jagger (1985) for a n excellent discussion of the photochemistry and photobiology involved]. Further evidence that DNA may be a direct antenna for NUV

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photons is that careful action spectra studies always show an abrupt 330- to 340-nm break in the slope for lethality, mutagenesis, and single-strand DNA breaks (SSBs) (Ananthaswamy et al., 1979; Jagger, 1985; Kubitschek et al., 1986; Mackay et al., 1976; Peak et al., 1983) for both phages and bacteria, even under strict anoxic conditions. Whether the 330- to 340-nm chromophore is a n unidentified configuration of the DNA or a non-DNA molecule is not fully resolved. The ability to detect mutations a t the base sequence level (Miller, 1985) might be a conclusive way of determining whether DNA can be a photoreceptor. By treating appropriate lac1 cells with NUV, both under aerobic and anaerobic conditions, l a d dmutants can be collected. By recombination with F1 phage, the lacl region can be sequenced to determine the specific base changes produced by NUV. Another technique for identification of direct DNA lesions by NUV might be by antibody reaction (Katcher and Wallace, 1983; Kow and Wallace, 1985). Although direct damage of DNA by NUV cannot be ignored, it is likely that oxygen in a n excited or radical state is responsible for most of the DNA damage (Breimer and Lindahl, 1985; Jagger, 1985; Martin et al., 1984; Nishida et al., 1981; Quintiliani, 1986; Sestili et al., 19861, since a number of quenching agents can protect transforming DNA against NUV. Although transforming DNA is altered more readily in the presence than in the absence of 0 2 (Cabrera-Juarez, 1981; Jagger, 1985; Nishida et al., 19811, there is a possibility that the excited or radical oxygen comes from DNA. Since xthA and sodAB mutants are sensitive to both NUV and H202, it is likely that part of the DNA damage occurs by altering the sugar moiety (Fenton reaction), which ultimately leads to base damage. Oxygen is required for most (but not all) biological reactions by NUV. In these photooxidations, if the target is the DNA or the membrane, this could have drastic consequences. The subject of photodynamic action has been extensively reviewed ( Jagger, 1985; Straight and Spikes, 1985), especially with regard to psoralens. It should be kept in mind that cells contain numerous photodynamic endogenous photosensitizers.

B. THIOLATED tRNA Another NUV chromophore reaction that affects DNA is the absorption of 334 nm by thiolated tRNA, as studied in E . coli by Favre and by Jagger and their associates (Blanchet et al., 1984; Caldeira de Araujo and Favre, 1986; Favre et al., 1985; Hajnsdorf and Favre, 1986; Jagger,

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1985; Salet et al., 1985; Peak et al., 1988) and in Salmonella typhimurium by Kramer et al. (1988). The primary biological effect is one of growth delay (see Section V1,A). NUV produces a cross-link of the 4-thiouracil with cytosine, thus inactivating the acetylation capacity of this tRNA. There is an enormous increase (100-fold) in ApppGpp and ppGpp, which may signal the induction of other proteins [this increase does not occur in nuu mutants (Kramer et al., 1988)I. Growth is resumed upon synthesis of new thiolated tRNA. It is intriguing to note that the altered tRNA may also initiate a signal that is involved in stopping normal protein synthesis and DNA metabolism, leaving numerous single-strand DNA gaps as part of the normal DNA replication process. However, it is hard to distinguish DNA gaps as a result of growth delay from direct NUV effects. The relationship between the rate of chromosome synthesis and NUV sensitivity is not clear-cut, and use of nuu mutants that lack the thiolated tRNA may be a way to distinguish between direct and indirect actions of NUV. Cells with no (or slowed) DNA synthesis (fewer chromosomal growing forks) are more resistant to NUV than are rapidly growing cells with several growing forks (Eisenstark, 1982; Hartman and Eisentstark, 19781, yet 4-thiouridine mutants that do not exhibit DNA growth delay (and thus have more growing forks when cells are in log growth phase) are more resistant t o high fluences of NUV than are the wild-type bacteria (Favre et al., 1985; Hajnsdorf and Favre, 1986; Jagger, 1985).This is an apparent paradox, since the growing cells should be more sensitive as a result of more chromosomal growing forks. However, the following, observations may help to resolve this inconsistency. At much lower fluences, results are quite different; Kramer et al. (1988) compared nuu' and nuu- mutants and found that the nuumutants, lacking thiouridine, is actually more sensitive. They argue that thiolated tRNA molecules in E . coli act as photoreceptors and initiate alarmone synthesis, e.g., ppGpp (Favre et al., 1985; Hajnsdorf and Favre, 1986) or AppppA (Bochner et al., 1986; Lee et al., 19831, which signals numerous protective, enzymatic activities. Although NUV fluences that cause growth delay are not lethal, if these fluences are maintained for several hours, cells may not recover. In nature, such low fluences may still have lethal and mutagenic effects on bacteria, especially if the bacteria are stressed by low (normally nonlethal) doses of other agents (Kramer and Ames, 1987). As noted, the action spectrum for inactivating T7 phage (or bacteria) fits neither the absorption spectrum of DNA nor that of protein. Further, if phage particles are irradiated in a nonlethal concentration

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of H202,the NUV effect is greatly amplified, with a peak of 340 nm for this synergistic effect (Ananthaswamy et al., 1979). Since phages contain no thiolated bases, there is obviously another (non-tRNA) effect of NUV a t -340 nm, which could be either direct DNA damage or DNA-protein cross-linking. AND OTHERMOLECULES C. PORPHYRINS OF THE RESPIRATORY CHAIN

Porphyrin components of the respiratory chain in E . coli are chromophores that may be involved in endogenous photodynamic effects on either DNA or cell membranes (Jagger, 1985; Kramer and Ames, 1987; Peters, 1977; Tuveson and Summartano, 1986). Some of the evidence is based on the observation that a hem mutant, which is defective in the ability to make porphyrin, is resistant to NUV; other mutants that accumulate heme are sensitive to NUV (Peak et al., 1987). The photodynamic behavior of porphyrins has been reviewed (Sies, 1985). Tuveson and Summartano (1986) point out that, since photodynamic action requires oxygen, additional components of the respiratory chain might be involved (Aliabadi et al., 1986; Gennis, 1987; Ingeldew and Poole, 1984; Jagger, 1985). Indeed, there are a number of molecules associated with the respiratory chain that absorb in the NUV range [e.g., riboflavin = (Amm = 375 nm), menaquinone (A = 330 nm), pyridoxal phosphate (A = 390 nm), and porphyrins (A = 380 nm)]. Of particular interest is the sensitivity of a mutant that lacks NADH dehydrogenase ( n d h ) to low concentrations of H202 (Imlay et al., 1988) and to NUV. Note also that heme-containing catalase may be a photosensitizer, as observed by increased sensitivity when cells contain increased catalase (Eisenstark and Perrot, 1987; Kramer and Ames, 1987). Also, NUV can inactivate catalase both in uitro and in uiuo (Cheng et al., 1981; Freierabend and Engel, 1986; Wilke, 1988). It should be noted that, although riboflavin synthesis and porphyrin biosynthesis involve different pathways, if the two were coordinately regulated, riboflavin might be an important endogenous photosynthesizer (Tuveson and Summartano, 1986). Indeed, it is an excellent exogenous photosensitizer, probably yielding 0 2 - and/or singlet oxygen. Kramer and Ames (1987) noted that cells that are ahp' (flavincontaining alkyl hydroperoxidase) are more NUV resistant than are ahp-. Storz et al. (1989) have analyzed mutant- and plasmidcontaining strains of ahp and found that one ahp gene is under oxyR control, but another one is not.

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D. AMINOACIDSAND POLYPEPTIDES Early in our studies, we proposed that most NUV damage, in contrast to FUV damage, might be the result of a secondary effect rather than of a direct hit on DNA (McCormick et al., 1976; Yoakum et al., 1975). Tryptophan was found to be a chromophore for NUV and yielded H202 as a photoproduct (McCormick et al., 19761, as well as N-formylkynurenine (A,, = 318 nm). H202, in turn, was assumed to produce lesions in DNA (Ananthaswamy and Eisenstark, 1976, 1977; Ananthaswamy et aZ.,1979; Hartman, 1986; Hartman and Eisenstark, 1978,1980; Hartman et al., 1979). H202 also may be a photoproduct of cysteine irradiation (Greenberg and Demple, 1986; McCormick et al., 1976, 1982; Owens and Hartman, 1986). We have since found that Hz02, in the presence of certain wavelengths of NUV, synergistically kills bacteria and phages (Eisenstark et al., 1980; Hartman and Eisenstark, 1978, 1980, 1982; Hartman et al., 1979). This synergistic action may be the result of NUV conversion of H202 to 0 2 - (Ahmad, 19811, and this action may generate a new NUV photoreceptor (McCormick et al., 1982). Another possibility is that NUV generates 02- to give a Fenton reaction ( 0 2 - + H202 + Fe2+),yielding OH.. Further support that HzO2 may be a critical photoproduct of NUV comes from the knowledge that certain mutations lead to greater sensitivity to both NUV and H202 (Eisenstark, 1985; Eisenstark and Perrot, 1987; Sammartano and Tuveson, 1983; Sammartano et al., 1986; Tyrrell, 1985). Studies of phage T7, which contains only DNA and protein, emphasize a paradox that could be explained by a polypeptide acting as a photoreceptor; peak absorption is 254 nm for DNA and 280 nm for protein, but action spectra for killing of phage T7 (Ananthaswamy et al., 1979) and S. typhimurium (Eisenstark, 1971; Mackay et al., 1976) show distinct shoulders a t -334 nm. The action spectrum for inactivating phage T7 (or bacteria) fits neither the absorption spectrum of DNA nor of protein. In a search for a NUV chromophore, we observed that the sulfhydryl in peptide-bound cysteine, in the presence of oxygen (or H202), is photochemically altered (McCormick et al., 1982). Treatment of reduced glutathione (a cysteine-containing tripeptide) with 2.5 mM H202 results in the formation of a NUV chromophore having a maximal absorption 25 nm above the absorption of the initial glutathione. From examination of related compounds, it is apparent that the N-acylcysteinamide of the peptide residue is the key element required for generation of the new chromophore. Although we have not

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yet determined the structure of the new chromophore, we do know that it is not simply the oxidized cysteinyl residue. Glutathione is a useful model molecule to show that a cysteine-containing polypeptide can be altered to generate a new chromophore, one that can absorb NUV and possibly result in biological damage (Greenberg and Demple, 1986; Owens and Hartman, 1986). There are a number of enzymes with absorption in the 290- to 400-nm range, but in only a few cases is i t known whether NUV (or a reactive oxygen molecule) will produce a critical biological change. In the case of one enzyme in particular, dihydroxy acid dehydratase, its sensitivity to 02-has been well characterized (Brown and Seither, 1983; Kuo et al., 1987). It is also sensitive to NUV; the amount of its activity in E . coli cells can be reduced by 50% with a fluence that will inactivate only 5% of population (Wilke, 1988). Although the absorption specturm of E . coZi DHAD has not been determined, DHAD isolated in the oxidized state from spinach has its major absorption between 290 and 450 nm (Flint and Emptage, 1988). The enzyme contains a n iron-sulfur cluster (which may account for its particular absorption spectrum); a shift from the oxidized to the reduced state greatly reduces enzyme activity and greatly reduces its absorption from about 370 to 450 nm. Primary studies with E . coli DHAD indicate that it has a similar iron-sulfur cluster (Flint and Emptage, 1988). In addition to its reputation of being the enzyme most sensitive to 0 2 - , DHAD inactivation may be important in triggering the stringent response (Brown and Seither, 1983; Cashel and Rudd, 1987). This sensitivity of DHAD is supported by the observation that sodAB mutants grow very poorly on minimal medium (Touati, 1988b). Since 02- inactivates DHAD (an important enzyme in amino acid biosynthesis), this failure could be due to starvation of branched-chain amino acids. DHAD catalyzes a step in the valine-isoleucine biosynthetic pathway. While there are mutants (ilvD)that accumulate this enzyme, it is difficult to study its NUV sensitivity resistance because the presence of branched amino acids reduces the DHAD in the cell and valine needs to be added to the medium for growth of the mutant to occur. In vivo NUV inactivation ofE. coli ribonucleotide reductase has been reported (Peters, 1977). Its role in coping with NUV deserves further study. Other examples of proteins that could be inactivated directly by NUV are noted in Section 111. Figure 1shows a schematic summary of the photoreceptors present in cells, as well as other key molecules involved in NUV damage and recovery.

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J

P

FIG. 1. (See the text, Section 1,B.) The photoreceptors are the outer and inner membrane, molecules in the periplasmic space, thiolated tRNA, the two catalases (HPI and HPII), dihydroxy acid dehydratase (DHAD), DNA, flavin- and hemecontaining molecules, and other molecules that absorb in the 300-to 400-nm range. Hydrogen peroxide, superoxide anion, hydroxy radical, and singlet oxygen are toxic photoproducts, a s well as DNA with photo alterations. Two superoxide dismutase enzymes (Mn and Fe) detoxify the superoxide anion.

E. PHOTOPROTECTIVE CHROMOPHORES When cells have been damaged by FUV, they may be rescued from death by NUV. This photoprotection is due to one of several different biological reactions, and deliniation of these may give us some understanding of NUV cellular chromophores and targets. There is a photoreactiving (PR) enzyme (DNA photolyase) with a n absorption peak of 380 nm; it complexes with the pyrimidine dimers produced by FUV, and NUV rapidly splits the complex, leaving pyrimidine monomers (Sancar et al., 1983).This is another example of a protein chromophore for NUV. The PR enzyme is -49 kDa (Sancar and Sancar, 1984)and has a flavin adenine dinucleotide component as a chromophore. If this enzyme action were the only explanation of photoprotection, a mutant lacking this enzyme should not display photoprotection. Such mutants and deletions of phr have been studied carefully, but even these strains are capable of some degree of dimer cleavage by NUV (Hussain and Sancar, 19871, even under conditions that would indicate involvement of a nonenzymatic reaction. It is possible that a C-C (6-4) dimer is the direct NUV photoreceptor, resulting in restoration of the

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monomer state upon irradiation. This is important, since it would further support the view that DNA configurations can be photoreceptors of NUV, as noted in Section II,A. It would also indicate that NUV may be an antimutagen, since such C-C dimers (6-4) are mutagenic (Glickman et al., 1986). Ill. Oxidative Photoproducts of NUV

There is considerable evidence that H202 is a photoproduct of NUV and that other reactive oxygen molecules are subsequently generated (McCormick et al., 1976). Also, it is possible that 0 2 - could be a direct photoproduct of NUV. At least two sets of enzymes, catalases and superoxide dimutases, are known to detoxify these reactive molecules; numerous endogeneous radical scavengers, such as glutathione, are also known. However, as the roles of these enzymes are characterized in stress experiments, it is obvious that they operate in a highly sophisticated manner, and these enzymes and radical scavengers may have functions in addition to being general detoxifiers in cells, such as involvement in metal ion metabolism. It would be expected that reactive oxidative species (and NUV) might be more damaging a t certain precise locations than at others, and that catalase and/or SOD might be expected to be located at these sites. Furthermore, it should be kept in mind that these enzymes could have effects at precise times that are critical in growth and development of cells. An example is the striking difference in the need for catalase at different stages of vegetative growth and sporulation in Bacillus subtilis (Dowds et al., 1987). As another example of a specific role for catalase, it is interesting to note that the katG gene product is located at the cytoplasmic membrane but the katE gene product is located in the cytoplasm (Heimberger and Eisenstark, 1988). Moreover, the role of catalase HPI in restoration of membrane transport is intriguing and raises a question of its importance in all transport systems (Farr et al., 1988). Additionally, HPI is important in overcoming the mutations in sodA sodB strains and thus is a n antimutagen (J. Hoerter and A. Eisenstark, unpublished). Another possible role for SOD, in addition to quenching 02-and involvement in Fe/Mn metabolism, might be to activate or inactivate enzymes, thus serving a n interesting cellular regulatory role. As noted

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above (Section I1,D) an example is dihydroxy acid dehydratase, which is inactivated by 02-(Brown and Seithers, 1983) and NUV (Wilke, 1988). The special role of the SOD/02- balance may be to modulate enzyme activity, as well as to signal the stringent response in E . coli, with the abrupt cessation of protein synthesis. Other examples include the 3’ to 5’ exonuclease activity of phage T7 that specifically inactivated by molecular oxygen (Tabor and Richardson, 1987). Of particular interest is a possible role of SOD in the activation of ribonucleotide reductase in E.coli (Peters, 1977). Flavin reductase generates superoxide radicals, which would inactivate ribonucleotide reductase (Fontecave et al., 1987). There are also examples in organisms other than E . coli, such as the oxidation of rhodanase in algae that renders them susceptible to proteolysis (Horowitz and Bowman, 19871, and a possible superoxide-dependent peptidyl deaminase for making citrulline in mouse bone marrow (Kamoun et al., 1988). A. HYDROGEN PEROXIDE There is evidence that the toxic effect of HzOz is primarily to damage DNA; this is attributed to a Fenton reaction that generates OH. from H202,DNA-bound iron, and a source of reducing equivalents (Imlay and Linn, 1988). In our earlier studies on the effect of NUV on bacteria, we found that a photoproduct of NUV was HzOz, which, in part, might have a role in NUV lethality. Since that time, numerous observations have been made of the similarities between Hz02 and NUV effects (Ahmad, 1981; Ananthaswamy and Eisenstark, 1976; Eisenstark, 1985; Hartman, 1986; Roth, 1981; Sammartano and Tuveson, 1983; Tyrrell, 19851, consistent with the view that biologically relevant quantities of HzOz may be generated in sztu following NUV irradiation of cells. But, again, we face a paradox. It would be expected that mutants that are devoid of catalase would be very sensitive to NUV; they are not. Although Sammartano et al. (1986) showed that katF mutants that lack catalase activity are sensitive to NUV, we now know that katF is a regulatory gene for katE and xthA and not a structural gene for catalase. The sensitivity of katF mutants to NUV and H202 is due to the lack of ex0111 enzyme and not the lack of catalase HPI (Sak et al., 1989). Consistent with the NUV/H202 relationship is the observation that small doses of either H202or NUV often induce proteins that protect against challenge doses of either (see Section V1,B).However, it should be noted that there are some exceptions to reciprocity of induction

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(Eisenstark and Perrot, 1987; Kramer and Ames, 1987). In addition, the fact that catalase mutants (katE and katG and even the katE katG double mutant) are void of enzyme activity but are not sensitive to NUV (Eisenstark, 1982) shows that a NUV/H202relationship is not simple. Also, the amount of endogenous catalase can be physiologically manipulated; when this is done t o reduce catalase drastically, cells actually become more resistant, rather than sensitive. The amount of catalase in a cell can also be increased via a multicopy plasmid with the katG gene. Cells with excess HPI are not more resistant to H202; they actually become more sensitive to NUV (Eisenstark and Perrot, 1987; Wilke, 1988). Upon comparison of new proteins that are synthesized and shut off following induction by H202 and NUV, there are numerous differences (Kramer et al., 1988; Pierceall, 19881, further supporting nonidentical effects. However, the oxyR regulon is involved in both HzOz and NUV stress, since a deletion of the oxyR gene results in hypersensitivity to both (Eisenstark and Perrot, 1987; Kramer and Ames, 1987). Also, mutations in glutathione reductase ( g s h ) and alkyl hydroperoxide reductase (ahp),both regulated by oxyR, result in sensitivity to NUV (Kramer and Ames, 1987; Storz et al., 1989). These observations, plus toxicity differences in mutants defective in porphyrin (Kramer and Ames, 1987; Tuveson and Sammartano, 1986) and tRNA synthesis (Favre et al., 19851, further indicate that H202 and NUV differ in their modes of killing cells. This further is supported by the synergistic (and not additive) action by NUV and H202 (Ahmad, 1981; Ananthaswamy and Eisenstark, 1976; Ananthaswamy et al., 1979; Hartman and Eisenstark, 1978, 1980; Hartman et al., 1979). Yet, there are important overlaps, particularly in the roles of enzymes regulated by the oxyR gene. From these comparisons of NUV and H202 toxicity, it would appear that H202 is only indirectly involved in NUV killing, but still may be a source of oxygen radicals. B. SUPEROXIDE ANION NUV irradiation results in 0 2 - increase (Ahmad, 1981) (see Sections I11 and V,B for discussion of superoxide dismutase). Not only can this be observed chemically, but the fact that sodA sodB strains are highly sensitive to NUV (A. Eisenstark, unpublished) would indicate that 0 2 - is produced. Perhaps a better explanation is that H202 is generated by NUV and this reacts with 0 2 - to yield OH., which damages DNA.

114

A. EISENSTARK

Before considering the effects of SOD deficiencies in mutants, it is of interest to note the effect of overproduction, since one of the explanations of human Down's syndrome is that excess SOD is synthesized as a result of triploid chromosome 21. Excess MnSOD in E. coli does not lead to increased sensitivity to NUV, but cells with plasmid FeSOD are more sensitive (A. Eisenstark, unpublished). Scott et al. (1987) reported that bacterial cells with excess FeSOD are more sensitive to paraquat and attributes this to the increased HzO2 (and perhaps subsequent hydroxy radicals) that results from the SOD action. The fact that the sodA sodB double mutant is hypersensitive to both NUV and H202 could result from generation of hydroxyl radicals through the iron-catalyzed Haber-Weiss reaction: Fez++ HzOz + H' + Fe3+ + HO. + H20 HO. + H202 -+ HO, + HZ0 HOi + Fe3+-+ O2 + Fez++ H' 2H202 + 0

2

+ 2Hz0

The sodA sodB double mutant has metabolic problems (e.g., it cannot grow on minimal medium) and is highly mutagenic. It is interesting to note that all of these deficiencies can be overcome by the addition of a plasmid containing either of the two E . coli SOD genes, or by the human Cu/Zn SOD plasmid (Natvig et al., 1987; Touati, 1988a). Also, it should be noted that mutagenesis is oxygen dependent and requires the ex0111 repair enzyme t o transform the premutational DNA lesion into a mutation. In assessing any correlation between the quantity of SOD and protection against oxidative damage, there is disagreement in the literature. Experiments utilize paraquat to generate 02- and to determine its effect on sod mutants and plasmid strains. Perhaps anomalous observations could be the result of other affects of paraquat, and not directly the result of 0 2 - . Another viewpoint is that the role of SOD may not be primarily for quenching 02-;rather, SOD is more important in metallic ion metabolism in cells (Fee et al., 1989; Niederhoffer et al., 1989).

C. HYDROXYL RADICAL The hydroxyl radical (OH.) reactivity is so great that it will react almost immediately with whatever molecules are in its vicinity. By the use of OH. scavengers, experiments have shown that excess of this radical may have critical consequences (Billen, 1984). Rowley and Hallewell (1982) showed, using thiol compounds that readily react with OH., that thiol compounds did not prevent 02--dependent formation of

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the OH radical in the presence of iron ions. The thiol compounds could produce the radical themselves. They also showed that addition of only OH. scavengers prevented DNA strand scission, and the addition of SOD and catalase prevented strand scission. They concluded that both 0 2 - and HzOz are needed for strand scission but the OH. is the actual attacking molecule. D. SINGLET OXYGEN Singlet oxygen ('Oz),the lowest electronically excited energy state of molecular oxygen, has a relatively long lifetime and the potential to react with a variety of biological substrates. There is disagreement, however, as to its biological significance. Dahl et al. (1987, 1988) state that singlet oxygen is highly toxic but not mutagenic. DecuyperDebergh et al. (1987), however, found that it produces a high frequency of mutations in the lac2 region upon exposure of RF (double-stranded) DNA of phage M13 mp19. Photosensitizing systems are used to implicate singlet oxygen involvement in biological effects such as bacteriophage 4x174 inactivation (Houba-Herin et al., 1982). Dahl et al. (1987, 1988) recently demonstrated directly that exogenous pure singlet oxygen is over 10,000-foldmore toxic to bacteria on a molar basis than is H202?but that it is not mutagenic. Biological sources of singlet oxygen may include photosensitization reactions involving endogenous sensitizers, decomposition or interconversion of other active oxygen species, electron transport systems, and some enzyme systems. In order to cope with adverse effects of singlet oxygen, living systems may use at least two defense strategies: (1) interception of singlet oxygen either by quenchers (Ames et al., 1981; Dahl et al., 1987, 1988) and/or (2) prevention of singlet oxygen formation by direct quenching of excited-state sensitizers (Dahl et al., 1987, 1988). Dahl et al. (1988) investigated the abilities of several biomolecules at physiologically relevant concentrations and neutral pH to protect against oxidation of a target substrate by singlet oxygen. Dahl et al. (1989) noted that bacterial species that contain carotenoids are more resistant to killing by singlet oxygen than those that are void of carotenoids. IV. DNA Damage

The critical DNA lesions produced by NUV and HzOz differ from

FUV lesions, despite some qualitative similarities in photoproducts

116

A. EISENSTARK

(Ferron et al., 1972). The evidence is as follows: (a) analyses of DNA damage by the different agents yield different profiles (Ananthaswamy and Eisenstark, 1976; Caimi and Eisenstark, 1986; Hartman and Eisenstark, 1982, 1987; Mitchell and Clarkson, 1984; Tuveson et al., 1983); (b) defense against these agents involves different regulons (Aliabadi et al., 1986; Christman et al., 1985; Eisenstark, 1985; Morgan et al., 1986) with induction of different proteins; (c) different sets of mutants are hypersensitive to these agents (Eisenstark, 19851, although some mutants are hypersensitive to both; (d) DNA repair and mutagenesis of FUV damage are influenced by u m u and muc genes, but not after NUV irradiation (Eisenstark, 1983); and (e) numerous physiological effects are different (Jagger, 1985) (Table 1). There is strong evidence that a NUV lesion is a substrate for exonuclease 111. The DNA lesion may be a single-strand break with a 3’-end-blocking group (phosphoglycoaldehyde ester), which may be activated by exonuclease I11 to allow synthesis by polymerase I (Demple et al., 19861. This is consistent with the observations that xthA (exoIII) and polA (pol11 mutants are sensitive to both H202 and NUV. Endonuclease IV may also be involved in the process by initiating the repair of ruptured 3’-dexoxyribose (Demple et al., 1986). Further support, although indirect, comes from the observation that, when ex0111 acts on DNA damaged by 02-,mutation frequency is increased (Farr et al., 1986). NUV is one of the ways that 02-might be generated (Ahmad, 1981; Fridovich, 1986; Kramer and Ames, 1987), thus the similarity with regard to exoIII. We have examined the possibility that an initial lesion might be a DNA-protein cross-link produced by NUV. DNA-protein cross-links occur particularly in phage (Hartman et al., 1979; Casas-Fimet et al., 1984) and mammalian cells (Eisenstark et al., 1982), where the DNA and protein are packaged geometrically in close quarters. Evidence with bacteria is far less striking, but only a small fraction of DNA and protein is tightly bound in these cells. The observation of DNAprotein cross-links as a critical NUV lesion may not conflict with the observation of DNA strand breaks (Ananthaswamy and Eisenstark, 1977; Caimi and Eisenstark, 1986; Demple et al., 1986; Eisenstark et al., 1982; Hartman and Eisenstark, 19801, since these single-strand and double-strand DNA breaks could be secondary consequences of photochemical damage, following the initial cross-link. Note that the number of DNA breaks is not correlated with the number of lethal NUV events (Ananthaswamy and Eisenstark, 1976, 1977; Hartman and Eisenstark, 1980; Tuveson et al., 1983). By comparison, the number of DNA-protein cross-links of phage (Hartman and Eisen-

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TABLE 1 Some Distinct Differences between Far- and Near-UV Damage in Bacteria and Phages Effect DNA degradation in recA mutants Mutation enhancement Growth inhibition (division delay) Use of SOS regulon Log cells more sensitive than stationary Sensitivity of katF mutant Sensitivity of r t h A mutant Sensitivity of nfo mutant Sensitivity of polA mutant Sensitivity of sodA sodB double mutant Production of DNAphage protein links Block phage DNA injection Sensitivity of Deinococcus radiodurans Enhancement of resistance and mutation by plasmid pKMlOl Synergistic action with nonlethal doses of HZO2 Weigel reactivation of A phage Oxygen demand for lethality Enzyme induction inhibition Liquid holding recovery

Far-UV

NUV

References

Yes

No

Ferron et al. (1972)

High Low

Limited High

Favre et al. (1985) Jagger (1985)

Yes

No

Slight

High

Turner and Eisenstark (1984) Eisenstark (1982)

Low

High

Eisenstark and Perrot (1987)

Low

High

Low

High

Sammartano and Tuveson (1983) A. Eisenstark (unpublished)

Low

High

Eisenstark and Perrot (1987)

No

Yes

A. Eisenstark (unpublished)

Low

High

Eisenstark et al. (1982)

No

Yes

Resistant

High

Hartman and Eisenstark (1982) Caimi and Eisenstark (1986)

High

No

Eisenstark (1983)

No

Yes

Ananthaswamy and Eisenstark 11976)

Yes

No

Low

High

Turner and Eisenstark (1984) Ferron et al. (1972)

No

Yes

Yes

No

Jagger 11985);Turner and Eisenstark (1984) Jagger (1985)

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A. EISENSTARK

stark, 1980; Tuveson et aZ.,1983) correlated very well with the number of phages inactivated, but DNA breaks did not show this correlation. One way to rationalize these conflicting results is t o consider that NUV damage may involve a cascade of events, including cross-links, followed by an alkylated base, with subsequent breakage of one DNA strand and then the other strand. Further disenchantment with SSBs as direct lethal events comes from knowledge that xthA and poZA mutants are very HzOzand NUV sensitive, but fewer SSBs occur than expected when compared to the number of SSBs in wild-type strains (Demple et al., 1983; Miguel and Tyrrell, 1986).The number of SSBs by NUV is much lower in E . coli than in Deinococcus radiodurans (Caimi and Eisenstark, 1986), an organism that is very sensitive to NUV but notoriously resistant to FUV. There are features of DNA damage that are common to both NUV and Hz02, but there are also some features that are unique to NUV. This may be due not only to a direct action of H202 on DNA, but also to an indirect photoeffect on DNA, perhaps by way of membrane (Klamen and Tuveson, 1982; Kelland et al., 1984; Kramer and Ames, 19871, tRNA (Blanchet et al., 1984; Cadet et aZ., 1986; Favre et al., 1985; Hajnsdorf and Favre, 1986; Klamen and Tuveson, 1982; Kelland et al., 1984; Peak et al., 1987a), and/or other photoreceptors (Jagger, 1985; Spitzer and Weiss, 1985; Tuveson and Summartano, 1986). The biological similarities between the DNA damage and repair produced by NUV and Hz02 could be accounted for if some DNA damage by NUV is the generation of H202 (and subsequent reactive oxygen molecules) (McCormick et al., 1976; Yoakum et al., 1975). To verify this, the DNA lesions after H2O2 treatment should be compared in detail with NUV lesions, both at low and high doses (Imlay and Linn, 1986; Kramer and Ames, 1987; Linn and Imlay, 19871, as well as by sequencing genes that have been mutated by the two agents. A t high concentrations of HzOz, it has been shown by alkaline sucrose sedimentation experiments that DNA is cleaved (Ananthaswamy and Eisenstark, 1977). In the presence of a low concentration of ferric chloride and low concentrations of H202, 4x174 supercoiled DNA is incised (Van Rijin et al., 1985). The H202 photoproduct of NUV inhibits replication gap closure (Yoakum et al., 1975) and also stimulates DNA repair synthesis (Hagensee and Moses, 1986);this repair synthesis requires polymerase I (polA) (Ananthaswamy and Eisenstark, 1977), exonuclease I11 (xthA) (Weiss and Cunningham, 19851, and probably polymerase I11 (poZC) (Hagensee and Moses, 1986). It also has been observed that all four of the bases may be released from the backbone (Breimer and

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Lindahl, 1985). Hagensee and Moses (1986) reported that cells with a temperature-sensitive polymerase I11 mutation are sensitive to H202, but cannot synthesize DNA after H202treatment, unlike WT cells. They suggest that pol111 may either be required for a small (but undetectable) amount of synthesis or may provide a terminus modification function for subsequent DNA polyI synthesis. The observation that certain mutants (i.e., xthA and poZA) are sensitive indicates that NUV lesions become substrates for the appropriate DNA repair enzymes. However, since the r t h A gene product may have four different enzymatic activities, there is some uncertainty as to the nature of the lesion. Analyses of DNA lesions by sequencing of appropriate mutated genes (Ito et al., 1988; Miller, 1985) are informative. Base-specific damage was determined by 4-thiouridine (4TU) photosensitization with 334-nm radiation in M13 phage DNA (Ito et al., 1988). Whether the same damages would occur i n vivo without 4TU is not known. Identification of endonuclease cleavage sites (Wei et al., 1986) andlor the use of monoclonal antibodies against NUV- and H202-damagedDNA (Mitchell and Clarkson, 1984) might be another approach to identify specific modifications. V. NUV-Sensitive Mutants

Table 2 lists genes that may be involved in cellular responses to NUV. Mutations in several of these genes render cells sensitive to NUV (see Section 111for discussion of effects of oxidative photoproducts of NUV). A. CATALASE Throughout our studies, we have considered catalse as a logical “NUV-recovery enzyme,” since NUV yields H202 (McCormick et al., 1976; Yoakum et al., 1975). However, as noted in Section III,A, the role of catalase is complex and may be minor in the capability of the cell to deal with NUV damage (Eisenstark and Perrot, 1987). Indeed, the H202 photoproduct of NUV may have only a minor role in the lethal process, particularly when compared to alkyl hydroperoxidases and other photoproducts (Kramer and Ames, 1987). The fact that katF (catalase) mutants are sensitive to NUV would support the argument that H202is a photoproduct of NUV (Eisenstark and Perrot, 1987; Samartano et al., 19861, since katF+ is necessary for the synthesis of HPII. katF+ is also a regulatory gene for ex0111

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A . EISENSTARK

TABLE 2 Genes of Escherichia coli That May Influence NUV Responses Gene

Map position

Phenotype

NUV sensitive

ahp

13

Flavin-containing hydroperoxidase

Yes

CYd

17

Cytochrome

Yes

darn gsh

74 58

DNA adenine methylase Glutathione reductase

Yes Yes

Heme synthesis

See the text

Dihydroxy acid dehydratase; isoleucine-valine synthesis Catalase activity

See the text

Regulatory gene for katE synthesis

Yes

Catalase activity

Yes

No

hemA -H

8-90

iluD

85

katE

38

katF

59

katG

7

led

92

ndh

22

Resistance or sensitivity to X-rays and UV; repressor of SOS proteins NADH dehydrogenase

nfo

60

Endonuclease IV

Yes

nrdl3

49

Ribonucleotide reductase

Yes

nth

36

Endonuclease I11

Yes

Near-ultraviolet radiation growth delav

Yes

nuuA

9

Yes

Yes

References Kramer et al. (1988); Storz et al. (1989) Sammartano and Tuveson (1987) Yallaly (1988) Kramer and Ames (1987) Tuveson and Sammartano (1986); Bachmann (1987) Bachmann (1987)

Loewen and Triggs (1984) Loewen and Triggs (1984); Sak (1989) Loewen and Triggs (1984); TriggsRaine and Loewen (1987); Triggs-Raine et al. (1988) Walker (1987)

Imlay and Linn (1988) Saporito et al. (1988) A. Eisenstark (unpublished); Fontecave et al. (1987) Weiss and Cunningham (1985) Favre et al. (1985)

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TABLE 2 (Continued) Gene

Map position

Phenotype

nuuC

45

oxyR

89

Phr

16

polA

87

recA

58

recB -C

61

relA

60

SOdA

88

sodB

37

umuC-D uurA

26 92

XthA

38

General recombination, repair of radiation damage; induction of phage A Recombination and repair of radiation damage; exonuclease V subunits Regulation of RNA synthesis; stringent factor; ATP : GTP 3' pyrophosphotransferase Mn superoxide dismutase Fe superoxide dismutase Induction of mutation Repair of UV damage to DNA; excision nuclease Exonuclease I11

gal-halt deletion (unknown gene)

16

NUV sensitivity

a

Near-ultraviolet radiation growth delay Regulator of oxidative stress Deoxypyrimidine photolysase; photoreactivation DNA polymerase I

NUV sensitive

References

Yes

Favre et al. (1985)

See the text No

Kramer and Ames (1987) Husain and Sancar (1987)

Yes

Yes

Sancar et al. (1983); Ananthaswamy and Eisenstark (1977) Walker (1987)

No

Walker (1987)

Yes

Kramer et al. (1988)

No"

Touati (1988a)

No"

Touati (1988a)

No Yes

Walker (1987) Turner and Webb (1981)

Yes

Eisenstark and Perrott (1987) Husain and Sancar (1987)

Yes

The sodA sodB double mutant is NUV sensitive (A. Eisenstark, unpublished data)

(product of xthA) (Sak et al., 1989; Schellhorn and Hassan, 19881, which is the basis for sensitivity of katF mutants. The lack of correlation between catalase content and NUV sensitivity is emphasized in experiments in which the amount of intracellular

122

A. EISENSTARK

catalase is manipulated in a number of ways (Eisenstark and Perrot, 1987): (1) by the use of mutant and plasmid strains with altered endogenous catalase; (2) physiologically, by the addition of glucose; and (3) by induction of catalase synthesis with oxidizing agents. Not only is there no correlation between NUV resistance and catalase activity, but in some cases the correlation is inverse. As noted above, mutants other than katF (i.e., katE, IzatG, and double mutant katE katG) that are defective in catalase activity (Loewen, 1984; Loewen et al., 1983, 1985a; Loewen and Triggs, 1984) are not particularly sensitive to NUV (Eisenstark and Perrot, 1987; Wilke, 1988). Also, a strain that carries the KatG plasmid (Loewen et al., 1983) and overproduces catalase is actually more sensitive to NUV (Eisenstark and Perrot, 1987). It thus appears that while catalase may decompose H202 produced by NUV, it may also act as a photosensitizer in the cell when it is present in excess (Wilke, 1988). It is possible that catalase may have a physiological role only under very special conditions of stress, since its protective role is less important than certain other factors (Carlsson and Carpenter, 1980; Eisenstark and Perrot, 1987; Sammartano et al., 1986). Despite the lack of sensitivity of kat mutants, a role in protection against NUV cannot be completely ignored, since bovine catalase in plating media will reduce the lethal effect of NUV (Kramer and Ames, 1987; Sammartano and Tuveson, 1984; and our own observations). Interestingly, we have found that if cells are stressed by a number of agents (including heat), catalase in the medium will give higher colony counts (unpublished); H202 may be a common product of generalized cellular stress (Ames, 1983; Hartman, 1986; Richter and Loewen, 1981; Tyrrell, 1985; Vassilyadi and Archibald, 1985; Winguest et al., 1984). As discussed elsewhere in this review, the observations that catalase HPI may be antimutagenic and that this enzyme has a role in permeation indicate that it has specific functions that are not visible in tests for survival alone.

B. SUPEROXIDE DISMUTASE Judging by the number of recent publications dealing with superoxide dismutase and 4ts effects on 0 2 - , perhaps no other enzyme has received more attention. The numerous theories of the role of 0 2 - in various disease and aging processes, together with searches for therapeutic uses of SOD, may be found in several reviews (Touati, 1988; Hassan, this volume; Fridovich, 1986; Petkau, 1986; Rotilio, 1986; Sies, 1985). A breakthrough in our ability to understand the mechanism of SOD

BACTERIAL GENES INVOLVED IN RESPONSE TO NUV

123

action came when Touati (1983; Sakamoto and Touati, 1984) first isolated SOD genes in E . coli. She succeeded in isolating these from a cosmid bank, of which clones were individually tested for SOD overproduction. Since that time, Touati and associates have obtained mutants that are defective in two SODSin E . coli, a n inducible MnSOD (sodA), and a constitutive FeSOD (sodB). MnSOD is present only under aerobic growth, whereas FeSOD is expressed both aerobically and anaerobically. The MnSOD DNA sequence (Carlioz et al., 1988), together with a detailed study of MnSOD regulation (Touati, 1988b), suggests two possible promotors, perhaps one that is active in normal aerobic growth and another that is active under oxidative stress conditions. Superoxide dismutase is a logical candidate as a NUV-recovery enzyme (Ahmad, 1981; Farr et al., 1986; Touati, 1983; Touati and Carlioz, 1986).We have found that the presence of a plasmid carrying an inducible Mn superoxide dismutase (sodA) (Bloch and Ausubel, 1986; Carlioz and Touati, 1986; Tajeda and Avila, 1986; A. Eisenstark, unpublished) endows the cell with some resistance to NUV, but not t o FUV. Although the amount of protection is small, it does implicate 02-as a lethal radical. This strain carries a low-copy-number plasmid in a cell that already has a n Mn sod gene. A plasmid containing the FeSOD gene (sodB)(Nettleton et al., 1984; Sakamoto and Touati, 1984) does not endow the cell with NUV resistance (Scott et al., 19871, although excess SOD is produced. We have tested sodA and sodB strains (Carlioz and Touati, 1986; Farr et al., 1986; Touati, 1983, 1988a,b; Touati and Carlioz, 1986), but found them t o be only slightly more sensitive than the wild type under aerobic conditions; however, the sodA sodB double mutant is very sensitive t o NUV. This is compatible with the results of Carlioz and Touati (1986), who found that among such mutants only the double mutant was sensitive to paraquat, a generator of superoxide anion. They conclude that the role of SOD was to handle 02-only when the cell is under special stresses. These double mutants have a number of metabolic problems, but an additional (suppressor) mutation can restore the ability to grow on minimal medium (Fee et al., 1989). This mutation might be involved in the branched-chain amino acid pathway, since DHAD could be depleted in the double mutants. Also, such double mutants probably contain specific DNA lesions, which, when acted on by exoIII, yield a higher mutation frequency (Farr et al., 1986) (see Section VIII). C. EXONUCLEASE I11 AND ENDONUCLEASE IV The observations that x t h A mutants are more sensitive to NUV but no more sensitive to FUV than are the wild type, and that there are

124

A. EISENSTARK

fewer single-strand DNA breaks in the xthA mutant, support the view that a major role of xthA (ex0111 enzyme) may be to recognize apurinic or apyrimidinic sites and to nick the DNA at the 3’ side of such sites (Demple et al., 1983, 1985, 1988; Kow and Wallace, 1985; Wallace, 1988; Weiss and Duker, 1986,1987).Since xthA mutants are sensitive to both NUV and H202, a logical conclusion might be that both produce apurinic sites. However, there are other enzymatic activities of the xthA gene protein, and thus other lesions may also be recognized. DNA protein cross-links might interfere with action by ex0111 enzyme; thus, there could be two actions in the case of a DNA-protein cross-link, proteinase action on the attached polypeptide, leaving a n exposed altered base, and direct action by ex0111 at the cross-link site. There are two mutations (nfo and nth) that influence the sensitivity of xth mutants. The nfo mutation increases sensitivity to H202 and NUV; the nfo gene codes for endonuclease IV. However, the addition of a third mutation (nth, coding for endonuclease 111) gives only small protection to the double mutant (Cunningham et al., 1986; our observations). EndoIV enzyme is induced by paraquat and other 0 2 - generating agents (Chan and Weiss, 1987). Although strains with xthA mutations are sensitive to NUV, strains that carry a plasmid with the xthA gene and thus overproduce the enzyme are not more NUV resistant than are the wild type. One possible mode of action on NUV lesions is that the enzyme (exoIII) recognizes the urea moiety that results from alteration of thymine in DNA (Katcher and Wallace, 1983; Kow and Wallace, 1985; Taylor and Weiss, 1982). Since mutants that lack the enzyme are sensitive to NUV, it would appear that NUV may create this urea moiety, which may be lethal to the cell unless it is repaired. In the absence of the ex0111 enzyme in the cell, this nick obviously would not take place. Ex0111 may have an important role in the mutational process (see Section VIII). D. OTHERNUV-SENSITIVEMUTANTS 1. polA. Strains with the polA mutation are sensitive to both H202 and NUV (Ananthaswamy and Eisenstark, 1977); these strains are deficient in the 5’ to 3’ exonuclease (but not in 3’ to 5‘) activity. The polA gene product is not induced by NUV (A. Eisenstark, unpublished). Also, a strain with a polA-bearing plasmid (an overproducer of the enzyme) is not resistant to NUV. The fact that polA cells are sensitive to NUV indicates that, following the production of the

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urea moiety by NUV action, there may be no ex0111 repair, but the gap that is left requires the polA gene product (Kow and Wallace, 1985). 2. dam. The product of the dam gene is a DNA adenine methylase which methylates deoxyadenosine in -GATC- sequences in doublestranded DNA (Marinus, 1987; Szyf et al., 1986). Strains with dam mutations are sensitive to NUV (Yallaly, 1988). Since the dam gene enzyme is not required for viability, it may be that NUV damage within this sequence may be lethal unless removed by the dam gene enzyme. Increasing the levels of dam gene enzyme in plasmid-carrying strains reduces this sensitivity slightly, as might be expected. However, increasing the levels of enzyme much above wild-type levels results in a sharp increase in the sensitivity of the cell. A possible explanation is that for wildtype levels of methylase, only one of the two DNA strands is methylated, as needed for appropriate activity, but excess methylation leads t o undesirable methylation of adenines on the other strand as well. 3. recA. This protein has a role in NUV recovery (Ferron et al., 1972; Miguel and Tyrrell, 19861, but whether the entire SOS system is involved is difficult to assess, because of some physiological factors that are invovled (see Section VII,A). Although NUV lesions do not lead directly to SOS induction, it should be emphasized that constitutive levels of certain SOS proteins, particularly recA recombinase, may still function to repair NUV damage (Ennis et al., 1985; Tessman et al., 1986; Turner and Eisenstark, 1984). Thus, recombinational repair may be critical for recovery from NUV damage. It is important to note that while recA mutants are very sensitive to NUV in rapidly growing cells, they are not particularly sensitive when cells are in stationary phase. 4. uvr. Cells that lack excision repair capacity are slightly more sensitive to killing by NUV, but become very sensitive to mutation. The double mutant recA uurA is hypersensitive t o NUV. It would be interesting to test mutagenicity in this strain,since recA strains are “mutation proof” t o FUV. 5. hem. Based on the theory that hematins in porphyrins are photoreceptors, and that NUV acts photodynamically to injure cells, it would be expected that a mutation that results in excess of these photoreceptors would render them sensitive and that a mutation that depletes these photoreceptors would make the cells more resistant. Indeed, this is the case for such hem mutants (Sammartano and Tuveson, 1987; Kramer et al., 1988). Probably for the same reasoning, flavin mutants are also sensitive (Kramer et al., 1988). 6. Deletion of gal-hatt. Cells that have the deletion gal-att are

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sensitive to NUV; the reason is not yet known, but this region contains both hem and chl genes and appropriate mutants need to be tested. 7. nuuA and reZA. Mutations that lead to the absence of 4thiouridine in tRNA (nuvA)were found to be sensitive when irradiated with NUV at a low fluence rate (Kramer et aZ., 19881, but Favre et al. (1985) found such mutants to be more resistant than wild type, using higher fluence rates and monochromatic illumination at 334 nm. His explanation was that such mutants did not undergo growth delay, and thus were physiologically healthier. 8. ahp. As noted in Section II,C, a mutation in the flavin hydroperoxidase gene makes the cell sensitive to NUV. 9. nrdB. Mutants that are defective in the synthesis of the B subunit of ribonucleotide reductase are very sensitive to NUV (A. Eisenstark, unpublished). This subunit of the enzyme contains an organic free radical at position tyrosine-122 of its polypeptide chain (Fontecave et al., 19871, has strong absorption in the NUV region of the spectrum, and has been shown to be inactivated by NUV (Peters, 1977). VI. Physiological Effects of NUV

At NUV fluences far below that which show lethal or mutagenic effects, dramatic physiological changes may be observed: (a) as little as 15 kJ/@ of NUV blocks induction of P-galactosidase synthesis by FUV in a recA ::lac fusion, and (b) uptake of [3Hlproline is blocked by the same dosage. The basis for such sharp changes with so little energy has not been fully resolved, but may rest on knowledge of the precise mechanism whereby the inactivation of a thiolated tRNA stimulates synthesis of an alarmone. A. GROWTH DELAY One of the striking effects of a nonlethal dose of NUV is the abrupt cessation of cell division, reduction in cell size, and cessation of protein synthesis. This growth delay phenomenon has been studied by Favre and associates and Jagger and associates. These cellular changes are triggered by photon action on thiolated tRNA as described in Section II,B.

B. NUV INDUCTION OF NEWPROTEINS Upon gel electrophoresis, a number of new [35Slmethionine-labeled proteins are observed following Hz02 and NUV stresses (Pierceall, 1988; Kramer et al., 1988; Christman et al., 1985; Morgan et al., 1986;

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Peters and Jagger, 1981; Van Bogelen et al., 19871, but the newly induced proteins are not identical for the two stresses. In both E . coli and S. typhimurium there is a positive regulator (oxyR) of H202inducible proteins (Christman et al., 1985). A mutation in this gene results in the constitutive synthesis of 30 proteins, including catalse HPI, alkyl hyperoxidase reductase, and the heat-shock protein, dnaK. Mutation in oxyR makes cells resistant to NUV and H202, but a deletion of this regulator gene makes them hypersensitive (Eisenstark and Perrot, 1987). Among the inducible genes that katG (catalase) (Demple and Halhrook, 1983) and sodA, which are induced by both H202 (Gregory and Fridovich, 1973; Morgan et al., 1986) and NUV (Eisenstark and Perrot, 1987); however, the s o d 4 gene may not be under oxyR control (Touati, 1988a). Neither agent induces synthesis of polA, sodB, or xthA gene products. There are distinct differences between NUV and H202 with regard to protein synthesis patterns, particularly during the first 10 minutes of H202 treatment (Pierceall, 1988). After that time, no differences were noted among the NUV and H202 protein patterns. The differences could he explained if (1)the damages are the same, hut NUV generates H202slowly (or, alternatively, NUV inactivates catalase, allowing an accumulation of metabolically generated H202); or (2) the two agents induce different proteins initially and may be under the control of two different regulons, but only for a short period of time. In answering these questions, it should be noted that NUV causes sharp growth delay, but the growth delay by H202is for a much shorter time (Pierceall, 1988). Experiments have been hampered by the fact that NUV normally stops protein synthesis (growth delay) as well as producing membrane changes that diminish the uptake of [35S]methionine.The method was improved by using the nuuA mutant, which lacks thiolated tRNA (Favre and Hafnsdorf, 1983; A. Eisenstark, unpublished observations) and does not have “growth delay.” J. Hoerter and A. Eisenstark (unpublished) observed that synthesis of numerous polypeptides (-44% of the 509 spots that can be clearly identified on a two-dimensional electrophoresis gel) is completely shut off after NUV treatment. Even fewer polypeptides could be seen after treatments with 1 mM H202.Thus, synthesis of numerous polypeptides continues after NUV treatment but are shut off after Ha02 treatment. Synthesis of 30 polypeptides (6%)that are shut off by NUV reappear after 10 minutes of NUV, even if NUV is continued; synthesis of 54 polypeptides (11%) that are turned off during NUV reappear within 10 minutes following discontinuation of the NUV treatment. Upon comparison of the polypeptides that remain after NUV with the polypeptides that remain after H202 treatment, only a few polypep-

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tides were found to migrate to the same position on the gels. This further supports the view that the polypeptides needed in the recovery process are different in the two treatments. There is a paradox with regard to induced protection by small doses of H202and NUV. Several reports would lead to the conclusion that there is complete reciprocity with regard to challenging doses and induction of resistance of both H202 and NUV (Brawn and Fridovich, 1985; Eisenstark and Perrot, 1987; Sammartano and Tuveson, 1985; Tyrrell, 1985). However, interpretation of numerous experiments performed in our laboratory leads to a different conclusion. We found that nonlethal doses of H202 sensitize (rather than protect) bacterial cells and phages following irradiation with NUV (Ananthaswamy and Eisenstark, 1976; Anasthaswamy et al., 1979; Hartman and Eisenstark, 1978,1980). We have also found that NUV may make cells more sensitive to H202 (Hartman and Eisenstark, 1978, 1980, 1982; Hartman et al., 19791, a n observation also made by Kramer and Ames (1987). Since NUV irradiation is carried out over 0-60 minutes in our studies of NUV-H202 synergistic effects, there would have been ample time for induction (Hartman and Eisenstark, 1978, 1980, 1982; Hartman et al., 1979). Thus, in our studies of the synergistic action of NUV and H202, not only did we fail to observe interchangeable induction by the two agents, but there is strong indication that NUV and H202 produce different kinds of damages, and that each of the damages sensitizes the cell to the other agent. C. MEMBRANE EFFECTS The NUV effects ofE. coli membranes are rather drastic (Farr et al., 1988; Klamen and Tuveson, 1982; Chamberlin and Moss, 1986; Kelland et al., 1984). The main questions are (1) whether nonlethal membrane changes are involved in lethality and mutagenesis, and (2) whether there is a relationship between growth delay and membrane changes (Pizzaro and Orce, 1988). The evidence that membrane damage may contribute less than DNA damage is based on the observation that certain mutants that are very sensitive to NUV (xthA, recA, polA, and darn) are all involved in DNA metabolism. It might be worthwhile to examine mutants with membrane protein defects for sensitivity to NUV. To weigh the relative importance of membrane damage to DNA damage, it should be determined whether cell death occurs before one can see a single DNA lesion. If membrane damage were more important than DNA damage, then death and severe membrane damage should occur at fluences that show no DNA lesions. It should be noted that membrane damage by NUV can be amplified greatly by salts in

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minimal media used for plating (Klamen and Tuveson, 1982; Kelland et al., 1984). In a careful action spectrum study by Kelland et al.(19841, considerable membrane damage was observed at wavelengths above 310 nm, peaking a t 334 nm. They also showed that this membrane damage could contribute to lethality in repair-proficient strains. However, in repair-deficient uurA or uurA recA strains, lethality due to membrane damage was not apparent. This further indicates that DNA damage overrides the membrane damage and thus is a more important contributor to lethality. Membrane damage can be monitored by growing cells in “RB’ as a substitute for K + and looking for Rb leakage after NUV (there is little or no leakage at wavelengths below 305 nm). Leakage occurs at fluences equal to or slightly less than fluences causing inactivation at wavelengths above 305 nm (Klamen and Tuveson, 1982). VII. Regulons Involved in Response to NUV Stress

A regulon has been defined as one or more operons under the control of a common regulatory protein (Neidhardt, 1987; Neidhardt and von Bogelen, 1987). Thus, a single mutation in a regulatory gene (e.g., recA, htp, oxyR, or rel) can alter synthesis of a battery of enzymes upon environmental stress (e.g., blockage of DNA synthesis, heat shock, excess oxidation, or starvation of a required amino acid). For each regulon, there may be a key molecule, known as a n “alarmone,” such as ppGpp, which is synthesized and triggers the reaction. When cells are stressed by NUV, some of the enzymes controlled by each of the above regulatory genes may be involved, but NUV stress may stimulate still another regulon, the characteristics of which are not yet known. There is evidence that response to NUV stress may involve genes that are under the control of a t least four known regulons, and perhaps another yet to be determined regulon. Oxygen-related stimulons have also been described (Aliabadi et al., 1986; Jamison and Adler, 1987). Also, a regulon for iron metabolism ( f u r ) has been described (Nettleton et al., 1984; Niederhoffer et al., 1989)that could be involved in NUV responses, but there is no information as yet. A. SOS REGULON When bacterial DNA is damaged by FUV, or when DNA synthesis is blocked by other means, recA protein cleaves the lexA repressor, which sits at about 20 promotor sites, and a battery of gene products are synthesized (Walker, 1987).

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Recovery from NUV stress involves some of the genes under SOS control, but there is also considerable evidence that there is not a complete overlap between the two. For example, if there were a n overlap, a n inducing dose of FUV (or nalidixic acid) should protect the cell against a challenge dose of NUV, since the SOS proteins would have been induced. Experiments show that there is no protection. When one looks at individual genes that are induced by NUV, there is evidence that SOS may not be operating. For example, plasmid pKM101, whose mucA and mucB genes endow cells with enhanced mutation frequency and enhanced resistance to FUV (Walker, 19841, has no influence on these properties when cells are damaged by NUV. Thus, NUV lesions do not induce SOS repair nor subsequent expression of mucA and mucB genes on plasmid pKM101. Further, when cells are preirradiated with NUV and subsequently irradiated with FUV, there is blockage of SOS repair, including the repair normally controlled by genes on pKMlOl (Eisenstark, 1983; Turner and Eisenstark, 1984). Note that this blockage may not occur in nuu mutants (Caldeira de Araujo and Favre, 1986; see next paragraph). When E. coli cells in which the recA promoter is fused to a lac structural gene Mud[(Ap,lac) ::recA1 are irradiated with selected monochromatic wavelengths (245, 313, 334, and 365 nm), only the 254-nm wavelength induces high yields of P-galactosidase, but there is no induction by any of the NUV wavelengths (Turner and Eisenstark, 1984). Also, A prophage induction and Weigle reactivation are observed with FUV but not with NUV. Further, prior exposure of the cells to the selected monochromatic NUV wavelengths inhibits (1)the induction of P-galactosidase by subsequent 254-nm radiation, (2) subsequent 254-nm induction of A prophage, (3) Weigle reactivation of FUV-damaged phage, and (4) increase in mutation frequency. These observations are consistent with the hypothesis that NUV blocks subsequent recA protease action, although other possibilities are not yet ruled out. Caldeira de Araujo and Favre (1986) presented an alternative explanation for the observed blockage of the SOS response by NUV. Their experiments support the view that the transient cessation of growth and protein synthesis produced by NUV prevents the expression of the inducible SOS response; in mutants (nuu)cells that escape this growth delay effect, NUV triggers the SOS response as assayed by induction of a n sfi ::lac2 fusion strain (sfi is one of the SOS genes). It is obvious that further tests are needed to see if recovery from NUV damage is completely under SOS control. The recA protein has multiple activities, one of which is genetic recombination (Tessman et al., 1986). NUV damage does not block

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recA recombinase activity (Turner and Eisenstark, 1984). To test whether the recombination function is influenced by NUV, host C600 cells were irradiated with a selected wavelength or a combination of wavelengths (254, 365, or 365 nm, then 254 nm) and were then infected with Xbio-11, a phage that requires a functional recA+ recombinase activity to form plaques. Plaque formation was observed after such treatment. Thus, recA recombinase activity is not blocked by FUV or NUV; indeed, recombination is stimulated (Turner and Eisenstark, 1984). Many of these tests for SOS response have been performed for H20z induction (Imlay and Linn, 1987); results differ somewhat from NUV induction. They found that very low (Mode 1) doses of H202 induce synthesis of the recA protein, but mutations in genes in the SOS pathway did not make cells more sensitive. Further, H202 failed to show Weigle recovery of treated phage, nor did it show mutagenesis via urnuCD. Since both are identified with the SOS response, this indicates that HzOZ (like NUV) does not involve all of the steps associated with the SOS response. There is a further matter that is yet to be clarified on a molecular basis. Certain mutants (e.g., recA) are more sensitive than wild type to NUV (Carlsson and Carpenter, 1980; Eisenstark, 1971; Eisenstark et al., 1980). Since inducible SOS does not account for NUV repair in wild-type bacteria, resistance to NUV is assumed to be due to the approximately 1000 molecules of constitutive recA recombinase protein present in each noninduced cell (Tessman et al., 1986). Also, recA cells are highly sensitive to NUV when in log phase, but not when in stationary phase (Peak et al., 1983; Tuveson et al., 1983); it is assumed that SOS induction is necessary for repair of log cells, but constitutive recA recombinase is sufficient for repair of stationary cells with completed chromosomes and no growing forks (Sharma and Smith, 1985; Smith and Sharma, 1987). Various roles of the recA protein can now be tested by use of the Tessman mutants of recA, one class of which has lost protease activity only and another class that has lost recombinase activity only (Tessman et al., 1986; Ennis et al., 1985).

B. OXIDATIVE STRESS REGULON( o x y R ) The oxidative stress regulon is controlled by the oxyR gene; a single mutation in oxyR will result in a shift from induced to constitutive synthesis of a battery of proteins, and a deletion in oxyR will result in absence of these (Christman et al., 1985; Kramer and Ames, 1987; Kramer et al., 1988). The oxyR regulon is a positive regulator. The oxyR regulates at least nine proteins involved in defense against

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oxidizing agents, i.e., HzOz. Some of the genes involved are katG, which codes for HPI; ahp, which codes for alkylhydroperoxidase reductase; and gshA, which codes for glutathione reductase. The E . coli oxyR2 mutant constitutively overexpresses 50-fold HPI and 20-fold alkylhydroperoxidase reductase. The oxyR deletion mutants are hypersensitive to killing by NUV. A number of mutants have been studied that affect sensitivity to oxygen ( Jamison and Adler, 1987), but the relationship of these to oxyR has not been reported. It should be emphasized that the induction of the oxyR regulon by NUV is not identical to induction by H202 (Kramer et al., 1988; Pierceall, 1988). Although there is some overlap, different sets of proteins are induced.

C. STRINGENT REGULON The stringent response is defined as a shutdown in the synthesis of rRNA and ribosomal protein operon expression during starvation for amino acids. The stringent response is mediated by guanosine tetraphosphate, ppGpp. ppGpp is a n alarmone, which is a regulatory molecule that alerts cells to the onset of oxidative stress and perhaps other stresses. Stringency is a regulatory response to unloaded tRNAs acting as the triggering device. This results in a sudden and complete shutoff of stable RNA synthesis followed by the cessation of protein synthesis. The cessation of protein synthesis leads to growth delay. The triggering device is a n 8-13 adduct in tRNAs, as discussed above. The relationship of the stringent response to NUV effects has been described (Hajnsdorf and Favre, 1986). D. HEAT-SHOCK REGULON Heat shock is regulated by the HtpR protein, which is a positive sigma-32 factor. Escherichia coli induces 17 proteins under the heatshock response. The induction occurs transcriptionally and requires RNA synthesis. The inducer could again be a small nucleotide, i.e., AppppA. Overlap between proteins induced by heat shock and by NUV has been noted (J. Hoerter and A. Eisenstark, unpublished; Pierceall, 1988). VIII. Mutation by NUV

Compared to FUV, NUV is relatively nonmutagenic on a per-lethalhit basis (Turner and Webb, 1981). Either NUV produces fewer mutagenic-type DNA lesions relative to FUV or a large portion of

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NUV lethality is due to non-DNA damage. Indeed, the frequency and the specificity of mutation (Eisenstadt, 1987; Levin and Ames, 1986) by NUV is an area that is poorly understood and in need of attention, especially considering the abundant exposure of organisms to these wavelengths of radiation. There are a number of reports of NUV mutagenesis, even under anoxic conditions (Cabrera-Juarez, 1964, 1981; Peak et al., 1983; Webb, 1977), although the frequency is much less than under aerobic conditions. Mutations by NUV must occur by a route different from FUV mutation, since only FUV mutation involves the umuDC gene products or that of their analog mucAB on the pKMlOl plasmid. It also requires recA protein in its activated form. When cells with mucAB genes are induced by an agent that turns on SOS repair, mutation frequency rises substantially. However, DNA damage by NUV fails to give this increased frequency of mutations in strains containing the mucAB plasmid (Eisenstark, 1983). Since NUV mutations may be via oxidative damage of DNA, then the SOS repair may not be involved (Farr et al., 1986).Note the lack of involvement of umu genes in H202 mutagenesis (Imlay and Linn, 19871, similar to the case with NUV. Kubitschek et al. (1986) presented a precise action spectrum for genetic mutations in E. coli, which was corrected for finite slit width when irradiating with a monochrometer attached to a light source. Their data verify that there is a plateau at -334 nm for mutations as well as for lethality, further supporting a unique aspect of mutagenesis by NUV. Assuming that 0 2 - is a product of NUV, NUV would be expected to have mutagenic effects similar to those observed following oxidative damage to DNA (Greenberg and Demple, 1988; Storz et al., 1987). 0 2 was found t o be mutagenic by Farr et al. (19861, but only in sodAB strains with ex0111 activity. The triple mutants, sodAB &A, lacked ex0111 activity and did not yield mutations. Although the evidence is only indirect, 0 2 - as a mutagen produced by NUV must be seriously considered (Farr et al., 1986; Greenberg and Demple, 1988; Fridovich, 1986; Moody and Hassan, 1982). It would be interesting to compare mutations in NUV-irradiated cells with and without a plasmid that overproduces exoIII. It should also be noted that endonuclease IV is induced by methyl viologen, a generator of 02-,and that this enzyme may have a role in mutation processing (Chan and Weiss, 1987; Cunningham et al., 1986). Some SOS mutation repair following irradiation at 365 nm has been observed in experiments with DNA-repairless mutants (Turner and Webb, 1981). However, distinct differences among the 254- and 365nm effects were seen and can be accounted for by proposing that there

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is selective damage a t 365 nm that inhibits error-free and error-prone repair systems. The fact that recA mutants are nonmutable at 365-nm radiation makes the SOS role confusing (Turner and Webb, 1981). Since the pKMlOl plasmid does not protect cells from NUV, and since it does not increase mutation frequency (Eisenstark, 1983) following NUV, one would assume that SOS is not involved. However, since the absence of recA proteins knocks out NUV mutagenesis, this indicates that the recA protein has some role (Turner and Webb, 1981). It may be that recA recombinase but not recA protease or another gene product of SOS induction is still necessary for NUV mutations. Also, preirradiation with NUV (sublethal dose) blocks both the SOS response and subsequent tryptophan reversions by FUV (Turner and Eisenstark, 1984). It should also be noted that NUV can be antimutagenic (see Section I1,E) by breaking C-C, 6-4 dimers. Additional examples of NUV mutagenic and antimutagenic effects remain a puzzle. For example, Leonard0 et aZ. (1984) showed that some enrichment in plating media is necessary for mutants to show up after NUV irradiation. Also, the number of mutations by NUV is higher in uvrA strains (Turner, 19841, as is the case for FUV and spontaneous mutations (Sharma and Smith, 1985). IX. Summary

A model of the possible pathways of activities following NUV treatment was presented in Section I and in Fig. 1. Some of the components are firmly established, some are speculative, and many are difficult to evaluate because of insufficient experimental information. Perhaps the most relevant experiments, especially concerning ozone depletion, would be t o determine the mutational specificity of NUV. By selecting ZacI mutants after exposing cells to NUV, and sequencing the bases of this gene, this is now feasible. There are some problems, however. The mutation frequency is normally so low that it might be difficult to distinguish NUV mutants from spontaneous mutants. However, by irradiating cells having a uvrA or uvrB mutation, the frequency of mutation above background can be increased considerably. There remains the problem as t o what fraction of the observed mutations results from oxidative damage. Some of this could be clarified by comparing mutation spectra of cells treated with NUV and cells subjected to excess oxidative damage and determining what fraction results from other avenues of lesion formation in DNA.

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Different species of reactive oxygen could cause different kinds of DNA lesions, and, fortunately, use of appropriate mutants should allow us to sort out any differences in specificity of lesions. Also, by appropriate manipulation of quantities of endogenous photosensitizers, it might be possible to sort out the specific mutations that are caused by photodynamic action. Another avenue of research is t o explore the pathways by which NUV lesions are repaired, and whether such repair is error prone or error free. Again, the use of mutants such as xthA, uur, and polA should assist in our understanding of the specificity of the mutational events. There are now a number of examples of global control mechanisms whereby cells abruptly shift their protein synthesis pattern under environmental stress. It is important to understand whether NUV stress results in induction of one or more of the known regulatory genes, or whether another regulon might be involved. One particular aspect of regulation that remains unsolved is the role of the katF gene, which is known to regulate the xthA and katE, but it may also regulate other genes as well. A number of striking physiological events occur even a t very low fluences of NUV irradiation of cells. In part, this may be related to regulon induction. However, some of these events are in need of special exploration, such as changes a t the membrane level. Also, there is a need to understand the requirement for balance among some of the enzymes involved in response to NUV stress. As was the case for sorting out various avenues of mutation by FUV, it would be important to identify which physiological events are due to each of the various reactive oxygen molecules, and which events are the result of direct photoreaction. Most intriguing is the ability of very low fluences of NUV to produce abrupt membrane changes. The results of many of these explorations may assist in understanding many metabolic processes (both normal and diseased), and perhaps in understanding certain development problems. There is obviously a triggering of certain events by modulation of reactive oxygen species that may result from NUV irradiation.

ACKNOWLEDGMENTS Research by the author was supported in part by the National Science Foundation (DMS-85027-08), by the University of Missouri Institutional Biomedical Research Support Grant RFR 07053 (National Institutes of Health), and by the National Institute of Environmental Health (DHHS ES04889).Assistance by Susan Bradford and Barb Owen, who typed several revisions of the manuscript, is gratefully acknowledged.

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REFERENCES Adelman, R., Saul, R. L., and Ames, B. N. (1988). Oxidative damage to DNA: Relation to species metabolic rate and life span. Proc. Natl. Acad. Sci. U.S.A. 85, 2706-2708. Ahmad, S. I. (1981). Synergistic killing of coliphage T7 by near-ultraviolet radiation plus hydrogen peroxide: Possible role of superoxide radicals. Photobiochem. Photobiophys. 2, 173-180. Aliabadi, Z., Warren, F., Mya, S., and Foster, J. W. (1986). Oxygen-related stimulons of Salmonella typhimurium identified by Mud (Ap lac) operon fusions. J . Bacteriol. 165, 780-786. Alonso-Moraga, A. A., Bocanegua, J . M., Torres, J., Lopez-Barea, J., and Peuyo, C. (1987). Glutathione status and sensitivity to GSH-reacting compounds of Escherichia coli strains deficient in glutathione metabolism and/or catalase activity. Mol. Cell. Biochem. 73, 61-68. Ames, B. N. (1983). Dietary carcinogens and anticarcinogens. Science 221, 1256-1264. Ananthaswamy, H. N., and Eisenstark, A. (1976). Near UV-induced breaks in phage DNA: Sensitization by hydrogen peroxide (a tryptophan photoproduct). Photochem. Photobiol. 24, 439-442. Ananthaswamy, H. N., and Eisenstark, A. (1977). Repair of H202-indueed single strand breaks in E . coli DNA. J . Bacteriol. 130, 187-191. Ananthaswamy, H. N., Hartman, P. S., and Eisenstark, A. (1979). Synergistic lethality of phage T7 by near-UV radiation and hydrogen peroxide: An action spectrum. Photochern. Photobiol. 29, 53-56. Anderson, R. (1983). Alkylamines: Novel lipid constituents in Deinococcus radiodurans. Biochim. Biophys. Acta 743, 266-268. Anderson, R., and Hansen, K. (1985). Structure of a novel phosphoglycolipid from Deinococcus radiodurans. J . Biol. Chem. 260, 12219-12223. Anderson, R., Daya, M., and Reeve, J. (1987). An evaluation of the contribution of membrane lipids to protection against ultraviolet radiation. Biochim. Biophys. Acta 905,227-230. Bachmann, B. (1987). Linkage map of Escherzchia coli K12. I n “Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology” (F. C. Neidhardt et al., eds.), 7th Ed. Am. SOC.Microbiol., Washington, D.C. Barbado, C. M., Ramirez, M. A., Blanco, J., Lopez-Barea, J., and Pueyo, C. (1983). Mutants of Escherichia coli sensitive to hydrogen peroxide. Curr. Microbiol. 8, 251-253. Billen, D. (1984). The role of hydroxyl radical scavengers in preventing DNA strand breaks induced by X irradiation of toluene-treated Escherichia coli.Radiat. Res. 97, 626-629. Bissett, D. L., Hannon, D. P., and Orr, T. V. (1987).An animal model of solar-aged skin: Histological physical, and visible changes in UV-irradiated hairless mouse skin. Photochem. Photobiol. 46, 367-378. Blanchet, T., Hajnsdorf, E., and Favre, A. (1984). Metabolism of tRNA in nearultraviolet-illuminated Escherichia coli. The tRNA repair hypothesis. Eur. J . Biochem. 139,547-552. Bloch, C. A,, and Ausubel, F. M. (1986). Paraquat-mediated selection for mutations in the manganese-superoxide dismutase gene sodA. J . Bacteriol. 168, 795-798. Blum, H. F. (1941). “Photodynamic Action and Diseases Caused by Light.” Van Nostrad-Reinhold, Princeton, New Jersey (reprinted in 1964 with an updated appendix by Harper, New York). Bochner, B. A,, Zylicz, M., and Georgopoulos, C. (1986). Escherzchia coli DnaK protein

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NUV

137

possesses a 5’-nucleotidase activity that is inhibited by AppppA. J . Bacteriol. 168, 931-935. Brawn, M. K., and Fridovich, I. (1985). Increased superoxide radical production evokes inducible DNA repair in Escherichia coli. J . Biol. Chem. 260, 922-925. Breimer, L. H., and Lindahl, T. (1985). Enzymatic excision of DNA bases damaged by exposure to ionizing radiation or oxidizing agents. Mutat. Res. 150, 85-89. Brown, 0. R., and Seither, R. L. (1983). Oxygen and redox-active drugs: Shared toxicity sites. Fundam. Appl. Toxicol. 3, 209-215. Cabrera-Juarez, E. (1964). “Black light” inactivation of transforming DNA from Haemophilus influenzae. J . Bacteriol. 87, 771-778. Cabrera-Juarez, E. (1981). Near-ultraviolet mutation of transforming DNA irradiated in uiuo. Mutat. Res. 83, 301-306. Cabrera-Juarez, E., and Setlow, J . K. (1976). Mutation of Haemophilus influenzae transforming DNA in uitro with near-ultraviolet irradiation: Action spectrums. Mutat. Res. 35, 199-206. Cadet, J., Berger, M., Decarroz, C., Wagner, J . R., Van Lier, J . E., Ginot, Y. M., and Vigny P. (1986). Photosensitized reactions of nucleic acids. Biochimie 68, 813-834. Caimi, P., and Eisenstark, A. (1986). Sensitivity of Deinococcus radiodurans to near-ultraviolet radiation. Mutat. Res. 162, 145-151. Caldeira de Araujo, A., and Favre, A. (1986). Near ultraviolet DNA damage induces the SOS response in Escherichia coli. EMBO J . 5, 175-179. Calkins, J., ed. (1982). “The Role of Solar Ultraviolet Radiation on Marine Ecosystems,” p. 724. Plenum, New York. Carlioz, A., and Touati, D. (1986). Isolation of superoxide dismutase mutants in E . coli:Is superoxide dismutase necessary for aerobic life? EMBO J . 5,623-630. Carlioz, A,, Ludwig, M. L., Stallings, W. C., Fee, J. A., Steinman, H. M., and Touati, D. (1988). Iron superoxide dismutase: Nucleotide sequence of the gene from Escherichia coli K12 and correlations with crystal structures. J . Biol. Chem. 3, 1555-1562. Carlsson, J., and Carpenter, V. S. (1980). The recA’ gene product is more important than catalase and superoxide dismutase in protecting Escherichia coli against hydrogen peroxide toxicity. J . Bacteriol. 142, 319-321. Casas-Fimet,J. R., Toulme, J . J., Cazenave, C., and Santus, R. (1984). Role of tryptophan and cysteine in binding of gene 32 protein from phage T4 to single-strand DNA. Modification of crucial residues by oxidation with selective free-radical anions. Biochemistry 23, 1208-1213. Cashel, M., and Rudd, K. E. (1987). The stringent response. Zn “Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology (F. C. Neidhardt et al., eds.), 7 Ed., pp. 1410-1438. Am. SOC.Microbiol., Washington, D.C. Chamberlin, J., and Moss, S. H. (1986). Lipid peroxidation and other membrane damage produced in E . coli K1060 by near-UV radiation and deuterium oxide. Photochem. Photobiol. 45, 625-630. Chan, E., and Weiss, B. (1987). Endonuclease IV o f E . coli is induced by paraquat. Proc. Natl. Acad. Sci. U.S.A. 84, 3189-3194. Cheng, L., Kellog, E. W., and Packer, L. (1981). Photoinactivation of catalase. Photochem. Photobiol. 34, 125-129. Christman, M. F., Morgan, R. W., Jacobson, F. S., and Ames, B. N. (1985). Positive control of a regulon for defenses against oxidative stress and some heat-shock proteins in Salmonella typhimurium. Cell 41, 753-762. Clare, D. A., Duong, M. N., Darr, D., Archibald, F., and Fridovich, I. (1984). Effects of molecular ozxygen on the detection of superoxide radical with nitroblue tetrazolium and an activity strain for catalase. Anal. Biochem. 140, 532-637.

138

A . EISENSTARK

Coulondre, C., and Miller, J . H. (1977). Genetic studies of the lac repressor. IV. Mutagenic specificity in the l a d gene ofEscherichia coli. J . Mol. Biol. 117,577-606. Cunningham, R. P., Saporito, S. M., Spitzer, S. G., and Weiss, B. (1986).Endonuclease IV (nfo)mutant of Escherichia coli. J . Bacteriol. 168, 1120-1127. Dahl, T. A., Midden, W. R., and Hartman, P. E. (1987). Pure singlet oxygen cytotoxicity for bacteria. Photochem. Photobiol. 46, 345-352. Dahl, T. A., Midden, R. W., and Hartman, P. E. (1988). Some prevalent biomolecules as defenses against singlet oxygen damage. Photochem. Photobiol. 47, 357-362. Dahl, T. A,, Midden, R. W., and Hartman, P. E. (1989). Comparison of killing of gram-negative and gram-positive bacteria by pure singlet oxygen. J . Bacteriol. 17, 2188-2194. Davies, K. J., Delsignore, M. E., and Lin, S. W. (1987). Protein damage by oxygen radicals: 11. Modification of amino acids. J . Biol. Chem. 262, 9902-9907. Decuyper-Debergh, D., Piette, J., and Van de Vorst, A. (1987). Singlet oxygen-induced mutations in M13 lac2 phage DNA. EMBO J . 6, 3161. Demple, B., and Halbrook, J . (1983). Inducible repair of oxidative DNA damage in E. coli. Nature (London)304, 466-468. Demple, B., Halbrook, J., and Linn, S. (1983). E . coli rth mutants are supersensitive to hydrogen peroxide. J . Bacteriol. 153, 11079-11082. Demple, B., Daikh, Y., Greenberg, J., and Johnson, A. (1985). Alkylation and oxidative damages to DNA: Constitutive and inducible repair systems. I n “Antimutagenesis and Anticarcinogenesis: Mechanisms” (D. M. Schenkel, P. E. Hartman, T. Kada, and A. Hollaender, eds.), pp. 205-217. Plenum, New York. Demple, B., Johnson, A., and Fung, D. (1986). Exonuclease I11 and endonuclease IV remove 3’ blocks from DNA synthesis primers in HzOz-damagedEscherichia coli. Proc. Natl. Acad. Sci. U S A . 83, 7731-7735. Dowds, B. C. A,, Murphy, P., McConnell, D. J., and Devine, K. M. (1987). Relationship among oxidative stress, growth cycle, and sporulation in Bacillus subtilis. J . Bacteriol. 169, 5771-5775. Droffner, M. L., and Yamamoto, N. (1983). Anaerobic cultures of Salmonella typhimurium do not exhibit inducible proteolytic function of recA gene and recBC function. J . Bacteriol. 156, 962-965. Droffner, M. L., and Yamamoto, N. (1985). Isolation of thermophilic mutants of B. subtilis and B . pumilus and transformation of the thermophilic trait to meosphilic strains. J . Gen. Microbiol. 131, 2791-2794. Eisenstadt, A. (1987). Analysis of mutagenesis. I n “Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology” (F. C. Neidhardt et al., eds.), 7th Ed., pp. 1016-1033. Am SOC.Microbiol., Washington, D.C. Eisenstark, A. (1971). Mutagenic and lethal effects of visible and near-ultraviolet light on bacterial cells. Adu. Genet. 16, 167-198. Eisenstark, A. (1982). Sensitivity to UV-B irradiation as related to bacterial life cycles. In “The Role of Solar Ultraviolet Radiation in Marine Ecosystems” ( J . Calkins, ed.), pp. 389-391. Plenum, New York. Eisenstark, A. (1983). Genetic damage in Salmonella typhimurium by near-ultraviolet radiation: Lack of repair by plasmid pKMlO1. Mutat. Res. 122, 267-272. Eisenstark, A. (1985). Recovery from near-ultraviolet radiation damage in bacteria. I n “Molecular Biology of Bacterial Growth” (M. Schaechter, F. C. Neidhart, J . L. Ingraham, and N. 0. Kjeldgaard, eds.), pp. 243-255. Jones & Bartlett, Boston, Massachusetts. Eisenstark, A. (1987). Mutagenic and lethal effects of near-ultraviolet radiation (290400 nm) on bacteria and phage. Enuiron. Mol. Mutagen. 10, 317-337.

BACTERIAL GENES INVOLVED IN RESPONSE TO NUV

139

Eisenstark, A., and Perrot, G. 119871. Catalase has only minor role in protection against near-ultraviolet radiation damage in bacteria. Mol. Gen. Genet. 207, 68-72. Eisenstark, A., Buzard, R. L., and Hartman, P. S. (1980). Inactivation of phage by near-ultraviolet radiation and hydrogen peroxide. Photochem. Photobiol. 44,603606. Eisenstark, A., Kovacs, S., and Terry, J. (1982). Cell damage by near-ultraviolet radiation. Role of DNA-protein cross-links. Natl. Cancer Inst. 60, 177-181. Ennis, D. G., Fisher, B., Edmiston, S., and Mount, D. W. (1985).Dual role forEscherichia coli RecA protein in SOS mutagenesis. Genetics 82, 3325-3329. Ewing, D. (1983).Radiation sensitization ofE. coli B/r by nitrous oxide. Radiat. Res. 96, 275-283. Farr, S. B., DAri, R., and Touati, D. (1986). Oxygen-dependent mutagenesis in Escherichia coli lacking superoxide dismutase. Proc. Natl. Acad. Sci. U.S.A. 83, 8268-8272. Farr, S. B., Touati, D., and Kogoma, T. (1988). Effects of oxygen stress on membrane functions in Escherichia coli: Role of HPI catalase. J . Bacteriol. 170, 1827-1842. Favre, A., and Hajnsdorf, E. (1983). Photoregulation of E. coli growth and the near ultraviolet photochemistry of tRNA. I n “Molecular Models of Photoresponsiveness” (G. Montagnoli and B. F. Erlanger, eds.), pp. 75-94. Plenum, New York. Favre, A,, Hajnsdorf, E., Thiam, K., and Caldeira de Araujo, A. (1985). Mutagenesis and growth delay induced in Escherchia coli by near-ultraviolet radiation. Biochimie 67, 335-342. Fee, J. A., Niederhoffer, E. C., and Naranjo, C. (1989). First description of a variant ofE. coli lacking superoxide dismutase activity yet able to grow efficiently on minimal, oxygenated medium. I n “Trace Element Metabolism in Man and Animals” (L. Hurley, C. Keen, B. Lonnerdal, and R. Rucker, eds.), pp. 239-243. Plenum, New York. Ferron, W. L., Eisenstark, A,, and MacKay, D. (1972). Distinction between far- and near-ultraviolet light killing of recombinationless (recA)Salmonella typhimuriurn. Biochim. Biophys. Acta 277, 651-658. Flint, D. H., and Emptage, M. H. (1988). Dihydroxy acid dehydratase from spinach contains a [ 2 Fe-2Sl cluster. J . Biol. Chem. 263, 3558-3564. Fontecave, M., Grusland, A., and Reichard, P. (1987). The function of superoxide dismutase during the enzymatic formation of the free radical of ribonucleotide reductase. J . Biol. Chem. 262, 12332-12336. Freierabend, J., and Engel, S. (1986).Photoinactivation of catalase in vitro and in leaves. Arch. Biochem. Biophys. 251, 567-576. Fridovich, I. (1986).Superoxide dismutases. Adu. Enzymol. 58, 61-85. Gameson, A. L. H., and Gould, D. J. (1975). Effects of solar radiation on the mortality of some terrestrial bacteria in sea water. I n “Discharge of Sewage from Sea Outfalls” (A. L. H. Gameson, ed.), pp. 209-219. Pergamon, New York. Gennis, R. B. (1987). The cytochromes of Escherichia coli. Microbiol. Reu. 46, 387399. Glickman, B. W., Schaaper, R. M., Haseltine, W. A., Dunn, R. L., and Brash, D.E. (1986). The C-C (6-4) UV photoproduct is mutagenic in Escherchia coli. Proc. Natl. Acad. SCZ. U.S.A. 83, 6945-6949. Greenberg, J. T., and Demple, B. (1986).Glutathione inEscherchia coli is dispensable for resistance to HzOzand gamma radiation. J . Bacteriol. 168, 1026-1029. Greenberg, J. T., and Demple, B. (1988). Overproduction of peroxide-scavenging enzymes in Escherichia coli suppresses spontaneous mutagenesis and sensitivity to redox-cycling agents in oxyR- mutants. EMBO J . 7, 2611-2617.

140

A. EISENSTARK

Gregory, E. M., and Fridovich, I. (1973).Induction of superoxide dismutase by molecular oxygen. J . Bacteriol. 114, 543-548. Hagensee, M. E., and Moses, R. E. (1986). Repair response of Escherichia coli to hydrogen peroxide. J . Bacteriol. 168, 1059-1065. Hajnsdorf, E., and Favre, A. (1986). Metabolism of tRNAs in growing cells ofEscherichia coli illuminated with near-ultraviolet light. Photochem. Photobiol. 43, 157-164. Halliwell, B. (1987). Oxidants and human disease: Some new concepts. FASEB J . I, 358-364. Hartman, P. S. (1986).In situ hydrogen peroxide production may account for a portion of NUV (300-400 nm) inactivation of stationary phase. E . coli. Photochem. Photobiol. 43, 87-89. Hartman, P. S., and Eisenstark, A. (1978). Synergistic killing of E. coli by near-UV radiation and hydrogen peroxide: Distinction between recA-repairable and recAnonrepairable damage. J . Bacteriol. 131, 769-779. Hartman, P. S., and Eisenstark, A. (1980). Killing of Escherichia coli K12 by nearultraviolet radiation in the presence of hydrogen peroxide: Role of double-strand DNA breaks in the absence of recombinational repair. Mutat. Res. 72, 31-42. Hartman, P. S., and Eisenstark, A. (1982). Alteration of bacteriophage attachment capacity by near-UV irradiation. J . Virol. 43, 529-532. Hartman, P. S., and Eisenstark, A. (1987). Synergistic killing of E . coli by near-UV radiation and hydrogen peroxide. Distinction between recA-repairable and recAnonrepairable damage. J . Bacteriol. 131, 769-779. Hartman, P. S., Eisenstark, A., and Pauw, P. G. (1979). Near-ultraviolet radiation plus Hz02 inactivation on phage T7: DNA-protein crosslinks prevent DNA injection. Proc. Natl. Acad. Sci. U.S.A. 76, 3228-3232. Hassan, H. M. (1984). Exacerbation of superoxide radical formation by paraquat. In “Methods in Enzymology” (L. Palker, ed.), Vol. 105, pp. 523-532. Academic Press, New York. Hassett, D. J., Britigan, B. E., Svendsen, T., Rosen, G. E., and Cohen, M. S. (1987). Bacteria form intracellular free radicals in response to paraquat and streptonigrin. J . Biol. Chem. 262,13404-13408. Heimberger, A., and Eisenstark, A. (1988). Compartmentalization of catalases in Escherichia coli. Biochem. Biophys. Res. Commun. 15, 392-397. Helene, C., Charlier, M., Montenay-Garestier, T., and Laustriata, G., eds. (1982). “Trends in Photobiology,” p. 820. Plenum, New York. Hollaender, A. (1943). Effect of long ultraviolet and short visible radiation (3500-4900 & on E . coli. J . Bacteriol. 46, 531-541. Horowitz, P. M., and Bowman, S. (1987). Oxidation increases the proteolytic susceptibility of a localized region in rhodanase. J . Biol. Chem. 262, 14544-14548. Houba-Herin, N., Calberg-Bacq, C. M., Piette, J., and VandeVorst, A. (1982). Mechanisms for dye-mediated photodynamic action: Singlet oxygen production, deoxyguanosine oxidation, and phage inactivation efficiencies. Photochem. Photobiol. 36, 297-308. Husain, I., and Sancar, A. (1987). Photoreactivation in phr mutants of Escherichia coli. J . Bacteriol. 169, 2367-2372. Imlay, J. A., and Linn, S. (1986). Bimodal pattern of killing of DNA-repair-defective or anoxically grown Escherichia coli by hydrogen peroxide. J . Bacteriol. 166,519-527. Imlay, J. A., and Linn, S. (1987). Mutagenesis and stress responses induced in Escherichia coli by hydrogen peroxide. J . Bacteriol. 169, 2967-2976. Imlay, J. A., and Linn, S. (1988). DNA damage and oxygen radical toxicity. Science 240, 1302-1309.

BACTERIAL GENES INVOLVED IN RESPONSE TO

NUV

141

Imlay, J. A., Chin, S. M., and Linn, S. (1988).Toxic DNA damage by hydrogen peroxide through the Fenton reaction i n vivo and in uitro. Science 240, 640-642. Ingledew, W. J., and Poole, R. K. (1984). The respiratory chain of Escherichia coli. Microbiol Rev. 48, 222-271. Ito, A., Robb, F. T., Plak, J. G., and Peak, M. J . (1988). Base-specific damage induced by 4-thiouridine photosensitization with 334-nm radiation in M13 phage. Photochem. Photobiol. 47, 231-240. Jagger, J. (1985). “Solar-UV Actions on Living Cells.” Praeger, New York. Jamison, C. S., and Adler, H. I. (1987). Mutations in Escherichia coli that effect sensitivity to oxygen. J . Bacteriol. 169, 5087-5094. Kamoun, P. P., Schneider, E., and Dy, M. (1988). Superoxide induced diimination of arginine in hematopoietic cells. FEBS Lett. 226, 285-286. Katcher, H. L., and Wallace, S. S. (1983). Characterization of the Escherchia coli X-ray endonuclease, endonuclease 11. Biochemistry 22, 4071-4081. Kelland, L. R., Moss, S. H., and Davies, D. J . G. (1984). Leakage of “RB+ after ultraviolet irradiation of E . coli K12. Photochem. Photobiol. 39, 329-335. Kirkman, H. N., and Gaetani, G. F. (1984). Catalase: A tetrameric enzyme with four tightly bound molecules of NADPH. Proc. Natl. Acad. Sci. U.S.A. 81, 4343-4347. Kitzler, J., and Fridovich, I. (1986).Effects of the lethality of paraquat. J . Bacteriol. 167, 346-349. Klamen, D. L., and Tuveson, R. W. (1982). The effect of membrane fatty acids composition on the near-UV (300-400 nm) sensitivity of Escherichia coli K1060. Photochem. Photobiol. 35, 167-173. Kogoma, T., Farr, S. B., Joyce, K. M., and Natvig, D. 0. (1988).Isolation of gene fusions ( s o i : :lacZ) inducible by oxidative stress in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 85,4799-4803. Kow, W. Y., and Wallace, S. S. (1985). Exonuclease I11 recognizes urea residues in oxidized DNA. Proc. Natl. Acad. Sci. U.S.A. 82, 8354-8358. Krabbenhoft, K. L., Anderson, A. W., and Elliker, P. R. (1967). Influence of culture media on the radiation resistance of Micrococcus radiodurans. Appl. Microbiol. 15, 178- 185. Kramer, G. F., and Ames, B. N. (1987). Oxidative mechanisms of toxicity of lowintensity near-UV light. J . Bacteriol. 169, 2259-2266. Kramer, G. F., Baker, J. C., and Ames, B. N. (1988). Near-UV Stress in Salmonella typhinurium: 4-Thiouridine in tRNA, ppGpp, and ApppGpp as components of an adaptive response. J . Bacteriol. 170, 2344-2351. Kubitschek, H. E., Baker, K. S., and Peak, M. J. (1986). Enhancement of mutagenesis and human skin cancer rates resulting from increased fluences of solar ultraviolet radiation. Photochem. Photobiol. 43, 443-447. Kuo, C. F., Mashino, T., and Fridovich, I. (1987). a,P-Dihydroxy-isovaleratedehydratase: A superoxide-sensitive enzyme. J . Biol. Chem. 262, 4724-4727. Lancy, P., and Murray, R. G. E. (1978). The envelope of Micrococcus radiodurans: Isolation, purification, and preliminary analysis of the wall layers. Can. J . Microbiol. 24, 162-176. Lang, H., Riesenberg, D., Zimmer, C. H., and Bergter, F. (1986). Fluence-rate dependence of monophotonic reactions of nucleic acids in vitro and in uiuo. Photochem. Photobiol. 44, 565-570. Laspia, M. F., and Wallace, S. S. (1988). Excision repair of thymine glycols, urea residues, and apurinic sites in E . coli. J . Bacteriol. 170, 3359-3366. Lee, P. C., Bochner, B. R., and Ames, B. N. (1983). AppppA, heat shock stress in cell oxidation. Proc. Natl. Acad. Sci.U.S.A. 80, 7496-7500.

142

A . EISENSTARK

Leonardo, J. M., Reynolds, P. R., and Tuveson, R. W. (1984). Mutation induction by 365-nm radiation and far-ultraviolet light in E . coli strains differing in near- and far-ultraviolet light sensitivity. Mutat. Res. 126, 1-8. Levin, D. E., and Ames, B. N. (1986). Classifying mutagens as to their specificity in causing the six possible transitions and transversions: A simple analysis using the Salmonella mutagenicity assay. Enuiron. Mutagen. 8, 9-28. Lewis, N. F., Alur, M. D., and Kumta, U. S. (1973). Role of carotenoid pigments in radio-resistant micrococci. Can. J . Microbiol. 20, 455-459. Lin, W. S., Wong, F., and Anderson, R. (1987). Role of superoxide in radiation-killing of Escherichia coli and in thymine release from thymidine. Biochem. Biophys. Res. Commun. 147, 778-786. Linn, S., and Imlay, J. A. (1987). Toxicity, mutagenesis, and stress responses induced in Escherichia coli by hydrogen peroxide. J . Cell Sci. (Suppl.)6, 289-301. Loewen, P. E. (1984). Isolation of catalase-deficient Escherichia coli mutants and genetic mapping of katE, a locus that affects catalase activity. J . Bacterial. 157, 622-626. Loewen, P. C., and Triggs, B. L. (1984). Genetic mapping of katF, a locus that with katE affects the synthesis of a second catalase species in Escherichia coli. J . Bacterial. 160, 668-675. Loewen, P. C., Triggs, B. L., Klassen, G. R., and Weiner, J. H. (1983). Identification and physical characterization of Col E l hybrid plasmid containing a catalase gene of Escherichia coli. Can J . Biochem. Cell Biol. 61, 1315-1321. Loewen, P. C., Switala, J., and Triggs-Raine, L. (1985a). Catalases HPI and HPII in Escherichia coli are induced independently. Arch. Biochem. Biophys. 243,144-149. Loewen, P. C., Triggs, B. L., George, C. S., and Hrabarchuk, B. E. (1985b). Genetic mapping of katG, a locus that affects synthesis of the bifunctional catalaseperoxidase hydroperoxidase I in Escherichia coli. J . Bacteriol. 162, 661-667. McCormick, J. P., Fisher, J. R., Pachlatko, J. P., and Eisenstark, A. (1976). Characterization of a cell-lethal tryptophan photoxidation product: Hydrogen peroxide. Science 191,468-469. McCormick, J. P., Klita, S., Terry, J., Schrodt, M., and Eisenstark, A. (1982). Formation by hydrogen peroxide or 254 nm radiation of a near-UV chromophore from peptide-bound cysteine. Photochem. Photobiol. 36, 367-369. Mackay, D., Eisenstark, A., Webb, R. B., and Brown, M. S. (1976). Action spectra for lethality in recombinationless strains of S. typhimurium and E . coli. Photochern. Photobiol. 24, 337-343. Marinus, M. G. (1987). Methylation of DNA. I n “Escherichia coli and Salmonella typhirnurium: Cellular and Molecular Biology” (F. C. Niedhardt et al., eds.), 7th Ed., pp. 697-702. Am. SOC.Microbiol., Washington, D.C. Martin, M. E., Strachan, R. C., Aranha, H., Evans, S. L., Salin, M. L., Welch, B., Arceneaux, J. E. L., and Byers, B. R. (1984). Oxygen toxicity in Streptococcus: Manganese, iron and superoxide dismutase. J . Bacteriol. 159, 745-749. Marx, J. L. (1987). Oxygen free radicals linked to many diseases. Science 235,529-530. Meir, E., and Yagil, E. (1984). Catalase-negative mutants of Escherichia coli. Curr. Microbiol. 11, 13-18. Miguel, A. G., and Tyrrell, R. M. (1986). Repair of near-ultraviolet (365nm)-induced strand breaks in E . coli DNA: The role of the recA andpolA gene products. Biophys. J . 49,485-491. Miller, J. H. (1985). Mutagenic specificity of ultraviolet light. J . Mol. Biol. 131,223-248. Mitchell, D. L., and Clarkson, J. M. (1984). Induction of photoproducts in synthetic polynucleotides by far and near ultraviolet radiation. Photochem. Photobiol. 40, 735-741.

BACTERIAL GENES INVOLVED IN RESPONSE TO

NUV

143

Moody, C. S., and Hassan, H. M. (1982). Mutagenicity of oxygen free radicals. Proc. Natl. Acad. Sci. U.S.A.79, 2855-2859. Morgan, R. W., Christman, M. F., Jacobson, F. S., Storz, G., and Ames, B. N. (1986). Hydrogen peroxide-inducible proteins in Salmonella typhimurium overlap with heat shock and other stress proteins. Proc. Natl. Acad. Sci. U.S.A. 83,8059-8063. Moseley, B. E. B. (1963). The variation in X-ray resistance of Micrococcus radioduruns and some of its less-pigmented mutants. Int. J . Radiat. Biol. 6, 489. Moseley, B. E. B., Mattingly, A., and Copland, H. J. R. (1972). Sensitization to radiation by loss of recombination ability in a temperature-sensitive DNA mutant of Micrococcus radiodurans held a t its restrictive temperature. J . Gen. Microbiol. 72, 329-338. Murphy, P., Dowds, B. C. A., McConnell, D. J., and Devine, K. M. (1987). Oxidative stress and growth temperature in Bacillus subtilis. J . Bacteriol. 169, 5766-5770. Natvig, D., Imlay, K., Touati, D., and Hallewell, R. A. (1987). Human copperhinc superoxide dismutase complements superoxide dismutase-deficient E . coli mutants. J . Biol. Chem. 262, 14697-14701. Neidhardt, F. C. (1987). Multigene systems and regulons. In “Escherichia coli and Salmonella typhirnurium: Cellular and Molecular Biology” (F. C. Neidhardt et al., eds), 7th Ed., pp. 1313-1317. Am. SOC. Microbiol., Washington, D.C. Neidhart, F. C., and van Bogelen, R. A. (1987). Heat shock response. In “Escherichia coli and Salmonella hyphimuriurn: Cellular and Molecular Biology” (F. C. Neidhardt et Microbiol., Washington, D.C. al., eds.), 7th Ed., pp. 1334-1345. Am. SOC. Nettleton, C. J., Bull, C., Baldwin, T. O., and Fee, J . A. (1984). Isolation of the Escherichia coli iron superoxide dismutase gene: Evidence that intracellular superoxide concentration does not regulate oxygen-dependent synthesis of the manganese superoxide dismutase. Proc. Natl. Acad. Sci. U.S.A. 81, 4970-4973. Niederhoffer, E. C., Naranjo, C. M., and Fee, J. A. (1989). Relationship of the superoxide dismutase genes, sodA and SodB, to the iron uptake ( f u r )Regulon in Escherichia coli K12. I n “Metal Ion Homeostasis: Molecular Biology and Chemistry” (D. Winge and D. Hamer, eds.), pp. 149-158. Liss, New York. Nishida, K. I., Takagi, M., and Yano, K. (1981). Escherichia coli inactivation by superoxide anion and hydrogen peroxide. J . Gen Microbiol. 27,447-485. Owens, R. A,, and Hartman, P. E. (19861. Glutathione: A protective agent in Salmonella typhimurium and Escherichia coli as measured by mutagenicity and by growth delay assays. Enuiron. Mutagen. 8, 659-673. Parsons, P. G., and Hayward, I. P. (1985). Inhibition of DNA repair synthesis by sunlight. Photochem. Photobiol. 42, 287-293. Peak, J . G., Peak, M. J., and Tuveson, R. W. (1983). Ultraviolet action spectra for aerobic and anaerobic inactivation of Escherichia coli strains specifically sensitive and resistant to near ultraviolet radiations. Photochem. Photobiol. 38, 541-543. Peak, M. J., Johnson, J . S., Tuveson, R. W., and Peak, J. G. (1987a). Inactivation by monochromatic near-UV radiation of a n Escherichia coli hemA8 mutant grown with and without aminolevulinic acid: The role of DNA vs membrane damage. Photochem. Photobiol. 45, 473-478. Peak, M. J., Ito, A., Foote, C. S., and Peak, J . G. (1987b). Photosensitized inactivation of DNA by monochromatic 334-nm radiation in the presence of 2-thiouracil: Genetic activity and backbone breaks. Photochem. Photobiol. 47, 809-814. Peak, M. J., Midden, R., Babasick, D. M., and Haugen, D. A. (1988). Photochemistry of 4-thiouridine and thymine, Photochem. Photobiol. 48, 229-232. Peters, J. (1977). In uiuo photoinactivation of E . coli ribonucleotide reductase by near-ultraviolet light. Nature (London)267, 546-548.

144

A. EISENSTARK

Peters, J., and Jagger, J . (1981).Inducible repair of near-UV radiation lethal damage in E . coli. Nature (London) 289, 194-195. Petkau, A. (1986). Scientific basis for the clinical use of superoxide dismutase. Cancer Treat. Rev. 13, 17-44. Pierceall, W. (1988). Comparison of induced proteins synthesized in hydrogen peroxideand near-ultraviolet-treated bacterial cells. M.A. thesis, Univ. of Missouri, Columbia. Pizarro, R. A., and Orce, L. V. (1988). Membrane damage and recovery associated with growth delay induced by near-UV radiation in Escherichia coli K-12. Photochem. Photobiol. 47, 391-397. Quintiliani, M. (1986).The oxygen effect in radiation inactivation ofDNA and enzymes. Int. J . Radiat. Biol. 4, 573-594. Rebeyrotte, N., Rebeyrotte, P., Maviel, M. J., and Montaudon, D. (1979). Lipides et lipopolyosides de Micrococcus radiodurans. Ann. Microbiol. (Inst. Pasteur). 130B, 407-414. Richter, H., and Loewen, P. C. (1981). Induction of catalase in E . coli by ascorbic acid involves hydrogen peroxide. Biochem. Biophys. Res. Cornmun. 100, 10391046. Roth, D. (1981). DNA-denaturing effect of irradiated hydrogen peroxide. Photochem. Photobiol. 33, 251-252. Rotilio, G. (1986). “Superoxide and Superoxide Dismutases in Chemistry, Biology and Medicine.” Elsevier, Amsterdam. Rowley, D. A., and Hallewell, B. (1982). Superoxide dependent formation of hydroxyl radicals in the presence of thiol compounds. FEBS Lett. 138, 33-36. Sak, B. (1989).Exonuclease I11 and catalase HP-I1 in Escherichia coli are both regulated by the katF gene product. M.A. dissertation. Univ. of Missouri, Columbia. Sak, B. D., Eisenstark, A,, and Touati, D. (1989). Exonuclease 111 and hydroperoxide I1 in Escherichia coli are both regulated by the katF gene product. Proc. Natl. Acad. Sci. U.S.A.86 (in press). Sakamoto, H., and Touati, D. (1984). Cloning of the iron superoxide dismutase gene (so&) in Escherichia coli K-12. J . Bacteriol. 159, 418-420. Salet, C., Bazin, M., Moreno, G., and Favre, A. (1985).4-Thiouridine as a photodynamic agent. Photochem. Photobiol. 41, 617-619. Sammartano, L. J., and Tuveson, R. W. (1983). Escherichia coli rthA mutants are sensitive to inactivation by broad-spectrum near-ultraviolet (300-400 nm) radiation. J . Bacteriol. 156, 904-906. Sammartano, L. J., and Tuveson, R. W. (1984). The effects of exogenous catalse on broad-spectrum near-UV (300-400 nm) treated Escherichia coli cells. Photochem. Photobiol. 40, 607-612. Sammartano, L. J., and Tuveson, R. W. (1985).Hydrogen peroxide induced resistance to broad-spectrum near-ultraviolet light (300-400 nm) inactivation in Escherichia coli. Photochem. Photobiol. 41, 367-370. Sammartano, L. J., and Tuveson, R. W. (1987). Escherichia coli strains carrying the cloned cytochrome d terminal oxidase complex are sensitive to near-UV inactivation. J . Bacteriol. 169, 5304-5307. Sammartano, L. J., Tuveson, R. W., and Davenport, R. (1986). Control of sensitivity to inactivation by HzOz and broad-spectrum near-UV radiation by the Escherichia coli katF locus. J . Bacteriol. 168, 13-21. Sancar, A., and Sancar, G. B. (1984).Escherichia coli DNA photolyase is a flavoprotein. J . Mot. Biol. 172, 223-227. Sancar, G. B., Smith, F. W., and Sancar, A. (1983).Identification and amplification ofthe E . coli phr gene product. Nucleic Acids Res. 11, 6667-6677.

BACTERIAL GENES INVOLVED IN RESPONSE TO

NUV

145

Saporito, S. M., Smith-White, B. J., and Cunningham, R. P. (1988).Nucleotide sequence of the xth gene of Escherichia coli K12.J.Bacteriol. 170,4542-4547. Sato, N., Ohnishi, T., Tano, K.,Yamamoto, K.,and Nozu, K.(1985).Induction of umuC gene expression in E . coli irradiated by near-ultraviolet light. Photochem. Photobiol. 42, 135-139. Schaaper, R. M., Danforth, B. N., and Glickman, B. W. (1985).Rapid repeated cloning of mutant lac repressor genes. Gene 39, 181-189. Schellhorn, H. E., and Hassan, H.M. (1988).Transcriptional regulation of kutE in Escherichia coli K12.J . Bucteriol. 170,4286-4292. Scott, M. D., Meshnick, S. R., and Eaton, J. W. (1987).Superoxide dismutase-rich bacteria. J. Biol. Chem. 262, 3640-3645. Sestili, P., Piedimonte, G., Flaminio, C., and Cantoni, 0. (1986).Induction of DNA breakage and suppression of DNA synthesis by the OH radical generated in a Fenton-like reaction. Biochem. Znt. 12,493-501. Setlow, J. K.,and Dugan, D. E. (1964).The resistance of Micrococcus radiodurans to ultraviolet radiation. I. Ultraviolet-induced lesions in the cell’s DNA. Biochim. Biophys. Acta 87, 664-668. Sharma, R. C., and Smith, K.C. (1985).A minor pathway of postreplication repair is independent of the recB, recC, and recF genes. Mutat. Res. 146,169-176. Sies, H., ed. (1985).“Oxidative Stress.” Academic Press, London. Slonczewski, J. L., Gonzalez, T. N., Bartholomew, F. M., and Holt, N. J. (1987).Mu d-directed lucz fusions by pH in Escherichia coli. J.Bacteriol. 169,3001-3006. Smith, K. C., and Sharma, R. C. (1987).A model for the recA-dependent repair of excision gaps in UV-irradiated E. coli. Mutat. Res. 183, 1-9. Sohal, R. S.,and Allen, R. G. (1986).Relationship between oxygen metabolism, aging and development. Adv. Free Radical Biol. Med. 2, 117-160. Spitzer, E. D., and Weiss, B. (1985).dfp gene of Escherichia coli K-12,a locus affecting DNA synthesis, codes for a flavoprotein. J.Bucteriol. 164, 994-1003. Storz, G., Christman, M. F., Sies, H., and Ames, B. N. (1987).Spontaneous mutagenesis and oxidative damage to DNA in Salmonella typhimurium. Proc. Natl. Acad. Sci. U.S.A.84,8917-8921. Storz, G.,Jacobson, F. S., Tartaglia, L. A,, Morgan, R. W., Silveira, L. A., and Ames, B. N. (1989).An alkyl hydroperoxide reductase induced by oxidative stress in Salmonella typhimurium and Escherichia coli: Genetic characterization and cloning of ahp. J. Bucteriol. 171,2049-2055. Straight, R. C., and Spikes, J . D. (1985).Photosensitized oxidation of biomolecules. Zn “Singlet Oxygen” (A. A. Frimer, ed.), Vol. 4,pp. 91-143. CRC Press, Boca Raton, Florida. Sweet, D. M., and Moseley, B. E. B. (1976).The resistance ofMicrococcus radioduruns to killing and mutation by agents which damage DNA. Mutat. Res. 34, 175-186. Szyf, M.,Meisels, E., and Razin, A. (1986).Biological role of DNA methylation: Sequence-specific single-strand breaks associated with hypomethylation of GATC site of Escherichia coli DNA. J . Bacteriol. 168, 1487-1490. Tabor, S.,and Richardson, C. (1987).Selective oxidation of the exonuclease domain of bacteriphage T7 DNA polymerase. J. Biol. Chem. 262, 15330-15333. Tajeda, Y.,and Avila, H. (1986).Structure and gene expression of the E. coli Mnsuperoxide dismutase gene. Nucleic Acids Res. 14,4577. Taylor, A. F., and Weiss, B.(1982).Role of exonuclease I11 in the base excision repair of uracil-containing DNA. J.Bacteriol. 151, 351-357. Tessman, E. S., Tessman, I., Peterson, P. K., and Forestal, J. D. (1986).Roles of RecA protease and recombinase activities of Escherichia coli in spontaneous and UVinduced mutagenesis and in Weigle repair. J.Bucteriol. 168, 1159-1164.

146

A . EISENSTARK

Thompson, B. G., Anderson, R., and Murray, R. G. E. (1980). Unusual polar lipids of Micrococcus radiodurans stain Sark. Can J . Microbiol. 26, 1408-1411. Thornley, M. J., Horne, R. W., and Glauert, A. M. (1965). The fine structure of Micrococcus radiodurans. Arch. Mikrobiol. 51, 267-289. Touati, D. (1983). Cloning and mapping of the maganese superoxide dismutase gene ( s o d ) of Escherichia coli K-12. J . Bacteriol. 155, 1078-1087. Touati, D. (1988a).Molecular genetics of superoxide dismutases. Free Radical Biol. Med. 5, 393-402. Touati, D. (1988b). Transcriptional and posttranscriptional regulation of manganese superoxide dismutase biosynthesis in E. coli, studied with operon and protein fusions. J . Bacteriol. 170, 2511-2520. Touati, D., and Carlioz, A. (1986).Superoxide dismutase mutants of Escherichia coli. I n “Superoxide and Superoxide Dismutase in Chemistry, Biology and Medicine” (G. Rotilo, ed.). Elsevier, Amsterdam. Triggs-Raine, B. L., and Loewen, P. C. (1987). Physical characterization of katG, encoding HPI of Escherichia coli. Gene 52, 121-128. Triggs-Raine, B. L., Dable, B. W., Mulvey, M. R., Sorby, P. A,, and Loewen, P. C. (1988). Nucleotide sequence of katG encoding catalase HPI of Escherichia coli. J . Bacteriol. 170,4415-4419. Turner, M. A,, and Eisenstark, A. (1984). Near-ultraviolet radiation blocks SOS responses to DNA damage in Escherichia coli. Mol. Gen. Genet. 193, 33-37. Turner, M. A., and Webb, R. B. (1981). Comparative mutagensis and interaction between near-ultraviolet (313-405 nm) and far-ultraviolet (254 nm) radiation in E. coli strains differing in repair capabilities. J . Bacteriol. 147, 410-417. Tuveson, R. W., and Sammartano, L. J. (1986).Sensitivity of hemA mutant Escherichia coli cells to inactivation by near-ultraviolet light depends on the level of supplementation with delta-aminolevulic acid. Photochem. Photobiol. 43, 621-626. Tuveson, R. W., Peak, J. G., and Peak, M. J. (1983).Single-strand DNA breaks induced by 365 nm radiation in E . coli strains differing in sensitivity to near- and far-UV. Photochem. Photobiol. 37, 109-112. Tyrrell, R. M. (1985). A common pathway or protection of bacteria against damage by solar UVA (334 nm, 365 nm) and oxidizing agents (HzO,). Mutat. Res. 145,129-136. Van Bogelen, R. A,, Kelly, P. M., and Neidhardt, F. C. (1987).Differential induction of heat shock, SOS, and oxidative stress regulons and accumulation of nucleotides in E . coli. J. Bacteriol. 169, 26-32. Van Rijin, K., Mayer, T., Blok, J.,Verberne, J. B., and Loman, H. (1985). Reaction rate of OH radicals with 4x174 DNA: Influence of salt and scavenger. Int. J . Radiat. Biol. 47,309-317. Vassilyadi, M., and Archibald, F. (1985). Catalase, superoxide dismutase, and the production of 02-sensitive mutants of Bacillus coagulans. Can. J . Microbiol. 31, 994-999. Walker, G. C. (1984). Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol. Rev. 48, 60-93. Walker, G. C. (1987). The SOS response of Escherichia coli. In “Escherichia coli and Salmonella typhimuriurn: Cellular and Molecular Biology” (F.C. Neidhardt et al., eds.), 7th Ed., pp. 1346-1357. Am. SOC.Microbiol., Washington, D.C. Wallace, S. S. (1988).AP endonucleases and DNA glycosylases that recognize oxidative DNA damage. Enuiron. Mol. Mutagen 12, 431-477. Webb, R. B. (1977). Lethal and mutagenic effects of near-ultraviolet radiation. Photochem. Photobiol. Rev. 2, 169-261.

BACTERIAL GENES INVOLVED IN RESPONSE TO

NUV

147

Wei, K., Tomita, S., and Suzuki, K.(1986). Block of cleavage of ultraviolet-irradiated DNA with restriction endonucleases. J . Radiat. Res. 27,247-253. Weiss, B., and Cunningham, R. P. (1985). Genetic mapping of nth, a gene affecting endonuclease I11 (thymidine glycol-DNA glycosylase) in Escherichia coli K-12. J . Bacterial. 162, 607-610. Weiss, R. B., and Duker, N. J. (1986). Photoalkylated DNA and ultraviolet-irradiated DNA are incised at cytosines by endonuclease 111. Nucleic Acids Res. 14,6621-6631. Weiss, R.B.,and Duke, N. J. (1987). Endonucleolytic incision of UVB-irradiated DNA. Photochem. Photobiol. 45, 763-768. Wilke, A. (1988). Effects of increased endogenous catalase on resistancehensitivity to near-ultraviolet radiation in E . coli. M.A. thesis, Univ. of Missouri, Columbia. Winguest, L., Rannung, U., Rannug, A., and Ramel, C. (1984).Protection from toxic and mutagenic effects of Hz02by catalase induction in Salmonella typhimurium. Mutut. Res. 141, 145-147. Yallaly, P. (1988). Influence of DNA adenine methylase on sensitivity of E . coli to near-ultraviolet radiation and hydrogen peroxide. M.A. thesis, Univ of Missouri, Columbia. Yamamoto, N., and Droffner, M. L. (1985). Mechanisms determining aerobic or anaerobic growth in the facultative anaerobe Salmonella typhimurium. Proc. Natl. Acad. sci.U.S.A.82,2077-2081. Yamamoto, N., Droffner, M. L., and Yamamoto, S. (1987). Conditional mutations of Salmonella typhimurium that are suppressed by anaerobic or aerobic environments. FEMS Microbiol. Lett. 42, 249-252. Yoakum, G.,Eisenstark, A., and Webb, R. B. (1975). Near-UV photoproduct(s) of L-tryptophan: Aninhibitor of medium-dependent repair of X-ray-induced singlestrand breaks in DNA which also inhibit replication-gap closures in Escherichia coli DNA. “Molecular Mechanisms for Repair of DNA, Part B” (P. C. Hanawalt and R. B. Setlow, eds.), pp. 453-458. Plenum, New York.