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Investigating phenotypes resulting from a lack of superoxide dismutase in bacterial null mutants
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[I 5] Investigating Phenotypes Resulting from a Lack of Superoxide D i s m u t a s e in Bacterial Null M u t a n t s By DANIELE TOUATI
Introduction Superoxide dismutases (SODs) are present in virtually all aerobes and in several anaerobes, 1 with both evolutionarily distinct SOD families found. Mn- and FeSODs (sodA and sodB genes), and cFe,MnSOD (cambialistic), are generally located in the cytoplasm, but a few are secreted. Cu,ZnSODs (sodC) have so far been found only in gram-negative bacteria and are located in the periplasm. As superoxide ( 0 2 - ) cannot cross the inner bacterial membrane at physiologically neutral pH, the protective effects of SODs should be related to SOD localization and are directed against endogenous or exogenous 02" --mediated oxidative stress. Studies of SOD null mutant phenotypes revealed that, despite a common enzymatic function, bacterial SODs display a broad spectrum of biological roles ranging from minor to essential, depending on specific bacterial metabolism, environmental conditions, and stage in the life cycle. O b t a i n i n g B a c t e r i a l Null M u t a n t s There is no positive selection system for SOD-deficient mutants, 2 and it is difficult to screen for negative phenotypes. This makes it virtually impossible to identify null mutants obtained by random insertional mutagenesis. However, improvements in genetic engineering techniques and the development of sequencing programs have provided powerful tools for constructing mutants. Strategies for the construction of sod mutants are not specific and depend on the available genetic tools in the bacterium of interest. Generally, the cloned gene is mutated (internal deletion and/or insertion of an antibiotic resistance cassette) and the mutated allele is exchanged with the wild-type chromosomal allele to produce stable mutants. It is important to check that exchange has actually occurred, as there is always a risk that the mutated allele will integrate into another part of the chromosome. The mutant should display a lack of the corresponding SOD activity and the expected changes in DNA digestion pattern as shown by Southern blotting or, if 1 D. Touati, in "Oxidative Stress and the Molecular Biology of Antioxidant Defenses" (J. Scandalios, ed.), p. 447. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1997. 2 A positive effect of SOD deficiency has just been described in Corynebacterium melassecola [M. Merkamm and A. Guyonvarch, J. Bacteriol. 183, 1284 (2001)], but the underlying metabolic mechanism is still unclear. Its understanding might provide a new useful tool to select sod mutants in some bacteria.
METHODS IN ENZYMOLOGY, VOL. 349
Copyright 2002, Elsevier Science (USA). All fights reserved~ 0076-6879/02 $35.00
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sequences flanking the cloned region are available, by polymerase chain reaction (PCR) amplification of the region. Although sodgenes have now been cloned and sequenced for a large number of bacteria, the number of sod mutants available is still small. This is partly because the genetic tools required to obtain mutants are not available for several bacteria, including pathogens such as mycobacteria. Other strategies, currently used predominantly in eukaryotes, such as the inhibition of gene expression by antisense RNA, may be necessary to determine the role of SOD in these difficult-to-handle microorganisms. POTENTIAL PROBLEMS
1. The sod mutation may be lethal. However, the failure to obtain a sod mutant is never sufficient in itself to conclude that the lack of SOD is lethal. In such a case, the chromosomal mutant should be constructed in the presence of expression of a wild-type gene (e.g., carded on a plasmid). To conclude that the mutation is lethal, it is necessary to demonstrate that it is maintained only when a wild-type copy is expressed. An example is the failure to obtain a mutant of the single cytoplasmic FeSOD of Legionella pneumophila, a strict aerobe living in a highly oxygenated environment. 3 2. There are several known examples of two SODs (Fe and Mn) being present in the same compartment of a bacterium. But one can fail to detect it on an activity gel. Indeed, some SODs are expressed only under particular conditions, such as the MnSOD from Bordetella pertussis, which is expressed only under strict iron starvation. 4'5 Despite the high level of conservation between Fe- and MnSOD amino acid sequences, differences in codon usage between bacterial species may result in failure to detect a new sod gene on Southern blot analysis. Only BLAST sequence analysis of a complete genome sequence, when available, can ensure that no cryptic SOD has been missed. C y t o p l a s m i c sod M u t a n t s Cytoplasmic SODs protect against the toxicity caused by excess 0 2 - generation in the cell. This may result from natural endogenous 0 2 - production, principally via the respiratory chain, or by metabolic transformation of a xenobiotic. Extensive studies have been carded out in Escherichia coli, from which the first cytoplasmic SOD mutants were isolated and characterized. 6 In those mutants,
3 A. B. Sadosky, J. W. Wilson, H. M. Steinman, and H. A. Shuman, J. Bacteriol 176, 3790 (1994). 4 N. Khelef, D. DeShazer, R. L. Friedman, and N. Guiso, FEMS MicrobioL Lett. 142, 231 (1996). 5 H. Graeff-Wohlleben, S. Killat, A. Banemann, N. Guiso, and R. Gross, J. Bacteriol. 179~ 2194 (1997). 6 A. Carlioz and D. Touati, EMBO J. 5, 623 (1986).
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lacking both cytoplasmic Fe- and MnSOD, steady-state 02"- levels were found to be 100 to 1000 times higher than in their SOD + counterpart. Some of the observed defects were clearly shown to be due to direct damage to specific targets, but most of the damage, including that to DNA and presumably also to membrane, was caused by hydroxyl radicals. These were produced by the Fenton reaction, which was triggered by the excess of 02" -, via an increase in the intracellular free iron pool. 7 Only a limited number of studies have been carded out in other bacteria and have tended to focus exclusively on the effect of SOD deficiency on a particular function of interest. As endogenous 02"- production and the targets sensitive to 0 2 - mediated stress depend on the specific metabolism and mode of life of the bacterium, there is no general method for assaying the physiological consequences of an SOD defect in bacterial species. General Remarks 1. Each of the experiments described below should be repeated at least three times to confirm the overall trend. 2. Any phenotype linked to an sod mutation should be oxygen dependent. This can easily be (and, indeed, has been) demonstrated with anaerobes and facultative anaerobes, in which the phenotype is abolished in anaerobiosis. Phenotypes Related to General 02"- Stress Conditions Impairment of Aerobic Growth and Aerotolerance 0 2 - is produced only in the presence of oxygen. Thus oxygen becomes toxic if 0 2 - is not eliminated at a high rate by SOD. Comparison of the growth rate and yield of sod mutants and wild type in rich medium in air supports this view. However, as illustrated in Table I, the sensitivity of sod mutants to oxygen varies greatly from one bacterial species to another. Sensitivity to Compounds Generating Endogenous 02 sod mutants generally show greater sensitivity to compounds producing O 2 by redox cycling (Table I). Paraquat is the most commonly used, because it does not generate other toxic by-products. A crude assay (disk assay) involves spotting l 0 / z l of various concentrations of paraquat on small paper disks placed over a lawn of bacteria (0.1/zl of saturated culture) spread on a Luria-Bertani (LB) agar plate, and measuring the diameter of the inhibition zone after a period of growth. A more precise measurement involves evaluating the effect on the exponentially growing cells. So far as possible, a protocol adapted from that described below (for E. coli) is recommended. Precultures in LB (or minimal) medium, inoculated with 7 K. Keyerand J. A. Imlay,Proc.Natl. Acad. Sci. U.S.A.93, 13635(1996).
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TABLE I SENSITIVITYTO OXYGENANDPARAQUATOFCYTOPLASMICsod MUTANTS
Species Escherichia coli
Pseudomonas aeruginosa and P. putida Bordetella pertussis Streptococcus pneumoniae Streptococcus pyogenes Legionella pneumophila Sinorhizobium meliloti Synechococcus species Haemophilus influenzae Lactococcus lactis Campylobacter jejuni, C. coli Porphoryonas gingivalis
sod gene
Impairment of aerobic growth or loss of aerotolerance
Sensitivity to paraquat
Refs.
sodA sodB sodA sodB sodA sodB sodA sodB sodB sodA sodA sod sodB e sodA e sodB e sodA e sodA e sodB e
No No Yes, slight No Yes Yes Yes No Yes, slight Yes, strong Lethal Yes, slight Yes, slight Yes, strong Yes, strong No
Yes Yes, low Yes, high Yes, low Yes, high Yes, high Yes ND Yes, high Yes, high -Yes, low Yes, high Yes, high ND ND
6 6 6 a, b a, b a, b 4 5 c d 3 17 f g h i
sodA e
Lysis
--
j
D. J. Hasset, H. E Schweizer, and D. E. Ohman, J. BacterioL 177, 6330 (1995). b y. C. Kim, C. D. Miller, and A. J. Anderson, Appl. Environ. Microbiol. 66, 1460 (2000). c H. Yesilkaya, A. Kadioglu, N. Gingles, J. E. Alexander, T. J. Mitchell, and P. W. Andrew, Infect. lmmun. 68, 2819 (2000). dc. M. Gibson and M. G. Caparon, J. Bacteriol. 178, 4688 (1996). e Reported as single cytosolic SOD. fG. Samson, S. K. Herbert, D. C. Fork, and D. E. Laudenbach, Plant Physiol. 105, 287 (1994). g R. A. D'Mello, P. R. Langford, andJ. S. Kroll, Infect. Immun. 65, 2700 (1997). h j. W. Sanders, K. J. Leenhouts, A. J. Haandrikman, G. Venema, and J. Kok, J. BacterioL 177, 5254 (1995). i E. C. P e s c i , D. t. Cottle, and C. L. Pickett, Infect. lmmun. 62, 2687 (1994). a
single colonies, are grown to an optical density (OD) o f 1 (end of exponential growth phase) and then rapidly chilled in a w a t e r - i c e mixture and kept cold for up to 40 hr. Cultures are prepared b y inoculating the same m e d i u m , prewarmed, with precultures. The dilution is chosen to allow the growth o f three generations before challenge with paraquat (at an O D of about 0.2). N o growth lag is observed under these conditions. Growth is recorded by O D m e a s u r e m e n t at various times. The range of paraquat concentrations used results in only mild growth inhibition for the wild-type strain (1 to 1 0 0 / z M for E. coli). A control, to ensure that the correspondence b e t w e e n O D and c o l o n y - f o r m i n g units per milliliter ( C F U / m l ) is maintained, should be done at several points by plating bacteria and c o u n t i n g colonies after growth.
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POTENTIALPROBLEMS 1. The initial volume of cultures should be sufficient compared with the total volume of samples withdrawn to maintain approximately constant aeration of the culture. 2. Paraquat penetrates cells by means of active transport (demonstrated in E. coli and presumably also true in other bacteria). Thus (a) the intracellular concentration depends on both the concentration of paraquat in the medium and the number of bacteria. Only experiments done at the same bacterial density can be compared; and (b) bacteria vary greatly in their wild-type sensitivity to paraquat because of differences in penetration. 3. Starting with a diluted overnight culture results in a growth lag that is not uniform from one cell to another. This may result in paraquat being added to a heterogeneous population giving less reproducible results unless the overnight culture is highly diluted and allowed to go through a large number of generations before challenge. Specific Characterized Damage and Associated Phenotypes [ 4 Fe-4S ] Cluster Sensitivity
The inactivation by 02' - of a group of dehydratases containing [4Fe-4S] clusters, via the oxidative excision of iron, was first demonstrated in vitro. 8 Enzymes containing these clusters (dihydroxy acid dehydratase, aconitase, and gluconate dehydratase), 6,9'1° are inactivated in E. coli mutants deficient in both cytoplasmic SODs, as shown by direct enzyme activity measurements and defective associated phenotypes (branched amino acid auxotrophy, growth impairment on succinate, gluconate). The presence and role of [4Fe-4S]-containing enzymes varies depending on the specific metabolism of each bacterium, and putative targets must be identified in each case. 11 POTENTIALPROBLEM.Impairment of aconitase activity can be measured in many bacterial sod mutants. However, some bacteria (such as E. coli) can partially compensate for the oxidative inactivation of aconitase by an increase in aconitase gene expression under oxidative stress conditions. DNA Damage
O:z- cannot directly react with DNA, but there is now considerable evidence that an increase in 02"- levels leads to DNA damage. Several studies provide strong support for the theory that 02"- triggers Fenton reaction (H202-q8 D. H. Flint, J. F. Tuminello,and M. H. Emptage,J. BioL Chem. 268, 22369 (1993). 9 p. R. Gardnerand I. Fridovich,J. Biol. Chem. 266, 1478 (1991). l0 p. R. Gardnerand I. Fridovich,J. Biol. Chem. 267, 8757 (1992). I1 S. I. Liochevand I. Fridovich, FreeRadic. Biol. Med. 16, 29 (1994).
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Fe2+----~OH'-FFe3++OH-) by the oxidative release of iron from iron-containing molecules, such as those containing [4Fe-4S] clusters, and thereby increasing the pool of free iron available to catalyze hydroxyl radical production, l° Studies of mutants lacking cytosolic SOD provided the first in vivo evidence that excess 0 2 ' causes DNA damage. 12,13 The hallmarks of DNA damage in SOD-deficient mutants are as follows. 1. An oxygen-dependent increase in spontaneous mutagenesis (methods for measuring mutagenesis in sod mutants are described in a previous volume of this series). 14 2. A lethal aerobic phenotype of strains lacking cytosolic SOD and unable to repair DNA double-strand breaks by homologous recombination.15 3. An increase in HzO2-mediated killing via the Fenton reaction. 6,13 MEASUREMENT OF KILLING BY H202. A midexponential phase culture (OD 0.3 to 0.5) in LB, prepared as for paraquat challenge, is dispensed into Erlenmeyer flasks and H202 is added to various concentrations (0 to 20 mM). After incubation with shaking for 20 min at growth temperature, the treatment is stopped by adding catalase (1000 U/ml; Boehringer Mannheim, Indianapolis, IN) and chilling. Surviving cells are counted by plating on LB plates, after dilutions into cold 10 -2 M MgSO4 [or phosphate-buffered saline (PBS)] containing catalase. To check that the increase in killing is mediated by Fenton chemistry, the experiment can be repeated in the presence of a cell-permeable iron chelator (1 mM 2,2-dipyridyl or diethylenetriaminepentaacetic acid (DTPA) added to the cell culture a few minutes before challenge and in the dilution buffer), which should abolish the increase. POTENTIALPROBLEMS 1. Killing should be determined as a function of HzO2 concentration. H202 kills cells in two modes (demonstrated in E. coli, 16 but also observed in other bacteria). Only mode 1 killing, which occurs at lower H202 concentration, is well characterized and has been shown to be due to DNA damage mediated by the Fenton reaction. The sensitivity of sod mutants to H202 should therefore be measured in the range of concentrations corresponding to mode 1 killing. 2. The sensitivity of wild-type strains differs greatly between species (e.g., similar levels of killing are observed with 250/zM in Salmonella typhimurium and 2 mM in E. coli). 3. The reliability of measurements of killing by H202 due to excess 0 2 - implies that 0 2 - does not interfere with catalase and peroxidase activities. Although 12S. B. Farr, R. D'Ari, and D. Touati,Proc. Natl. Acad. Sci. U.S.A. 83, 8268 (1986). 13K. Keyer,A. StrohmeierGort, and I. A. Imlay,J. Bacteriol. 177, 6782 (1995). 14D. Touati and S. B. Farr, Methods Enzymol. 186, 646 (1990). 15D. Touati,M. Jacques, B. Tardat, L. Bouchard, and S. Despied,J. BacterioL 177, 2305 (1995).
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this is generally true, exceptions exist. In Sinorhizobium meliloti catalase and presumably a global defense against H202 is strongly induced by O2-, leading to higher resistance to H202 in the sodA mutant than in the wild typeJ 7 Metabolic Defects, Impairment of Growth in Minimal Medium Growth in minimal medium requires numerous synthethic pathways and presents many targets susceptible to oxidative damage. Thus, sod mutants display growth impairment. However, targets are not easy to identify and differ depending on the metabolic pathway affected. Escherichia coli sod mutants were found to be unable to grow on minimal medium unless provided with all amino acids. 6 Studies in several laboratories demonstrated that this general auxotrophy was due to several types of damage: a defect in branched amino acid synthesis due to dehydratase inactivation, 18 interference with aromatic biosynthesis by transketolase inactivation, 19which acts at an early step of biosynthesis, and cell envelope defects leading to sulfite leakage and cysteine auxotrophy.2° Similar general amino acid auxotrophy has been observed in sod mutants in other bacteria, suggesting similar defects. However, in some cases the growth impairment on minimal medium was not reversed by providing amino acids, indicating other (or additional) metabolic defectsJ 6 Membrane Damage Although there is evidence of membrane damage in sod mutants,21 the nature of the lesions has not been identified and is difficult to investigate. Lipid peroxidation is unlikely to be solely responsible for the damage, because there are almost no unsaturated lipids in most bacterial membranes. M u t a n t s in N o n c y t o p l a s m i c S u p e r o x i d e D i s m u t a s e s No sources of 02"- have yet been found in the periplasm and outer bacterial membranes. Thus, periplasmic, secreted, and outer membrane-associated SODs are thought to protect against external 0 2 - . In particular, SODs are expected to protect against the oxidative burst produced by the host during infection, thereby contributing to pathogenicity and virulence. No mutants have yet been obtained for secreted Fe- and MnSODs. Mutants have been obtained only for the periplasmic Cu,ZnSODs (sodC). The expected phenotypes were, as follows: resistance to endogenous 02'- (as generated by paraquat), sensitivity to exogenous 02"(as generated by hypoxanthine-xanthine oxidase, or pyrogallol), and attenuation 16 j. A. lmlay and S. Linn, J. Bacteriol. 166, 519 (1986). 17 R. Santos, D. H6rouart, A. Puppo, and D. Touati, Mol. Microbiol. 38, 750 (2000). 18 C. E Kuo, T. Mashino, and I. Fridovich, J. Biol. Chem. 262, 4724 (1987). 19 L. Benov and I. Fridovich, J. Biol. Chem. 274, 4202 (1999). 20 L. Benov, N. M. Kredich, and I. Fridovich, J. Biol. Chem. 271, 21037 (1996). 21 j. A. Imlay and I. Fridovich, J. Bacteriol. 174, 953 (1992).
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of virulence and pathogenicity (sensitivity to killing by macrophages, attenuated virulence in animal models). All sodC mutants display levels of resistance to paraquat similar to those of the wild type, consistent with periplasmic SOD affording no protection against cytoplasmic O2'-. In pathogens, sodC mutants generally display attenuation of virulence (reviewed in Lynch and Kuramitsu), 22 consistent with a lower level of protection against oxidative burst (02"-, H202, NO'). sodC mutants have been reported to be sensitive to exogenous 0 2 -. However, the sources of O2' - used produced both 02" - and H202 and most experiments were done in the absence of addition of catalase. The addition of this enzyme is necessary to ensure that the observed effects are attributable to exogenous O2 -. Reports that the sensitivity of E. coli sodC mutant to hypoxanthine-xanthine oxidase was abolished by catalase raises further doubt about the mechanism of sodC protection. 23 The phenotypes of sodC mutants observed to date suggest that periplasmic SOD may protect against both endogenous and exogenous O 2 - . The source of endogenous O2' - remains unclear, but this aspect should not be neglected in future studies of sodC mutants. B a c t e r i a l sod Null M u t a n t s a s T o o l s Cloning sod Genes from Other Organisms It has been found that the production of SOD from any organism in the cytoplasm of the sodA sodB mutant of E. coli fully complements the deficiencies of this mutant. Thus, complementation assays have been widely used to clone sod genes from other organisms. This strategy is still useful in the absence of a genomic sequence. Such assays also led to the isolation of another O2' --scavenging enzyme, 02"- reductase (see Ref. 24 and [13] in this volume25). Two tests are available. Complementation of Lack of Growth on Minimal Medium. A sodA sodB strain is transformed with a genomic library. Transformants are scraped from the selection plates and pooled, suspended in 10 -2 M cold MgSO4 buffer, washed twice, and plated on minimal glucose medium (M63 or M9) to which 10 -6 M paraquat and appropriate antibiotic (used for transformant selection) have been added. Putative sod + colonies appear after 2 to 3 days of incubation at 37 °. Note: External suppressors of the amino acid auxotrophy phenotype occur with high frequency, but these colonies are nonetheless usually sensitive to paraquat. 21'26 A preliminary assay should be done to evaluate the maximum number (usually at least 105 ) of 22 M. Lynch and H. Kuramitsu, Microbes Infect. 2, 1245 (2000). 23 A. Strohmeier Gort, D. M. Ferber, and J. A. Imlay, Mol. Microbiol. 32, 179 (1999). 24 M. J. Pianzzola, M. Soubes, andD. Touati, J. Bacteriol. 178, 6736 (1996). 25 V. Nivi~re and M. Lombard, Methods Enzymol. 349, [13], 2002 (this volume). 26 S. Maringanti and J. A. Imlay, J. Bacteriol. 181, 3792 (1999).
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TABLE II OTHER SPECIfiCPHENOTYPESASSOCIATEDWITH sod MUTANTS Defect condition Normal growth
Mutant sodB sodA sodB sodB
Stationary phase
sodC sodC sodA sodB sodA
sodA
Infection
Cytosolic or periplasmic
Symbiosis
sodA
Chilling
sodB
Acid stress
sodA
Phenotype Sensitivity of photosystem to moderate light Pyocyanin production abolished Reduced production of toxin and adhesin (adenylate cyclase, hemolysin, pertactin) Rapid decrease in survival Sensitivity to peroxynitrite Does not survive long-term starvation Loss of viability Less resistant to oxidative stress; growth resumes slowly from stationary phase Decrease in sporulation frequency; defect in spore coat assembly Attenuated virulence depending on mode and location of infection, and/or resistance to killing by macrophages Reduced nodulation; defect in bacteroid differentiation; drastic decrease in nitrogen fixation Sensitivity to moderate chilling in light Increase in sensitivity to acid stress
Species
Refs.
Cyanobacterium
a
Pseudomonas aeruginosa Bordetella pertussis
b 4
Legionella pneumophila Salmonella typhimurium Staphylococcus aureus
c d e
Campylobacter coli Pseudomonas aeruginosa
f g
Bacillus subtilis
h
Numerous pathogens
22
Sinorhizobium meliloti
17
Cyanobacterium
i
Staphylococcus aureus
e
a G. Samson, S. K. Herbert, D. C. Fork, and D. E. Laudenbach, Plant Physiol. 105, 287 (1994). b D. J. Hasset, H. P. Schweizer, and D. E. Ohman, J. BacterioL 177, 6330 (1995). c G. St:. John and H. M. Steinman, J. Bacteriol. 178, 1578 (1996). d M. A. De Groote, U. A. Ochsner, M. U. Shiloh, C. Nathan, J. M. McCord, M. C. Dinauer, S. J. Libby, A. Vasquez-Torres, Y. Xu, and E C. Fang, Proc. Natl. Acad. Sci. U.S.A. 94, 13997 (1997). e M. O. Clements, S. P. Watson, and S. J. Foster, J. Bacteriol. 181, 3898 (1999). f D. Purdy, S. Cathraw, J. H. Dickinson, D. G. Newell, and S. E Park, Appl. Environ. Microbiol. 65, 2540 (1999). g B. Polack, D. Dacheux, I. Delic-Attree, B. Toussaint, and P. M. Vignais, Infect. lmmun. 64, 2216 (1996). h A. O. Hentiques, L. R. Melsen, and C. P. Moran, Jr., J. Bacteriol. 180, 2285 (1998). i D.J. Thomas, J. B. Thomas, S. D. Prier, N. E. Nasso, and S. K. Herbert, Plant Physiol. 120, 275 (1999).
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sodA sodB bacteria that can be plated without residual growth. A slightly higher concentration of paraquat can be used if there is residual growth. Restoration of Aerobic Growth of sodA sodB recA Strain. The test requires access to an anaerobic chamber. A sodA sodB recA strain (air sensitive) is transformed with the genomic library under anaerobiosis. Pooled transformants are plated on LB plates under aerobiosis and incubated overnight at 37 ° under aerobiosis. The plating of 104 to 105 SOD- bacteria should result in no survival under these conditions. POTENTIAL PROBLEM. The aerobic growth of a sodA sodB recA strain also can be restored by recombination (cloning of a rec + gene). Thus, the putative sod + clones should be further tested for complementation in minimal medium. Identifying New 02"- Sensitive Targets and Situations of Endogenous Oxidative Stress Other defects observed in sod mutants of various bacteria under various conditions are listed in Table II. In all cases, the specific damage has not been clearly characterized. Defects may result from the vulnerability to O2"--mediated stress of a particular function or from an increase in oxidative stress in particular conditions. Thus, the low level of nitrogen fixation in the sodA S. meliloti mutant is probably due to direct inactivation of the nitrogenase complex, but the reasons for the defect in nodulation remain unclear. 16 The attenuation of virulence in various cytoplasmic sod mutants may result from damage due to the high metabolic rate during multiplication of bacteria in their host and associated endogenous 0 2 production, whereas attenuation in periplasmic sod mutants seems to be related to protection against oxidative burst. 22 Concluding Remarks Studies of bacterial sod null mutants have already identified several 0 2 - targets and shed light on the mechanism of 0 2 ' - toxicity. The effects of SOD deficiency in bacteria differ greatly according to SOD location and also because of the diversity of bacterial modes of life. For a particular bacterium, the lack of SOD may have minor effects in one phase of the life cycle, but drastic effects in another (e.g., free-living and symbiotic S. meliloti). 17 Many of the effects of SOD deficiency are still unclear. Studies of additional sod null mutants in various bacteria will help uncover new protective effects, to identify new 02'--sensitive targets and possibly new sources of 02"-, and to obtain further insight into the mechanisms, both direct and indirect, of 02" - toxicity. Acknowledgments We thank the Association pour la Recherche sur le Cancer for supporting our work on bacterial superoxide dismutase mutants (Grant 558 l).
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[I 5] Investigating Phenotypes Resulting from a Lack of Superoxide D i s m u t a s e in Bacterial Null M u t a n t s By DANIELE TOUATI
Introduction Superoxide dismutases (SODs) are present in virtually all aerobes and in several anaerobes, 1 with both evolutionarily distinct SOD families found. Mn- and FeSODs (sodA and sodB genes), and cFe,MnSOD (cambialistic), are generally located in the cytoplasm, but a few are secreted. Cu,ZnSODs (sodC) have so far been found only in gram-negative bacteria and are located in the periplasm. As superoxide ( 0 2 - ) cannot cross the inner bacterial membrane at physiologically neutral pH, the protective effects of SODs should be related to SOD localization and are directed against endogenous or exogenous 02" --mediated oxidative stress. Studies of SOD null mutant phenotypes revealed that, despite a common enzymatic function, bacterial SODs display a broad spectrum of biological roles ranging from minor to essential, depending on specific bacterial metabolism, environmental conditions, and stage in the life cycle. O b t a i n i n g B a c t e r i a l Null M u t a n t s There is no positive selection system for SOD-deficient mutants, 2 and it is difficult to screen for negative phenotypes. This makes it virtually impossible to identify null mutants obtained by random insertional mutagenesis. However, improvements in genetic engineering techniques and the development of sequencing programs have provided powerful tools for constructing mutants. Strategies for the construction of sod mutants are not specific and depend on the available genetic tools in the bacterium of interest. Generally, the cloned gene is mutated (internal deletion and/or insertion of an antibiotic resistance cassette) and the mutated allele is exchanged with the wild-type chromosomal allele to produce stable mutants. It is important to check that exchange has actually occurred, as there is always a risk that the mutated allele will integrate into another part of the chromosome. The mutant should display a lack of the corresponding SOD activity and the expected changes in DNA digestion pattern as shown by Southern blotting or, if 1 D. Touati, in "Oxidative Stress and the Molecular Biology of Antioxidant Defenses" (J. Scandalios, ed.), p. 447. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1997. 2 A positive effect of SOD deficiency has just been described in Corynebacterium melassecola [M. Merkamm and A. Guyonvarch, J. Bacteriol. 183, 1284 (2001)], but the underlying metabolic mechanism is still unclear. Its understanding might provide a new useful tool to select sod mutants in some bacteria.
METHODS IN ENZYMOLOGY, VOL. 349
Copyright 2002, Elsevier Science (USA). All fights reserved~ 0076-6879/02 $35.00
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sequences flanking the cloned region are available, by polymerase chain reaction (PCR) amplification of the region. Although sodgenes have now been cloned and sequenced for a large number of bacteria, the number of sod mutants available is still small. This is partly because the genetic tools required to obtain mutants are not available for several bacteria, including pathogens such as mycobacteria. Other strategies, currently used predominantly in eukaryotes, such as the inhibition of gene expression by antisense RNA, may be necessary to determine the role of SOD in these difficult-to-handle microorganisms. POTENTIAL PROBLEMS
1. The sod mutation may be lethal. However, the failure to obtain a sod mutant is never sufficient in itself to conclude that the lack of SOD is lethal. In such a case, the chromosomal mutant should be constructed in the presence of expression of a wild-type gene (e.g., carded on a plasmid). To conclude that the mutation is lethal, it is necessary to demonstrate that it is maintained only when a wild-type copy is expressed. An example is the failure to obtain a mutant of the single cytoplasmic FeSOD of Legionella pneumophila, a strict aerobe living in a highly oxygenated environment. 3 2. There are several known examples of two SODs (Fe and Mn) being present in the same compartment of a bacterium. But one can fail to detect it on an activity gel. Indeed, some SODs are expressed only under particular conditions, such as the MnSOD from Bordetella pertussis, which is expressed only under strict iron starvation. 4'5 Despite the high level of conservation between Fe- and MnSOD amino acid sequences, differences in codon usage between bacterial species may result in failure to detect a new sod gene on Southern blot analysis. Only BLAST sequence analysis of a complete genome sequence, when available, can ensure that no cryptic SOD has been missed. C y t o p l a s m i c sod M u t a n t s Cytoplasmic SODs protect against the toxicity caused by excess 0 2 - generation in the cell. This may result from natural endogenous 0 2 - production, principally via the respiratory chain, or by metabolic transformation of a xenobiotic. Extensive studies have been carded out in Escherichia coli, from which the first cytoplasmic SOD mutants were isolated and characterized. 6 In those mutants,
3 A. B. Sadosky, J. W. Wilson, H. M. Steinman, and H. A. Shuman, J. Bacteriol 176, 3790 (1994). 4 N. Khelef, D. DeShazer, R. L. Friedman, and N. Guiso, FEMS MicrobioL Lett. 142, 231 (1996). 5 H. Graeff-Wohlleben, S. Killat, A. Banemann, N. Guiso, and R. Gross, J. Bacteriol. 179~ 2194 (1997). 6 A. Carlioz and D. Touati, EMBO J. 5, 623 (1986).
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lacking both cytoplasmic Fe- and MnSOD, steady-state 02"- levels were found to be 100 to 1000 times higher than in their SOD + counterpart. Some of the observed defects were clearly shown to be due to direct damage to specific targets, but most of the damage, including that to DNA and presumably also to membrane, was caused by hydroxyl radicals. These were produced by the Fenton reaction, which was triggered by the excess of 02" -, via an increase in the intracellular free iron pool. 7 Only a limited number of studies have been carded out in other bacteria and have tended to focus exclusively on the effect of SOD deficiency on a particular function of interest. As endogenous 02"- production and the targets sensitive to 0 2 - mediated stress depend on the specific metabolism and mode of life of the bacterium, there is no general method for assaying the physiological consequences of an SOD defect in bacterial species. General Remarks 1. Each of the experiments described below should be repeated at least three times to confirm the overall trend. 2. Any phenotype linked to an sod mutation should be oxygen dependent. This can easily be (and, indeed, has been) demonstrated with anaerobes and facultative anaerobes, in which the phenotype is abolished in anaerobiosis. Phenotypes Related to General 02"- Stress Conditions Impairment of Aerobic Growth and Aerotolerance 0 2 - is produced only in the presence of oxygen. Thus oxygen becomes toxic if 0 2 - is not eliminated at a high rate by SOD. Comparison of the growth rate and yield of sod mutants and wild type in rich medium in air supports this view. However, as illustrated in Table I, the sensitivity of sod mutants to oxygen varies greatly from one bacterial species to another. Sensitivity to Compounds Generating Endogenous 02 sod mutants generally show greater sensitivity to compounds producing O 2 by redox cycling (Table I). Paraquat is the most commonly used, because it does not generate other toxic by-products. A crude assay (disk assay) involves spotting l 0 / z l of various concentrations of paraquat on small paper disks placed over a lawn of bacteria (0.1/zl of saturated culture) spread on a Luria-Bertani (LB) agar plate, and measuring the diameter of the inhibition zone after a period of growth. A more precise measurement involves evaluating the effect on the exponentially growing cells. So far as possible, a protocol adapted from that described below (for E. coli) is recommended. Precultures in LB (or minimal) medium, inoculated with 7 K. Keyerand J. A. Imlay,Proc.Natl. Acad. Sci. U.S.A.93, 13635(1996).
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TABLE I SENSITIVITYTO OXYGENANDPARAQUATOFCYTOPLASMICsod MUTANTS
Species Escherichia coli
Pseudomonas aeruginosa and P. putida Bordetella pertussis Streptococcus pneumoniae Streptococcus pyogenes Legionella pneumophila Sinorhizobium meliloti Synechococcus species Haemophilus influenzae Lactococcus lactis Campylobacter jejuni, C. coli Porphoryonas gingivalis
sod gene
Impairment of aerobic growth or loss of aerotolerance
Sensitivity to paraquat
Refs.
sodA sodB sodA sodB sodA sodB sodA sodB sodB sodA sodA sod sodB e sodA e sodB e sodA e sodA e sodB e
No No Yes, slight No Yes Yes Yes No Yes, slight Yes, strong Lethal Yes, slight Yes, slight Yes, strong Yes, strong No
Yes Yes, low Yes, high Yes, low Yes, high Yes, high Yes ND Yes, high Yes, high -Yes, low Yes, high Yes, high ND ND
6 6 6 a, b a, b a, b 4 5 c d 3 17 f g h i
sodA e
Lysis
--
j
D. J. Hasset, H. E Schweizer, and D. E. Ohman, J. BacterioL 177, 6330 (1995). b y. C. Kim, C. D. Miller, and A. J. Anderson, Appl. Environ. Microbiol. 66, 1460 (2000). c H. Yesilkaya, A. Kadioglu, N. Gingles, J. E. Alexander, T. J. Mitchell, and P. W. Andrew, Infect. lmmun. 68, 2819 (2000). dc. M. Gibson and M. G. Caparon, J. Bacteriol. 178, 4688 (1996). e Reported as single cytosolic SOD. fG. Samson, S. K. Herbert, D. C. Fork, and D. E. Laudenbach, Plant Physiol. 105, 287 (1994). g R. A. D'Mello, P. R. Langford, andJ. S. Kroll, Infect. Immun. 65, 2700 (1997). h j. W. Sanders, K. J. Leenhouts, A. J. Haandrikman, G. Venema, and J. Kok, J. BacterioL 177, 5254 (1995). i E. C. P e s c i , D. t. Cottle, and C. L. Pickett, Infect. lmmun. 62, 2687 (1994). a
single colonies, are grown to an optical density (OD) o f 1 (end of exponential growth phase) and then rapidly chilled in a w a t e r - i c e mixture and kept cold for up to 40 hr. Cultures are prepared b y inoculating the same m e d i u m , prewarmed, with precultures. The dilution is chosen to allow the growth o f three generations before challenge with paraquat (at an O D of about 0.2). N o growth lag is observed under these conditions. Growth is recorded by O D m e a s u r e m e n t at various times. The range of paraquat concentrations used results in only mild growth inhibition for the wild-type strain (1 to 1 0 0 / z M for E. coli). A control, to ensure that the correspondence b e t w e e n O D and c o l o n y - f o r m i n g units per milliliter ( C F U / m l ) is maintained, should be done at several points by plating bacteria and c o u n t i n g colonies after growth.
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POTENTIALPROBLEMS 1. The initial volume of cultures should be sufficient compared with the total volume of samples withdrawn to maintain approximately constant aeration of the culture. 2. Paraquat penetrates cells by means of active transport (demonstrated in E. coli and presumably also true in other bacteria). Thus (a) the intracellular concentration depends on both the concentration of paraquat in the medium and the number of bacteria. Only experiments done at the same bacterial density can be compared; and (b) bacteria vary greatly in their wild-type sensitivity to paraquat because of differences in penetration. 3. Starting with a diluted overnight culture results in a growth lag that is not uniform from one cell to another. This may result in paraquat being added to a heterogeneous population giving less reproducible results unless the overnight culture is highly diluted and allowed to go through a large number of generations before challenge. Specific Characterized Damage and Associated Phenotypes [ 4 Fe-4S ] Cluster Sensitivity
The inactivation by 02' - of a group of dehydratases containing [4Fe-4S] clusters, via the oxidative excision of iron, was first demonstrated in vitro. 8 Enzymes containing these clusters (dihydroxy acid dehydratase, aconitase, and gluconate dehydratase), 6,9'1° are inactivated in E. coli mutants deficient in both cytoplasmic SODs, as shown by direct enzyme activity measurements and defective associated phenotypes (branched amino acid auxotrophy, growth impairment on succinate, gluconate). The presence and role of [4Fe-4S]-containing enzymes varies depending on the specific metabolism of each bacterium, and putative targets must be identified in each case. 11 POTENTIALPROBLEM.Impairment of aconitase activity can be measured in many bacterial sod mutants. However, some bacteria (such as E. coli) can partially compensate for the oxidative inactivation of aconitase by an increase in aconitase gene expression under oxidative stress conditions. DNA Damage
O:z- cannot directly react with DNA, but there is now considerable evidence that an increase in 02"- levels leads to DNA damage. Several studies provide strong support for the theory that 02"- triggers Fenton reaction (H202-q8 D. H. Flint, J. F. Tuminello,and M. H. Emptage,J. BioL Chem. 268, 22369 (1993). 9 p. R. Gardnerand I. Fridovich,J. Biol. Chem. 266, 1478 (1991). l0 p. R. Gardnerand I. Fridovich,J. Biol. Chem. 267, 8757 (1992). I1 S. I. Liochevand I. Fridovich, FreeRadic. Biol. Med. 16, 29 (1994).
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Fe2+----~OH'-FFe3++OH-) by the oxidative release of iron from iron-containing molecules, such as those containing [4Fe-4S] clusters, and thereby increasing the pool of free iron available to catalyze hydroxyl radical production, l° Studies of mutants lacking cytosolic SOD provided the first in vivo evidence that excess 0 2 ' causes DNA damage. 12,13 The hallmarks of DNA damage in SOD-deficient mutants are as follows. 1. An oxygen-dependent increase in spontaneous mutagenesis (methods for measuring mutagenesis in sod mutants are described in a previous volume of this series). 14 2. A lethal aerobic phenotype of strains lacking cytosolic SOD and unable to repair DNA double-strand breaks by homologous recombination.15 3. An increase in HzO2-mediated killing via the Fenton reaction. 6,13 MEASUREMENT OF KILLING BY H202. A midexponential phase culture (OD 0.3 to 0.5) in LB, prepared as for paraquat challenge, is dispensed into Erlenmeyer flasks and H202 is added to various concentrations (0 to 20 mM). After incubation with shaking for 20 min at growth temperature, the treatment is stopped by adding catalase (1000 U/ml; Boehringer Mannheim, Indianapolis, IN) and chilling. Surviving cells are counted by plating on LB plates, after dilutions into cold 10 -2 M MgSO4 [or phosphate-buffered saline (PBS)] containing catalase. To check that the increase in killing is mediated by Fenton chemistry, the experiment can be repeated in the presence of a cell-permeable iron chelator (1 mM 2,2-dipyridyl or diethylenetriaminepentaacetic acid (DTPA) added to the cell culture a few minutes before challenge and in the dilution buffer), which should abolish the increase. POTENTIALPROBLEMS 1. Killing should be determined as a function of HzO2 concentration. H202 kills cells in two modes (demonstrated in E. coli, 16 but also observed in other bacteria). Only mode 1 killing, which occurs at lower H202 concentration, is well characterized and has been shown to be due to DNA damage mediated by the Fenton reaction. The sensitivity of sod mutants to H202 should therefore be measured in the range of concentrations corresponding to mode 1 killing. 2. The sensitivity of wild-type strains differs greatly between species (e.g., similar levels of killing are observed with 250/zM in Salmonella typhimurium and 2 mM in E. coli). 3. The reliability of measurements of killing by H202 due to excess 0 2 - implies that 0 2 - does not interfere with catalase and peroxidase activities. Although 12S. B. Farr, R. D'Ari, and D. Touati,Proc. Natl. Acad. Sci. U.S.A. 83, 8268 (1986). 13K. Keyer,A. StrohmeierGort, and I. A. Imlay,J. Bacteriol. 177, 6782 (1995). 14D. Touati and S. B. Farr, Methods Enzymol. 186, 646 (1990). 15D. Touati,M. Jacques, B. Tardat, L. Bouchard, and S. Despied,J. BacterioL 177, 2305 (1995).
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this is generally true, exceptions exist. In Sinorhizobium meliloti catalase and presumably a global defense against H202 is strongly induced by O2-, leading to higher resistance to H202 in the sodA mutant than in the wild typeJ 7 Metabolic Defects, Impairment of Growth in Minimal Medium Growth in minimal medium requires numerous synthethic pathways and presents many targets susceptible to oxidative damage. Thus, sod mutants display growth impairment. However, targets are not easy to identify and differ depending on the metabolic pathway affected. Escherichia coli sod mutants were found to be unable to grow on minimal medium unless provided with all amino acids. 6 Studies in several laboratories demonstrated that this general auxotrophy was due to several types of damage: a defect in branched amino acid synthesis due to dehydratase inactivation, 18 interference with aromatic biosynthesis by transketolase inactivation, 19which acts at an early step of biosynthesis, and cell envelope defects leading to sulfite leakage and cysteine auxotrophy.2° Similar general amino acid auxotrophy has been observed in sod mutants in other bacteria, suggesting similar defects. However, in some cases the growth impairment on minimal medium was not reversed by providing amino acids, indicating other (or additional) metabolic defectsJ 6 Membrane Damage Although there is evidence of membrane damage in sod mutants,21 the nature of the lesions has not been identified and is difficult to investigate. Lipid peroxidation is unlikely to be solely responsible for the damage, because there are almost no unsaturated lipids in most bacterial membranes. M u t a n t s in N o n c y t o p l a s m i c S u p e r o x i d e D i s m u t a s e s No sources of 02"- have yet been found in the periplasm and outer bacterial membranes. Thus, periplasmic, secreted, and outer membrane-associated SODs are thought to protect against external 0 2 - . In particular, SODs are expected to protect against the oxidative burst produced by the host during infection, thereby contributing to pathogenicity and virulence. No mutants have yet been obtained for secreted Fe- and MnSODs. Mutants have been obtained only for the periplasmic Cu,ZnSODs (sodC). The expected phenotypes were, as follows: resistance to endogenous 02'- (as generated by paraquat), sensitivity to exogenous 02"(as generated by hypoxanthine-xanthine oxidase, or pyrogallol), and attenuation 16 j. A. lmlay and S. Linn, J. Bacteriol. 166, 519 (1986). 17 R. Santos, D. H6rouart, A. Puppo, and D. Touati, Mol. Microbiol. 38, 750 (2000). 18 C. E Kuo, T. Mashino, and I. Fridovich, J. Biol. Chem. 262, 4724 (1987). 19 L. Benov and I. Fridovich, J. Biol. Chem. 274, 4202 (1999). 20 L. Benov, N. M. Kredich, and I. Fridovich, J. Biol. Chem. 271, 21037 (1996). 21 j. A. Imlay and I. Fridovich, J. Bacteriol. 174, 953 (1992).
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of virulence and pathogenicity (sensitivity to killing by macrophages, attenuated virulence in animal models). All sodC mutants display levels of resistance to paraquat similar to those of the wild type, consistent with periplasmic SOD affording no protection against cytoplasmic O2'-. In pathogens, sodC mutants generally display attenuation of virulence (reviewed in Lynch and Kuramitsu), 22 consistent with a lower level of protection against oxidative burst (02"-, H202, NO'). sodC mutants have been reported to be sensitive to exogenous 0 2 -. However, the sources of O2' - used produced both 02" - and H202 and most experiments were done in the absence of addition of catalase. The addition of this enzyme is necessary to ensure that the observed effects are attributable to exogenous O2 -. Reports that the sensitivity of E. coli sodC mutant to hypoxanthine-xanthine oxidase was abolished by catalase raises further doubt about the mechanism of sodC protection. 23 The phenotypes of sodC mutants observed to date suggest that periplasmic SOD may protect against both endogenous and exogenous O 2 - . The source of endogenous O2' - remains unclear, but this aspect should not be neglected in future studies of sodC mutants. B a c t e r i a l sod Null M u t a n t s a s T o o l s Cloning sod Genes from Other Organisms It has been found that the production of SOD from any organism in the cytoplasm of the sodA sodB mutant of E. coli fully complements the deficiencies of this mutant. Thus, complementation assays have been widely used to clone sod genes from other organisms. This strategy is still useful in the absence of a genomic sequence. Such assays also led to the isolation of another O2' --scavenging enzyme, 02"- reductase (see Ref. 24 and [13] in this volume25). Two tests are available. Complementation of Lack of Growth on Minimal Medium. A sodA sodB strain is transformed with a genomic library. Transformants are scraped from the selection plates and pooled, suspended in 10 -2 M cold MgSO4 buffer, washed twice, and plated on minimal glucose medium (M63 or M9) to which 10 -6 M paraquat and appropriate antibiotic (used for transformant selection) have been added. Putative sod + colonies appear after 2 to 3 days of incubation at 37 °. Note: External suppressors of the amino acid auxotrophy phenotype occur with high frequency, but these colonies are nonetheless usually sensitive to paraquat. 21'26 A preliminary assay should be done to evaluate the maximum number (usually at least 105 ) of 22 M. Lynch and H. Kuramitsu, Microbes Infect. 2, 1245 (2000). 23 A. Strohmeier Gort, D. M. Ferber, and J. A. Imlay, Mol. Microbiol. 32, 179 (1999). 24 M. J. Pianzzola, M. Soubes, andD. Touati, J. Bacteriol. 178, 6736 (1996). 25 V. Nivi~re and M. Lombard, Methods Enzymol. 349, [13], 2002 (this volume). 26 S. Maringanti and J. A. Imlay, J. Bacteriol. 181, 3792 (1999).
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153
TABLE II OTHER SPECIfiCPHENOTYPESASSOCIATEDWITH sod MUTANTS Defect condition Normal growth
Mutant sodB sodA sodB sodB
Stationary phase
sodC sodC sodA sodB sodA
sodA
Infection
Cytosolic or periplasmic
Symbiosis
sodA
Chilling
sodB
Acid stress
sodA
Phenotype Sensitivity of photosystem to moderate light Pyocyanin production abolished Reduced production of toxin and adhesin (adenylate cyclase, hemolysin, pertactin) Rapid decrease in survival Sensitivity to peroxynitrite Does not survive long-term starvation Loss of viability Less resistant to oxidative stress; growth resumes slowly from stationary phase Decrease in sporulation frequency; defect in spore coat assembly Attenuated virulence depending on mode and location of infection, and/or resistance to killing by macrophages Reduced nodulation; defect in bacteroid differentiation; drastic decrease in nitrogen fixation Sensitivity to moderate chilling in light Increase in sensitivity to acid stress
Species
Refs.
Cyanobacterium
a
Pseudomonas aeruginosa Bordetella pertussis
b 4
Legionella pneumophila Salmonella typhimurium Staphylococcus aureus
c d e
Campylobacter coli Pseudomonas aeruginosa
f g
Bacillus subtilis
h
Numerous pathogens
22
Sinorhizobium meliloti
17
Cyanobacterium
i
Staphylococcus aureus
e
a G. Samson, S. K. Herbert, D. C. Fork, and D. E. Laudenbach, Plant Physiol. 105, 287 (1994). b D. J. Hasset, H. P. Schweizer, and D. E. Ohman, J. BacterioL 177, 6330 (1995). c G. St:. John and H. M. Steinman, J. Bacteriol. 178, 1578 (1996). d M. A. De Groote, U. A. Ochsner, M. U. Shiloh, C. Nathan, J. M. McCord, M. C. Dinauer, S. J. Libby, A. Vasquez-Torres, Y. Xu, and E C. Fang, Proc. Natl. Acad. Sci. U.S.A. 94, 13997 (1997). e M. O. Clements, S. P. Watson, and S. J. Foster, J. Bacteriol. 181, 3898 (1999). f D. Purdy, S. Cathraw, J. H. Dickinson, D. G. Newell, and S. E Park, Appl. Environ. Microbiol. 65, 2540 (1999). g B. Polack, D. Dacheux, I. Delic-Attree, B. Toussaint, and P. M. Vignais, Infect. lmmun. 64, 2216 (1996). h A. O. Hentiques, L. R. Melsen, and C. P. Moran, Jr., J. Bacteriol. 180, 2285 (1998). i D.J. Thomas, J. B. Thomas, S. D. Prier, N. E. Nasso, and S. K. Herbert, Plant Physiol. 120, 275 (1999).
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sodA sodB bacteria that can be plated without residual growth. A slightly higher concentration of paraquat can be used if there is residual growth. Restoration of Aerobic Growth of sodA sodB recA Strain. The test requires access to an anaerobic chamber. A sodA sodB recA strain (air sensitive) is transformed with the genomic library under anaerobiosis. Pooled transformants are plated on LB plates under aerobiosis and incubated overnight at 37 ° under aerobiosis. The plating of 104 to 105 SOD- bacteria should result in no survival under these conditions. POTENTIAL PROBLEM. The aerobic growth of a sodA sodB recA strain also can be restored by recombination (cloning of a rec + gene). Thus, the putative sod + clones should be further tested for complementation in minimal medium. Identifying New 02"- Sensitive Targets and Situations of Endogenous Oxidative Stress Other defects observed in sod mutants of various bacteria under various conditions are listed in Table II. In all cases, the specific damage has not been clearly characterized. Defects may result from the vulnerability to O2"--mediated stress of a particular function or from an increase in oxidative stress in particular conditions. Thus, the low level of nitrogen fixation in the sodA S. meliloti mutant is probably due to direct inactivation of the nitrogenase complex, but the reasons for the defect in nodulation remain unclear. 16 The attenuation of virulence in various cytoplasmic sod mutants may result from damage due to the high metabolic rate during multiplication of bacteria in their host and associated endogenous 0 2 production, whereas attenuation in periplasmic sod mutants seems to be related to protection against oxidative burst. 22 Concluding Remarks Studies of bacterial sod null mutants have already identified several 0 2 - targets and shed light on the mechanism of 0 2 ' - toxicity. The effects of SOD deficiency in bacteria differ greatly according to SOD location and also because of the diversity of bacterial modes of life. For a particular bacterium, the lack of SOD may have minor effects in one phase of the life cycle, but drastic effects in another (e.g., free-living and symbiotic S. meliloti). 17 Many of the effects of SOD deficiency are still unclear. Studies of additional sod null mutants in various bacteria will help uncover new protective effects, to identify new 02'--sensitive targets and possibly new sources of 02"-, and to obtain further insight into the mechanisms, both direct and indirect, of 02" - toxicity. Acknowledgments We thank the Association pour la Recherche sur le Cancer for supporting our work on bacterial superoxide dismutase mutants (Grant 558 l).