Vol. 174, No. 3

JOURNAL OF BACTERIOLOGY, Feb. 1992, p. 953-961 0021-9193/92/030953-09$02.00/0 Copyright X 1992, American Society for Microbiology

Suppression of Oxidative Envelope Damage by Pseudoreversion of a Superoxide Dismutase-Deficient Mutant of Escherichia coli JAMES A. IMLAY AND IRWIN FRIDOVICH* Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 Received 28 June 1991/Accepted 25 November 1991

Mutants of Escherichia coli that are devoid of superoxide dismutase (SOD) fail to grow in aerobic minimal medium. This is largely because of the O2 sensitivities of several amino acid biosynthetic pathways, since amino acid supplements can restore growth, albeit at a slow rate. We now report that growth in amino acid-supplemented medium can be further stimulated by the presence of extracellular osmolytes. Osmolytes also partially suppress the amino acid requirements of the SOD mutant. These data suggest that the combination of oxidative inj]ury and turgor pressure permeabilizes the cell envelope and that critical metabolites, including the limiting products of damaged biosynthetic pathways, escape from the cell. External osmolytes may offer protection by countervailing the usual turgor pressure and thus stabilizing the damaged envelope. This model is consistent with the previous observation that deficiency of cell wall components is lethal to SOD mutants. A pseudorevertant that can grow at a moderate rate in normosmotic medium without amino acid supplementation has been obtained (J. A. Imlay and I. Fridovich, Mol. Gen. Genet. 228:410-416, 1991). Analysis suggests that the suppressor mutation allows the envelope either to resist or to tolerate oxidative lesions. Study of the pseudorevertant may illuminate the molecular basis of this oxidative envelope injury.

The discovery of superoxide dismutase (SOD) (29, 30) rapidly led to the view that 2- was an important agent of oxygen toxicity and that SOD played a defensive role (31). This view has been well supported by a variety of observations, the most recent and most convincing of which are the oxygen-dependent phenotypic deficits imposed by mutational defects in the genes encoding SODs (3,7, 34, 40) and by the suppression of these deficits by insertion of SOD genes from other species (5, 6, 18, 33, 39). Among the phenotypic consequences of SOD deficiency in Escherichia coli are oxygen-dependent auxotrophies for several amino acids (7). These are explicable in terms of 02--sensitive enzymes on the relevant biosynthetic pathways. Thus, the dihydroxyacid dehydratase, which catalyzes the penultimate step in the synthesis of branched-chain amino acids, is known to be inactivated by 02 (27). However, amino acid supplementation is not sufficient to restore these mutants to rapid growth, indicating that additional growth-inhibitory oxidative lesions remain to be identified. The SOD-deficient strains of E. coli (11) and yeasts (4) give rise to rare pseudorevertants that can be selected on the basis of growth in aerobic minimal medium. The E. coli pseudorevertants fall into two classes, the hardier of which has been subjected to genetic analysis (22a). In these pseudorevertants both the oxygen-dependent amino acid auxotrophies and the additional growth deficiency have been suppressed. We now describe investigations which indicate that oxidative damage to the cell envelope is responsible for the general growth deficiency and also contributes to the amino acid auxotrophies. The pseudoreversion may suppress these phenotypes because of a change in the cell envelope which renders it resistant to attack by 02-

*

MATERIALS AND METHODS Chemicals and enzymes. Paraquat, reduced glutathione, DL-serine hydroxamate, 5-methyl-DL-tryptophan, -(2-thienyl)-DL-alanine, D-(+)-cellobiose, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), xanthine, NADH, NAD+, horse heart type III cytochrome c, E. coli manganese-containing SOD, yeast alcohol dehydrogenase, and horseradish peroxidase were obtained from the Sigma Chemical Co. Hydrogen peroxide (30%) and sucrose were from Mallinckrodt, riboflavin was from Eastman, phenol was from the J. T. Baker Chemical Co., 4-aminoantipyrine was from Aldrich, and the protein assay dye reagent was from Bio-Rad. [5-3H]uridine, L-[4,5-3H]leucine, and L-[4,5-3H]isoleucine were purchased from Amersham. Xanthine oxidase was a gift of K. V.

Rajagopalan. Bacterial strains. The strains used in this study are listed in Table 1. J1201 and JI202 were created by P1 transduction of markers from J1199 into AB1157, and JI215 and J1216 were created by P1 transduction from JI199 into X478 (22a). Transductants were selected by their resistance to 15 ,ug of tetracycline per ml on LB plates. Pantothenate proficiency was tested by growth in vitamin-free minimal medium supplemented with 1% Casamino Acids. Because no phenotype could be predicted for the ssa-l allele in SOD' backgrounds, its presence or absence in an SOD' transductant was determined by the ability of that strain to donate the ssa-J allele to SOD- mutants in subsequent P1 transductions. This procedure has been described previously (22a). Growth media. LB medium contained (per liter) 10 g of bactotryptone, 5 g of yeast extract, 10 g of sodium chloride, and 2 g of glucose. Minimal medium consisted of minimal A salts (32) and (per liter) 3 mg of pantothenate, 5 mg of thiamine, and 2 g of glucose. Except where indicated, any L-amino acid supplements were to final concentrations of 0.5 mM and necessary vitamin supplements were to 3 mg/liter. All media were made with tap water to ensure the availability of trace minerals. In attempts to stimulate cell growth, the following supplement concentrations were used: 15 ,ug of pantothenic acid per ml; 3 ,ug (each) of p-aminobenzoic acid,

Corresponding author. 953

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TABLE 1. Bacterial strains used in this study Strain

Genotype

AB1157

F- thr-1 IeuB6 proA2 his4 argE3 thr-l lacY) galK2 rpsL supE44 ara-14 xyl-15 mtl-l tsx-33 Same as AB1157 plus (sodA::Mud PR13)25 (sodB-kan)1-A2 Same as J1200 plus flzuA468::TnIO pan-6 Same as J1132 plus ssa-l Same as AB1157 plusfhuA468::TnlO pan-6 Same as AB1157 plus fhuA468::TnlO pan-6 ssa-I Same as X478 plus JhuA468::TniO pan-6 Same as X478 plus fhuA468::TnlO pan-6 ssa-l F- ara-14 leuB6 azi-6 tonA23 lacZ36 proC32 tsx-67 purE42 supE44 trpE38 lysA23 rpsLI09 xyl-S mtl-l metE70 thi-l Hfr P03 relAl spoTI metBI

J1132 JI199 JI200 J1201

JI202 JI215

JI216

X478 BW113

Source

or

reference

B. Bachmann 23 22a 22a This work

This work This work

This work

B. Bachmann

B. Bachmann

p-hydroxybenzoic acid, nicotinamide, riboflavin, pyridoxine HCl, biotin, folic acid, 8-aminolevulinic acid, and cyanocobalamin per ml; 0.1 mM (each) inosine, cytidine, thymidine, and uridine; 15 p.g of inositol per ml; 0.3 mM oleic acid, 0.3 mM palmitic acid, and 1 mM glycerol-3-phosphate in 1 mg of Brij per ml; 0.3 mM N-acetylglucosamine; 1 mM dl-glutamate; and 50 ,ug of diaminopimelic acid per ml. Growth studies. Aerobic incubations were performed in flasks in a shaking water bath, while anaerobic incubations were in a Coy chamber under 85% N2-10% H2-5% CO2. To transfer cells from nutritionally rich to restricted media, cultures were centrifuged and washed twice with the restricted medium before final suspension in it. The centrifugation was conducted at room temperature to avoid temperature shocks, and the walls of the centrifuge tubes were wiped with Kimwipes after each spin to minimize the transfer of medium. The stringency of these washes was a critical determinant of subsequent growth behavior. In order to accurately determine stable growth rates in terms of optical density, we attempted to monitor growth for at least five generations, starting with a cell density of 1 x 106 to 3 x 106 CFU/ml. Growth rates of cells in trace amounts of required amino acids were also measured by colony enumeration; the initial culture density was only 1 x 103 to 2 x i03 CFU/ml to prevent the cells from consuming a significant fraction of the supplied amino acids. Determination of rates of endogenous superoxide production. Measurements of superoxide production by inverted membrane vesicles and cytosolic enzymes were performed as previously described (22). Briefly, log-phase cells were lysed by passage through a French pressure cell, and membrane and cytosolic fractions were separated by repeated high-speed centrifugations. The inverted vesicles were resuspended and incubated with physiological concentrations of NADH and NAD+ in the presence of a regenerating systemr with ethanol plus alcohol dehydrogenase. Superoxide evolution was measured as SOD-inhibitable cytochrome c reduction, and oxygen consumption was followed with a Clarke electrode. These measurements reveal the rate of per respiratory electron flux. The

superoxide production

respiration rates of intact cells were also determined with a Clarke electrode. Cytosolic 02 generation was similarly assayed in the presence of NAD(P)H and NAD(P)+; this activity was normalized to the extract protein concentration and accordingly extrapolated to obtain the rate pertaining in vivo. Determination of acid-soluble thiols. Cells were grown aerobically to log phase in LB medium, centrifuged and washed at room temperature, and resuspended to 4% of the original volume in cold minimal medium containing 1 mM EDTA. Cells were lysed by passage through a French pressure cell, a small aliquot of the resultant extract was set aside for the determination of protein, and cold trichloroacetic acid was added to a final concentration of 2.5%. After 10 min, the samples were centrifuged at 20,000 x g for 10 min. The supernatant was added to an assay solution (280 mM potassium orthophosphate [KPj], pH 7.8, 17 ,ug of DTNB per ml) and the A412 was determined. Reduced glutathione was used as a standard. Assay of catalase. Cells were grown aerobically to log phase in LB medium, centrifuged, washed, and resuspended to 4% of the original volume in 50 mM KPi (pH 7.0). The bacteria were lysed with a French press, the extract was centrifuged at 20,000 x g for 20 min, and the supernatant was dialyzed overnight at 4°C against 500 volumes of KPi, pH 7.0. The catalase activity was too low to measure by direct spectrophotometric determination of H202 at 240 nm, since protein in the extract interfered. The supernatant was diluted 1:5 into 10 mM hydrogen peroxide in 50 mM KPi, pH 7.0, at room temperature. At intervals, small aliquots of the reaction mix were diluted 1:150 into a horseradish peroxidase mix containing 2.3 mg of phenol per ml, 1 mg of 4-aminoantipyrine per ml, 1 mM KP1, 0.5 p,M H202, and 50 ,ug of peroxidase per ml (16). The resultant A505 is proportional to peroxide concentration, and a series of time points allows visualization of the peroxide decomposition by the catalase in the cell extract. One unit of catalase degrades 1 ,umol of H202 per min when acting on 8 mM H20&. Assay of SOD. Cells were grown to about 1 x 10 CFU/ml in aerobic minimal glucose medium supplemented with 0.5 mM of all 20 of the amino acids. The cultures were centrifuged, washed, and resuspended in 0.5% of the original volume of KPi (pH 7.8). Extracts were assayed for SOD-like activity by using the cytochrome c-xanthine oxidase assay as previously described (30), except that EDTA was omitted and cytochrome c was used at a final concentration of only 2 ,uM in order to amplify the effects of any SOD activity. Extracts were warmed to room temperature before assay. Miscellaneous. Cell killing by H202 was studied with log-phase cultures in LB medium as previously described (23). Protein was measured by the method of Bradford (6a). Intracellular concentrations of dissolved oxygen and reduced dinucleotides were determined as previously described (22). Measurements of RNA synthesis. Cells were grown to log phase in aerobic minimal medium supplemented with all 20 amino acids, pantothenate, 0.1 mM inosine, and 0.1 mM uridine. To initiate starvation, cultures were centrifuged, washed, and resuspended at -3 x 107 CFU/ml in the same medium lacking methionine. The cultures were then split, and methionine was restored to half the samples. [3H]uridine (1 ,uCi/ml; 10 p.Ci/umol) was added to all samples, and at various times 25 ,ul was spotted on Whatman 0.33-mm-thick chromatography paper as previously described (41). A drop of aqueous 5% trichloroacetic acid was immediately spotted onto the sample spot, and the filter was dried with a hair

VOL. 174, 1992

SUPPRESSION OF OXIDATIVE ENVELOPE DAMAGE

dryer. The unincorporated [3H]uridine was eluted from the sample spots with 5% trichloroacetic acid by descending chromatography. Sample spots were cut out, immersed in Aquasol-2 scintillation fluid, and counted. All samples were prepared in duplicate. Amino acid uptake. This procedure was adapted from that of Anderson and Oxender (1). Cells were grown to about 108

CFU/ml in aerobic minimal medium supplemented with 0.5 mM of all 20 amino acids. The culture was centrifuged, washed three times with 4°C minimal salts, and resuspended in minimal salts containing 0.5% glucose but devoid of amino acids. After 10 min of incubation at 3°C, 3 ml of the suspended cells was mixed with the desired concentration of tritiated amino acid (35 IxCi/4mol). At designated intervals over the course of 1 min, 0.5 ml of the cell suspension was removed and filtered under vacuum through Millipore HA filters. Filters were washed with 5 ml of 0.45-,um-pore-size 10 mM KPi (pH 7.2) at 37°C, dried, and counted. Amino acid uptake exhibited typical saturation kinetics (1), and transport of labelled amino acids could be competitively inhibited by nonradioactive amino acids.

RESULTS Strains of E. coli that are devoid of SOD grow in aerobic rich medium but fail in minimal medium (7). When cultures of the SOD-deficient strain J1132 were densely spread upon minimal medium plates, rare colonies appeared within 2 to 3 days at a frequency of 10-7 to 10-6. The progeny of a given colony replated on minimal medium at a frequency of 102 to lo-3, indicating that a mutation had partially suppressed the inability to grow on minimal medium. One such colony was selected for extensive characterization and is denoted JI200. The minimal medium tolerance of J1200 is due to a single mutation at 4 min on the chromosome (22a). Twenty-seven independently derived pseudorevertants were classified by their growth on minimal medium as either fast growing (doubling time [tD] of 2 h) or slowly growing (tD = 3 h). Members of the fast-growing class invariably bore the responsible mutation at 4 min. We have designated this gene ssa (suppressor of superoxide-dependent auxotrophy). The growth behavior of JI132 and J1200 in aerobic minimal medium are contrasted in Fig. 1. Upon dilution of the parent JI132 from rich medium into minimal glucose medium, cell growth stopped immediately, and the number of viable cells declined slowly over subsequent hours. In comparison, the pseudorevertant JI200 exhibited a much more rapid loss of viability during the first 12 h, but then either recovery of the remaining cells or outgrowth of a subpopulation allowed the culture to recover. The SOD-proficient relative, AB1157, grew rapidly under these conditions. The growth response to transfer into minimal medium is distorted by the requirement for induction of many biosynthetic enzymes, which are repressed in rich medium. To avoid this complication, the growth of both strains was examined after growth to exponential phase in anaerobic minimal medium and the subsequent introduction of air without any other change in medium composition (Fig. 2). Upon aeration the parent strain abruptly stopped growing. However, the growth of the pseudorevertant continued without pause and with only a slight reduction in rate. The inability of SOD-deficient E. coli to grow in minimal medium has been ascribed to the oxygen-dependent loss of amino acid biosynthesis (7). Indeed, supplementation with all 20 amino acids permits slow growth in aerobic medium (Fig. 2A). In these studies exclusion from the medium of

955

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JI132

o

0

0

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4

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12 16 20 24 Hours FIG. 1. Cell death and regrowth upon transfer to minimal medium. Cultures growing logarithmically in aerobic LB medium were washed and resuspended in minimal medium containing only the five amino acids for which these strains are genetically auxotrophic, as described in Materials and Methods; viability was determined at intervals by spreading aliquots on LB plates and counting colonies after 24 h. Symbols: 0, AB1157 (SOD' ssa+); A, J1132 (SODssa+); 0, JI200 (SOD- ssa-1).

branched-chain or sulfur-containing amino acids was sufficient to prohibit growth upon aeration. Without aromatic amino acid supplements, some slow growth occurred before stasis. In contrast, the pseudorevertant continued to grow without the addition of either sulfur-containing or aromatic amino acids (Fig. 2B). (We note that aeration stopped the growth of pseudorevertant cultures lacking only branchedchain amino acids but not of cultures lacking all the amino acids, suggesting that the former condition may retard growth because of amino acid imbalance.) Comparisons of growth rates in various media are presented in Table 2. The oxygen-dependent auxotrophies of the SOD mutant suggest that enzymes requisite for the biosynthesis of these amino acids are inactivated by the high level of superoxide that accumulates inside the cell. The vulnerable enzyme in the branched-chain amino acid biosynthetic pathway has been shown to be dihydroxyacid dehydratase (27); sensitive enzymes in the other two pathways have not yet been identified. Because a single mutation largely restored the function of three independent pathways, it is unlikely to have conferred its effect by altering the critical biosynthetic enzymes in a way that would shield their putative superoxide-sensitive sites. The SOD-deficient strain J1132 apparently is deficient at functions other than amino acid biosynthesis, since the tD was still 100 min when all the amino acids were supplied.

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Suppression of oxidative envelope damage by pseudoreversion of a superoxide dismutase-deficient mutant of Escherichia coli.

Mutants of Escherichia coli that are devoid of superoxide dismutase (SOD) fail to grow in aerobic minimal medium. This is largely because of the O2- s...
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