INTRODUCTION

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The discovery of superoxide dismutase (SOD) by McCord and Fridovich introduced biology to the field of free radical chemistry (37). The presence of this enzyme in virtually all aerobic organisms (38) forced the conclusion that superoxide (O₂) must be formed as a by-product of aerobic metabolism and, if not scavenged, must damage critical biomolecules. Left unknown were the identities of those target biomolecules. This point was controversial, since chemical studies indicated that amino acids, nucleotides, and sugars are essentially not reactive with O₂ (6, 7, 15, 43).

In 1986 Carlioz and Touati reported the construction of mutants of Escherichia coli that lacked both the manganese- and iron-containing isozymes of SOD (10). These mutants were capable of aerobic growth only if branched-chain, aromatic, and sulfurous amino acids were provided in the medium, and if a fermentable carbon source was available. They also displayed a high rate of spontaneous mutagenesis (14). Similar phenotypes were reported for wild-type cells during exposure to hyperbaric oxygen, presumably because of the accelerated pace of O₂ formation (8). The requirement for branched-chain amino acids was traced to the inactivation by O₂ of dihydroxyacid dehydratase, an enzyme in the common biosynthetic pathway (31). This enzyme utilizes a [4Fe-4S]²⁺ cluster as a Lewis acid to catalyze substrate dehydration. The solvent exposure of the cluster allows it to be accessible to O₂, which can univalently oxidize it. The resultant [4Fe-4S]³⁺ cluster is unstable and disintegrates to the [3Fe-4S]⁺ form, which is catalytically inactive, with loss of a ferrous iron atom to the cytosol (16, 17). Several reports following this discovery showed that other dehydratases in the cell, including the tricarboxylic acid (TCA) cycle enzymes aconitase and fumarase, contain [4Fe-4S]²⁺ clusters that suffer the same damage when exposed to superoxide (20, 21, 34). E. coli can repair these enzymes by a process that is presently undefined but which must entail the reduction of the cluster and replacement of the lost iron atom. The amounts of SOD and of repair enzymes in E. coli appear to be calibrated to precisely balance the rate of dehydratase damage by endogenous O₂, so that the dehydratases retain near-maximal activity during aerobic growth (22).

Interestingly, the hypermutagenesis of SOD mutants arises from the iron that is released by the damaged clusters. This iron accumulates in the cytosol, where it catalyzes the oxidation of DNA (28, 30, 35). Thus, several phenotypes of SOD mutants have been traced to oxidation by O₂ of iron-sulfur clusters.

However, the auxotrophies for sulfur-containing and aromatic amino acids and the slow growth in rich medium are not directly attributable to cluster damage. It appears that neither amino acid biosynthetic pathway contains labile dehydratases. The sulfur requirement has been linked to the apparent leakage of sulfur from SOD mutants (5), although the specific lesion that causes this phenomenon is unknown. The aromatic amino acid biosynthetic defect has recently been ascribed to the oxidation of the intermediate 1,2-dihydroxyethyl thiamine pyrophosphate of transketolase (3). In vitro, superoxide oxidized and released this compound with progressive inactivation of the enzyme, and catabolic processes that require transketolase function were shown to be defective in SOD mutants. A resultant deficiency in erythrose-4-phosphate production could plausibly diminish aromatic amino acid biosynthesis.

A pleiotropic suppressor which relieves all the auxotrophies of SOD mutantsthose for sulfurous and aromatic as well as branched-chain amino acidswas isolated and characterized by Imlay and Fridovich (25, 26). It was originally hoped that this suppressor might protect multiple targets by curbing the rate of endogenous O₂ production. However, the original analysis indicated that the mutation did not act to change the internal O₂ concentration (26). The goal of the present study was to decipher the mechanism of suppression and thereby gain further understanding of the nature of O₂ toxicity.

	MATERIALS AND METHODS

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Chemicals and enzymes. o-Nitrophenyl--D-galactopyranoside, fluorocitrate, deferoxamine mesylate (desferrioxamine), isopropyl--D-thiogalactopyranoside, 6-phosphogluconate, NADP⁺, NADH, diethylenetriaminepentaacetic acid (DTPA), hydrogen peroxide, dipicolinate, diaminopimelic acid, lactate, succinate, glutathione, ATP, horse heart cytochrome c, xanthine, xanthine oxidase, porcine heart isocitrate dehydrogenase, bovine erythrocyte Cu,ZnSOD, and rabbit muscle lactic dehydrogenase were purchased from Sigma. Coomassie protein reagent was obtained from Pierce. Magnesium sulfate heptahydrate, ferrous sulfate heptahydrate, sodium nitrite, and manganese chloride were obtained from Aldrich. -Mercaptoethanol and sodium citrate dihydrate were obtained from Fisher Scientific. Restriction enzymes were purchased from Gibco BRL. Ready-to-go PCR beads were obtained from Pharmacia Biotech. Shrimp alkaline phosphatase was obtained from Boehringer Mannheim. Water was purified from a Labconco Water Pro PS system by using house deionized water as the feedstock.

Growth media. Luria-Bertani (LB) medium contained (per liter) 10 g of Bacto Tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 2 g of glucose. Minimal medium consisted of minimal A or E salts medium (39) with 1 mM MgSO₄ · 7H₂O and with 5 mg of thiamine and 2 g of glucose per liter. Casamino Acids medium was additionally supplemented with 0.2% Casamino Acids. L-Amino acid supplements were used at a final concentration of 0.5 mM, and necessary vitamin supplements were used at 3 µg/ml. Spectinomycin, tetracycline, and ampicillin were used at 150, 12, and 100 µg/ml, respectively. Where indicated, diaminopimelic acid was added to a final concentration of 50 µg/ml.

Growth studies. Aerobic cultures were routinely grown in flasks at 37°C in a shaking water bath. Anaerobic cultures were grown in a Coy chamber (Coy Laboratory Products, Inc.) under 85% N₂-10% H₂-5% CO₂. Optical densities (OD) of cultures were measured at 600 nm. For studies of auxotrophies, cells were grown anaerobically in minimal A medium supplemented with all amino acids except sulfurous amino acids (cysteine and methionine) or aromatic amino acids (tyrosine, phenylalanine, and tryptophan). Cultures were grown for at least four generations to reach an OD at 600 nm (OD₆₀₀) of 0.2 before they were diluted in an aerobic medium of the same composition to an OD₆₀₀ of 0.01. The ability to grow on amino acid-supplemented minimal medium lacking the sulfurous amino acids was used in general as a diagnostic feature to identify the suppressor strains. When the cells were grown on minimal medium alone, they were routinely supplemented with arginine, histidine, leucine, proline, and threonine in order to meet the amino acid requirement of strain AB1157. Where specifically indicated, cells were grown to log phase in LB medium, centrifuged, and washed stringently with minimal salts before they were diluted to an OD₆₀₀ of 0.01 in minimal medium supplemented with all but the sulfurous amino acids. To test aerobic growth on nonfermentable carbon sources, cells were pregrown in anaerobic minimal medium that contained 20 amino acids, 0.4% lactate or succinate, and 40 mM sodium nitrate.

Strain construction. All strains were K-12 derivatives (Table 1). The dapD and dapB mutations were transduced (39) into sodA sodB strains with selection for the linked fhuA468::Tn10 and thr::Tn10, respectively, on LB plates containing tetracycline and diaminopimelic acid. Transductants were screened for diaminopimelate auxotrophy on LB plates. In general, in order to avoid suppressor mutations, transductions were performed under anaerobic conditions. Strain AS237 was rendered chloramphenicol sensitive by penicillin enrichment in the presence of chloramphenicol. The fur mutation was transduced by linkage to zbf-507::Tn10. Strain SM1106 was constructed by excising the Tn10 (36) from SM1093, thereby generating the tetracycline-sensitive derivative SM1104, and then transducing the fur null allele with the linked Tn10. To verify that the fur mutation was inherited with the Tn10, the Tn10 was transduced back into AN387, and transductants were screened for coinheritance of the kanamycin marker from the fur::Tn5 allele. tonB mutants were screened by their ability to confer resistance to infection by bacteriophage 80.

Recombinant DNA techniques. Standard cloning techniques were used (42). Oligonucleotide synthesis and sequencing were performed at the genetic engineering facility of the University of Illinois. PCR was performed directly on overnight cultures or on purified DNA by using Ready-to-Go PCR beads from Pharmacia. The primers for dapD, dapB, and the open reading frame (ORF) encoding dipicolinate synthase were designed by using the sequence available from GenBank and are as follows: dapD 5' end, GAT GGA TCC CGA ATT ACA ACC ATT; dapD 3' end, AAC CGA ATT CTG AGC TCG TGG; dapB 5' end, GGA TCC ATG CAT GAT GCA AAC ATCC G; dapB 3' end, AAG CTT TTA CAA ATT ATT GAG ATCA A; dipicolinate synthase ORF 5' end, GTT TAC CAT GGT AAC CGG ATT; dipicolinate synthase ORF 3' end, GAC CGG ATC CTT TAG TTT GGG. The dapD8 allele was sequenced and found to contain the missense mutations R164G, G167S, and M178I within the dapD locus. It was not determined whether additional mutations lie outside the gene.

Biochemical assays. -Galactosidase assays (39) were performed in triplicate. For 6-phosphogluconate dehydratase assays, the cells were grown in Casamino Acids medium with 0.2% gluconate as a carbon source. Cultures (250 ml) were grown to an OD of 0.1, centrifuged, and resuspended in 1 ml of ice-cold Tris-HCl (pH 7.65). The cells were lysed by passage through a French press. The lysates were immediately centrifuged in a microcentrifuge for 1 min at 12,000 rpm, and the supernatants were frozen immediately in a dry-ice-ethanol bath to prevent the inactivation of these labile enzymes by air. The enzyme activity of rapidly thawed extract was determined by the two-step method of Fraenkel and Horecker (18). A 100-µl reaction mixture containing extract, 8 mM 6-phosphogluconate, and 10 mM MgCl₂ was incubated at room temperature for 5 min. The reaction mixture was then diluted into 2 ml of 50 mM Tris (pH 7.65) and boiled for 2 min. The tubes were centrifuged to remove the particulates, and the supernatant was then assayed for pyruvate at 340 nm with lactate dehydrogenase and 0.2 mM NADH. To show that the active 6-phosphogluconate dehydratase recovered from suppressor strains is still superoxide sensitive, the extracts were exposed to superoxide that was generated by xanthine and xanthine oxidase (22). Extracts for aconitase assays were prepared in a similar way except that the cultures were grown in Casamino Acids medium containing 0.2% glucose. The lysis buffer contained 50 mM Tris (pH 7.4), 0.6 mM MnCl₂, and 20 µM fluorocitrate to block damage to iron-sulfur clusters (19). Aconitase activity was assayed as described previously (20). One milliliter of assay mixture consisted of extract, 30 mM citrate, 0.2 mM NADP⁺, 0.6 mM MnCl₂, 50 mM Tris-HCl (pH 7.4), and 1 U of isocitrate dehydrogenase. The production of NADPH was monitored at 340 nm. Protein was measured by the Bradford dye-binding assay by using ovalbumin as a standard.

Sensitivity to hydrogen peroxide. Overnight cultures grown in LB medium were diluted into fresh medium and were grown for at least four generations to reach an OD₆₀₀ of 0.2. One-milliliter aliquots were exposed to 2.5 mM H₂O₂, and the cultures were shaken at 37°C for 10 min. Killing was stopped by diluting the cultures 625-fold into LB medium containing 130 U of catalase/ml. Cells were plated onto LB plates in 2 ml of LB top agar, and the plates were incubated at 37°C overnight. Diaminopimelic acid auxotrophs were plated on LB plates containing 5 µg of diaminopimelic acid/ml.

Miscellaneous methods. Electron paramagnetic resonance (EPR) measurements were performed as described previously (30). Peak areas were normalized to those of iron standards, and intracellular iron concentrations were calculated by correlating OD to intracellular volumes (24). For iron starvation experiments, cells were grown anaerobically to log phase in minimal A medium supplemented with all amino acids. Cells were then diluted into an aerobic medium of the same composition containing various concentrations of DTPA, and growth was monitored. Measurement of expression of the SoxRS regulon was made possible by the introduction of a  bacteriophage carrying a soxS::lacZ fusion (22) into control and suppressor strains.  infections and screens for lysogens were carried out as described previously (44). Lysogens were identified by their ability to lyse a -sensitive strain after brief exposure to UV light. -Galactosidase assays were performed on the lysogens to determine the expression of soxS. RpoS induction was tested by introducing a  construct carrying a bolA::lacZ fusion into the control and suppressor strains. The dapB null mutation was also transduced into a strain containing a marO_II::lacZ fusion (12), and -galactosidase activity was measured.

HPLC quantitation of intracellular dipicolinic acid. Cultures were grown overnight in minimal E salts medium containing 0.2% glucose and 0.05% Casamino Acids. They were then subcultured into 2.5 ml of minimal medium containing all amino acids except cysteine and containing 10 µM [¹⁴C]aspartate (Amersham). Cysteine was omitted from the medium because it inhibits the growth of dap mutants, presumably by competing with diaminopimelic acid for its transport. At an OD₆₀₀ of 0.8 the cells were pelleted by centrifugation, immediately resuspended in 5% trichloroacetic acid, and incubated on ice for 10 min. The precipitated material was separated by centrifugation, and the clear supernatant was lyophilized to dryness. The dried sample was resuspended in 0.2 ml of buffer A (5 mM Tris-HCl [pH 7]). The sample was injected onto a Beckman system gold high-pressure liquid chromatograph (HPLC) using a Waters SAX column with a constant flow rate of 1 ml of buffer A/min. The column was washed with buffer A for 5 min and then eluted on a linear gradient to 100% buffer B (Tris-HCl [pH 7], 5 mM; NaCl, 1 M) over 20 min. The column was then washed with 100% buffer B for an additional 5 min. Eluate from the column was passed through a Beckman 168 UV/visible spectrophotometer set at 270 nm and an in-line scintillation counter (Beckman detector 171).

SOD assays of dipicolinate-metal chelates. In vitro solution assays for SOD (37) were performed with few modifications. Reaction mixtures included 100 µM dipicolinate and either 100 µM FeSO₄ · 7H₂O, 20 µM CuSO₄, or 1 to 4 µM MnCl₂. EDTA was omitted in order to avoid metal binding.

Measurements of dipicolinate-iron binding and oxidation rates. Binding, titration, and oxidation studies of the ferrous iron-dipicolinate complex relied upon an  of 1,510 M¹ cm¹ at 484 nm. The ferric complex does not absorb at this wavelength. In these studies water was used without buffers, since buffering anions displace equilibria by competing for free iron; instead, reagents were independently adjusted to pH 7 before mixing, and postreaction pH measurements verified that the pH did not change. The binding constant of the complex was determined by titrating 10.0 µM ferrous iron with dipicolinate. The 2:1 stoichiometry of the dipicolinate-Fe²⁺ complex was demonstrated by similar titrations of 100 and 400 µM ferrous iron. Binding competition experiments between dipicolinate and citrate, ATP, and reduced glutathione were conducted by adding 50 µM FeSO₄ · 7H₂O to a solution containing 200 µM dipicolinate and either 1 to 150 mM citrate, 0.167 to 42 mM ATP, or 0.5 to 86 mM glutathione. Time courses of A₄₈₄ were observed to ensure that equilibrium concentrations of the ferrous iron-dipicolinate complex had been obtained. Solutions were assembled from anaerobic stock solutions and monitored in sealed anaerobic cuvettes to prevent any oxidation of the ferrous complexes by molecular oxygen. The rates of oxidation of the ferrous iron-dipicolinate, -citrate, and -ATP complexes by dissolved oxygen in air-saturated water were determined by monitoring the disappearance of the 484-nm peak (for dipicolinate) or the appearance of the 330-nm absorbance of the ferric chelates (for citrate and ATP). The absorbance maxima of authentic ferric chelates were demonstrated by adding H₂O₂.

	RESULTS

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The suppressor mutation creates a stop codon in dapD. An earlier study showed that the ssa-1 mutation suppressed the auxotrophies for branched-chain, aromatic, and sulfurous amino acids that are otherwise imposed by O₂ stress. Mapping experiments had placed the ssa-1 mutation in the 4-min region of the chromosome but had not identified the altered gene (25).

View larger version (11K): [in this window] [in a new window]   FIG. 1.   Complementation of the suppressor mutation with dapD. (A) Subclones of pDB2 are represented. pSM904, carrying dapD alone, complements the ssa-1 mutation. (B) Aerobic growth of JI200 carrying pSM902. Anaerobic log-phase cultures in minimal medium containing all amino acids except Met and Cys were shifted to an aerobic medium of same composition. Symbols: diamonds, JI200 (SOD ssa-1); rectangles, JI132 (SOD); circles, SM506 (SOD ssa-1/pSM904 [dapD+]).

View larger version (14K): [in this window] [in a new window]   FIG. 2.   Pathway of diaminopimelate and lysine biosynthesis. The conversion of dihydrodipicolinate to dipicolinate by dipicolinate synthase is represented at the bottom.

Accumulation of the intermediates upstream of dapD is important for protection. The sequence data from the PCR products suggested that lowering the amount of tetrahydrodipicolinate synthetase in the cell is useful in a SOD mutant. To verify this, we introduced a null mutation of dapD into a SOD mutant. This mutation also relieved the auxotrophies (Fig. 3). The suppression of auxotrophies was reproduced in each genetic background that we examined (AB1157, GC4468, and AN387).

View larger version (23K): [in this window] [in a new window]   FIG. 3.   The dapD mutation allows SOD strains to grow without branched-chain, aromatic, and sulfurous amino acids. Cultures were grown to log phase in anaerobic minimal medium supplemented with five amino acids (see Materials and Methods) and were diluted into an aerobic medium of same composition. Dap mutants were supplemented with diaminopimelic acid. Symbols: rectangles, AS237 (SOD); circles, JI200 (SOD ssa-1); diamonds, SM1004 (SOD dapD); triangles, SM1006 (SOD dapD dapA).

The suppressors exhibit an increase in the activities of labile dehydratases. Superoxide destroys the iron-sulfur clusters of a subfamily of dehydratases, thereby eliminating the function of the pathways to which they belong. The relaxed requirement in suppressed strains for branched-chain amino acid supplements suggested that the activity of dihydroxyacid dehydratase might be increased. The suppressed strains also showed better growth on nonfermentable carbon sources such as lactate (Fig. 4) and succinate (data not shown). This growth requires functional aconitase and fumarase and suggested a general improvement in dehydratase activities. In fact, while SOD-deficient strains usually show very low aconitase activity in airabout 10% of wild-type levelsthe suppressor strains exhibited about 70% of the wild-type level (Table 2). This was also true for 6-phosphogluconate dehydratase, another labile dehydratase. The residual 6-phosphogluconate dehydratase activity in the cell extracts remained fully vulnerable to O₂, confirming that the increased activity was due to stabilization of the labile Fe-S dehydratase rather than its replacement by a O₂-resistant isozyme (data not shown).

View larger version (20K): [in this window] [in a new window]   FIG. 4.   Growth on a nonfermentable carbon source. Cultures were grown to log phase in an anaerobic medium containing all amino acids with lactic acid as a carbon source. They were then diluted into an aerobic medium of the same composition. Symbols: circles, AN387 (SOD+); rectangles, AS237 (SOD); diamonds, SM1008 (SOD dapB).

The reductive properties of the intermediates are not important for suppression. The structures of tetrahydrodipicolinate and dihydrodipicolinate suggested two possible scenarios by which their accumulation could benefit SOD mutants. The first possibility was that these compounds could act as reductants in the cell. Superoxide destabilizes 4Fe-4S clusters by univalently oxidizing them; a reductant might prevent their inactivation by rereducing the cluster before its iron atom is lost. Tetrahydrodipicolinic acid, in particular, is expected to be a good reducing agent, since its oxidation product is aromatic (32). In fact, tetrahydrodipicolinic acid spontaneously oxidizes in solution. Spontaneous disproportionation of dihydrodipicolinic acid creates tetrahydrodipicolinic acid, leading to the same result.

View larger version (14K): [in this window] [in a new window]   FIG. 5.   Accumulation of dipicolinate. Fifty microliters of the sample (see Materials and Methods) was analyzed on an HPLC anion-exchange column. An unlabelled internal standard of dipicolinate was monitored at 270 nm, and the 14C-labelled dipicolinate was counted on a flowthrough scintillation counter attached in series to the HPLC. (A) SM1128 (SOD+ dapA/SM912 [encoding dipicolinate synthase]). (B) SM1127 (SOD+ dapB/SM912). Peak a represents [14C]aspartate, and peak b represents 14C-labelled dipicolinate. Authentic dipicolinate and aspartate standards coeluted with these peaks (data not shown).

View larger version (23K): [in this window] [in a new window]   FIG. 6.   Dipicolinate synthase overexpression in a SOD-deficient strain relieves the auxotrophies. Cultures were grown to log phase in minimal medium supplemented with all but the sulfurous amino acids and were diluted into an aerobic medium of same composition. Symbols: circles, AS237 (SOD); rectangles, SM1024 (SOD plus vector); triangles, AN387 (SOD+); diamonds, SM1025 (SOD/pSM912 [encoding dipicolinate synthase]).

Dipicolinate binds and stabilizes ferrous iron in vitro. The structure of dipicolinate should predispose it to be a cation chelator, and its iron-binding ability has been used in assays of the dipicolinate content of spores. However, its function in spores is thought to arise from an ability to bind calcium rather than iron. In order to serve as an effective iron chelator in vivo, dipicolinate must outcompete other suspected intracellular iron chelators, including citrate, ATP, and glutathione.

View larger version (14K): [in this window] [in a new window]   FIG. 7.   Dipicolinate binds ferrous iron with greater avidity than does citrate. (A) Titration of 10.0 µM ferrous iron with dipicolinate. Half of all ferrous iron is in the Fe2+-dipicolinate complex in 11 µM free dipicolinate. The amount of free dipicolinate was calculated based on the stoichiometry of 2 dipicolinate atoms/complex. (B) Citrate-stimulated Fe2+ oxidation is inhibited by the addition of dipicolinate. Top curve, 330 µM Fe2+ in water. Citrate (33 mM) was added at the time indicated by the arrow, and the appearance of Fe3+ was monitored at 331 nm. The reaction slows due to depletion of Fe2+. For the bottom curve, a mixture of 33 mM citrate and 1.7 mM dipicolinate was added to 330 µM Fe2+ at the time indicated by the arrow.

The accumulation of dipicolinates causes the derepression of the Fur regulon in suppressor strains. The chelation of intracellular iron was tested by measurement of the expression of genes in the Fur regulon. In iron-replete cells, the Fur regulatory protein binds ferrous iron and gains DNA-binding capacity. Iron starvation, through either exhaustion of extracellular iron or the presence of extracellular chelators, prevents DNA binding by Fur, apparently by diminishing the pool of iron available for it to bind. The occasional appearance of extracellular chromophores similar to enterochelin suggested that the Fur regulon might be induced in the suppressed strains.

Suppressed strains accumulate high levels of intracellular iron. The dapD and dapB mutations and dipicolinate synthase overproduction did not suppress the characteristic sensitivity of SOD mutants to hydrogen peroxide; in fact, sensitivity was somewhat increased (data not shown). This effect was more obvious in SOD-proficient cells: exposure of wild-type cells to 2.5 mM H₂O₂ for 15 min typically permitted survival of 80% of the cells, whereas only 2% of dapB mutants survived. This suggested that dapB mutants might accumulate high levels of free intracellular iron that could catalyze oxidative DNA damage. EPR spectroscopy of whole cells confirmed that dapB mutants contained 300 µM free iron, in contrast to the 30 µM iron detected in their dapB⁺ parents (Fig. 8).

View larger version (20K): [in this window] [in a new window]   FIG. 8.   The dapB mutation increases the intracellular iron concentration. EPR scans of AN387 (SOD+) (wild type) and SM1095 (SOD+ dapB) are shown. Scans are normalized to the intracellular volume.

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SOD-deficient strains are starved for iron. The presence of high concentrations of iron chelates in the suppressed strains suggested that in unsuppressed SOD mutants the rate of cluster repair might be limited by the availability of intracellular iron. Although SOD-deficient cells are replete with free iron which is released from the iron-sulfur clusters by O₂, this iron may not be available to repair the damage to the iron-sulfur clusters; in fact, it appears to be rapidly removed from the cytosol by an undefined process (29). Thus, the constant turnover of iron-sulfur clusters could plausibly lead to iron starvation in SOD-deficient strains. To test whether this was the case, cells were exposed to the cell-impermeant chelator DTPA in order to abruptly terminate iron uptake. When 10 mM DTPA was added to cultures of SOD-proficient cells in amino acid-supplemented medium, a sevenfold increase in cell mass occurred before growth stopped. Under the same conditions SOD mutants stopped growing sooner, after only a 1.6-fold increase. This result suggested that either SOD mutants had lower levels of iron reserves or they had a higher iron demand. Subsaturating concentrations of DTPA were also far more inhibitory to SOD mutants than to wild-type strains (Fig. 9A).

View larger version (15K): [in this window] [in a new window]   FIG. 9.   SOD mutants are sensitive to iron limitation. (A) Subsaturating concentrations of DTPA can inhibit the growth of a SOD-deficient strain. Log-phase cultures grown under anaerobic conditions in minimal medium supplemented with all amino acids were diluted into an aerobic medium with or without 50 µM DTPA. Growth in 50 µM DTPA is represented. Symbols: triangles, AN387 (SOD+); circles, AS237 (SOD); diamonds, AN387 plus DTPA; rectangles, AS237 plus DTPA. (B) A tonB mutation slows the growth of a SOD-deficient strain. Log-phase cultures grown under anaerobic conditions in minimal medium supplemented with all amino acids were diluted into an aerobic medium of same composition. Symbols: diamonds, AN387 (SOD+); rectangles, SM1120 (SOD+ tonB); circles, AS237 (SOD); triangles, SM1122 (SOD tonB).

Fur derepression alone is not enough for suppression. Fur derepression and consequent rapid iron uptake can be achieved by introducing a null mutation in the Fur repressor protein. A fur mutation significantly relieved the aromatic amino acid auxotrophy of the SOD strain (Fig. 10A), but the sulfur-containing and branched-chain amino acid auxotrophies persisted.

View larger version (17K): [in this window] [in a new window]   FIG. 10.   (A) A fur mutation relieves the aromatic amino acid auxotrophy of a SOD-deficient strain. Log-phase cultures grown under anaerobic conditions in minimal medium supplemented with all but the aromatic amino acids were diluted into an aerobic medium of the same composition. Symbols: rectangles, SM1105 (SOD fur); diamonds, AS237 (SOD). (B) Fur derepression alone is not sufficient for suppression. Cultures growing logarithmically in LB medium were washed and resuspended in minimal medium containing all but the sulfurous amino acids. Symbols: solid rectangles, AN387 (SOD+); solid circles, AS237 (SOD); open circles, SM1093 (SOD dapB); triangles, SM1116 (SOD dapB fur); diamonds, SM1105 (SOD fur); open rectangles, SM1123 (SOD dapB fur/pSM915 [dapB+]).

Suppression is not mediated by induction of antioxidant defenses. The SoxR sensor protein, which activates the response to superoxide-generating agents, may be activated in vivo by removal of iron from its [2Fe-2S] cluster. However, assays of soxS::lacZ expression determined that this regulon was not activated in the suppressor background, despite the accumulation of dipicolinates. Further, neither bolA::lacZ nor marO_II::lacZ alleles were induced, indicating that the RpoS and Mar responses also remained inactive (data not shown).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

SOD deficiency imposes upon E. coli an inability to use nonfermentable carbon sources, auxotrophies for several families of amino acids, and slow growth even in glucose- and amino acid-supplemented medium. It is remarkable that pleiotropic suppression of these disparate phenotypes is achieved by the accumulation of a single metabolite, dipicolinate, or its reduced derivatives. This observation is of interest not only with respect to cell physiology but also because of its possible utility in pharmaceutical research.

The original report of the isolation of these mutants mapped the suppressor locus near but outside dapD. The data presented here show that that placement was erroneous. In retrospect, it is apparent that some of the mapping analyses were confounded by the fact that the suppressor allele was a nonsense mutation in a gene that, in the media used, was essential. Partial suppression of the mutation was achieved by the supE44 allele of the strain in which the mutation was isolated but was variable in the genetic backgrounds used for mapping. This resulted in incorrect phenotyping, since too little suppression of the nonsense allele would prohibit growth of the mutants, and too much would preclude suppression of the SOD auxotrophies. Furthermore, we have observed that growth of the dapD mutants in cysteine-supplemented media is also strain dependent, presumably manifesting some aspect of the competition between cysteine and diaminopimelate for the cysteine transporter. In any case, the sequence and complementation data reported here definitively establish that the original suppression occurred by mutation of dapD.

We do not yet understand the details of the connection between dipicolinate accumulation and suppression of SOD phenotypes. The restoration of branched-chain amino acid synthesis and of TCA cycle function is clearly due to the increased activity of the labile dehydratases in these pathways. Because dipicolinate chelates iron both in vitro and in vivo and maintains it in a reduced form, we have suggested that it may capture iron lost from the clusters and recycle it into a cluster repair process. This idea is speculative and, since the repair process has not been biochemically defined, has not been easily testable yet in vitro. However, previous work demonstrated that in nonsuppressed strains the iron lost from clusters is not available to rebuild them (29). A predictable consequence is that the cycles of enzyme damage and remetallation catalytically deplete the cells of usable iron, until there is little iron left to remetallate them. This would explain the hypersensitivity of SOD mutants to tonB mutations and to extracellular chelators, both of which reduce the rate of new iron import. Thus, iron recycling mediated by dipicolinates would benefit SOD cells. This concurs with Benov and Fridovich's observation that by providing additional iron in the growth medium they could increase the activities of the dehydratases and the cell growth rate in SOD mutants (4). While we were unable to see a similar effect (data not shown), that discrepancy could simply reflect differences in the abundance of trace metals in the salts used in growth media.

It is intriguing that an intracellular chelator which restores the function of the dehydratases also suppresses the sulfur and aromatic auxotrophies. This seems to comprise circumstantial evidence that these phenotypes as well are somehow connected to cluster damage. This idea is not easily reconciled with the recent demonstration that the aromatic auxotrophy may derive from the inactivation of transketolase (3). It is not clear how accumulation of iron-dipicolinate might enhance transketolase activity. However, some association of the aromatic auxotrophy with iron metabolism is implied by the observations that the auxotrophy is suppressed both by excessive iron supplements and by fur mutations. There is no evidence that either of the E. coli tkt genes is regulated by fur, and we do not recognize any Fur binding site in their promoter regions.

Although the connections between these phenotypes and cluster damage remain to be unravelled, this idea also dovetails with observations made in other studies. First, the amount of intracellular O₂ needed to confer sulfur and aromatic auxotrophies is about the same as that necessary to inactivate the dehydratases (22). Second, the ability of Mycoplasma pneumoniae (23) to thrive in an aerobic habitat without any SOD correlates with the absence of any of the known labile dehydratases. Finally, Culotta and colleagues discovered that mutations in genes suspected of being involved in cluster assembly can suppress the sulfur auxotrophy of SOD yeast (45).

That SOD-deficient strains are iron starved, despite the fact that their cytosol is replete with iron released from the iron-sulfur cluster enzymes, seems odd. However, one can imagine that a pipeline must deliver iron from transporters and storage proteins to the cluster assembly process and that iron adrift in the cytosol might not easily reenter that pipeline. The pipeline idea has an appeal, both because free iron is so sticky that it would seem unwise to rely upon its random diffusion through the cell and because of its extreme toxicity. Indeed, it has been found that other, less-toxic metals such as copper and nickel are carried through cells by chaperones (13, 40, 41). No such system has yet been uncovered for iron, but mutants would presumably have escaped simple genetic screens because of their inviability.

These experiments suggested that the Fur protein perceives a different pool of iron than do the dehydratases. For example, while the dehydratases were demetallated in the SOD mutants, the Fur protein remained metallated and active as a repressor. In contrast, the accumulation of the intracellular chelator caused Fur demetallation at the same time that it improved the assembly of dehydratase clusters. One explanation might be that Fur monitors the abundance of free iron in the medium and perceives SOD mutants as being iron rich. This could seem surprising: one might think that Fur would measure the iron content of the putative pipeline that delivers iron to the clusters. However, Touati et al. (46) showed that the import of iron in excess of the cellular iron storage capacity causes free iron to spill into the cytosol. Therefore, under normal conditions the presence of free iron would signal that iron import systems should be shut down, and Fur would respond accordingly. Only when free iron arises from cluster disintegration would this sensing system be inappropriate.