Proc. Natl. Acad. Sci. USAVol. 90, pp. 2310-2314, March 1993Biochemistry

Induction of manganese-containing superoxide dismutase inanaerobic Escherichia coli by diamide and 1,10-phenanthroline:Sites of transcriptional regulationCHRISTOPHER T. PRIVALLE, STEPHANIE E. KONG, AND IRWIN FRIDOVICH*Department of Biochemistry, Duke University Medical Center, Durham, NC 27710

Contributed by Irwin Fridovich, December 14, 1992

ABSTRACT Transcriptional regulation of the sodA gene,a member of the soxRS regulon encoding the manganese-containing superoxide dismutase (MnSOD; superoxide:super-oxide oxidoreductase, EC 1.15.1.1) of Escherichia coli, wasexamined in a variety of regulatory mutants. Diamide, anoxidant that causes the anaerobic biosynthesis of the MnSODpolypeptide and also facilitates insertion of manganese at theactive site, was found to anaerobically induce MnSOD in bothsoxRS and fur arcA fnr strains. Metal chelating agents alsocaused anaerobic induction of MnSOD in afur arcA fnr triplemutant; however, this induction of MnSOD and of glucose-6-phosphate dehydrogenase (G6PD) by 1,10-phenanthroline wasdependent on an intact soxRS locus. A strain ofE. coli bearinga fusion of the soxS promoter to lacZ was used to demonstratethat both diamide and 1,10-phenanthroline caused anaerobicactivation of soxS transcription. These results indicate that (a)both diamide and 1,10-phenanthroline induce the soxRS reg-ulon anaerobically by stimulation of soxS transcription; (ii)diamide, but not metal chelators, also induces MnSOD bio-synthesis by a soxRS-independent mechanism, perhaps medi-ated by effects onfur, arcA, orfnr-mediated repression ofsodA;and (iii) the soxRS locus contains a metal-binding componentand is responsive to the redox status of the cell.

those encoding MnSOD, glucose-6-phosphate dehydroge-nase (G6PD), endonuclease IV, and fumarase C (5, 6, 12).

It has been previously demonstrated that anaerobic elec-tron sinks such as nitrate (13-16), a variety of oxidants (15,17), and diamide (18-20), a thiol oxidant (21), elicit anaerobicbiosynthesis of MnSOD. Electron sinks result in anaerobicproduction of an inactive iron-containing MnSOD. In con-trast, diamide has been shown to exert two distinct effects:anaerobic induction of the biosynthesis of the MnSOD poly-peptide and facilitation of the insertion ofmanganese into thenascent polypeptide, resulting in active MnSOD (22). Theavailability of well-characterized regulatory mutants, whichexpress sodA under anaerobic conditions, allowed the tran-scriptional effect of diamide to be examined in greater detail.The results reported here suggest the existence of more thanone regulator of sodA expression which is influenced bydiamide. Furthermore, sodA, but not zwf (encoding G6PD),can be anaerobically induced in the absence of the soxRSlocus. Diamide and metal chelators have been shown todirectly activate the soxRS locus, evidence supportive of thehypothesis that SoxR is a metal-binding gene regulator thatacts as an intracellular sensor for superoxide (23) or cellularredox state (12).

The expression of sodA (MnSOD; superoxide:superoxideoxidoreductase, EC 1.15.1.1) in Escherichia coli is regulatedby several distinct global control systems. These transcrip-tional effectors include products of the fur (1-4), arc (1, 2),fnr (2), and soxRS (5, 6) genes. Fur protein, the product of thefur (ferric uptake regulation) gene, negatively controls tran-scription of several genes of E. coli involved in iron assim-ilation (7, 8). The arc (aerobic respiration control) system ofE. coli involves two gene products: ArcA (encoded by thearcA gene) and ArcB (encoded by the arcB gene), whichbelong to a family of two component signal transducingsystems in which ArcB is a membrane-bound sensor andArcA is a cytoplasmic DNA-binding regulator (9, 10). The arcsystem represses the genes of aerobic respiration duringanaerobic growth, and, in conjunction with Fur, negativelyregulates sodA (1, 2), thus coupling sodA expression to bothintracellular iron concentration and respiration. Fnr (fuma-rate-nitrate reduction) protein, the product of thefnr gene, isan iron-containing protein involved in anaerobic activation orrepression of several genes involved in anaerobic respiration(11). The soxRS (superoxide response) locus regulates, in apositive manner, the global response to superoxide (5, 6). Atwo-stage control system has been demonstrated for thesoxRS regulon in which an intracellular redox signal convertsthe SoxR protein into a transcriptional activator of the soxSgene (24, 42). The resulting induction of SoxS activatestranscription of nine genes of the soxRS regulon, including

MATERIALS AND METHODSStrains. The following strains were obtained for use in this

study: GC4468 (F-lacU169 rpsL), JTG936 (same as GC4468plus soxR105), DJ901 (same as GC4468 plus AsoxRS901), andTN530 (a GC4468 lysogen of A-pTN1530) (AsoxRsoxS'::lacZ) from B. Demple (5, 24) (Harvard School ofPublic Health); RK4353 (AlacYl69 araDJ39 non thi rpsLgyrA) and RK5279 (same as RK4353 plus fnr250) from S.Linn (25) (University of California, Berkeley); QC1062 (sameas GC4468 plus fur arcA sodB) from D. Touati (1) (InstitutJacques Monod, Universite Paris); and NC522 (same asGC4468 plus fur arcA fnr) from H. Hassan (2) (NorthCarolina State University, Raleigh).Growth Conditions. E. coli was grown anaerobically at

37°C in trypticase soy/yeast extract (TSY medium) (pH 7.0)containing, per liter, 30 g of trypticase soy and S g of yeastextract. Media were preequilibrated in a Coy anaerobicchamber (Coy Laboratory Products, Ann Arbor, MI) for atleast 24 h before inoculation, and growth was carried outunder an atmosphere of 85% N2/10% H2/5% CO2. Experi-mental cultures were typically initiated with a 2% inoculumfrom overnight cultures. Iron sulfate, diamide, and phenan-throlines were added to anaerobic media from sterile, anaer-obically equilibrated stock solutions.Enzyme Assays. Cells from 25-ml cultures were aerobically

harvested by centrifugation at 10,000 x g for 10 min at 4°C,washed once with 50 mM potassium phosphate buffer/0.1

Abbreviations: MnSOD, manganese-containing superoxide dismu-tase; G6PD, glucose-6-phosphate dehydrogenase.*To whom reprint requests should be addressed.

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mM EDTA, pH 7.8, and resuspended in 1.0 ml of this buffer.Cells were lysed by two passages through a French pressurecell at 20,000 psi (1 psi = 6.9 kPa). Lysates were clarified bycentrifugation at 14,000 x g for 10 min at 4°C. Cell-freeextracts were dialyzed overnight at 4°C against 500 vol ofKPi/EDTA buffer, with one change ofbuffer. Protein contentwas estimated colorimetrically with bovine serum albuminserving as the standard (26). SOD electromorphs were sep-arated by electrophoresis on 7.5% polyacrylamide gels (27)and bands of SOD activity were visualized by activity stain-ing (28). Western blot analysis was performed as described(19). G6PD was assayed spectrophotometrically as theG6PD-dependent reduction of NADP+ (29). f3Galactosidasewas assayed in cells permeabilized with SDS/chloroform asdescribed by Miller (30).

Materials. Trypticase soy broth was from Baltimore Bio-logical Laboratories. Yeast extract was from Difco. Acryl-amide, methylene bisacrylamide, gelatin, and goat anti-rabbitIgG-horseradish peroxidase conjugate were from Bio-Rad.NADP+, diamide, 1,10-phenanthroline, and 2,2'-dipyridylwere purchased from Sigma. Ultrapure FeSO4-7H2O wasobtained from Aesar (Johnson Matthey Inc.). 1,7-Phenan-throline was from Alpha Products (Ward Hill, MA).

RESULTS AND DISCUSSIONEffect of Diamide on MnSOD and G6PD in soxRS Mutants.

Diamide resulted in anaerobic induction of MnSOD in wild-type cells (Fig. 1) as reported (19). Mutants constitutive withrespect to soxRS (JTG936) lack detectable MnSOD underanaerobic growth conditions and respond to diamide with a3-fold increase in MnSOD compared to the parental strain.The lack of MnSOD polypeptide in the anaerobic soxRSconstitutive strain, in the absence of diamide, suggests thatsodA expression remains under repression control, whichcan be relieved by diamide. Diamide also induces MnSOD ina AsoxRS strain (DJ901), indicating that this response is notdependent on the soxRS locus. However, the somewhatdiminished effect of diamide on MnSOD induction in thisstrain (15-20%) suggests that some component of the actionof diamide is mediated by soxRS.The response to diamide of G6PD, another member of the

soxRS regulon (5, 6), was also examined. As shown in Table1, G6PD was anaerobically induced 2-fold by diamide in theparental strain. Anaerobically grown soxRS constitutive mu-tants contained elevated levels of G6PD, which remainedinducible by diamide. In contrast, G6PD was not induced bydiamide in the AsoxRS mutant. Thus, unlike MnSOD, thediamide-mediated induction of G6PD was dependent on thesoxRS locus. These results suggest that anaerobic biosyn-

1 2 3 4 5 6 71 2 3 4 5 6 7~~~~.L

_ _ .aI +.1 + I + I

GC4468 JTG936 DJ901

FIG. 1. Effect of diamide on MnSOD biosynthesis in soxRSmutants. E. coli K-12 cells were grown under anaerobic conditionsat 37°C for 2 h in TSY medium. Diamide (0.5 mM) was then addedas indicated (lanes +), and cells were grown an additional 60 min at37°C. Soluble extracts (15 ,ug of protein per lane) were applied to aSDS/10o polyacrylamide slab gel and electrophoresis was carriedout at 25 mA per slab. Western blot analysis was performed asdescribed (19). Lanes: 1 and 2, GC4468 (wild type); 3 and 4, JTG936(soxRIOS); 5 and 6, DJ901 (AsoxRS); 1, 3, and 5, no addition; 2, 4,and 6, 0.5 mM diamide; 7, 0.1 ,ug of purified E. coli MnSOD.

Table 1. Effect of diamide on G6PD activity in soxRS mutants

Specific activity,Strain unit/mg

GC4468 0.10GC4468 + diamide 0.20JTG936 (soxR05) 0.24JTG936 (soxRO05) + diamide 0.42DJ901 (AsoxRS) 0.10DJ901 (AsoxRS) + diamide 0.10

E. coli K-12 cells were grown as described in the legend to Fig. 1.

thesis of MnSOD is regulated by at least one additional site,which can be affected by diamide.

Effects of Diamide and of 1,10-Phenanthroline on an arcAfur Double Mutant. Tardat and Touati (1) have utilizedsodA-lacZ operon fusions to search for mutants that expresssodA under anaerobic conditions. These studies revealedthat simultaneous mutations in genes for two different globalregulatory systems, fur and arc, are required for the anaer-obic expression of sodA (1, 2). The effect of these mutationson diamide-mediated induction of MnSOD was thus exam-ined in an arcA fur mutant. As shown in Fig. 2, the arcA furmutant expressed the MnSOD polypeptide anaerobically,although it lacks detectable MnSOD activity (lanes 5). Di-amide induced the MnSOD polypeptide and caused theappearance of active MnSOD in both the parental strain andthe arcA fur mutant (lanes 2 and 6). Specifically, diamideresulted in an -8-fold induction ofMnSOD polypeptide overthe basal level found in the arcA fur mutant.These results demonstrate that the effect of diamide is not

dependent on fur or arcA and suggest the existence of an

1 2 3 4 5 6 7 8 9A

B

01

-Mn

-Hy

- Fe

%x x

10

FIG. 2. Effect of 1,10-phenanthroline and diamide on MnSODbiosynthesis and activation in an arcA fur mutant. E. coli K-12 cellswere grown anaerobically at 37°C for 1.75 h in TSY mediumsupplemented as indicated with 0.1 mM 1,10-phenanthroline. Di-amide (0.5 mM) was then added as indicated, and cells were grownan additional 30 min. (A) Soluble extracts (3.5 p.g of protein per lane)were applied to a SDS/10%o polyacrylamide slab gel and electropho-resis was carried out at 25 mA per slab. Western blot analysis wasperformed as described (19). (B) Soluble extracts (50 U.tg of proteinper gel) were applied to 7.5% polyacrylamide gels and stained forSOD activity. Lanes: 1-4, GC4468; 5-8, QC1062 (arcA fur sodB); 1and 5, no addition; 2 and 6, 0.5 mM diamide; 3 and 7, 0.1 mM1,10-phenanthroline; 4 and 8, 0.1 mM 1,10-phenanthroline plus 0.5mM diamide; 9 (A) 0.05 ,ug of purified E. coli MnSOD.

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additional regulatory locus. Initial insight into the nature ofsuch a site was provided by the finding that 1,10-phenanthroline also resulted in the anaerobic induction ofMnSOD polypeptide and activity in the arcAfur mutant (Fig.2, lanes 7 vs. lanes 5). Tardat and Touati (1), utilizing asodA-lacZ operon fusion, have previously noted the tran-scriptional effects of 1,10-phenanthroline on the arcA furstrain. In addition to effects at the transcriptional level, bothdiamide and 1,10-phenanthroline have effects at the matura-tional level (22). The MnSOD polypeptide made under an-aerobic conditions is inactive because of the insertion of ironinto the nascent MnSOD polypeptide. Diamide, perhaps byaffecting valence and subsequent availability of iron and/ormanganese, and 1,10-phenanthroline, by chelation of iron,result in formation, under anaerobic conditions, of the activeMnSOD (22).

Effects of Diamide and of 1,10-Phenanthroline on an fnrMutant. The anaerobic induction of MnSOD in the arcA furmutant by diamide, coupled with the induction by 1,10-phenanthroline, suggested the possibility that another iron-responsive repressor protein was involved in the transcrip-tional control of sodA expression. Consistent with this hy-pothesis is the recent report by Hassan and Sun (2) thatmutations in the fnr gene were found to affect aerobic andanaerobic expression of sodA-lacZ. FNR, the gene regulatorof anaerobic respiratory genes of E. coli, is converted in vivoby 02 and by chelating agents to an inactive state (31, 32).Conversely, incubation of metal-depleted bacteria with Fe2+resulted in active FNR (31, 32). The purified FNR proteincontains a cysteine-rich N-terminal segment, which mayform an iron-binding redox-sensing domain (33, 34), and,when isolated in the presence of iron, contains -1 mol of ironper monomer (35). We have found that the activity of nitratereductase, an enzyme under control of the fnr regulon, isinhibited by =80% in the presence of 1,10-phenanthroline(data not shown), suggesting that this chelator can directlyaffect fnr function.As shown in Fig. 3, anfnr strain (RK5279) contains much

less (but detectable at higher protein applications) MnSODpolypeptide anaerobically than the arcA fur strain but re-mained strongly inducible by diamide and weakly inducibleby 1,10-phenanthroline (lanes 14-17). These results excludefnr as the sole site of action of diamide and suggest that the

GC4468 QCl 062-l

1 2 3 4 5 6 7 8

RK4353 RK52799 1 1 I 1 19 lo 11 12 13 14 15 16 17

- a. a. ~~~~~~~~~~4 -- -dvf

C' ~~070.

'4, '4, '4

FIG. 3. Effect of diamide and 1,10-phenanthroline on MnSODbiosynthesis in arcA fur and fnr mutants. E. coli K-12 cells weregrown under anaerobic conditions at 37'C for 1.75 h in TSY mediumsupplemented as indicated with 0.1 mM 1,10-phenanthroline. Di-amide (0.5 mM) was then added as indicated, and cells were grownan additional 30 min. Soluble extracts (7 ,g of protein per lane) wereapplied to a SDS/10% polyacrylamide slab gel and electrophoresiswas carried out at 25 mA per slab. Western blot analysis wasperformed as described (19). Lanes: 1-4, GC4468; 5-8, QC1062(arcA fur sodB); 10-13, RK4353 (fnr+); 14-17, RK5279 (fnr250); 1,5, 10, and 14, no addition; 2, 6, 11, and 15, 0.5 mM diamide; 3, 7, 12,and 16, 0.1 mM 1,10-phenanthroline; 4, 8, 13, and 17, 0.1 mM1,10-phenanthroline plus 0.5 mM diamide; 9, 0.05 Ag of purified E.coli MnSOD.

induction of MnSOD by 1,10-phenanthroline involves an-other site(s) in addition tofnr, perhaps attributable to effectsat the fur locus. To further define the existence of anadditional regulatory site, the effects of diamide and metalchelators on anaerobic MnSOD biosynthesis were examinedin a strain bearing simultaneous mutations in the arcA, fur,and fnr genes.

Anaerobic Expression of sodA in Differentfur arcA fnr andsoxRS Mutants. Anaerobic production of the MnSOD poly-peptide was examined in several mutant strains. As shown inFig. 4, the fur arcA fnr triple mutant contained higher levelsofMnSOD than thefur arcA orfnr strains. Diamide increasedthe level of MnSOD in all of these strains. It follows that thefur arcA fnr mutant is not fully derepressed with respect tothe anaerobic expression of sodA and that an additionalsite(s) of action of diamide exists.These results are in contrast to those previously reported

(2), which have suggested that thefur arcAfnr mutant is fullyderepressed for anaerobic sodA expression. This conclusionwas based on the finding that sodA-lacZ expression in theanaerobically grown triple mutant was not further induced by2,2'-dipyridyl (2). We have therefore reexamined the effect ofmetal chelators on MnSOD biosynthesis in the triple mutant.As shown in Fig. 5, 1,10-phenanthroline was more effectivethan 2,2'-dipyridyl in eliciting anaerobic production of theMnSOD polypeptide in both the triple mutant (lanes 7-9) andthe parental strain (lanes 1-3). No effect of these chelatingagents on MnSOD biosynthesis could be discerned in aAsoxRS mutant (lanes 4-6), suggesting both that the induc-tive effect of chelating agents on the fur arcA fnr strain isexerted at the soxRS locus and that metal chelation activatessoxRS anaerobically.The soxRS locus consists of two divergently arranged

genes, soxR and soxS (23, 36). The predicted SoxR protein ishomologous to the MerR family of proteins (23, 24), whichactivate transcription of the mer operon in response to Hg2+(37). This sequence includes a cluster of cysteine residues,which bind Hg2+ in the case ofMerR (38) and may bind Fe2+in the case of SoxR. This site could respond to oxidation ineither oftwo ways: (i) by direct oxidation of pairs of cysteineresidues to disulfides, or (ii) by oxidation of the bound Fe2+to Fe3+. The SoxR protein may not function as a transcrip-tional activator of soxS when it contains bound Fe2+; how-ever, oxidation or chelation ofbound iron, or oxidation ofthecysteine residues, may convert SoxR into a transcriptionalactivator of soxS. Diamide could effect oxidation of thecysteine cluster of SoxR, while chelating agents would re-move bound iron from the cluster, thus allowing anaerobicsoxS expression.

Effects of Diamide and of 1,10-Phenanthroline on Expres-sion of soxS. Both diamide and 1,10-phenanthroline result in

1 2 3 4 5 6 7 8

-..w __ _ _

' 0

vs>

&

-'- -IQc-dt -

- 1-,

,-24'94-

FIG. 4. Effect of diamide on MnSOD biosynthesis in differentfurarcA fnr mutants. E. coli K-12 cells were grown anaerobically for 2h at 37°C in TSY medium. Diamide (0.5 mM) was then added asindicated (+), and cells were grown an additional 30 min. Solubleextracts (7 ,ug of protein per lane) were applied to a SDS/10opolyacrylamide slab gel and electrophoresis was carried out at 25 mAper slab. Western blot analysis was performed as described (19).Lanes: 1, 3, 5, and 7, no addition; 2, 4, 6, and 8, 0.5 mM diamide.

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Proc. Natl. Acad. Sci. USA 90 (1993) 2313

1 2 3 4 5 6 7 8 9 10

~~~~~~~~~~~~~~~~~~~~~~~~~~~~.... .....................

9,5/0 ~ '( 94-°oV~~~~

FIG. 5. Effect of iron chelators on MnSOD biosynthesis in soxRSand fur arcA fnr mutants. E. coli K-12 cells were grown underanaerobic conditions for 4.5 h at 37°C in TSY medium supplementedas indicated with 0.1 mM 1,10-phenanthroline or 0.25 mM 2,2'-dipyridyl. Soluble extracts (5 ,ug of protein per lane) were applied toa SDS/Ilo polyacrylamide slab gel and electrophoresis was carriedout at 25 mA per slab. Western blot analysis was performed asdescribed (19). Lanes: 1-3, GC4468 (wild type); 4-6, DJ901(AsoxRS); 7-9, NC522 (fur arcA fnr); 1, 4, and 7, no addition; 2, 5,and 8, 0.1 mM 1,10-phenanthroline; 3, 6, and 9, 0.25 mM 2,2'-dipyridyl; 10, 0.05 ug of purified E. coli MnSOD.

anaerobic induction of MnSOD and G6PD. The diamide-mediated induction of G6PD and the chelator-mediated in-duction of MnSOD and G6PD are dependent on an intactsoxRS locus. To determine the effects of these agents on thesoxRS locus, we used a strain of E. coli constructed bylysogenization of the lac deletion mutant GC4468 with Aprophages bearing the AsoxRsoxS'::lacZ fusion (24), thusallowing the expression of 3galactosidase to serve as ameasure of soxS transcription. As shown in Fig. 6, diamideresulted in a 6-fold anaerobic induction of soxS, while 1,10-phenanthroline resulted in an -2-fold induction. The effect of1,10-phenanthroline was eliminated by addition of Fe2+ anda nonchelating phenanthroline was without effect. Aerobicexpression of soxS was 2.3-fold greater than the anaerobiclevel. These results indicate that the expression of soxS,which is regulated by SoxR, can be stimulated by diamide orby iron chelators.

In summary, MnSOD is regulated at the transcriptionallevel by several global regulators: Fur, Arc, FNR, and Sox.Fur couples MnSOD expression to intracellular iron concen-

600-

500-

c 400_

(U 30010

100 |_l

control diamide 1,10-phen 1,10-phen 1,7-phen aerobic+ Fe control

FIG. 6. Induction of soxS'::lacZ by diamide and 1,10-phenanthroline. E. coli K-12 cells TN530, a GC4468 lysogen ofA-pTN1530 (AsoxR soxS'::1acZ), were grown anaerobically for 1 h at37°C in TSY medium. Diamide (0.5 mM), 1,10-phenanthroline (0.1mM) with or without FeSO4 7H2O (0.033 mM), or 1,7-phenanthroline(0.1 mM) was then added as indicated, and cells were grown anadditional 2 h. Aerobic controls were grown at 37°C (200 rpm) for 2h. /-Galactosidase activity was determined in cells permeabilizedwith SDS/chloroform (29).

tration (1, 4), Arc and FNR couple it to respiration andcellular redox state (1, 2), while Sox couples it to intracellularsuperoxide concentration (5, 6) or redox state (12) dependingon MnSOD content and growth conditions of the cells. Theiron in FNR and Fur appears to act as a sensor of the redoxstate of the cell. Thefnr gene product is produced under bothaerobic and anaerobic conditions (39), and reversible inter-conversion of the anaerobic (active) state to the aerobic(inactive) state has been demonstrated by the anaerobicaddition of chelating agents (31, 33). Thus, the valence stateof iron or its availability can modulate the function of bothFur and FNR. SoxR may also be a redox sensor that utilizesiron, based on the observation that the chelator-mediatedinduction of MnSOD appears to be dependent on the soxRSlocus. The four cysteines of SoxR may constitute a metalbinding site, which could accommodate a redox active metalsuch as iron. In the reduced state, such a SoxR-Fe2+ complexmay render SoxR inactive as a transcriptional activator.Oxidation of the cysteine residues to disulfides, oxidation ofbound Fe2+, or removal of the iron by chelation may, in turn,allow activation of SoxR.The anaerobic induction of MnSOD in a fur arcA fnr

mutant by diamide or 1,10-phenanthroline suggests the ex-istence of another mechanism(s) of anaerobic repression ofsodA, which can be removed by oxidation or by iron chela-tion. Evidence for additional regulatory mechanisms is cur-rently emerging. Integration host factor, a DNA-bindingprotein that induces a bend in the DNA at its target-bindingsite (40), has been implicated in the regulation ofsodA (41, t).Further work will be required to determine whether integra-tion host factor can be affected by diamide or by ironchelators. Additional studies also will be necessary to deter-mine whether the induction of MnSOD in the fur arcA fnrmutant by diamide or 1,10-phenanthroline is mediated solelyby the soxRS locus or through other, as yet undefined, sites.

tSchrum, L. W. & Hassan, H. M., American Society for Microbi-ology, 92nd General Meeting, New Orleans, May 26-30, 1992,p. 221 (abstr. H-227).

This work was supported by research grants from the Council forTobacco Research-U.S.A., Inc., and from the Johnson and JohnsonFocused Giving Program.

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