Study of Redox-Regulated Transcription Factors in Prokaryotes

METHODS: A Companion to Methods in Enzymology 11, 267–278 (1997)Article No. ME960421

Study of Redox-Regulated TranscriptionFactors in Prokaryotes

Bruce Demple

Department of Molecular and Cellular Toxicology, Harvard School of Public Health,Boston, Massachusetts 02115

methods employed in the study of these responses,Several prokaryotic regulatory proteins that respond to we consider three systems in Escherichia coli for

changes in oxygen tension or the presence of oxidative which the key regulatory proteins have been identi-agents have now been identified. The Fnr protein governs the fied and characterized.expression of numerous genes during anaerobic growth, bothas a transcriptional activator and as a repressor. OxyR proteinresponds to cellular exposure to H2O2 to stimulate transcrip-tion of several defense proteins. SoxR protein is triggered by

GENETIC SYSTEMS IN ADAPTATION TOsuperoxide or nitric oxide to activate a multigene regulon for

OXIDATIVE STRESSantioxidant defense and antibiotic resistance. Each of theseproteins has been purified and characterized for DNA bindingand transcriptional activity in vitro. Fnr, OxyR, and SoxR all The fnr system of E. coli acts under anaerobic con-seem to respond directly to redox signals generated in the ditions to regulate the transcription of at least 31cell, and their in vitro properties support this view: Fnr has an genes in total (1). Fnr protein represses many genesoxygen-sensitive [4Fe-4S] center essential for DNA binding; of aerobic metabolism, such as ndh (encoding NADHOxyR may be activated via oxidation of a key cysteine residue; dehydrogenase II) and sdh (encoding succinate dehy-and SoxR activation depends on redox-sensitive [2Fe-2S] drogenase), and activates anaerobic metabolismcenters. Basic methods for genetic and biochemical analysis genes, such as glpACB (an operon encoding the an-in these systems are presented, with emphasis on detailed aerobic form of glycerol-3-phosphate dehydrogenase)methods for SoxR that illustrate general approaches for all and narGHJI (an operon encoding nitrite reductase).the systems. q 1997 Academic Press Many fnr-regulated genes are under additional neg-

ative or positive control by arcA or narX/narL (1).Fnr protein is homologous to the catabolite activatorprotein Cap, but contains in addition near the N-terminus a cluster of three cysteine residues (num-bers 20, 23, and 29) that, together with cysteine-122,

Bacteria have evolved adaptive responses to tran- are thought to anchor an oxygen-sensitive [4Fe-4S]center. A current model (Fig. 1; see (3)) proposessitions between anaerobic and aerobic growth (1), to

imbalances in the production and disposal of reac- that formation of the [4Fe-4S] center at low oxygentension promotes dimerization and consequent spe-tive oxygen species such as superoxide (Or0

2 ) orH2O2, and to environmental free radicals such as cific DNA binding by Fnr to exert its transcriptional

effects; oxygen exposure destroys the [4Fe-4S] cen-nitric oxide generated by macrophages (2). These re-sponses are mediated by the coordinate regulation ters and eliminates DNA binding by Fnr.

The oxyR system acts in response to H2O2 to in-of groups of genes (regulons), each group under thecontrol of a common regulator. For this review of the duce at least eight genes of oxidative defense. These

2671046-2023/97 $25.00Copyright q 1997 by Academic PressAll rights of reproduction in any form reserved.

AID Methods A 421H / 670d$$$$41 01-30-97 18:24:24 metaal AP: Methods A

268 BRUCE DEMPLE

include katG-encoded catalase-hydroperoxidase, tabolism genes such as fumC (encodes fumarase),acnA (encodes aconitase), and fpr (encodes ferri-ahpFC-encoded alkyl hydroperoxidase, glutathione

reductase encoded by gor, and a protective DNA doxin:NADPH oxidoreductase); and resistance genesfor antibiotics, organic solvents, and heavy metalsbinding protein encoded by dps. In this system, ex-

isting OxyR protein is thought to be activated by a such as micF (encoding an antisense RNA thatdown-regulates OmpF porin expression) and acrAredox signal to bind and stimulate transcription of

the various target promoters (Fig. 2; see (4)). The and acrB (encoding efflux pumps for antibiotics andperhaps other compounds). This diversity of func-redox signal may involve a reaction with cysteine-

199 near the OxyR C-terminus (5). Many of the oxyR tions could reflect multiple avenues of oxidativestress or perhaps a more general resistance role forregulon genes are also regulated by the stationary

phase/starvation response system programmed by the soxRS regulon. The soxRS regulon is activatedby exposure of E. coli to agents that generate intra-the s38 protein encoded by rpoS (6–9).

The soxRS system includes a dozen or more induc- cellular superoxide, such as paraquat or quinones,or to nitric oxide, which can penetrate cell mem-ible transcription units for antioxidant defense

genes such as sodA (encoding Mn-containing super- branes. The system operates in two stages of tran-scriptional activation in which existing SoxR proteinoxide dismutase), nfo (encoding a DNA repair endo-

nuclease for oxidative damages), and zwf (encoding is converted to an active form and triggers transcrip-tion of the soxS gene; the resulting SoxS proteinglucose-6-phosphate dehydrogenase); aerobic me-binds and activates the various target promoters(Fig. 3). As the redox-sensitive component in thissystem, SoxR will be the focus of comments regard-ing the soxRS system below. The activation of SoxRseems to involve the protein’s [2Fe-2S] centers, per-haps an oxidation (10) or a metal assembly (11) reac-tion. Many soxRS regulon genes are also controlledas part of other systems, such the marRAB antibioticresistance regulon (12) or the fur Fe-assimilationregulon (13). Such multiregulation may allow fine-tuning of the soxRS response to specific conditions,as is likely the case for the additional regulation thatacts on fnr or oxyR regulon genes.

The methods discussed below have been used gen-erally in the analysis of redox-regulated transcrip-tion factors from prokaryotes. Methods that applyspecifically to one protein are indicated. I have alsoconsidered general aspects of experimental strategyfor defining redox-regulated genetic systems and thebiochemistry of the proteins that control them.

IN VIVO METHODS

Two-Dimensional (2D) Gels

2D gel analysis has proved generally useful for theFIG. 1. A model for regulation via Fnr. (A) Proposed structure ofidentification and analysis of complex responses inFnr based on homology to CAP protein. Mutation of Asp154 to

alanine generates an oxygen-insensitive protein. The four cysteine bacteria (14). 2D gels were employed in the initialresidues thought to anchor the [4Fe-4S] centers to the protein are studies of the oxyR and soxRS regulons to demon-indicated. (B) Proposed mechanism: active (DNA-binding) Fnr pro- strate the induced synthesis of numerous proteinstein contains a pair of [4Fe-4S] centers and is predominantly di- in response to hydrogen peroxide (15, 16) or superox-meric; oxygen causes loss of the iron center and monomerization

ide-generating compounds (15–18). In combinationof Fnr, which eliminates DNA binding. Reprinted with permissionfrom Lazazzera et al. (1996) J. Biol. Chem. 271, 2762–2768. with genetic analysis (see below), 2D gels helped es-


269REDOX-REGULATED TRANSCRIPTION FACTORS IN PROKARYOTES

tablish the groups of gene products under the control fully removed from their tubes and incubated inSDS-containing buffer (19). Each tube gel is thenof these regulators.

The basic methodology for 2D gels (19) was worked laid across the top of an individual SDS–polyacryl-amide slab gel in the Laemmli (22) buffer system.out by O’Farrell (20) and has been employed exten-

sively in the study of bacterial gene expression by Molecular size (Mr) markers are usually placed in alane at one side of the slab. After electrophoresis,Neidhardt and colleagues (14, 21). In the first dimen-

sion, polypeptides denatured in a neutral chaotropic the gel is removed for analysis of proteins.Two fundamental approaches are used to identifysolvent, usually 6–8 M urea, are subjected to isoelec-

tric focusing in tube gels, usually at relatively high and quantitate proteins displayed on 2D gels (19).Most commonly, a cell sample is pulse-labeled withvoltage and for extended times. The polyacrylamide

matrix is generated in the presence of ampholyte [35S]methionine and the radioactive proteins areidentified by autoradiography after electrophoresis.mixtures, which are combinations of buffers with

different pKa values over a selected range. Ampho- This procedure has the advantage of high sensitivityowing to the high specific activity of commerciallylytes are available for pH ranges from about 3 to 10,

which covers the range of most proteins; the actual available [35S]methionine (§1000 Ci/mmol). More-over, discovering changes in expression is favoredpH gradient is estimated after electrophoresis by

placing gel slices in a minimal volume of water and because the pulse label is incorporated in proportionto the differential synthesis rate. For example, themeasuring the pH. During isoelectric focusing, the

ampholytes, which vary in their pKa , electrophorese pulse-labeling method allowed a total of nearly 100inducible proteins to be identified in E. coli treateduntil they reach isoelectric points, where they act as

local buffers to generate a pH gradient between the with superoxide-generating agents (Fig. 4) (17). Analternative method is to visualize unlabeled proteinsanode and the cathode. In standard isoelectric focus-

ing, the denatured polypeptides move in the gel until in some general way, such as silver staining. In thelatter approach, identification is favored for proteinsthey reach the pH of their isoelectric points, with

net neutral charges. In some cases, nonequilibrium with dramatically increased levels, and individualspots can be readily subjected to microsequencing toisoelectric focusing may be used to resolve highly

basic or acidic proteins (19). obtain molecular information.Several considerations need to be borne in mindFollowing isoelectric focusing, the gels are care-

FIG. 2. A model for regulation via OxyR. In the reduced state, OxyR (proposed from footprinting data to be a tetramer) can bindonly the sites shown to repress oxyR transcription. After oxidation (perhaps at cysteine-199), an allosteric change in the proteinshifts the contacts at oxyR (which remains repressed) and allows binding to the positively regulated katG, ahpC, and oxyS promoters.Reprinted with permission from Toledano et al. (1994) Cell 78, 897–909.


270 BRUCE DEMPLE

in performing analyses on 2D gels. First, the timing is an important complement to studies of proteinof samples is usually important. Some proteins may expression by 2D gel analysis: the regulatory levelbe induced rapidly after exposure to an agent, while (transcriptional vs posttranscriptional) for a giventhe increased synthesis of others occurs more slowly. gene can be readily determined, and quantitativeThis is clearly the case for the H2O2-inducible pro- induction studies over a range of conditions can beteins in Salmonella typhimurium and E. coli (15). readily achieved.Second, developing a complete picture of the constel- Known genes can be readily analyzed by theselation of proteins with regulated expression usually methods. For cloned genes, vectors are availablerequires multiple gel runs, even for the same condi- (see (25)) that allow the insertion of promoter frag-tions of exposure and timing, due to variation in the ments to direct transcription of the reporter gene.degree of labeling or absolute amount of induction in A powerful application of fusion technology is ina given experiment. Such differences are especially the identification of novel genes based on their reg-important to bear in mind in studies of agents of ulated expression under defined conditions. Widelyoxidative stress, whose biological effects can depend used systems are based on the transposable phageexquisitely on the growth medium, the cell density, Mu (e.g. (26, 27)). Derivatives that carry an antibi-and the growth state of the bacteria. otic resistance marker and a promoterless lacZ

gene have been engineered, such that Mu insertionin a transcription unit in the correct orientationUse of Gene Fusionsplaces b-galactosidase expression under new tran-A simple but powerful approach to the analysis ofscriptional control. A collection of cells containinggene expression is the use of transcriptional (operon)such insertions is analyzed by replica plating onor translational (gene) fusions to a reporter geneLac-indicator media with and without the agent(23). The E. coli lacZ (b-galactosidase) gene has beenin question to identify cells containing regulatedespecially widely used, thanks to a set of simplefusions. This approach allows thousands of individ-assays for this enzyme that range from modest toual isolates to be screened and was used to identifyhigh sensitivity and that can be applied either to

whole cells or to cell extracts (24). This methodology a number of paraquat-inducible promoters under

FIG. 3. A model for regulation via SoxR. An oxidative stress signal activates the existing SoxR protein to trigger transcription ofthe soxS gene. Elevation of the SoxS protein concentration activates the regulon promoters and down-regulates soxS. Not shown isthe binding of nonactivated SoxR to the soxS promoter with affinity about equal to that of activated SoxR. Reprinted with permissionfrom Nunoshiba (1996) Crit. Rev. Euk. Gene Expr., in press.



soxRS control (28–30). Curiously, extensive efforts simple but direct means to demonstrate the depen-dence of soxS transcription on soxR (34, 35). Fusionsto identify novel H2O2-inducible promoters using

several variations of this methodology were unsuc- to soxS also enabled a systematic exploration of theability of many agents as potential inducers ofcessful (our unpublished data). One possibility is

that the effectiveness of H2O2 on solid media is soxRS. These studies also demonstrated specific ac-tivation of soxRS by superoxide-generating com-more difficult to control than in liquid culture (e.g.,

owing to effect scavenging by bacterial catalase). pounds (34, 35) and by nitric oxide (36), but not byother agents such as H2O2.Such difficulties might well apply to other types of

oxidative stress.Transcriptional fusions to individual genes have

been useful in the exploration of the conditions thatIN VITRO METHODSactivate various regulons, including fnr (31, 32),

oxyR (5), and soxRS (18, 33). An especially importantcase has been the analysis of transcriptional fusions Although the physiological conditions that regu-

late the activity of Fnr, OxyR, and SoxR in vivo sug-of lacZ to the E. coli soxS gene, which provided a

FIG. 4. Two-dimensional gel analysis of oxidative stress proteins in E. coli. The power of 2D gels to resolve large numbers ofproteins whose expression is changed by oxidative stress. (A) Untreated E. coli. (B) E. coli treated with 1 mM menadione (MD in D),a superoxide-generating agent. (C) Summary of regulated proteins identified in this and similar experiments. (D) Symbols for C.Reprinted with permission from Greenberg and Demple (1989) J. Bacteriol. 171, 3933–3939.


272 BRUCE DEMPLE

gest that each of these proteins responds to an oxida- needed to purify the protein, perhaps even em-ploying strict anaerobic conditions throughout astive signal of some sort, the most convincing

evidence for this idea has accrued from experiments has been used for nitrogenase (40).with the purified proteins in vitro. For purification,each of these proteins was expressed from a cloned Analysis of DNA Bindinggene; obtaining sufficient material for physical and The most commonly used methods for studyingactivity analysis from wild-type cells would have protein–DNA binding are electrophoretic mobilityproved arduous, to say the least. Another key ele- shift assays (EMSA, or ‘‘band-shifts’’) and DNasement for elucidating the likely control mechanisms footprinting assays. Both methods can achieve highfor these proteins and demonstrating their roles as sensitivity and specificity. Although EMSA is verytranscription factors was the availability in cloned widely used owing to its simplicity, quantitative in-form of genes that they regulate. Consequently, fun- terpretation of the results from this method must bedamental molecular genetic studies necessarily pre- done with caution because the electrophoretic sepa-ceded analysis of biochemical regulatory mecha- ration and the ‘‘caging’’ effect of the gel matrix cannisms. produce significant nonequilibrium effects. The sen-

sitivity of the EMSA method means that specificbinding activity may even be observed using crudeComments on Protein Purificationextracts. For example, the first direct indication that

The choice of protein expression system and gen- SoxR protein binds the soxS promoter came fromeral methods for protein purification have been experiments with crude cell extracts (34).widely discussed (37). Here I will briefly focus onissues that may matter particularly for these redox- Fnr and OxyRregulated transcription factors. Expression vectors

Specific binding has been explored for Fnr usingthat add oligopeptide or protein ‘‘tags’’ to the proteinboth DNA fragments, such as the ndh promoter (41),of interest are popular because they can greatly easeand a synthetic 48-bp oligonucleotide containing thethe purification process by allowing affinity chroma-consensus operator site (3, 32, 42, 43). For OxyR,tography on a predetermined matrix (38). However,binding has been demonstrated using restrictionit is a general concern with this methodology thatfragments of the katG, ahpC, oxyS, and oxyR promot-the added material may also change the biochemicalers (4, 44).properties of the protein of interest, particularly in

regard to redox reactions, which can be subtle. Con-SoxRsiderable thought should thus be given to whether

such tags are to be employed, and it is probably most For SoxR, only a single target is known, the soxSpromoter (Fig. 3). DNA substrates for in vitro bind-useful to carry out initial studies using vectors that

express the native protein. Indeed, the results ob- ing reactions have been generated by PCR, whichallows versatility: one primer is 5*-labeled with [32P]-tained with the tagged species should be verified

using the native protein; such repetition can be phosphate (transferred from [g-32P]ATP by poly-nucleotide kinase) and used with the unlabeledavoided by first attempting purification and charac-

terization of the native protein. primer to generate products with strand-specific la-beling. The labeled, double-stranded DNA (180 bp;For proteins that are overproduced to a high level,

conventional purification techniques such as ion ex- (10)) is isolated using commercially available col-umns (Qiagen) and may be further purified by elec-change chromatography usually suffice for their iso-

lation. These methods have been greatly stream- trophoresis and extraction from the gel ((19); com-mercial materials are also available (e.g., fromlined by the development of fast protein liquid

chromatography systems. For example, although we Qiagen) to streamline this process). We have alsosuccessfully analyzed SoxR-specific binding usingoriginally developed a protocol for SoxR isolation

(10) involving an affinity column containing soxS synthetic DNA fragments as short as 40 bp con-taining the SoxR binding site (see below) in the cen-promoter DNA (see below), similar purity was later

achieved without an affinity chromatography step ter (unpublished data).Binding reactions are set up in 20-ml binding(39). However, it should be noted that purification

conditions may have dramatic effects on the in vitro buffer (10 mM Tris–HCl, pH 7.5, 75 mM KCl, 2 mM

dithiothreitol (DTT), 10% glycerol; 0.1 mg of po-activities of redox-sensitive transcription factors(see below). In such cases, special measures may be ly(dI)–poly(dC) can be included for cruder samples,



but is unnecessary for the purified protein; 2 mM aerobic growth, indicates that the transcriptional ac-MgCl2 can be added for subsequent binding reactions tivity of these proteins is oxidatively modulated.with RNA polymerase) containing 0.5–1.0 fmol DNA Testing the specific transcriptional activities ofand protein samples containing 1–100 fmol (17 pg– these proteins in vitro has provided key evidence1.7 ng) SoxR and incubated at 257C up to 10 min supporting this concept, and this approach will cer-(10). Electrophoresis sample buffer (19) is then tainly be instrumental in the analysis of other tran-added and the samples are applied to and electro- scription factors suspected to respond to the cellularphoresed in 5% polyacrylamide gels in 20 mM Tris– redox state.HCl, pH 8.0, 3 mM sodium acetate, pH 7.9, 1 mM Common features apply to the proteins consideredEDTA. After autoradiography (Fig. 5A) or phos- here: specific genes regulated by each had alreadyphorimaging, binding affinity is expressed conserva- been identified and cloned, and the regulated tran-tively as the concentration of the protein required to scription of these genes appeared to be mediated byachieve a 50% ‘‘shift’’ of the DNA probe from the s70-containing RNA polymerase (RNAP), the pre-bound to the free form. This descriptive approach dominant enzyme in exponentially growing cells (6).does not fall victim to the possible confounding ef- These features enabled the relatively simple recon-fects of monomer–multimer equilibria of the pro- stitution of regulated gene expression in vitro. Thetein, multiple binding sites in the DNA probe, or specific target genes for OxyR, SoxR, and Fnr varystabilization or destabilization of protein–DNA com- considerably, but in each case the promoter borneplexes during electrophoresis. (See (45) for a more on a plasmid proved to program transcription suit-detailed summary of DNA binding methodology for ably; in some cases, linear templates could also beSoxR.) used (see below). An accurate and sensitive method

Specific SoxR–DNA interactions can also be for quantitating in vitro transcription is the ‘‘primerreadily analyzed by footprinting methods (summa- extension’’ method, in which a specific oligonucleo-rized in Fig. 5B). (For DNase I footprinting methods tide complementary to a region in the desired tran-see (10, 45).) We have also used 5-phenyl-1,10-phen- script is used to prime DNA synthesis by reverseanthroline-Cu(I) to footprint SoxR–DNA complexes transcriptase and generate a product of predictable(see (46) for details). This method does not produce size for subsequent analysis. This method (describedstrong cleavage at most nucleotides in B-form DNA, in detail in (46) and below) has the advantages ofbut is quite sensitive to protein-induced DNA struc- specificity deriving from base pairing by the primerstural distortions. and sensitivity because the oligonucleotides used for

this purpose can be labeled to a high level by addingIn vitro Transcription Assays a 5*-[32P]phosphate (transferred from [g-32P]ATP by

polynucleotide kinase). After the reverse transcrip-The in vivo activation of OxyR and SoxR by agentsof oxidative stress, and the inactivation of Fnr by tion reaction, the labeled RNA product is displayed

FIG. 5. Specific DNA binding by SoxR in vitro. (A) Electrophoretic mobility shift assay of SoxR binding a soxS promoter fragment.Reprinted with permission from Hidalgo and Demple (1994) EMBO J. 13, 138–146. (B) Summary of footprinting studies with SoxRon the soxS promoter. The inverted repeat is shown in boldface; the outlined areas are protected by bound SoxR. The arrows representhypersensitive or unprotected sites. CuPPA, 5-phenyl-1,10-phenanthroline-Cu(I). Reprinted with permission from Hidalgo et al. (1995)J. Biol. Chem. 270, 20908–20914.


274 BRUCE DEMPLE

on a sequencing-type gel (11, 47) and its position tion directly, but the abortive assay with SoxRseemed to be quite sensitive to unknown variablesdetermined and the amount of product quantitated

using film (autoradiography or enzyme-generated in different RNAP preparations (E. Hidalgo and B.Demple, unpublished data). Nevertheless, it waslight) or imaging cassettes for subsequent analysis,

as in phosphorimaging. General aspects of the in possible in this way to demonstrate strong activationof initiation by Fe-containing SoxR that was virtu-vitro transcription with Fnr and OxyR are men-

tioned below, with a more detailed description for ally undetectable with the apo-protein ((10); see be-low for preparation of apo-SoxR).the SoxR system.

An alternative approach uses a primer extensionassay to quantify products generated from a su-Fnrpercoiled plasmid containing the entireÇ1-kb soxRSIn vitro transcription studies with Fnr proteinlocus along with a control gene (bla, encoding b-lac-have had only limited success, probably owing to thetamase) not expected to be under SoxR control (Fig.instability of the [4Fe-4S] center in the presence of6) (46). The DNA template (200–400 ng) and SoxRoxygen. Promising results have been obtained usingprotein (1–100 ng) are first combined in binding/the ndh gene as an in vitro target (41, 48, 49).transcription buffer (10 mM Tris–HCl, pH 7.5, 75mM KCl, 15 mM MgCl2, 2 mM DTT, 10% glycerol;OxyR total volume 19 ml in 500-ml Eppendorf tubes) and

Four target gene promoters have so far proved incubated 5 min at room temperature. E. coli s70-useful for in vitro transcription modulated by OxyR containing RNAP is then added to achieve a final(4, 44): oxyR itself, which is negatively regulated, concentration of 0.1 mM (e.g., 1 ml of a Ç300 units/and katG, ahpC, and oxyS, which are all positively ml stock), and the incubation is continued at 377Cregulated (50). for 15 min. Transcription is initiated by the addition

of all four NTPs (1 ml of a stock containing 25 mMSoxR each) and further incubation for 5 min at 377C. The

reactions are stopped and the nucleic acids precipi-Genetic analysis indicated only one direct targetfor SoxR activity, namely the soxS promoter (Fig. 3). tated by the addition of 300 ml of a mixture con-

taining 73% (v/v) ethanol, 7 mg/ml yeast tRNA, 0.11The first in vitro transcription studies with SoxRemployed anÇ180-bp linear DNA fragment with the M sodium acetate and chilling at 0207C for 45 min.

The precipitates are then collected by centrifugationsoxS promoter approximately in the center (10).Initiation of transcription activated by SoxR was for 20 min at 10,000g at room temperature and re-

suspended in 10 ml diethyl pyrocarbonate-treatedfirst determined using an abortive initiation assay(10). After incubation of binding reactions of SoxR, water.

Primer extension reactions are then carried outthe soxS promoter fragment, and 1 ml s70-containingRNAP (300 units/ml stock from Promega Corp. or on the recovered precipitates. We have improved this

assay over our original procedure (46) by using thePharmacia; an alternative supplier is EpicentreTechnologies of Madison, WI) in 20 ml binding buffer soxS-specific primer ‘‘soxS-1’’ (5*-GCGATAAGA-

TCCTGAATAAT-3 *), which hybridizes 75 bp 3* ofcontaining MgCl2 (see above), transcription is initi-ated by the addition of 10 ml containing 300 mg/ml the first mRNA nucleotide (11). This primer or the

bla-specific primer pBR-1 (5*-GGGTGAGCAAAA-heparin, 0.3 M potassium glutamate, 30 mM Tris–HCl, pH 8.0, 3 mM MgCl2, 0.3 mg/ml BSA, 3 mM CAGGAA-3 *) (46) is 5*-labeled with [g-32P]ATP and

polynucleotide kinase (New England Biolabs), andCaCl2, 3 mM DTT, 15% (v/v) glycerol, 0.6 mM ApG(a dinucleotide corresponding to the first two resi- a 50-fmol aliquot is added to a 5-ml sample of the

recovered transcription products and incubated withdues of the soxS transcript; (10)), 0.1 mM ATP, 0.25mM [a-32P]UTP (ú600 Ci/mmol; transcription grade avian myeloblastosis virus reverse transcriptase in

reactions as recommended by the supplier, Promegafrom ICN), and the next two nucleotides. After 15min at 377C, the reactions are stopped by the addi- (Madison, WI). These samples are then electropho-

resed alongside size markers (a HinfI digest oftion of 3 ml 60 mM EDTA, 30% (v/v) glycerol, and 5 mlformamide-containing loading buffer. This reaction fX174 RFI DNA (Promega), 32P-labeled at the 5*-

termini, is appropriate) on 8% polyacrylamide gelsresults in the synthesis of a labeled trinucleotideproduct readily detected upon electrophoresis in a in 6 M urea (51), followed by autoradiography. The

resulting bands (Fig. 6) can be quantified by densito-denaturing polyacrylamide gel (51). This method hasthe advantage of measuring transcriptional initia- metric scanning or phosphorimager analysis.



This method allowed the detection of both the soxS the cell; in each case, the protein itself seems tosense the particular oxidative stress that alters itsand the control (bla) transcripts produced in the

same transcription reaction (Fig. 6). Moreover, es- activity specifically. In line with this view, the invitro activities of Fnr, OxyR, and SoxR can be modu-sentially the same result was obtained using small

linear DNA fragments (õ200 bp each for both the lated by ‘‘redox signals’’ in various ways, as outlinedbelow. Although the effects do not prove unambigu-soxS and the control promoters) in a run-off tran-

scription assay (unpublished data). Both methods ously that the modulatory mechanisms observed invitro duplicate those that operate in vivo, thedescribed above can allow multiple rounds of tran-

scription, which can increase the signal in the changes for all three proteins are quite consistentwith their behavior in the cell.assays. However, if initiation is not the rate-limiting

step for transcription, misleading results could beobtained. Weiss and colleagues used an alternative

Fnrapproach to generate single-round transcriptionSpecific binding of Fnr protein to DNA dependsproducts in vitro using templates similar to those

on the presence of Fe in the protein. Guest and co-described above (39). The essential difference hereworkers (41, 48) demonstrated strongly increasedwas to add heparin (Ç100 mg/ml final concentration)binding of the protein to the ndh promoter in theafter the initial binding reaction with SoxR andpresence of added Fe under anaerobic conditions.RNAP, such that inactive ‘‘closed’’ complexes are de-These observations indicated instability of the Festabilized, transcriptionally competent ‘‘open’’ com-center in the protein on exposure to oxygen, whichplexes remain, and new protein–DNA binding isis quite consistent with the hypothesis that Fnr inblocked; heparin does not block the RNAP elongationvivo is inactivated by O2 (52) (see Fig. 1). An im-reaction. Thus, only a single round of transcriptionportant advance was the identification of mutantoccurs, which gives a direct measure of initiationforms of Fnr altered in a putative dimer interfaceactivity. Since the multiple-round (10, 11, 46) andand active under aerobic conditions (53). One suchsingle-round (39) transcription results are in goodmutation was combined with a second alterationagreement, there is no reason to think that stagesproximal to the protein’s metal binding site (at posi-other than initiation effect the overall in vitro tran-tion 28; see Fig. 1) to demonstrate that active Fnrscription rate.contains a probable [4Fe-4S] cluster (43). This metal

Modulating the Activity of Redox-Sensitive center is hypothesized to be anchored by an N-termi-Transcription Factors nal group of cysteine residues (Fig. 1).

The presence of the [4Fe-4S] cluster stabilizes aA characteristic of the proteins discussed here istheir apparent responsiveness to conditions within dimeric form of the wild-type Fnr protein, isolated

FIG. 6. In vitro transcription assay for SoxR. Plasmid pBD100 contains both a SoxR-regulated promoter (soxS) and a controlpromoter (bla). Products identified by primer extension are shown on the right.


276 BRUCE DEMPLE

under anaerobic conditions and in the presence of tion or reactivation of redox-sensitive transcriptionfactors during complex, multistep procedures.sodium dithionite, which then binds its target DNA

sites (3). The metal center itself is destroyed in the Such pitfalls have been overcome through the useof a mutant form of OxyR with cysteine 199 changedpresence of oxygen by an unknown mechanism, thus

inactivating Fnr (3, 52). It is noteworthy that a num- to alanine (5). This protein no longer exhibits tran-scriptional activation in vivo in response to H2O2 orber of proteins, particularly hydrolyases such as

aconitase, contain oxidant-sensitive [4Fe-4S] clus- in vitro upon exposure to oxygen, but still binds andrepresses oxyR itself in all circumstances.ters (54), suggesting that this may be a general fea-

ture of this subclass of [4Fe-4S] moieties due to theirexposure to solvent. SoxR

The transcriptional activity of SoxR protein canbe modulated in several ways. Recombinant, wild-OxyRtype Fe-SoxR isolated by standard methods from E.

Recombinant wild-type OxyR protein isolated coli is highly active in in vitro transcription assaysfrom E. coli is active in transcription of katG and (10, 39). This result is reminiscent of the initial ob-other oxyR-regulated genes. This result suggested servations for OxyR, suggesting the fortuitous acti-that the isolation process itself might oxidize the vation of the protein during purification.protein to trigger its activity (44), perhaps through The inclusion of 1 mM 2-mercaptoethanol in purifi-the generation of small amounts of H2O2 in the buff- cation buffers leads to the isolation of transcription-ers. To inactivate OxyR DNA-binding and transcrip- ally inactive apo-SoxR: this form of the protein lackstion activity under anaerobic conditions, OxyR pro- the reddish-brown color characteristic of Fe-SoxRtein is first incubated with 100 mM DTT (it is unclear and has no significant bound Fe (10). Notably, apo-from published results (44) whether this incubation SoxR binds with unchanged affinity to its site in theis at 47C or a higher temperature), then introduced soxS promoter (Fig. 5A), but is without detectableinto a mixture containing the appropriate template activity in transcription assays (Fig. 7). The SoxRDNA (0.2 mg in 42 ml (total) of 40 mM Tris–HCl, pH [2Fe-2S] centers can also be destabilized by very7.9, 100 mM KCl, 10 mM MgCl2, 100 mM DTT), and high concentrations (75–100 mM) of DTT (46).incubated 10 min at 377C. RNAP (0.5 mg) is then Insight into the mechanism by which apo-SoxR isadded, the incubation at 377C continued for 10 min, generated has come from recent studies with theand 1 ml of a mixture of the 4 NTPs (25 mM each) biological thiol glutathione (55). This work hasadded to initiate transcription. After 5 min at 377C, shown that the SoxR [2Fe-2S] centers are destroyedthe reactions are terminated by phenol addition and by glutathione in an oxygen-dependent process thatextraction with phenol–chloroform. Primer exten-sion analysis is carried out directly on these samples.

Active OxyR remains if the above reactions arecarried out in buffers with the DTT concentrationreduced to 1 mM. The activity of OxyR can also bemodulated by controlling the oxygen concentration:under anaerobic conditions, only 10 mM DTT is re-quired to inactive OxyR, and the protein isolatedunder anaerobic conditions also lacked transcrip-tional activity. The original presentation of these re-sults (44) also demonstrates a pitfall due to the oxy-gen sensitivity of OxyR and provides a generalcaution for working with redox-regulated transcrip-tion factors. It appeared that oxygen affected onlythe transcriptional activity of OxyR and not its bind-ing affinity for DNA. In retrospect, this interpreta-tion evidently arose because of exposure of the pro-tein to oxygen in the course of the analysis, which

FIG. 7. Transcriptional activity of Fe-SoxR vs apo-SoxR. Tran-reactivated it; in fact, nonactivated OxyR does not scripts were detected by primer extension assays. Reprinted withbind its positively regulated targets significantly (4). permission from Hidalgo et al. (1995) J. Biol. Chem. 270, 20908–

20914.Caution is needed to prevent the accidental inactiva-



2. Hidalgo, E., and Demple, B. (1996) in Regulation of Genegenerates Or02 and H2O2 and probably proceeds

Expression in Escherichia coli (Lin, E. C. C., and Lynch, A. S.,through the production of thiol-derived free radicalsEds.), pp. 433–450, Landes, Austin, TX.that attack the metal center (55). Fe-SoxR can be

3. Lazazzera, B. A., Beinert, H., Khoroshilova, N., Kennedy,completely inactivated in a simple reaction by the M. C., and Kiley, P. J. (1996) J. Biol. Chem. 271, 2762–2768.addition of 1 mM glutathione to a simple buffer (50 4. Toledano, M. B., Kullik, I., Trinh, F., Baird, P. T., Schneider,mM Hepes–KOH, pH 7.6, 500 mM NaCl), aerobic T. D., and Storz, G. (1994) Cell 78, 897–909.incubation at room temperature for 30 min, and then 5. Kullik, I., Stevens, J., Toledano, M. B., and Storz, G. (1995)dilution into transcription reactions. This inactiva- J. Bacteriol. 177, 1285–1291.tion can also be followed physically by monitoring 6. Loewen, P. C., and Hengge-Aronis, R. (1994) Annu. Rev. Mi-

crobiol. 48, 53–80.absorbance at 332 nm, one of the maxima in the7. Becker-Hapak, M., and Eisenstark, A. (1995) FEMS Micro-SoxR UV-visible spectrum (55).

biol. Lett. 134, 39–44.Active Fe-SoxR can also be reconstituted from apo-8. Ivanova, A., Miller, C., Glinsky, G., and Eisenstark, A. (1994)SoxR in vitro. Apo-SoxR in 50 mM HEPES–NaOH,

Mol. Microbiol. 12, 571–578.pH 7.6, 0.1 M NaCl, 0.14 M 2-mercaptoethanol is9. Altuvia, S., Almiron, M., Huisman, G., Kolter, R., and Storz,deoxygenated by bubbling gently with 100% argon G. (1994) Mol. Microbiol. 13, 265–272.

gas, then incubated under anaerobic conditions 10. Hidalgo, E., and Demple, B. (1994) EMBO J. 13, 138–146.(£0.5 ppm O2) with the addition of 0.15 mM each

11. Hidalgo, E., and Demple, B. (1996) J. Biol. Chem. 271, 7269–FeCl3 and Na2S (from previously deoxygenated 150 7272.mM stocks). These mixtures are incubated at room 12. Ariza, R. R., Cohen, S. P., Bachhawat, N., Levy, S. B., andtemperature for 1–24 h; maximal reactivation oc- Demple, B. (1994) J. Bacteriol. 176, 143–814.curs in 6 h (11). Samples from these reactions can be 13. Touati, D., Jacques, M., Tardat, B., Bouchard, L., and Des-

pied, S. (1995) J. Bacteriol. 177, 2305–2314.transferred directly to transcription reactions. This14. VanBogelen, R. A., Abshire, K. Z., Pertsemlidis, A., Clark,noncatalytic reactivation process can be greatly ac-

R. L., and Neidhardt, F. C. (1996) in Escherichi coli and Sal-celerated by the Azotobacter vinelandii NifS proteinmonella: Cellular and Molecular Biology, (Neidhardt, F. C.,acting on L-cysteine (56) to generate S20 in the reac-Ed.), pp. 2067–2117, ASM Press, Washington, DC.

tion (rather than by adding Na2S). These modified15. Christman, M. F., Morgan, R. W., Jacobson, F. S., and Ames,

reactions contain 4 mM apo-SoxR in 50 mM Hepes– B. N. (1985) Cell 41, 753–762.NaOH, pH 7.6, 100 mM NaCl, 1 mM L-cysteine, 5 16. Morgan, R. W., Christman, M. F., Jacobson, F. S., Storz, G.,mM DTT, 2 mM ferrous ammonium sulfate. Under and Ames, B. N. (1986) Proc. Natl. Acad. Sci. USA 83, 8059–

8063.these conditions, full reactivation was observed in17. Greenberg, J. T., and Demple, B. (1989) J. Bacteriol. 171,£2 min (11). Interestingly, a limited amount of reac-

3933–3939.tivation also occurs if the NifS protein is omitted18. Greenberg, J. T., Monach, P., Chou, J. H., Josephy, P. D., and(unpublished data), indicating that apo-SoxR retains

Demple, B. (1990) Proc. Natl. Acad. Sci. USA 87, 6181–6185.some bound sulfide, perhaps as persulfides.19. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seid-The SoxR [2Fe-2S] centers are reducible in vitro

man, J. G., Smith, J. A., and Struhl, K. (1995) in Currentby dithionite (10, 11, 46), with a redox potentional Protocols in Molecular Biology, Chapter 10, Wiley, New York.about 0300 mV (H. Ding and B. Demple, unpub- 20. O’Farrell, P. H., and O’Farrell, P. Z. (1977) Methods Cell.lished data). It is currently being explored whether Biol. 16, 407–420.such reduction can directly inactivate transcription 21. Phillips, T. A. (1988) DNA Prot. Eng. Techniques 1, 5–9.by Fe-SoxR. This experiment is technically challeng- 22. Laemmli, U. K. (1970) Nature 227, 680–685.ing, owing to the possibility that oxygen may be rein- 23. Silhavy, T., Berman, M. L., and Enquist, L. W. (1984) Experi-troduced during the transcription reactions; more- ments with Gene Fusions, Cold Spring Harbor Laboratory

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Study of Redox-Regulated Transcription Factors in Prokaryotes