hlyx, the fnr homologue of actinobacillus pleuropneumoniae, is a [4fe–4s]-containing...

HlyX, the FNR homologue of Actinobacilluspleuropneumoniae , is a [4Fe–4S]-containingoxygen-responsive transcription regulator thatanaerobically activates FNR-dependent Class Ipromoters via an enhanced AR1 contact

Jeffrey Green * and Mandy L. BaldwinKrebs Institute for Biomolecular Research, Department ofMolecular Biology and Biotechnology, University ofSheffield, Western Bank, Sheffield S10 2UH, UK.

Summary

The hlyX gene of the pig pathogen Actinobacillus pleu-ropneumoniae encodes HlyX, a homologue of FNR, theanaerobic transcription regulator of Escherichia coli .The hlyX gene complements the anaerobic respiratorydeficiencies of E. coli fnr mutants but also induces theexpression of an otherwise latent haemolysin. There-fore, FNR and HlyX have distinct but overlappingregulons. The hlyX gene has been overexpressed asa gst ::hlyX fusion and the HlyX protein purified. Simi-lar to FNR, HlyX can acquire a [4Fe–4S] cluster, whichpromotes binding to the FNR box ( Kd of 20–30 nM)under anaerobic conditions. Expression of hlyX in E.coli induced the anaerobic production of at least fivepolypeptides, including the yfiD gene product, whichwere not induced by fnr . Analysis of the yfiD promoterregion revealed the presence of two FNR boxes situ-ated at ¹61.5 and ¹114.5. Consistent with this obser-vation, expression from the semi-synthetic Class Ipromoter FFþ20pmelR was efficiently activated byHlyX but not by FNR. The weaker level of FNR-mediated activation of Class I promoters suggeststhat there is a poorer activating contact (activatingregion 1 (AR1) equivalent) between FNR and RNA poly-merase at these promoters and that HlyX possessesan additional or improved AR1. The AR1 of HlyX ispartially characterized by a surface-exposed regionaround amino acid A187, which confers the alteredspecificity and provides an explanation for the exist-ence of distinct but overlapping HlyX and FNR regu-lons.

Introduction

Actinobacillus pleuropneumoniae is an important pigpathogen, causing acute and often fatal pleuropneumonia(Nicolet, 1992). Three related RTX (repeats in the struc-tural toxin) toxins, ApxI, ApxII and ApxIII, have been impli-cated in the virulence of this Gram-negative bacterium(Frey et al., 1993a; Inzana, 1991). The RTX toxins candamage neutrophils and macrophages (Frey et al., 1993b),and ApxI and ApxII can lyse erythrocytes from a numberof species (Frey and Nicolet, 1988; Rosendal et al.,1988). How these virulence factors are regulated isunclear, but A. pleuropneumoniae possess a gene, hlyX,which, when expressed in Escherichia coli K-12 strains,endows a haemolytic phenotype (Lian et al., 1989; Greenet al., 1992). The HlyX protein does not have haemolyticactivity but seems to induce an otherwise latent haemo-lysin in E. coli. Indeed, HlyX is a homologue of FNR, theanaerobic transcription regulator of E. coli, and as suchis a member of the CRP/FNR family of transcription regu-lators (Spiro, 1994). All the members are structurallyrelated to CRP, the cyclic AMP receptor protein, and havea C-terminal helix-turn-helix DNA-binding domain and anN-terminal sensory domain, although this domain arrange-ment is reversed for Bacillus subtilis FNR (Ramos et al.,1995). The FNR-like members of the family are character-ized by the presence of four essential cysteine residues(Sharrocks et al., 1990; Melville and Gunsalus, 1990) andby the amino acid sequence E--SR in the DNA bindinghelix which confers specificity for the FNR consensussequence TTGAT----ATCAA (Guest et al., 1996).

The amino acid sequence of HlyX is 73% identical toFNR, and HlyX retains all the essential cysteine residuesthat form the ligands for the redox-sensitive [4Fe–4S] clus-ter of FNR (Khoroshilova et al., 1995; Green et al., 1996a,b;Lazazzera et al., 1996). The anaerobic incorporation of theiron–sulphur cluster into FNR increases the affinity ofFNR for its DNA target (Khoroshilova et al., 1995; Greenet al., 1996a,b). It is likely that the incorporation of the[4Fe–4S] cluster is accompanied by dimerization of FNRand that transition promotes DNA binding (Lazazzera etal., 1993; 1996; Khoroshilova et al., 1995; Green et al.,

Molecular Microbiology (1997) 24(3), 593–605

Q 1997 Blackwell Science Ltd

Received 1 November, 1996; revised 13 March, 1997; accepted 17March, 1997. *For correspondence. E-mail [email protected]; Tel. (0114) 2224403; Fax (0114) 2728697.

m

1996b), although there is still some evidence to suggestthat FNR may act as a monomer (Melville and Gunsalus,1996).

The amino acid sequence of the predicted DNA-bindinghelix of HlyX contains all the residues required for recog-nition of FNR-binding sites. Accordingly, when HlyX isexpressed in E. coli, it recognizes and regulates transcrip-tion from FNR-dependent genes in response to anaerobio-sis (MacInnes et al., 1990; Soltes and MacInnes, 1994).However, as well as complementing the anaerobic growthdeficiencies of fnr mutants, HlyX activates the expressionof a haemolysin in E. coli K-12 strains which FNR doesnot. Studies with hybrid FNR–HlyX proteins suggeststhat unique features of both the N- and C-terminal seg-ments of HlyX are required for the activation of the haemo-lysin (Green et al., 1992). Recently, homologues of HlyX(and hence also of FNR) have been identified in four oralfacultative anaerobes (Hattori et al., 1996). Among theseare the pathogens Haemophilus aphrophilus and Actino-bacillus actinomycetemcomitans in which the FNR homo-logues may act as regulators of virulence determinants aswell as of anaerobic respiratory functions.

In the present work, the purified HlyX protein has beenshown to possess an oxygen-sensitive [4Fe–4S] clusterafter anaerobic reconstitution. Acquisition of the iron–sul-phur cluster results in enhanced DNA binding to an FNRbox. Expression of hlyX in E. coli caused the anaerobicinduction of at least five polypeptides which were notdetectably induced by FNR, as judged by 2-D gel electro-phoresis. One of these has been identified as the yfiDgene product, and the yfiD promoter has been shown tobe a Class I promoter with FNR boxes at ¹61.5 and¹114.5. HlyX-mediated activation of Class I promotersrequires a unique surface-exposed region of HlyX whichcontains amino acid A187. By analogy with CRP, thisregion probably makes an activating contact with RNApolymerase. Thus the molecular basis for the distinct butoverlapping regulons of HlyX and FNR lies in the abilityof HlyX to efficiently activate transcription from Class Ipromoters.

Results

Overproduction and purification of HlyX

Previous attempts to overproduce and purify HlyX wereunsuccessful. There was no significant amplification whenthe hlyX gene was expressed from the tac promoter ofptac85 (Green et al., 1992). Therefore, a different strategywas adopted in which HlyX was expressed as a glu-tathione-S-transferase (GST) protein fusion (GST–HlyX)from pGS903. The GST–HlyX fusion formed up to 13%of soluble cell protein with yields of 3 mg l¹1 of culture.However, the yield of HlyX after release from the fusionby cleavage with the protease thrombin was disappointing.The isolated HlyX protein was prone to precipitation andsuffered degradation which reduced the yield of pureHlyX to only 6% of the theoretical maximum (Table 1).However, HlyX was purified to near homogeneity (Fig.1) and the N-terminal amino acid sequence (GSLMKIVS-DAKHTGR) confirmed that the purified protein was HlyXand that the putative redox-sensing region was intact. Puri-fied HlyX and the GST–HlyX fusion protein cross-reacted

Q 1997 Blackwell Science Ltd, Molecular Microbiology, 24, 593–605

Table 1. A typical purification of HlyX.Step Vol (ml) Protein (mg ml¹1) Total protein (mg) HlyX (%)a Yield (%)b

Crude extract 3 20.8 62.4 5.2 100Glutathione agarose 20 0.4 8.0 22.6 56Heparin agarose 3 1.1 3.3 33.5 34Thrombin cleavage 7.5 0.3 2.2 50.4 34Bio-Scale Q 2.5 0.04 0.1 95.0 2.9

a. The percentage of HlyX in each fraction was calculated after quantitative densitometry ofgels stained with Coomassie brilliant blue. When HlyX was present as a GST–HlyX fusion,the figure obtained was divided by two to correct for the GST component.b. The yield of HlyX was calculated from the percentage of HlyX estimated by densitometry ofSDS–polyacrylamide gels gels stained with Coomassie brilliant blue, and the total proteinrecovered.

Fig. 1. SDS–PAGE analysis of overproduced HlyX. Lane 1, crudeextract (10 mg) visualized by staining with Coomassie brilliant blue;lane 2, pure HlyX protein (0.8 mg) visualized by silver staining. Theposition of the molecular weight markers, GST–HlyX fusion proteinand HlyX are indicated.

594 J. Green and M. L. Baldwin

with anti-FNR serum in Western blots. Sufficient HlyX pro-tein was produced to study HlyX–DNA interactions but, forstoicheiometric and spectral characterizations, the GST–HlyX fusion protein had to be used.

Physicochemical properties of GST–HlyX

The anaerobic, active form of FNR contains two [4Fe–4S]clusters per dimer (Green et al., 1996a; Lazazzera et al.,1996). As HlyX retains all the cysteine residues that actas ligands for the cluster, it was predicted that HlyXwould possess a similar [4Fe–4S] cluster. Anaerobic incu-bation of the GST–HlyX fusion protein with the NifS-basedreconstitution mixture (cysteine, dithiothreitol (DTT) and10 ferrous ions per monomer) developed for FNR* andFNR (Khoroshilova et al., 1995; Green et al., 1996a)resulted in a straw-brown product with a broad absorptionat 420 nm (Fig. 2a). The model [4Fe–4S] compound

[Fe4S4(S–Et)4]2¹ has an e420 of 17 200 M¹1 cm¹1 and,based on this figure, at least 80% of the HlyX present inthe reconstitution mix contained a [4Fe–4S] cluster. Thisis similar to the value obtained with FNR, indicating thatthe reconstitution procedure is efficient and that the pre-sence of GST in the GST–HlyX fusion protein does notaffect cluster formation. The stoicheiometries of Fe:S:pro-tein (Table 2) indicated that the GST–HlyX protein as pre-pared can contain up to 1.1 atoms of Fe per monomer.After reconstitution with NifS in the presence of 10 ferrousions per monomer, GST–HlyX contains one [4Fe–4S]cluster per monomer.

Scanning the reconstitution reaction at intervals re-vealed the presence of a transient intermediate with anabsorption maximum around 338 nm (Fig. 2b). Occasion-ally this species was observed as a shoulder in the‘fully reconstituted’ HlyX. Reconstitution reactions con-taining only 1 ferrous ion (rather than 10) per GST–HlyX


Fig. 2. Absorption spectra of GST–HlyX. All spectra were obtained with anaerobic samples in sealed cuvettes.a. GST–HlyX (7.5 mM monomer) after reconstitution by the NifS-catalysed procedure with excess ferrous ions.b. Time course of the reconstitution of GST–HlyX (5.3 mM monomer). The reconstitution mix (10 ferrous ions per monomer) was incubated at378C and a spectrum was obtained at time 0 and then after 2 and 4 h.c. Time course of the NifS reconstitution (1 ferrous ion per monomer) of GST–HlyX (4 mM monomer). Spectra were obtained at hourlyintervals.

Table 2. Iron and acid-labile sulphur content ofGST–HlyX. Fe and S contents

(atoms per GST–HlyX monomer)

GST–HlyX Fe S Fe:S ratio

As prepared 0.02–1.10 # 0.02–0.08 –Reconstituted with 10 ferrous ions per monomer 3.4–3.5 2.8–3.0 1.1–1.2Reconstituted with 1 ferrous ion per monomer 0.75–0.80 ND –

The amounts of iron and acid-labile sulphur incorporated into the GST–HlyX fusion proteinafter reconstitution by the NifS-based procedure with 10 or 1 ferrous ions added per monomer.Protein was determined using the Bio-Rad protein reagent and corrected by reference to astandard of GST–HlyX of known amino acid composition. ND, not determined.

The HlyX protein of Actinobacillus pleuropneumoniae 595

monomer generated an absorbance band at 338 nm,failed to produce the 420 nm absorbance (Fig. 2c) andresulted in the incorporation of only 0.8 Fe atoms permonomer (Table 2). This suggests that the 338 nmabsorbing species may represent an intermediate in theformation/degradation of the active [4Fe–4S] cluster.

The stoicheiometric and spectroscopic analyses stronglysuggest that, like FNR, HlyX contains one [4Fe–4S] clus-ter per monomer, i.e. two per dimer.

Oxygen-responsive binding of HlyX to an FNR box

Sufficient HlyX protein was available to investigate HlyX–DNA interactions. Oxygen-responsive DNA binding wasdemonstrated in gel retardation assays using end-labelledndh promoter DNA and reconstituted HlyX protein (Fig. 3).Target DNA (ndh promoter) was bound with a Kd ofapproximately 20 nM (Kd is the concentration of HlyXrequired to retard 50% of the added DNA) if the reconsti-tuted HlyX was maintained under strictly anaerobic condi-tions. However, exposure of the reconstituted HlyX to airfor 30 min prior to the retardation assay resulted in theloss of DNA-binding activity (Fig. 3). Aerobically, unrecon-stituted HlyX failed to retard ndh DNA at concentrations upto 300 nM. Therefore, HlyX binds target DNA with an affi-nity similar to that observed for FNR–DNA interactions(Green et al., 1996a) and there is a greater than 13-foldincrease in DNA binding upon incorporation of the [4Fe–4S] cluster.

DNase I footprinting reactions showed that HlyX protec-ted both FNR sites in the ndh promoter (Fig. 4). The sizesof the protected regions (24 bp) were similar to thoseobserved previously with FNR (Sharrocks et al., 1991),indicating that HlyX can bind as a dimer to the same targetDNA sequence as FNR. Even though the FNR II site isessentially a half site, the size of the protected region isconsistent with the presence of a HlyX dimer at this site,supporting results from previous studies of FNR–ndhcomplexes (Sharrocks et al., 1991).

Anaerobic expression of HlyX in E. coli

Analysis of the polypeptides in soluble cell-free extractsproduced from anaerobic cultures of JRG3350 (fnr þ) orJRG2269 (hlyX þ) by 2-D gel electrophoresis revealedthe HlyX-mediated induction of one major (14 kDa) andtwo minor (55 kDa and 50 kDa) polypeptides (Fig. 5).These polypeptides were not observed in aerobic culturesof JRG1728 (fnr ¹) or the parental strain MC1000 (fnr þ).Similar analysis of the spent media of these cultures indi-cated that three polypeptides (55 kDa, 35 kDa and 27 kDa)were present only when HlyX was active. The major HlyX-dependent cytosolic protein was identified, by N-terminalamino acid sequence analysis (MITGIQITKA), as the pro-duct of an open reading frame, yfiD (Borodovsky et al.,1994). The yfiD gene is located in the srmB–ung intergenicregion (situated at 58 min in the E. coli linkage map) andencodes a protein of 14.3 kDa with strong similarity tothe C-terminal region of pyruvate formate-lyases.

Inspection of the nucleotide sequence upstream of theyfiD coding region revealed a ribosome-binding site (5 of9 consensus match, CGGGGAGGC, centred at ¹10 rela-tive to the initiating methionine) and two FNR boxes( TTGATTTAAATCAA centred at ¹115.5, and TTGATG-TAAAACAA centred at ¹168.5, with respect to the initiating


Fig. 3. Site-specific DNA-binding is enhanced by the incorporationof [4Fe–4S] clusters into HlyX. Gel retardation assays withradiolabelled ndh DNA and increasing amounts of reconstitutedHlyX kept anaerobically (lanes 1–5) or after exposure to air for30 min (lanes 6–10). The concentrations of HlyX dimers were:lanes 1 and 6, 0 nM; lanes 2 and 7, 5.5 nM; lanes 3 and 8, 11 nM;lanes 4 and 9, 22 nM; and lanes 5 and 10, 44 nM. The positions offree ndh DNA (ndh) and the HlyX–DNA complexes (ndh-HlyX,ndh-2HlyX) are indicated by arrows.

Fig. 4. DNase I footprint of the ndh–HlyX complex. DNase Idigestion of ndh DNA in the presence (þ, 100 nM) and absence (¹)of HlyX. The regions of HlyX protection (open boxes) whichcorrespond to the two FNR sites are indicated. Lane M is acalibrating Maxam and Gilbert G track.


methionine). Several potential ¹10 and ¹35 elementscould be identified; however, transcript mapping by primerextension indicated that the yfiD transcript is initiated froma position 61.5 base pairs downstream of the consensusFNR site (Fig. 6a). Gel retardation analysis indicatedthat HlyX binds to two sites in the yfiD promoter with aKd of approx. 30 nM (Fig. 6b). Footprinting analysis ofthe HlyX–yfiD and FNR–yfiD promoter binary complexesrevealed similar protection patterns with both regulatorsoccupying the regions ¹78 to ¹48 and ¹128 to ¹96 cov-ering the FNR boxes centred at ¹61.5 and ¹114.5 (Fig. 6,c and d). Some distortion of promoter DNA was indicatedby the presence of hypersensitive bases both within andbetween the protected regions. Thus it would appearthat HlyX is able to efficiently recognize and activate tran-scription from FNR boxes centred at ¹61.5 and beyond,whereas FNR cannot. This was confirmed by estimatinganaerobic yfiD promoter activity using a yfiD ::lacZ repor-ter plasmid (Fig. 7a). Expression of yfiD was anaerobi-cally induced 170-fold by HlyX but only 17-fold by FNR.

Identification of regions of HlyX required fortranscription activation

The footprinting analyses indicate that FNR and HlyXform similar complexes with the yfiD promoter in vitro.However, it appears that only HlyX has the capacity to effi-ciently activate transcription from this promoter in vivo,indicating that HlyX is capable of making an activatingcontact with RNA polymerase that FNR cannot. This pos-sibility was tested by estimating the relative efficiencies ofFNR- and HlyX-mediated anaerobic transcription activa-tion of FFpmelR, FF þ20pmelR and FF-71.5pmelR (Winget al., 1995). The use of the semi-synthetic promoters sim-plifies the interpretation of the results by eliminating any

possible effects caused by the presence of multiple regu-lator-binding sites in yfiD. Transcript mapping by primerextension indicated that both FNR and HlyX initiated tran-scription from the same position, i.e. 41.5 and 61.5 bpdownstream of the FNR binding site of FFpmelR andFF þ20pmelR, respectively (Fig. 7b). Experiments esti-mating the anaerobic induction of b-galactosidase activityof cultures expressing either HlyX or FNR (Fig. 7c) unequi-vocally show that FNR has a strong preference for pro-moters with an FNR site positioned at ¹41.5 (FFpmelR)except when the promoter has an improved ¹35 element(FF-71.5pmelR) as previously reported (Wing et al.,1995). However, HlyX is not as discriminating in so faras it will efficiently activate transcription from all the pro-moters tested (Fig. 7). The molecular basis of this abilitywas investigated after inspection of amino acid sequencealignments of the known FNR-like proteins and correlationof specific amino acid residues with the ability to endowthe haemolytic phenotype. Three regions of HlyX wereselected for investigation. First, there is a section of b7

(104MNMKHV109) which is very different from the equiva-lent region of FNR (105GSGHHP110) and contains K107which is conserved in another ‘haemolytic’ FNR, Btr fromBordetella pertussis. Second, there is Y180 in the loopbetween aD and b9 which is replaced in by F181 in FNR,and, finally, A187 in the loop between b9 and b10 isreplaced by P188 in FNR (Fig. 8). Site-directed mutagen-esis was used to replace selected residues, and the effectof the substitutions on the transcription activation proper-ties of the mutants was compared to those of HlyX andFNR (Fig. 7c, Table 3).

Substitutions in b7 had no effect on the ability of HlyX toactivate transcription from FF þ20pmelR and yfiD, or tocomplement the anaerobic respiratory deficiency of anfnr mutant (Fig. 7, Table 3). One substitution, K107→A


Fig. 5. The influence of HlyX on the cellular protein composition of E. coli JRG1728. Two-dimensional electrophoretic analysis of proteins inanaerobically grown E. coli (a) JRG3350 (fnr þ) or (b) JRG2269 (hlyX þ). Polypeptides were visualized by staining with Coomassie brilliantblue. The polypeptides unique to HlyXþ bacteria are indicated (arrows). The major HlyX-dependent polypeptide running at 14 kDa (largearrow) was identified as the yfiD gene product. The positions of the molecular mass markers (kDa) are indicated.


did reduce the induction of haemolytic activity and maythus identify the one of the regions responsible for endow-ing the haemolytic phenotype (Green et al., 1992). In thesame region, the substitution N105→A improved the abil-ity of HlyX to activate transcription from FFpmelR (Fig. 7)and thus this region may form part, or alter the presenta-tion, of the previously defined activating region 3 (AR3;Fig. 8) which makes a positive contact with RNA polymer-ase when FNR is positioned at ¹41.5 (Bell and Busby,1994). Alternatively it may define a new regulator–RNApolymerase contact patch.

The substitution Y180→F resulted in a marginally weakerhaemolytic phenotype, reduced expression of yfiD, and

slightly reduced FF þ20pmelR and FF-71.5pmelR activa-tion (Fig. 7, Table 3). However, the substitution A187→Presulted in an HlyX protein compromised in the ability to:(i) activate transcription from FFpmelR, FF þ20pmelRand FF-71.5pmelR; (ii) induce expression of yfiD; and (iii)endow the haemolytic phenotype (Fig. 7, Table 3). Tran-scription activation by HlyX–A187P from FF þ20pmelRwas 3.5-fold lower than that observed with HlyX and two-fold lower from FF-71.5pmelR such that the level of b-galactosidase expression was similar to that observedwith FNR. Activation of transcription from FFpmelR byHlyX–A187P was 1.9-fold lower than that observed withHlyX. Therefore, the turn between b9 and b10 contributes


Fig. 6. Interactions of HlyX and FNR with the yfiD promoter.a. Location of the transcript start site of the yfiD promoter by primer-extension analysis. The 58 end of the anaerobic yfiD transcript wasdetermined by comparing the size of the primer-extended molecules (lane 1, HlyX-expressing cells) with two sets of calibrating Maxam andGilbet G track markers (lane 3, ndh promoter DNA; lane 4, yfiD promoter DNA).b. Gel retardation analysis of with anaerobically reconstituted HlyX and the yfiD promoter. Increasing concentrations of HlyX dimer (nM) wereused as follows: lane 1, 0; lane 2, 3; lane 3, 6; lane 4, 15; lane 5, 30; lane 6, 60; lane 7, 120; lane 8, 240. The positions of free DNA and theHlyX–yfiD complexes are indicated.c. DNase I footprints of HlyX–yfiD and FNR–yfiD binary complexes. Lane 1, no protein; lane 2, 200 nM HlyX; lane 3, 400 nM HlyX; lane, 4;200 nM FNR; lane 5, 400 nM FNR; lane M, calibrating Maxam and Gilbert G track generated from yfiD promoter DNA. The regions ofprotection afforded by FNR or HlyX (FNR boxes I and II) and hypersensitive bands (arrowed) are indicated.d. Nucleotide sequence of the yfiD promoter region. The following features are identified: the FNR-binding sites (boxed); the ¹35 and ¹10hexamers (italic and overlined); the transcript start point (þ1); the ribosome-binding site (bold); and the initiating methionine (Met).



Fig. 7. Anaerobic activation of Class I andClass II promoters by FNR and HlyX.a. Regulation of yfiD expression by FNR andHlyX. Aer, aerobic; Ana, anaerobic; openbars, FNR; closed bars, HlyX.b. Location of the transcript start site of theFFpmelR and FF þ20pmelR promoters byprimer-extension analysis. The 58 ends of thetranscripts were determined by comparing theprimer-extended molecules (lane 1, FFpmelRfrom FNR-expressing cells; lane 2,FF þ20pmelR from FNR-expressing cells;lane 5, FFpmelR from HlyX-expressing cells;lane 6, FF þ20pmelR from HlyX-expressingcells) with two sets of calibrating Maxam andGilbert G tracks (lane 3, ndh promoter DNA;lane 4, yfiD promoter DNA).c. The effect of various amino acidsubstitutions on the anaerobic activation ofthe FF series of promoters by HlyX. Promoteractivity was estimated by measuring b-galactosidase (Miller units) produced afteranaerobic growth of JRG1728 (Dfnr) carryingthe indicated pRW50 derivatives encoding thelac operon under the control of different FNR-dependent promoters (FFpmelR, FNR box at¹41.5, closed bars; FF þ20pmelR, FNR boxat ¹61.5, open bars; and FF-71.5pmelR, FNRbox at ¹71.5 and an improved ¹35 element,shaded bars). FNR, HlyX and HlyXderivatives were introduced on a secondplasmid encoding FNR (pGS330), HlyX(pGS415), HlyX–M104A (pGS967), HlyX–N105A (pGS968), HlyX–K107A (pGS990),HlyX–Y180F (pGS993) and HlyX–A187P(pGS1003). The values quoted are theaverage of duplicate assays from at leastthree independent cultures.

Fig. 8. Diagram of the three-dimensionalstructure of the FNR/HlyX subunit based onthe crystal structure of CRP. The a-helices,A–F (cylinders) b-strands 1–12 (arrows),essential cysteines (ringed), the DNA-bindinghelix and its cognate recognition sequenceare indicated. The positions of four regionsinvolved in activation of RNA polymerase areshown: AR3, characterized by FNR–G85A,which is impaired in the activation of Class IIpromoters, and FNR–S73F, which is defectivein activation at both Class I and II promoters,identified previously are shown stippled; andthe extended AR3/AR2 of HlyX, characterizedby HlyX–N105A, which is improved in theactivation of Class II promoters, and AR1 ofHlyX, defined by HlyX–A187P which isimpaired in the activation of both Class I andClass II promoters, identified here are shownfilled. The numbering is for FNR unlessotherwise indicated.


to transcription activation irrespective of the position of theFNR/HlyX-binding site, but is more important when theHlyX binds upstream of ¹41.5.

Discussion

The HlyX protein of the pig pathogen A. pleuropneumo-niae is very similar to FNR, the anaerobic transcriptionregulator of E. coli. The present work clearly shows that,similar to FNR, HlyX responds to oxygen availability viaa redox-sensitive [4Fe–4S] cluster, incorporation of whichresults in enhanced site-specific DNA binding (Green etal., 1996a; Lazazzera et al., 1996).

Analysis of the kinetics of the reconstitution reactionrevealed the formation of an intermediate single-iron spe-cies associated with an absorbance at 338 nm during theformation of the [4Fe–4S] cluster. It may be similar tothe single-iron form of FNR that is often isolated after aero-bic purification in the presence of ferrous ions (Green etal., 1991) and could represent the portion of aerobicFNR preparations that is capable of chemical reconstitu-tion to an active form (Green and Guest, 1993a,b). A simi-lar absorbance band has been observed in some FNRmutants (E. T. Ralph, J. Green and J. R. Guest, unpub-lished) and in non-enzymically reconstituted FNR (Greenet al., 1996b), and thus the 338 nm absorption does notarise as a consequence of using the GST–HlyX fusion.It is tempting to speculate that the single iron atom is asso-ciated with the equivalent of C122 of FNR (C121 in HlyX)which appears to be particularly important for iron bindingin FNR (Green et al., 1993). It is probably safe to assumethat the one-iron species is not of the rubredoxin typebecause such proteins have very distinctive visible spectra(Lovenburg, 1972). The pathway leading to the formationof iron–sulphur clusters is poorly understood; however,more information is available regarding the destruction ofthe [4Fe–4S]2þ cluster of aconitases which undergo

oxidative degradation. The four-iron cluster first forms a[3Fe–4S]þ cluster which can then pass through a [2Fe-2S]þ stage before final cluster destruction (Busch et al.,1996; Kennedy et al., 1984). Thus far, a one-iron inter-mediate has not been identified but the spectral and stoi-cheiometric data presented here suggest that such aspecies may exist and further investigation is merited.

Recently, a latent haemolysin of E. coli K-12 has beenidentified as the 34 kDa protein, cytolysin A (Oscarssonet al., 1996). Cytolysin A is a contact-dependent haemoly-sin secreted by some strains of E. coli with particulargenetic backgrounds, including hns mutants and strainsbearing clyA- or slyA-expressing plasmids. The SlyAprotein is thought to be a regulator of clyA expression,although the exact nature of the regulation is unclear(Oscarsson et al., 1996). It is possible that ClyA is theHlyX-activated haemolysin. Analysis of the polypeptidessecreted by anaerobically grown hlyX-expressing culturesindicated the presence of a 35 kDa protein that could beClyA, but too little was produced for a positive identifica-tion; indeed, it is has been shown that the HlyX-inducedhaemolysin is a very low abundance protein (Soltes andMacInnes, 1994).

The major polypeptide induced by HlyX was a cytosolicprotein identified as the yfiD gene product. The role of yfiDin E. coli is unknown but its product is similar to the C-terminal region of another FNR- and HlyX-induced protein,pyruvate formate lyase. A yfiD mutant is being constructedand its anaerobic growth characteristics will be deter-mined. Analysis of the promoter region of this gene allowedthe identification of FNR binding sites centred at ¹61.5and ¹114.5. The architecture of the yfiD promoter is there-fore unlike any other previously analysed FNR-activatedpromoter (Guest et al., 1996) and the contribution ofeach FNR site to the overall regulation of yfiD expressionis being investigated. The genes regulated by the CRP/FNR family can be grouped into three classes based on


Table 3. Transcription activation properties of FNR, HlyX, and HlyX mutants.

Complementation of an Anaerobic production Activation of Class I Activation of Class IIRegulator fnr lesiona of yfiD (%)b Haemolytic phenotypec promoters (%)d promoters (%)e

FNR þþþþþ ND ¹ 17 100HlyX þþþþþ 100 þþþþþ 100 50HlyX–M104A þþþþþ 111 þþþþþ 113 52HlyX–N105A þþþþþ 89 þþþþþ 100 105HlyX–K107A þþþþþ 82 þþþ 99 50HlyX–Y180F þþþþþ 111 þþþþ 84 49HlyX–A187P þþþþþ 48 þ 29 25

a. The capacity of the regulators to anaerobically complement an fnr lesion. Growth of transformants on glycerol-nitrate minimal medium wasscored on a þþþþþ (extent of growth with FNR) to ¹ (extent of growth of JRG1728 (Dfnr)) scale.b. The capacity of the regulators to anaerobically induce expression of yfiD, i.e. the relative amount of the yfiD gene product observed after quan-titative densitometry of 2-D gels (HlyX equivalent to 100%; ND, not detected).c. The capacity of the regulators to anaerobically endow a haemolytic phenotype, scored on a scale of þþþþþ (HlyX) to ¹ (FNR).d. The capacity of the regulators to anaerobically activate Class I promoters (HlyX equivalent to 100% ¼ 780 Miller units).e. The capacity of the regulators to anaerobically activate Class II promoters (FNR equivalent to 100% ¼ 3800 Miller units).


the position of the DNA site for the regulator and therequirements for transcription activation (Ebright, 1993).Class I promoters have CRP/FNR sites centred at ornear ¹61.5, ¹72.5, ¹82.5 or ¹92.5, and thus the yfiDpromoter is a variant of the Class I type. Class I FNR-dependent promoters are rare and, with the exception ofaeg-46.5, where the FNR site is centred at ¹64.5 (Darwinand Stewart, 1995), the evidence for their existence is notrigorous (Guest et al., 1996). This is consistent with theweak FNR-dependent activation of yfiD and other Class Ipromoters, and the finding that FNR-activated promotersare generally Class II promoters with an FNR site closeto ¹41.5 (Guest et al., 1996).

Transcription is activated from CRP-dependent Class IIpromoters by direct CRP contacts with both the a and s

subunits of RNA polymerase (Williams et al., 1996).CRP contains three activating regions (AR1, AR2 andAR3) that make direct contact with RNA polymerase(Savery et al., 1996). AR1 of the upstream CRP monomercontacts the C-terminal domain of the a subunit of RNApolymerase, whereas AR2 and AR3 of the downstreamsubunit contact the N-terminal domain of the a subunitof RNA polymerase and region 4 of s70, respectively(Savery et al., 1996; Niu et al., 1996). The AR2 and AR3contacts are not possible and are not required for tran-scription activation from Class I promoters where a singlecontact between the C-terminal domain of a and AR1 ofthe downstream CRP subunit is sufficient. Genetic analy-sis of FNR has identified a region between b4 and b5 whichis largely conserved in HlyX, corresponding to AR3 andcharacterized by the mutation G85→A (Williams et al.,1991; Bell and Busby, 1994). The altered HlyX proteinsstudied here suggest that AR3 of HlyX may also includea region of b7, or that b7 may contribute to AR2 of HlyX.The substitution N105→A improved activation of Class IIpromoters by HlyX such that the level of activation wassimilar to that observed with FNR (Fig. 7), suggestingthat HlyX has a weaker AR2/3 compared to FNR. Interest-ingly, the FNR mutation F112→ l isolated by Bell andBusby (1994) also lies in b7 but, in contrast to the HlyXmutations described here, impairs the activation of tran-scription at FFpmelR and may thus also form part of theAR2 or AR3 of FNR/HlyX. Further experiments with alteredRNA polymerases will be necessary to define the b7 con-tact (the N-terminus of the a subunit, an AR2 contact; orregion 4 of the s70 subunit, an AR3 contact).

Crucially, no FNR equivalent of AR1 has yet been iden-tified, although the properties of the FNR mutant S73→Fsuggested that FNR has an AR1 (Bell and Busby, 1994)and it is this region that is required for activation fromClass I promoters such as yfiD. FNR can activate expres-sion of synthetic promoters with FNR boxes 41, 61, 71, 82and 92 bp upstream of the transcription start point (Gastonet al., 1990; Wing et al., 1995). However, for efficient

FNR-dependent activity from the remote sites (–61 andbeyond) an improved ¹35 hexamer ( TAAAGA improvedby a single base insertion to TTAAAG) was required(Wing et al., 1995). The natural FNR Class I promoter(Darwin and Stewart, 1995) similar to yfiD has a relativelypoor ¹35 element (aeg-46.5, 2 out of 6 match the con-sensus, TGCATC; yfiD, 1 out of 6 match the consensus,GAGGAG). However, maximum expression of aeg-46.5requires an additional transcription regulator, NarP, whichbinds between RNA polymerase and FNR. Analysis ofthe sequence of the yfiD promoter region reveals no evi-dence for NarP- or NarL-binding sites. Thus, it is likelythat HlyX has a much better AR1 than does FNR and isable to make an improved activating contact with RNApolymerase at Class I promoters. This conclusion wassupported by studies with the FF series of lacZ reporterfusions.

The AR1 of HlyX was partially defined as a surface-exposed region which includes A187 (Fig. 8). A previouslycharacterized FNR protein (FNR–S73F) is defective inactivation at both Class I and Class II synthetic FNR-dependent promoters (Bell and Busby, 1994; Wing et al.,1995). However, the substitution S73→A did not affectthe ability of FNR to activate transcription; therefore, it islikely that FNR possesses an AR1 equivalent but thatS73 does not form part of it. This work suggests that, byanalogy with HlyX, the AR1 of FNR is located, at least inpart, in the loop between b9 and b10 and makes a relativelypoor activating contact with RNA polymerase. Partial pro-teolysis of FNR with trypsin results in the release of a poly-peptide initiating at G185 indicating that this region isaccessible and readily available for protein–protein inter-actions (J. Green, unpublished). AR1 may extend as faras Y180 since HlyX–Y180F is slightly compromised inthe ability to activate transcription from FF þ20pmelR.

In conclusion, it would appear that the molecular basisof the ability of HlyX to activate the anaerobic expressionof a number of E. coli genes that are apparently notinduced by FNR is, at least in part, a consequence ofthe effectiveness of the respective AR1 contacts withRNA polymerase. FNR has a weaker AR1 and requiresadditional elements (such as an improved ¹35 hexameror an additional transcription regulator) to efficiently acti-vate transcription from Class I promoters, but is an effec-tive activator of transcription from Class II promoters viaAR3–RNA polymerase contacts. HlyX has the ability toactivate transcription from both Class I and Class II pro-moters by virtue of having a better AR1 as well as anAR3. The AR1 region of HlyX (and FNR) is defined bythe substitution A187→P and resides in the loop betweenb9 and b10. The HlyX substitution (N105→A) in b7 maydefine an additional region of AR3 or may define theequivalent of the CRP AR2. There is a need to furthercharacterize the exact nature of FNR–RNA polymerase



and HlyX–RNA polymerase contacts. It was previouslyestablished that the expression of the latent haemolysinof E. coli required elements from both the N- and C-termi-nal segments of HlyX (Green et al., 1992). Two of thealtered HlyX proteins studied here, HlyX–K107A andHlyX–A187P, were compromised in their ability to endowa haemolytic phenotype and thus both AR1 and AR2/3contacts appear to be important for the anaerobic induc-tion of haemolytic activity. It will be particularly interestingto determine whether the K107 (AR2/3) region is a specificcontact for RNA polymerase or if it can interact with otherregulators such as SlyA (Oscarsson et al., 1996) to allowhaemolysin expression. However, as anaerobiosis appearsto be sufficient for the induction of E. coli clyA and A.pleuropneumoniae hlyA (Hattori et al., 1996) expressionin the presence of HlyX, it is predicted that these promo-ters will contain FNR boxes centred at or upstream of¹61.5. Similarly, it is predicted that virulent strains of E.coli may possess FNR proteins capable of enhancedtranscription activation at Class I promoters.

Experimental procedures

Bacterial strains and plasmids

E. coli BL21 (F¹, ompT, hsdS (rB¹, mB

¹), gal ) transformed withpGS903, constructed by ligating the hlyX-containing BamHI–Sal I fragment from pGS415 (Green et al., 1992) into pGEX-KG (Guan and Dixon, 1991), was used to overproduce theGST–HlyX fusion protein. Plasmids used to test the effectsof the anaerobic expression of fnr, hlyX and the hlyX mutantswere the ptac85 derivatives: pGS330, FNR (Green et al.,1991); pGS415, HlyX (Green et al., 1992); pGS967, HlyX–M104A; pGS968, HlyX–N105A; pGS990, HlyX–K107A;pGS993, HlyX–Y180F; pGS1003, HlyX–A187P. StrainJRG1728 (D(lacIPOZYA)X74 galU galK rpsL D(ara–leu)D(tyrR–fnr–rac–trg)17 zdd-230 ::Tn9) was transformed withthe ptac85 derivatives to create strains carrying fnr (JRG3350),hlyX (JRG2269) or mutated hlyX genes encoding HlyX–M104A (JRG3384), HlyX–N105A (JRG3385), HlyX–K107A(JRG3428), HlyX–Y180F (JRG3430) and HlyX–A187P(JRG3455) for use in testing the effects of the different regu-lators on protein content and haemolytic activity.

The source of the ndh promoter DNA used in the gel retar-dation and footprinting analyses was pGS418 (Sharrocks etal., 1991).

The ability of FNR, HlyX and the altered HlyX proteins toactivate transcription from Class I and Class II promoterswas determined using the semi-synthetic lacZ reporter fusions,FFpmelR (Class II, FNR site at ¹41.5), FF þ20pmelR (ClassI, FNR site at ¹61.5) and FF-71.5pmelR (Class I, FNR site at¹71.5) (Wing et al., 1995) in strain JRG1728 (Dfnr) containingeither: pGS330 to generate JRG3402, 3403 and 3404;pGS415 to generate JRG3405, 3206 and 3407; pGS967 togenerate JRG3408, 3409 and 3410; pGS968 to generateJRG3411, 3412 and 3413; pGS990 to generate JRG3414,3415 and 3416; pGS993 to generate JRG3431, 3432 and3433; and pGS1003 to generate JRG3452, 3453 and 3454.The yfiD–lac operon fusion reporter plasmid pGS1000 was

formed by ligating the polymerase chain reaction-amplifiedyfiD promoter region (–240 to þ62) into EcoRI/BamHI-digested pRW50. This plasmid was introduced into JRG3350and JGR2269 to generate JRG3450 and JRG3451, respec-tively. Cultures were grown aerobically in Luria (L) brothwith vigorous shaking or anaerobically in L broth supplemen-ted with 0.4% glucose to mid-exponential phase (OD600 0.07–0.12) in sealed bottles at 378C. Beta-galactosidase activitywas determined according to Miller (1972). The EcoRI–BamHI yfiD promoter fragment of pGS1000 was transferredto pUC118 to create pGS1036 for sequencing and wasused as a source of promoter DNA for footprinting and gelretardation reactions.

Western blotting with anti-FNR serum indicated that theexpression of HlyX and the altered HlyX proteins was similaralthough they were all expressed at lower levels than FNR,consistent with the poor codon usage found in the hlyX gene.

Protein purification and GST–HlyX/HlyX reconstitution

HlyX was initially purified as a GST–HlyX fusion proteinamplified in aerobically grown E. coli BL21(pGS903). Cultureswere grown at 378C with vigorous shaking until OD600 0.5–0.7when the expression of the GST–HlyX fusion was initiated byaddition of IPTG to a final concentration of 30 mg l¹1. Incuba-tion was continued for a further 3 h before the cultures wereharvested by centrifugation. Clarified French pressure cellextracts (up to 150 mg total protein) were applied to a column(50 ×15 mm) of glutathione agarose (Sigma) equilibrated with50 mM Tris-HCl, pH 6.8, containing 0.5 mM DTT. The columnwas washed with 10 vols of the same buffer before eluting theGST–HlyX fusion with 50 mM Tris-HCl, pH 8.0, containing10 mM reduced glutathione. Fractions containing the fusionprotein were pooled and applied to a column (40 ×15 mm) ofheparin agarose equilibrated with 50 mM Tris-HCl, pH 8.0.After washing the column with 10 vols of the same buffer,the GST–HlyX protein was eluted with 50 mM Tris-HCl,pH 6.8, containing 0.4 M ammonium sulphate. At this stage,the fusion protein was essentially pure and, after desaltingon Sephadex G25, could be used for reconstitution and physi-cochemical characterization. Pure HlyX protein was obtainedby cleavage of the desalted GST–HlyX fusion with thrombin(14 U) for 3 h at 378C followed by anion-exchange chromato-graphy on a Bio-Scale Q (2 ml) column. HlyX was eluted witha linear gradient (22 ml) of 0–0.5 M NaCl in 20 mM Tris-HCl,pH 8.0. The product contains an additional two amino acidsderived from the thrombin-sensitive linker, whose presencewas confirmed by total amino acid analysis and N-terminalsequencing. Protein concentrations were estimated usingthe Bio-Rad protein reagent (Bradford, 1976). Bovine serumalbumin served as the standard, with a correction factor of1.1 being applied to allow for the difference between this stan-dard and a reference sample of GST–HlyX (analysed by AltaBioscience, University of Birmingham). NifS protein was puri-fied from E. coli BL21(lDE3) containing the nifS expressionplasmid pDB551 (Zheng et al., 1993).

The incorporation of iron and sulphur into HlyX and GST–HlyX was achieved by a modification of the method of Greenet al. (1996a). HlyX or GST–HlyX solutions (0.1–2.0 ml;approx. 0.04–0.8 mg ml¹1 protein) were incubated under N2

with 0.14 mM NifS protein, 1 mM L-cysteine (Sigma) and either



10 or 1 mol Fe2þ (as (NH4)2Fe(SO4)2; A.C.S. grade, Sigma)per mol of HlyX monomer and 2.5 mM DTT (Sigma), for 3 hat 378C in a sealable vessel in a Whitley Anaerobic Worksta-tion. The reaction products were applied to a 10 ml SephadexG25 (Pharmacia) column that had been equilibrated withanoxic 50 mM Tris-HCl buffer (pH 8.0) containing 1 mM DTTand 0.1 M NaCl, in the anaerobic glovebox, and the reconsti-tuted protein was eluted in the same buffer. For experimentsin which a series of spectra were obtained during the courseof a reconstitution, the reaction was allowed to proceed at378C in a sealed cuvette.

Iron contents were determined with bathophenanthroline onwet-ashed samples, and acid-labile sulphur of denaturedsamples of FNR protein was determined by the method ofBeinert (1983) in an anaerobic glovebox.

Spectroscopy

Samples of reconstituted GST–HlyX were removed from theanaerobic glovebox in sealed quartz cuvettes, incubated at378C in a Unicam UV-2 spectrophotomoter (Pye-Unicam),and spectra were obtained at the indicated intervals.

HlyX–DNA interactions

Anaerobic gel retardation assays with the ndh or yfiD promo-ters (0.05 pmol in 10 ml) were essentially as described (Greenet al., 1996a) except that ndh or yfiD replaced the FFmelRDNA and HlyX replaced FNR. After electrophoresis, the per-centage of total DNA retarded was estimated from autoradio-graphs by quantitative densitometry using a Vilber–LourmatBioprofil imaging system.

DNase I footprinting was carried out essentially as des-cribed previously (Green et al., 1991) except that the reac-tions were performed anaerobically and contained ndh oryfiD promoter DNA (10–100 ng), end-labelled at the BamHIsite (non-coding strand) of the EcoRI–BamHI fragment ofpGS418 or pGS1036, respectively; HlyX or FNR (100–800 nM); 2 ml of 5× binding buffer (0.1 M Tris-HCl pH 8.0,0.05 M MgCl2, 50 mM DTT and 25% glycerol); in a totalvolume of 10 ml. The mixtures were incubated for 10 min at258C followed by digestion with DNase I (1 ml of 1 U ml¹1 for60 s at 258C). Reactions were stopped by addition of 200 mlof 0.3 M sodium acetate containing 10 mM EDTA, followedby phenol–chloroform extraction. The DNA was ethanol preci-pitated and resuspended in 7 ml of loading buffer (40% (v/v)formamide, 5 M urea, 5 mM NaOH, 1 mM EDTA, 0.03% w/vbromophenol blue and 0.03% (w/v) xylene cyanol) for electro-phoretic fractionation on polyacrylamide–urea gels and auto-radiographic analysis. Maxam and Gilbert G tracks were usedto provide a calibration.

Two-dimensional gel electrophoresis

A Pharmacia Multiphor II two-dimensional gel electrophoresisapparatus was used with pre-cast gels. The soluble fraction ofsonic extracts or the spent media of anaerobic, stationaryphase (16 h growth) cultures were applied (10–100 mg totalprotein) to Imobiline drystrips IEF 3–10 gels (Pharmacia)and electrophoresed according to the manufacturer’s instruc-tions. The second dimension was run immediately after

completion of the first on Excelgel SDS (Pharmacia), 8–18% gradient gels according to the manufacturer’s instruc-tions. Polypeptides were then visualized using the Pharmaciaplusone silver-staining kit or using Coomassie brilliant blue.For amino acid sequencing, the second-dimension gel wasblotted onto Problot membrane (Applied Biosystems), andthe polypeptides were visualized by light staining with Coo-massie brilliant blue. The relative abundance of selectedspots was estimated by quantitative densitometry using aVilber–Lourmat imaging system. Spots of interest wereexcised and analysed on an Applied Biosystems Inc. 476Aamino acid analyser.

Transcript mapping

The transcription start point of the yfiD, FFpmelR andFF þ20pmelR promoters was determined by RNA extractionand primer-extension analysis. Total RNA was preparedfrom JRG1728 (pGS415), JRG3402, JRG3403, JRG3405and JRG3406 after anaerobic growth in the presence ofIPTG (30 mg l¹1) as described by Aiba et al. (1981). For pri-mer extension, the method of Gerischer and Durre (1992)was used with 10 pmol of primer S377 (58-GTAATCTG-GATCCCTGTAATCATG-38, yfiD co-ordinates 7652–7675)or S421 (58-GTCGCTGCTGCACATAAAC-38, FF(þ20)pmelRco-ordinates ¹291 to ¹273, numbered according to Web-ster et al., 1987), 100 mg RNA and AMV reverse transcriptase(50 U) (Nbl). After ethanol precipitation, the cDNA was frac-tionated on urea–polyacrylamide gels and analysed byautoradiography. The gels were calibrated using Maxamand Gilbert G tracks of ndh and yfiD DNA.

Other methods

The altered HlyX protein HlyX–M104A was generated usingthe Altered Sites II System (Promega) with the following muta-genic oligonucleotide: S378, 58-GGTCGGATTTGATGCGAT-TgcGAATATGAAACATGTCGG-38 (hlyX co-ordinates 477–515). The presence of the directed M104→A substitutionwas confirmed by automated DNA sequencing. Similarly: forHlyX–N105A, the mutagenic oligonucleotide S379, 58-GA-TGCGATTATGgcTATGAAACATGTCGGTTTCGC-38 (hlyXco-ordinates 488–521) was used; for HlyX–K107A, S380, 58-GCGATTATGAATATGgcACATGTCGGTTTC-38 (491–519)was used; for HlyX–Y180F, S381, 58-TATCTCAACGTTtTG-CGGCACCG-38 (713–735) was used; and for HlyX–A187P,S389, 58-GTTTTTCCcCTCGTGAATTC-38 (737–756) wasused.

The ability of FNR, HlyX and the altered HlyX proteins toconfer a haemolytic phenotype was assessed after anaerobicincubation for 16 h at 378C on blood (7% horse blood) agar, ofE. coli JRG1728 expressing fnr, hlyX or the mutant hlyXgenes from the tac promoter of ptac85 (Green et al., 1992).The ability of the regulators to complement the JRG1728 fnrmutation was assessed by their capacity to restore anaerobicgrowth on leucine-supplemented glycerol nitrate agar (Lamb-den and Guest, 1976).

Acknowledgements

We would like to thank Prof. J. R. Guest for many helpful dis-cussions, Prof. S. J. W. Busby and Dr H. Wing for useful



discussions and the gift of the FF–lacZ reporters, Dr J.MacInnes for help in identifying ‘haemolytic’ FNR proteins,and Dr A. J. Moir for amino acid and DNA sequencing. Thiswork was supported by the BBSRC.

References

Aiba, H., Adhya, S., and Decrombrugghe, B. (1981) Evidencefor two functional gal promoters in intact Escherichia colicells. J Biol Chem 256: 1905–1910.

Beinert, H. (1983) Semi-micro methods for the analysisof labile sulfide and of labile sulfide plus sulfane sulfur inunusually stable iron–sulfur proteins. Anal Biochem 131:373–378.

Bell, A., and Busby, S.J.W. (1994) Location and orientation ofan activating region in the Escherichia coli transcriptionfactor, FNR. Mol Microbiol 11: 383–390.

Borodovsky, M., Rudd, K.E., and Koonin, E.V. (1994) Intrin-sic and extrinsic approaches for detecting genes in a bac-terial genome. Nucl Acids Res 22: 4756–4767.

Bradford, M.M. (1976) A rapid and sensitive method for thequantitation of microgram quantities of protein utilisingthe principle of protein-dye binding. Anal Biochem 72:248–254.

Busch, J.H.L., Breton, J., Bartlett, B.M., James, R., Hatchi-kian, E.C., and Thomson, A.J. (1996) Expression inEscherichia coli and characterisation of a reconstitutedrecombinant 7Fe ferredoxin from Desulfovibrio africanus.Biochem J 314: 63–71.

Darwin, A.J., and Stewart, V. (1995) Nitrate and nitriteregulation of the Fnr-dependent aeg–46.5 promoter ofEscherichia coli K-12 is mediated by competition betweenhomologous response regulators (NarL and NarP) for acommon DNA-binding site. J Mol Biol 251: 15–29.

Ebright, R.E. (1993) Transcription activation at Class I CAP-dependent promoters. Mol Microbiol 8: 797–802.

Frey, J., and Nicolet, J. (1988) Regulation of hemolysinexpression in Actinobacillus pleuropneumoniae serotype1 by Ca2þ. Infect Immun 56: 2570–2575.

Frey, J., Stucki, U., and Nicolet, J. (1993a) Analysis of hemo-lysin operons in Actinobacillus pleuropneumoniae. Gene123: 51–58.

Frey, J., Bosse, J.T., Chang, Y-F., Cullen, J.M., Fenwick, B.,Gerlach, G.F., Gygi, D., Haesebrouck, F., Inzana, T.J.,Jansen, R., Kamp, E.M., Macdonald, J., MacInnes, J.I.,Mittal, K.R., Nicolet, J., Rycroft, A., Segers, R.P.A.M.,Smits, M.A., Stenbaek, E., Struck, D.K., van den Bosch,J.F., Willson, P.J., and Young, R. (1993b) Actinobacilluspleuropneumoniae RTX toxins: uniform designation of hae-molysins, cytolysins pleurotoxin and their genes. J GenMicrobiol 139: 1723–1728.

Gaston, K., Bell, A., Kolb, A., Buc, H., and Busby, S. (1990)Stringent spacing requirements for transcription activationby CRP. Cell 62: 733–743.

Gerischer, V., and Durre, P. (1992) Messenger-RNA analysisof the adc gene region of Clostridium acetobutylicumduring the shift to solventogenesis. J Bacteriol 174: 426–433.

Green, J., and Guest, J.R. (1993a) A role for iron in transcrip-tion activation by FNR. FEBS Lett 329: 55–58.

Green, J., and Guest, J.R. (1993b) Activation of FNR-dependent transcription by iron: an in vitro switch forFNR. FEMS Microbiol Lett 113: 219–222.

Green, J., Trageser, M., Six, S., Unden, G., and Guest, J.R.(1991) Characterization of the FNR protein of Escherichiacoli, an iron binding transcriptional regulator. Proc R SocLond B 244: 137–144.

Green, J., Sharrocks, A.D., MacInnes, J.I., and Guest, J.R.(1992) Purification of HlyX, a potential regulator of haemo-lysin synthesis, and properties of HlyX: FNR hybrids. ProcR Soc Lond B 248: 79–84.

Green, J., Sharrocks, A.D., Green, B., Geisow, M., andGuest, J.R. (1993) Properties of FNR proteins substitutedat each of the five cysteine residues. Mol Microbiol 8:61–68.

Green, J., Bennett, B., Jordan, P., Ralph, E.T., Thomson,A.J., and Guest, J.R. (1996a) Reconstitution of the [4Fe–4S] cluster in FNR and demonstration of the aerobic-anaerobic transcription switch in vitro. Biochem J 316:887–892.

Green, J., Irvine, A.S., Meng, W., and Guest, J.R. (1996b)FNR–DNA interactions at natural and semi-syntheticpromoters. Mol Microbiol 19: 125–137.

Guan, K and Dixon, J.E. (1991) Eukaryotic proteinsexpressed in Escherichia coli : an improved thrombincleavage and purification procedure of fusion proteinswith glutathione S-transferase. Anal Biochem 192: 262–267.

Guest, J.R., Green, J., Irvine, A.S., and Spiro, S. (1996) TheFNR modulon and FNR-regulated gene expression. InRegulation and Gene Expression in Escherichia coli. Lin,E.C.C., and Lynch, A.S. (eds). Austin Texas: R.G. Landes& Co, pp. 317–342.

Hattori, T., Takahashi, K., Nakanishi, T., Ohta, H., Fukui, K.,Taniguchi, S., and Takigawa, M. (1996) Novel FNR homo-logues identified in four representative oral facultativeanaerobes. FEMS Microbiol Lett 137: 213–220.

Inzana, T.J. (1991) Virulence properties of Actinobacilluspleuropneumoniae. Microb Pathog 11: 305–316.

Kennedy, M.C., Kent, T.A., Emptage, M.H., Merkle, H.,Beinert, H., and Munck, E. (1984) Evidence for the forma-tion of a [3Fe–4S] cluster in partially unfolded aconitase.J Biol Chem 259: 14463–14471.

Khoroshilova, N., Beinert, H., and Kiley, P.J. (1995) Associa-tion of a polynuclear iron–sulfur center with a mutant FNRprotein enhances DNA-binding. Proc Natl Acad Sci USA92: 2499–2505.

Lambden, P.R., and Guest., J.R. (1976) Mutants of Escheri-chia coli K-12 unable to use fumarate as an anaerobicelectron acceptor. J Gen Microbiol 97: 145–160.

Lazazzera, B.A., Bates, D.M., and Kiley, P.J. (1993) Theactivity of the Escherichia coli transcription factor FNR isregulated by a change in oligomeric state. Genes Dev 7:1993–2005.

Lazazzera, B.A., Beinert, H., Khoroshilova, N., Kennedy,M.C., and Kiley, P.J. (1996) DNA-binding and dimeriza-tion of the Fe–S containing FNR protein Escherichiacoli are regulated by oxygen. J Biol Chem 271: 2762–2768.

Lian, C-J., Rosendal, S., and MacInnes, J.I. (1989) Molecularcloning and characterisation of a haemolysin gene from



Actinobacillus (Haemophilus) pleuropneumoniae. InfectImmun 57: 3377–3382.

Lovenburg, W. (1972) Clostridial rubredoxin. MethodsEnzymol 24: 477–480.

MacInnes, J.I., Kim, J.E., Lian, C.-J., and Soltes, G.A. (1990)Actinobacillus pleuropneumoniae hlyX gene homology withthe fnr gene of Escherichia coli. J Bacteriol 172: 4587–4592.

Melville, S.B., and Gunsalus, R.B. (1990) Mutations in fnrthat alter anaerobic regulation of electron transport-associ-ated genes in Escherichia coli. J Biol Chem 265: 18733–18736.

Melville, S.B., and Gunsalus, R.B. (1996) Isolation of an oxy-gen sensitive FNR protein of Escherichia coli : Interactionat activator and repressor sites of FNR-controlled genes.Proc Natl Acad Sci USA 93: 1226–1231.

Miller, J.H. (1972) Experiments in Molecular Genetics. ColdSpring Harbour, New York: Cold Spring Harbour Labora-tory Press.

Nicolet, J.H. (1992) Actinobacillus pleuropneumoniae. InDiseases of Swine. 7th edn. Leman, A.D., Straw, B.E.,Mengeling, W.L., d’Allaire, S., and Taylor, D.J. (eds).Ames, Iowa: Iowa State University Press, pp. 401–408.

Niu, W., Kim, Y., Tau, G., Heyduk, T., and Ebright, R.H.(1996) Transcription activation at Class II CAP-dependentpromoters: two interactions between CAP and RNApolymerase. Cell 87: 1123–1134.

Oscarsson, J., Mizunoe, Y., Uhlin, B.E., and Haydon, D.J.(1996) Induction of haemolytic activity in Escherichiacoli by the slyA gene product. Mol Microbiol 20: 191–199.

Ramos, H.C., Boursier, L., Moszer, I., Kunst, F., Danchin, A.,and Glaser, P. (1995) Anaerobic transcription activation inBacillus subtilis : identification of distinct FNR-dependentand -independent regulatory mechanisms. EMBO J 14:5984–5994.

Rosendal, S., Devenish, J., MacInnes, J.I., Lumsden, D.H.,

Watson, S., and Xun, H. (1988) Evaluation of heat-sensitiveneutrophil-toxic and hemolytic activity of Actinobacilluspleuropneumoniae. Am J Vet Res 49: 1053–1058.

Savery, N., Rhodius, V., and Busby, S. (1996) Protein–protein interactions during transcription activation: thecase of the Escherichia coli cyclic AMP receptor protein.Phil Trans R Soc Lond B 351: 543–550.

Sharrocks, A.D., Green, J., and Guest, J.R. (1990) In vivoand in vitro mutants of FNR, the anaerobic transcriptionalregulator of E. coli. FEBS Lett 270: 119–132.

Sharrocks, A.D., Green, J., and Guest, J.R. (1991) FNR acti-vates and represses transcription in vitro. Proc R Soc LondB 245: 219–226.

Soltes, G.A., and MacInnes, J.I. (1994) Regulation of geneexpression by the HlyX protein of Actinobacillus pleuro-pneumoniae. Microbiology 140: 839–845.

Spiro, S. (1994) The FNR family of transcription regulators.Antonie van Leeuwenhoek 66: 23–36.

Webster, C., Kempsell, K., Booth, I., and Busby, S. (1987)Organisation of the regulatory region of the Escherichiacoli melibiose operon. Gene 59: 253–263.

Williams, R.A., Bell, A., Sims, G., and Busby, S.J.W. (1991)The role of two surface exposed loops in transcription acti-vation by the Escherichia coli CRP and FNR proteins.Nucleic Acids Res 19: 6705–6712.

Williams, R.A., Rhodius, V.A., Bell, A.I., Kolb, A., and Busby,S.J.W. (1996) Orientation of functional activating regions inthe Escherichia coli CRP protein during transcription acti-vation at Class II promoters. Nucleic Acids Res 24: 1112–1118.

Wing, H.J., Williams, S.M., and Busby, S.J.W. (1995) Spac-ing requirements for transcription activation by Escherichiacoli FNR protein. J Bacteriol 177: 6704–6710.

Zheng, L., White, R.H., Cash, V.L., Jack, R.F., and Dean,D.R. (1993) Cysteine desulfurase activity indicates a rolefor NifS in metallocluster biosynthesis. Proc Natl AcadSci USA 90: 2754–2758.



hlyx, the fnr homologue of actinobacillus pleuropneumoniae, is a [4fe–4s]-containing...

Documents