Molecular Microbiology (1992) 6(2), 267-275

Purification and initial characterization of AhrC: the regulator of arginine metabolism genes in Bacillus subtilis L. G. Czaplewski,^ A. K. North * M. C. M. Smith,' S. Baumberg and P. G. Stockley*^ Department of Genetics. University of Leeds, Leeds LS2 9JT. UK. Summary The argjnine-dependent repressor-activator from Bacillus subtilis, AhrC, has been overexpressed in Escherichia coli and purified to homogeneity. AhrC, expressed in £. coli, is able to repress a Bacillus promoter {argC^, which lies upstream of the argC gene. The purified protein is a hexamer with a subunit molecular mass of 16.7 kDa. Its ability to recognize DNA has been examined in vitro using argCp in both DNase I and hydroxyl radical protection assays. AhrC binds at two distinct sites within the argCp fragment. One site, argCoi, with the highest affinity for protein, is located within the 5' promoter sequences, whilst the other, argCo2, is within the coding region of argC, The data are consistent with the binding of a single hexamer of AhrC to argCoi via four of its subunits, possibfy allowing the remaining two subunits to bind at argCo2 in vivo forming a repression loop similar to those observed for the £ coii Lac repressor. Introduction Sequence-specific recognition of DNA by proteins is fundamental to the control of gene expression. In prokaryotes, one class of DNA-binding proteins, namely those containing the helix-turn-helix motif (h-t-h), has been extensively studied (Pabo and Sauer, 1984; Brennan and Matthews, 1989; Harrison and Aggarwal, 1990). The molecular details of their interaction with DNA are now beginning to emerge from the results of structural studies of crystals of protein-oligonucleotide complexes (Otwinowski etai, 1988; Jordan and Pabo, 1988; Aggarwal et

Received 12 September, 1991: accepted 30 September, 1991. Present addresses: fBritish Biotechnology Ltd, Thames Court, Watlington Road. Cowiey, Oxford OX4 5LY, UK; ^Department of Plant Pathology, 147 Hilgard Hall, University of Califomia, Berkeley, CA 94720. USA; §Depart" ment of Biological and Molecular Sciences, University of Stirling, Stirling FK9 4LA, UK. 'For correspondence. Tel. (0532) 333092; Fax (0532) 441175.

ai. 1988). Recently, however, another class of prokaryotic DNA-binding proteins has been characterized structurally (Raffertyefa/., 1989; Breg efa/., 1990; Phillips, 1991). One of these is exemplified by the Eseherichia eoli methionine repressor, MetJ, which does not contain the h-t-h motif and binds to its operator DNA via a pair of 3-strands (Somers, 1990). These results emphasize the importance of extending studies on prokaryotic regulators to other systems in which novel modes of DNA recognition may be found. The Baeillus subtilis regulatory protein, AhrC, and its E coli homologue, ArgR, display several unusual features which suggest such a novel mode of DNA recognition. AhrC and ArgR display functional multiplicity: in the presence of arginine, AhrC represses the B. subtilis arginine biosynthesis genes and activates the arginine catabolism genes (Mountain and Baumberg, 1980), whereas ArgR represses the £ coli arginine biosynthesis genes (Lim ef ai, 1987) and is an essential accessory protein in the resolution of plasmid ColEI multimers (Stirling ef ai. 1988). Although AhrC and ArgR share only 27% amino acid identity (North, 1989; North etai. 1989) AhrC can functionally complement ArgR for both repression and its role in recombination (Smith efa/., 1989; Stirling et al., 1988), presumably by binding to simitar regulatory sequences. No precedent exists for homologies between regulatory proteins of analogous function and their operator sites in such taxonomicatly distinct prokaryotes. Indeed, there are currently only a few structurally characterized examples of repressors in bacilli (Ebbole and Zaikin, 1989). AhrC has been crystallized and structural studies are proceeding (Boys etai. 1990). Similar studies are in progress on the E eoii repressor and should eventually allow detailed molecular comparison of the two systems (W. Maas, personal communication). Transcription of a promoter upstream of argC within the B. subtilis arginine biosynthesis gene cluster, argCAEBDcpa-argP, is repressed by the ahrC gene product in the presence of arginine {Smith ef ai. 1986). The sequences responsible for this control have been localized to a 276 bp £coRI-Sau3A fragment (Smith ef ai, 1989). The ahrC gene has now been cloned and overexpressed in E eoli. in which it represses the 6. subtilis argC promoter, argCp. The protein has been purified to homogeneity and characterized by A/-terminal amino acid sequencing, chemical

268

L. G. Czaplewski et al. induction, suggesting that high-level a/irCexpression was deleterious (data not shown), possibly because of expression of part of a second open reading frame (ORF) with homology to recN downstream of ahrC. In order to exclude this possibility, to improve expression from the ahrC clone, and to flank the ahrC coding sequence with unique restriction sites, a further plasmid (pUL2202) was constructed (see the Experimental procedures for details), although upon IPTG induction this plasmid was also lost from the culture.

Purification and proteoiytic digestions of AhrC 0

1 2 3 4 Time post-IPTG(h)

Fig. 1. In vivo actrvrty of the ahrC gene product, p-galactosidase specific activities {nmol mg 'min ') determined in aftrC ' fplasmid pUL2033, A , A) or ahrC (plasmid pGLW11. • , O) E. coli TGI cells, carrying the compatible fusion plasmid pUL714, in the presence (solid symbols) or absence (open symbols) of 1 mM IPTG.

cross-linking and analytical ultracentrifugation. AhrC is a hexamer with a subunit molecular nnass of 16.7 kDa. Together with the E. eoti homologue, ArgR, these are the only examples of prokaryotic regulators which are hexameric (dimers and tetramers being the norm in systems analysed so far), with the exception of the deoR repressor. which is an octamer (Mortensen et ai, 1989). The binding of AhrC to argCp. studied by DNase I and hydroxyl radical footprinting, suggests that the details of this interaction will be novel. Results arhC activity in vivo and overexpression in E. coli The activity of the cloned ahrC gene was demonstrated in vivo in E. coli by using an a^rC-overexpressing plasmid (pUL2033; Smith et ai, 1989) and an a/-gC::/ac2fusion on the compatible plasmid pUL714. The p-galactosidase activity of E coli TGI oells containing both plasmids {ahrC ^) was compared with the activity in cells containing pUL714 and the control plasmid pGLW11 (ahrC~}. in the presence and absence of isopropyl-p-o-thiogalactoside {IPTG), which induces the expression of ahrCor\ pUL2033, The (i-galactosidase activities were dramatically tower in ahrC^ cultures (Fig. 1), Implying that the cloned a/irCgene product was able to regulate the expression of the B. subtilis promoter in E. eoli and that repressor purified from E coli would function in binding assays in vitro. Laclmediated repression of ahrC expression from the tae promoter of pUL2033 was sufficiently leaky to repress transcription at argCp even in the absence of IPTG. Analysis of the plasmid DNA present showed that pUL2033 was unstable and was lost rapidly after IPTG

E. coli TG1 PUL2033 or pUL2202 were used to provide crude extracts for protein purifications. The protein was purified in two steps from a cell sonicate, first by virtue of its differential solubility in buffers of differing ionic strength, and then by ion-exchange chromatography on an S-Sepharose column. Under the buffer conditions used. AhrC is one of the few proteins in the extract to bind to the column and was eluted with an increasing gradient of NaCI as a chromatographically homogeneous fraction. The final preparation was essentially pure as determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 2). The details of the purification procedure are described in the Experimental procedures. The A/-terminal amino acid sequence of the purified AhrC protein was identical, over the first 30 amino acids, to that predicted from the DNA sequence of the ahrC gene encoded on pUL2202. Incubation of the purified protein with proteases of differing specificities, i.e. subtilisin and V8, resulted in rapid cleavage of the intact AhrC subunit. The digestion did not go to completion. Both proteases resulted in the appearance of two apparently stable proteoiytic fragments of approximately similar sizes (data not shown). These results suggest that AhrC may be organized into domains; this is a common feature of many prokaryotic regulators (Rashne, 1987). Molecular weight of native AhrC protein The absolute technique of low-speed sedimentation equilibrium in the analytical uitracentrifuge was used to determine the molecular weight of AhrC apo-protein; the equilibrium distribution of solute was recorded using the Rayleigh interference optical system as described below. A linear plot of log concentration (expressed in terms of fringe displacement units. J) versus the normalized radial displacement squared parameter was obtained using the M* procedure described by Creeth and Harding (1982). Because of the low loading concentration used («0.4mg ml"') the effects of thermodynamic non-ideality can be regarded as negligible.

Purification and Initial eharaeterization of AhrC 269

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examples in Kim et ai, 1977) we would expect that the monomeric units are fairly tightly bound into the intact hexamer, The organization of the AhrC molecule was also investigated using chemical cross-linking with bisimido esters (Thomas, 1978) and gel filtration. Analysis of the crosslinked protein on SDS-PAGE predominantly showed a species corresponding to a dimer of AhrC subunits, with very little material corresponding to higher aggregates (data not shown). The position of elution from gel filtration columns was consistent with a hexamer. The cross-linking result may reflect the organization of the apo-AhrC hexamer, perhaps into a trimer of dimers. Similar experiments with E. coli ArgR also suggest that it is a hexamer (Lime(a/., 1987). Footprinting protection analysis

Fig. 2. Purification of AhrC from E. coli. a. Eiution profile of S-Sepharose column used in the final AhrC purification step. The main peak (iii) is essentially pure AhrC, as judged by SDS-PAGE, The smallest peak (ii) represents AhrC which has been partially degraded by proteases (data not shown) and the first peak (i) is the flow through of proteins that do not bind to the column. b, SDS-PAGE of thB stages of the AhrC purification procedure. Lanes: 1, total cell sonicate: 2, supernatant after centrifugation; 3, pellet after cantrifugation; 4, supernatant after DNase I and NaCI addition; 5, pellet after same, 6, low-salt supernatant: 7, low-salt pellet; 8. S-Sepharose flow through (peak i in part a); 9, peak iii from S-Sepharose column; 10. molecular weight standards.

A plot of point weight average molecular weights M^^, (obtained by sliding strip quadratic fits to the observed fringe data; Harding, 1984) versus (fringe) concentration, J, showed no evidence of any dissociation of the native AhrC protein at low concentration. Our estimated value for the native molecular weight of the apo-AhrC protein of (96 000 ± 4000) is (5.8 ± 0.3 times) bigger than that of the monomer (16673, calculated from the amino acid composition) and, so, assuming that there is no significant post-translational modification we conclude that native AhrC apo-protein behaves as a hexamer in solution. Furthermore, because the point average molecular weight shows no significant decrease at low concentrations (cf.

The 276bp EcoR\-BamH\ argCp DNA fragment radiolabelled at either 5' end was used to determine the regions of DNA bound by AhrC using DNase I footprinting. Figure 3a shows autoradiographs of portions of the nuclease footprints of the argCp DNA on both strands. The areas protected from DNase I cleavage are summarized in Fig. 3b. Two regions of DNA were protected by AhrC in an L-arginine-dependent manner — one in the 5' flanking sequence of argC and the other within the coding sequence; we have termed these argCoi and argCo2' respectively. Protection oi argCoj occurs at lower protein concentrations than at argCo2 and is largely complete at 4.7 X 10"^ M AhrC (monomer). The argCop site required roughly eight times this protein concentration to achieve a similar degree of protection. The requirement of additional protein for binding to argCo2 implies that separate AhrC hexamers bind to each site in vitro. There was no change in the DNase I sensitivity of the unbound bases between the two operators, indicating that looping did not occur under the conditions used. Footprinting of the 3' half (H/nfl/SamHI) of the argC fragment, which only contains the argCo2 site (Fig. 3), showed protection of the same region as in the intact fragment and at the same repressor concentration, suggesting that binding to argCop was independent of binding to argCoi (data not shown). Footprinting of an argCp fragment containing a mutant argCop operator which contained a 4bp deletion of bases +118 to +121 {argCo2d) showed that binding to argCoi was unaffected. Remarkably, even though the deletion is in the centre of the argCo2 operator, the mutant argCo2d S'te was still protected by AhrC, but the region protected had shrunk by 4bp so that the footprint boundaries were identical in both operators (Fig. 3c). In order to map the protein-DNA contacts in more detail, a similar series of footprinting experiments was

270

L G. Czaplewski et al.

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Rg. 3. DNase I footprints of the 276bp £coRI-SamHI argCp-containrng fragment. a. Autoradiographs ot the top strand (U) or bottom strand (L) eiectrophoresed on 8% (w/v) denaturing acrylamide gels. Lanes: a, 2 x 10 "^M; b. 1 x 10 *M;c, 8 X 10 ' M i d , 4 x 10-'M:e, 2 x 1 0 " ' M ; t, 1 x 1 O ' M ; g 8 x 10 ^M;h, 4 x 10 " M ; i. 2 x 10 »M;), 1 x 10 *M;k. 8 x 10 «M;I,4 x 10 ^M m, 2 X 10" ^M; n. 1 x 10 ^ M ; o, 8 x 10 '°M: p, 4 x 10"'°M; q. O.OM. M, Maxam and Gilbert sequence laddera. Concentrations are in terms of AhrC monomer. b. Summary of the regions protected on the DNA sequence of argCp. The boundaries of both argCot and argCcs are indicated by the inverted arrows. Diamonds indicate the positions of unique restriction sites mentioned in the text. The position of the centre of the deletion in argCo^a is marked by an inverted triangle. Possible regulatory sequences for both transcription and translation are underlined. The amino acid sequence of the encoded ArgC is shown below the sequence in the one-letter code. Sequence numbers here are the same as Ihose throughout the text. c. Comparison of the DNase I footprints of the argCo2 (top) and argC(xa (bottom) operators. Arrowheads indicate the boundaries of the footprinted regions.

carried out using hydroxyl radical protection. This has the distinct advantage that every base-pair position in the protein-free DNA is sampled. The results of these experiments are shown in Fig. 4a. Discrete regions of protection can be seen in both argCoi and argCo2- A number of bases within argCoi show apparent hyper-reactivity towards the reagent, even in the absence of protein. In order to quantify the extent of protection from hydroxyl radical cleavage, autoradiographs similar to those in Fig. 4a were scanned by densitometry (Fig. 4b) and the relative change in cleavage between free DNA and the proteinDNA complex at intermediate protein concentrations calculated: the results are shown in Fig. 4c. The regions of argCoi protected from hydroxy radical cleavage are similar to the sites identified by DNAse 1 footprinting. The hydroxyl radical cleavage patterns on both strands show an approximate 10-base repeat between the maxima or minima of cleavage, with the

patterns being displaced by approximately 5bp between the strands. Detailed analysis of these cleavage patterns will be given in the Discussion. The question of the in vivo function of argCo2 has been addressed by replacing argCo2 in Bacillus with argCo2d and measuring argF activity. Previous experiments have shown that insertional mutagenesis of the argC gene results in almost complete abolition of ArgF activity, suggesting that the argCAEBD-cpa-argF cluster is expressed as a single operon {M. C. M. Smith and S. Baumberg. unpublished). Replacement of argCo2 with a^Co^tf was achieved by generating a partial diploid using an integration vector and isolating segregants which have deleted the vector sequences, leaving the argCo2a mutation in the chromosome. B. subtilis containing 3''gCo2d shows a reproducible fourfold decrease in repression ratio for ArgF. suggesting that argCosa is functionally important (L. G. Czaplewski, unpublished).

Purification and initiai characterization ofAhrC 271

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At \i L Ml Protection trandl

Rg. 4. Hydroxyl radical footprints of argCo,. a. Autoradiographs of the top strand (U) labelled at the 5' end and the bottom strand (LJ labelled at the 3'end. Lanes: a, 9 x 10 ^M; b. 3 x 1 0 * ' M ; c , 9 x 1 0 - ' M ; d , 3 x 10 ' M ; e , 9 x 10 «M:f, 3 x 10 * M ; 9 . 9 X 10 -^M; h. 3 X 10 ^ M ; i, 9 x 1O-"'M; i, O.OM. M. Maxam and Gilbert sequence ladders. Concentrations are in twms of AhrC monomer. b. Densitometer tracings of hydroxy! radical footprint patterns of the argCoi in the presence of either 9 x 1 0 ' " M (A) or 3 x 10 "M (B) AhrC. c. Results of 4b normalized as a percentage of the most protected base on each strand (100%) and ploned as a histogram,

Discussion As expected from its behaviour in vivo in E coli, AhrC binds in vitro to DNA fragments encompassing the B. subtiiis argC promoter region in an arginine-dependent manner as judged by gel retardations (Smith ef ai, 1989) and DNase I footprinting. Two regions of the argCp fragment are footprinted by AhrC. The first is located in the promoter region of the gene and overlaps the sequences identified as possible arginine operators by sequence homology to the E coli 'ARG boxes' (Smith e( ai. 1986). This site has the higher affinity for repressor in vitro and vi/e have termed the entire region (-60 to -9) argCoi- The lower-affinity site {argCo2) is located within the coding region of the argC gene (+100 to +133) and, like argCou shows similarity to the E coii ARG box consensus sequence (Fig. 3). The occurrence of two operator regions organized in this way is reminiscent of the E coli Lac repressor, which has a high-affinity site in the promoter

region and several lower affinity sites, one of which is located within the IacZ stnjotural gene. Recent work shows that the minor lac operator sites are functional in wVo(Oehlerefa/.. 1990). Although DNase I footprinting is useful in identifying the general regions of DNA bound by AhrC. the enzyme does not cleave readily in certain regions of the operator sequence, with the result that a detailed interpretation of the interaction is impossible. We therefore used hydroxyl radical footprints which have the advantage that in protein-free DNA every base pair can be cleaved. The results in Fig. 4 show that it is possible to quantify the extent of protection at every nucleotide position. For technical reasons only argCoi has been analysed in this way. The hydroxyl radical footprint encompasses the region footprinted by DNase I but extends beyond it slightly on the non-template strand. The extent of protection, on both strands, varies with a periodicity of approximately 10 bases and the pattern Is shifted by roughly five bases

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L G. Czaplewsiii et al.

a.

mmm b. argCo argC02

Fig. 5. a. Summary of protection patterns at argCoi- Black lines along the exploded helix show regions of protection (>50% compared to protein free pattern) from hydroxyl radical cleavage, the boxed nucleolides are hypersensitive sites. Open bars show the extent of protection by AhrC compared with regions of rough dyad symmetry (inverted arrows). b. Csrtoon showing how a repression loop might form in vivo.

between the two strands. Thus the protection is consistent with a continuous interaction between the protein and one face of the DNA over a distance of some five helical tums (52 bases). Previously, up to three putative operator sites within argCp were identified because of sequence homologies with the E coii ARG box consensus sequence (Smith ef al., 1986). However, the footprinting data suggest that only some of the sequence homology is functionally important. Gel retardation experiments with argCp (Smith et ai., 1986; L. G. Czaplewski, unpublished) are consistent with the binding of a single AhrC hexamer to the argCoi site. Preliminary stoichiometry measurements with E. coii ArgR are also consistent with such a complex (W. Maas, personal communication). Four major blocks of AhrC protection from hydroxyl radical cleavage are observed at argCoi, which also contains two sets of sequences with approximate dyad symmetry. Our current model of repressor binding is as follows: two out of three dimers of a single repressor hexamer contact argCoi in the major groove. The two right-hand blocks of hydroxyl radical protection are produced by one dimer that recognizes a region of dyad symmetry with homology to E. coli ARG boxes (Fig. 5a). The two left-hand blocks are protected by a second dimer lying two helical tums (20 bp) to the left of the centre of the protection of the adjacent dimer. However, the protection pattern of the left-hand side of argCoi does not coincide with sequences which contain dyad symmetry, suggesting that they are either non-functional or that binding affinity is dominated by contacts to the right half of the site. Experiments with synthetic constructs encompassing portions of the argCoi site

suggest that the latter explanation is correct (J. Baker et ai, in preparation). The E. co//ARG boxes described to date consist of tandem repeats of a 9bp imperfect palindrome separated by a variable spacer of either 2 or 3bp{Cunin etai., 1986; Lim etai, 1987). The S. subtiiis operators identified here do not fit easily into this model but the complementation of ArgR functions by AhrC in E. coii suggests that the sequence similarity between the operators is functionally significant. The AhrC-DNA contacts at argCoi are consistent with a model of interaction in whioh the DNA bends around the surface of the repressor hexamer. This may impose some sequence requirement to facilitate the necessary bending/ kinking. The hydroxyl radical cleavage pattern of proteinfree argCo, linear DNA reveals the hypersensitivity of three groups of nucleotides towards the reagent. This hypersensitivity is largely unaffected by AhrC binding. The detailed chemistry of hydroxyl radical cleavage is not straightforward. However, the generally accepted points of attack by the reagent are the carbon atoms of the ribose ring (Tullius and Dombroski, 1986). This is followed by internal rearrangements leading to eventual cleavage of the DNA backbone at this point. The hypersensitivity of positions within argCoi must therefore reflect changes in the ribose sugars at these positions, perhaps by changes in sugar pucker. This, in turn, may reflect changes in DNA duplex conformation at these positions, i.e. bending or kinking. The argCoi site is extremely rich in A-T base pairs and includes two A5 tracts which, both in solution and in oligonucleotide crystal structures, have been shown to lead to distortions from B-type DNA conformations (Nelson etai, 1987). The site of the most intense hyper-reactivity coincides with the argCp - 3 5 sequence and may reflect a DNA conformation that is important for transcription, whereas the other two sites are located at either end of an A5 tract and could therefore reflect the conformational effects of the junction between the A-tract and more regular B-type DNA. Since the hyper-reactivity is present in protein-free argCoi DNA it would appear that whatever the conformiation causing this behaviour it is stable in the absence of bound protein and may be a factor in DNA recognition. If argCoi is being bound by a single hexamer of 96 kDa, such a protection pattern could be produced by wrapping the DNA around a globular protein in a similar fashion to the wrapping of DNA around the histone octamer in the nucleosome (Satchwell et ai, 1986). In the case of the hexameric AhrC protein bound to two separate arginine operators, such a wrapped complex could result in the juxtaposition of two duplexes, thus explaining the function of arg repressors In the plasmid resolution reaction (Stirling etai, 1988). Evidence showing that B. subtiiis arginine operators are characterized by reasonably conserved sequence blocks whioh flank the sequences bound by repressor comes

Purification and initiai characterization of AhrC from DNase I footprinting of argCo2 and its deletion derivative argCo2d in which the sequence boundaries of the footprint are maintained despite the loss of 4bp between them. This result requires that the repressor be sufficiently flexible that it can recognize the conserved sequences even though they have altered their spacing by 4bp, placing them 120° away from their initial position in the duplex. Such flexibility might be reflected in the proteolytic sensitivity of the repressor. DNA-binding domains located on extended, flexible (and hence proteolytically sensitive) arms of polypeptide might well be able to accommodate the necessary changes in sequence position. In vitro the requirement for higher protein concentrations to obtain the footprint at argCo2 and the fact that the cleavage pattern of the DNA between argCoi and argCo2 does not alter when repressor binds suggests that separate hexamers are bound at the different operators. However, this does not exclude the possibility that, in vivo, on supercoiled DNA a repression loop might form, thus saturating the DNA-binding domains of a single AhrC hexamer (see Fig. 5b). Such loops have been proposed as important factors in transcriptional control of a number of E. coli operons including lac (Oehler et al, 1990), araC (Schlief, 1987), aroF-tyrA (Cobbett, 1988) and deoC (Mortensen et ai., 1989). Further experiments are proceeding to explore this possibility, which, if confirmed, would for the first time extend the E. coli lac repressor paradigm to Gram-positive organisms.

273

Klenow. pUL322d3 was constnjcted by Itgating the fcoRI-C/al ahfC fragment from pUL31d3 into fcoRI- and C/al-cut pBR322 (Bolivar and Backman. 1979). pUL2202 was constructed by ligating the Eco RI-/^/ndlll aftrC fragment from pUL322d3 into the polylinker of pGLWn (described by Smith efa/.. 1989). pUL3001, which contained the mutant argC operator (argCo^a). was constructed by cutting of pUL3000 with Nsi\, deletion of bases with nuciease Sa/31. Klenow end-filling to blunt the ends, and religation prior to transformation. The Ba/31 treatment was brief (15s) to limit the extent of the deletions. Plasmid DNA was prepared from pooled transformants and was restricted with an excess of /Vs/I. Uncut cccDNA was separated from cut linear plasmid by agarose gel electrophoresis and the cccDNA purified and used to transform E. coli TGI. Four independent Nsi\ site deletions were cloned into M13mp18 (Messing. 1983) and sequenced. All four contained the same four base-pair deletion (bases +118 to +121, Fig. 3b), and one clone pUL3001 was selected for further analysis.

Ceil growth and induction E. coli strain DS903(pUL2202) was grown at 37°C in rich medium (yeastextractiOgl \KH2P045.6gl-'.K2HP04.3H2037.8gl ', glucose lOg I ', thiamine lOmg I '. MgSO4.7HpO 492mg I ') supplemented with lOOmgl ' ampicillin. At an optical density at 600nm of 1.5 the cells were induced by the addition of IPTG (400mg I"') and the incubation continued for 3h. Cells were harvested by centrifugation and were washed once with Arg buffer (20mM Tris-HCI, lOmM MgCb. lOmM 2-mercaptoethanol. i m M phenylmethylsulphonyl fluoride (PMSF), 25p.M W-tosyl-L-phenylalanine chloramethyl ketone (TPCK), pH 7.5; Lim etai, 1987)then frozen at -2O''C.

Purification of AhrC protein Experimental procedures Bacteriai strains E. coii strains TG 1 and DS903 were as described in Smith et a/. (1989).

Piasmids Plasmid pUL712 was constructed by ligating the 276bp EcoRISau3Aaf£fCpromoterfragment from pUL710 (Mann etai., 1964} into pJF751 (Ferrari ef a/., 1985). A transiationai fusion of the first 53 codons of the argC gene, fused in the correct reading frame to the iacZ gene of pJF751, was formed. pUL714 was constructed by ligating the argC::/acZ gene fusion containing the Pst\-Bal\ fragment of pUL712 into Psfl-Haell-cut pACYCI 77 (Chang and Cohen. 1978) and inactivating the ampicillin resistance gene by cutting with Pst\ and end-filling with Klenow and dNTPs prior to religation. pUL3000 was constructed by ligating the 276bp EcoRI-SamHI argC promoter containing fragment of pUL714 into the polylinker of pUC18 (Yanisch-Pen-on et ai, 1985). pUL31d3 was constnjcted as follows: pUL2030 (Smith ef ai. 1986) was digested with /-//ndlll and the fragment ends blunted using nuciease Sa/31 and Klenow. The 0.8 kbp H/ndill-blunted fragment was gel-purified and digested with Cla\. The bluntended Cia\ ahrC fragment was cloned into Nco\- and C/al-cut pGCI (Myers ef ai, 1985) after the Wcol end had been blunted by

DS903(pUL2202) cells harvested from 101 of culture which had been grown and induced as above were thawed and resuspended in 160ml of ice-cold Arg buffer. The coll suspension was sonicated on ice to prepare a cell extract. The insoluble material was harvested by centrifugation at 15000 x g for 30 min at 4°C. The pellet was resuspended in 160ml of Arg buffer and treated with DNase I (1 ji-g ml ' final concentration) at 37°C for 15 min. Solid NaCI was added to the suspension to a final concentration of 0.5 M and incubated at 37''C for 15 min. The supernatant after DNase I and NaCI treatments was obtained by centrifugation at 15000 X g for 30 min at 4''C. The supernatant was extensively dialysed against Arg buffer containing 75mM NaCI at 4''C. The precipitate which formed was harvested by centrifugation at 10000 X gfor 30 min at 4''C and was resuspended in 0.5 M NaCI Arg buffer. Prior to loading onto an S-Sepharose cation-exchange column the protein was diluted with an equal volume of Arg buffer. The protein was loaded onto the column with 25QmM NaCI Arg buffer until the front of unbound protein had eluted from the column. The column was developed using a 250-600mM NaCt linear gradient in 11 of Arg buffer. Purification of AhrC was monitored at each step by SDS-PAGE. The major protein species was harvested and made 20% (w/v) in glycerol and stored at -20°C. A minor protein species separated by chromatography was also AhrC which appeared to be proteolytically nicked. The protein concentration was determined by measuring the absorbance at 280 nM, assuming that an OD2ao of 1.0 corresponds to a protein concentration of 1 mg m r ' .

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Molecular mass determination of apo AhrC A Beckman Model E analytical ultracentrifuge was used employing Rayleigh interference optics, an He-Ne laser light source 95mW. wavelength = 632.18nm) and an RTIC temperature measurement system. AhrC samples were dialysed for at least 24h against the phosphate-NaCI buffer (NajHPO^.12H20, 5.79g I '; KH2pO4, a i 9 7 g I '; NaCI 26.307g I " \ lOnM 2-mercaptoethanol. 0.1 mM PMSF; 2.5^LM TPCK). The 'low'- {or 'intermediate'-} speed method was used (Creeth and Harding, 1982). The concentration of solute left at the meniscus at sedimentation equilibrium was determined by mathematical manipulation of the fringe data, To minimize effects of non-ideality, a low loading concentration (=0.4mg ml ') using s:3mm columns in double sector cells of 30mm optical pathlength were used. Care was taken to monitor for possible dissociative phenomena at this concentration. The root (root J) speed was 10581 r.p.m. at 12.0''C. A partial specific volume of 0.751 ml g ' andamonomermolecularmassof 16673 Da were determined from the amino acid composition {Cohn and Edsall. 1943). Data from the Rayleigh interference patterns were captured off-line on an LKB Uttroscan XL (2D) scanner interfaced to a PC. Fringe concentration versus radial displacement plots were obtained using the UXSD PASCAL program ArjALYSE (Rowe ef al., 1989). These data were then transferred via the joint UK Computer Network for full data anslysis on the mainframe IBM 3081/B at the University of Cambridge, UK.

FB buffer (lOmM Tris-HCl. 5mM MgCb, 2.5mM CaCb. 250mM KCI. O.5mfvl dithiothreitol. 50|ig ml ' sonicated salmon-sperm DNA. pH 7.4) including L-arginine if appropriate in a total volume of 18 p-l for 30 min at 37^C. DNase 1 was added (2 M.1 of 40 jig ml ' DNase I solution in FB buffer) and incubated at 37°C for 1 min. An ice-cold stop solution (20PLI of 3 M ammonium acetate, 250mM EDTA. 300 |xg ml ' yeast tRNA, pH 8.5), 85jil of ice-cold water and 325 )j.l of cold ethanol were then added. The DNA fragments were precipitated in a dry ice/ethanol bath for 15 min prior to centrifugation. The precipitated DNA was washed with 70% (v/v) ethanol and then dried. The DNA was resuspended in 2.5|j.l of formamide sequencing gel buffer and the fragments separated by electrophoresis on an 8% (w/v) sequencing gel together with the Maxam and Gilberi G+A and C+T sequencing reactions of the same fragments (Maniatis e* al., 1982).

Hydroxyi radical footprinting Hydroxyl radical footprinting was carried out according to the protocol of Tullius and Dombroski (1986). The repressor-DNA complexes were formed in the same buffer used for DNase I footprinting. with the exceptions that the final KCI concentration was 200mM and the mixture contained only half the amount of heterologous DNA used previously. The repressor solutions used for hyroxyl radical footprinting were free of glycerol, which inhibits the reactions.

Amino acid sequencing protocol

Acknowledgements

AhrC protein and polypeptides produced by protease digestion were subjected to amino acid sequence analysis according to the procedures of Findlay et ai. (1989).

We would like to thank Professors Wemer Maas, Nicholas Glansdorff and David Sherratt and their colleages for many helpful discussions of their results with the E. coli argfl/xerA/cer systems. Isobel Parsons and Dr Jeff Keen helped with aspects of the protein chemistry. We would also like to thank Mr M. S. Ram2an of the SERC Hydrodynamics Unit (University of Nottingham, UK) for skilled technical assistance with the analytical ultracentrifuge measurements. This work has been supported, in part, by MRC grants G84 09250CB (to S.B. and Dr A, Mountain) and G85 16091 (to S.B.). the University of Leeds Research Fund, and by SERC Grant GR/E53842 (to M.C.M.S.. P.G.S. and S.B.). We are grateful to Ms Claire Marsland for help with preparation of the manuscript.

Preparation of operator fragments for binding studies Plasmid pUL3000 containing the 276bp EcoRI-SamHl argC promoter region was used for the production of wild-type argC operator containing radiolabelled DNA fragments. DNA fragments containing the mutant argCo2^ operator were obtained from pUL3001. Fragments were end-labelled with T4 polynucleotide kinase or filled in with Klenow enzyme according to the enzyme supplier's instructions. Radiolabelted fragments were separated from the vector DNA by agarose gel electrophoresis (0.8% (w/v) low melting-point agarose in TBE (90 mM Tris-borate, 1 mM ethyienediaminetetraacetic acid (EDTA)). The position of the radiolabelled DNA fragment was determined by autoradiography and the radioactive material excised. The fragment was purified from the gel slice by melting the agarose in 0.5M NaCI, 1 mM EDTA. 0.1 % (w/v) SDS at 65"C for 30 min. The DNA was extracted once with phenol and once with phenol/chloroform, ethanol-precipitated, washed with 70% (v/v) ethanol, and vacuum-dried. The DNA was resuspended in 500 p-l of TE (10 mM Tris-HCl. 1 mM EDTA. pH 8.0) and further purified by ionexchange chromatography using a Qiagen tip according to the manufacturer's instructions.

Footprinting with DNase I About 0.5ng of the 276bp EcoR\-BamH\ argCp DNA fragment from pUL3000 radiolabelled as above was incubated with AhrC in

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Purification and initial characterization of AhrC: the regulator of arginine metabolism genes in Bacillus subtilis.

The arginine-dependent repressor-activator from Bacillus subtilis, AhrC, has been overexpressed in Escherichia coli and purified to homogeneity. AhrC,...
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