mapping the dna-binding domain and target sequences of the streptomyces peucetius daunorubicin...

© 2002 Blackwell Science Ltd

Mapping the DNA-binding domain and targetsequences of the Streptomyces peucetiusdaunorubicin biosynthesis regulatory protein, DnrI

for expression of that pathway, as dictated by the naturalenvironment of the cell. A prime example of an organismthat rigorously adheres to this paradigm is the free-livingfilamentous soil bacterium of the genus Streptomyces.Bioactive secondary metabolite production within thesemicroorganisms is under the precise control of an intricatecascade of regulatory events. A large number of thesebioactive chemical agents have been developed intopharmaceutically useful drugs. Thus, there is consider-able interest in understanding the molecular mechanismsinvolved in the control of the biosynthetic genetic path-ways. In many species of streptomycetes that producediverse chemical compounds, the final checkpoint in tran-scriptional regulation is controlled by a family of proteinsrecently coined Streptomyces antibiotic regulatory pro-teins (SARPs) (Wietzorrek and Bibb, 1997). This family ofproteins has been characterized as transcriptional activa-tors, and their overproduction has often been associatedwith a concomitant increase in titres of the correspond-ing antibiotic. Until very recently, little was known aboutthe mechanism by which these proteins exerted theireffect on gene expression, except that they bound DNA sequences within the promoters of antibiotic bio-synthetic pathway genes and operons. Bioinformaticalanalyses using the primary amino acid sequences of theSARPs failed to reveal any significant similarities to thecanonical helix–turn–helix (HTH) motifs commonly foundin microbial DNA-binding regulatory proteins. Recentdatabase queries have now revealed that the SARPsshare sequence similarity with several members of theOmpR family of DNA-binding domains (Wietzorrek andBibb, 1997). OmpR is a protein that regulates the expres-sion of outer membrane porin proteins in enteric bacteriaand belongs to a large family of transcriptional factors thatbind DNA and interact productively with RNA polymerase(RNAP) (Makino et al., 1996; Kondo et al., 1997). Inter-estingly, the OmpR family of DNA-binding domains alsofails to contain the canonical HTH motif (Mizuno andTanaka, 1997). Regarding the DNA recognition domain(s)of the OmpR family, the recently determined X-ray struc-ture reveals two helices creating a structure that super-imposes well on those of other canonical HTH motifs(Martinez-Hackert and Stock, 1997a). However, this pre-sumed structure is unique, in that the intervening loopsequence of the two helices is inordinately long and may

Molecular Microbiology (2002) 44(2), 449–460

Paul J. Sheldon,† Sara B. Busarow and C. Richard Hutchinson*‡

School of Pharmacy, University of Wisconsin, 425 N. Charter Street, Madison, WI 53706, USA.

Summary

Streptomyces antibiotic regulatory proteins (SARPs)constitute a novel family of transcriptional activatorsthat control the expression of several diverse anti-biotic biosynthetic gene clusters. The Streptomycespeucetius DnrI protein, one of only a handful of theseproteins yet discovered, controls the biosynthesis ofthe polyketide antitumour antibiotics daunorubicinand doxorubicin. Recently, comparative analyseshave revealed significant similarities among the pre-dicted DNA-binding domains of the SARPs and the C-terminal DNA-binding domain of the OmpR familyof regulatory proteins. Using the crystal structure ofthe OmpR-binding domain as a template, DnrI wasmapped by truncation and site-directed mutagenesis.Several highly conserved residues within the N-terminus are crucial for DNA binding and proteinfunction. Tandemly arranged heptameric imperfectrepeat sequences are found within the –35 promoterregions of target genes. Substitutions for eachnucleotide within the repeats of the dnrG–dpsABCDpromoter were performed by site-directed mutage-nesis. The mutant promoter fragments were found tohave modified binding characteristics in gel mobilityshift assays. The spacing between the repeat targetsequences is also critical for successful occupationby DnrI and, therefore, competent transcriptional activation of the dnrG–dpsABCD operon.

Introduction

Inducibility of gene expression is predicted by the‘demand’ theory of gene regulation, which relates themode of regulation of a pathway to the level of demand

Accepted 14 January, 2002. *For correspondence. E-mail [email protected]; Tel. (+1) 510 732 8400, ext. 219; Fax (+1)510 732 8401. Present addresses: †Acera Biosciences, Inc., 1479Gortner Ave., Suite 240, Gortner Building, St Paul, MN 55108, USA.‡Kosan Biosciences, Inc., 3832 Bay Center Place, Hayward, CA94545, USA.

be the cause of previous searches failing to detect a puta-tive HTH motif in the OmpR family and the novel SARPfamily of DNA-binding regulatory proteins.

Here, we concentrate on the daunorubicin/doxorubicin(DNR/DXR) pathway-specific SARP, DnrI. The DnrIprotein is the final, and most significant, connection of aco-ordinate cascade composed of three pathway-specificregulatory proteins that together maintain precise controlof DNR/DXR biosynthetic gene cluster expression. Thefirst player within the regulatory network is the DnrOrepressor/activator. DnrO binds to the divergent promoterseparating the dnrO and dnrN genes and maintainsrepression of dnrO while activating dnrN expression(Otten et al., 2000; H. Jiang, personal communication). Inturn, DnrN activates the expression of dnrI in a processthought to be comparable with that of redZ–redD (Furuyaand Hutchinson, 1996), the pathway-specific regulatoryproteins of undecylprodigiosin production in Strepto-myces coelicolor (Narva and Feitelson, 1990; Guthrieet al., 1998). We show that the DNA-binding domain ofDnrI resides within the N-terminus of the protein. Moreprecisely, a region of residues has been mapped by site-directed mutagenesis (SDM), which appears to be criticalfor recognition and/or interaction with the target DNA. In

addition, the stoichiometry and kinetics of DnrI–DNA inter-action are characterized. Together, these findings suggesta functional three-dimensional structural make-up that iscomparable with the OmpR family.

It has been recognized that the DNA-binding domainsof OmpR family members bind to direct repeat sequenceswithin the regulatory regions of target genes (Aoyama andOka, 1990; Pratt and Lilhavy, 1995; Harrison-McMonagleet al., 1999; Okamura et al., 2000). Interestingly, genepromoter elements within several antibiotic biosyntheticgene clusters contain similar direct repeat sequences pro-posed to be important for DNA recognition and binding bytheir cognate SARPs (Tang et al., 1996; Wietzorrek andBibb, 1997; McDowall et al., 1999). Using the DNR/DXRbiosynthetic gene cluster as a model system, we haveexplored the effect of mutating the putative DnrI targettandem repeats found within the dnrG–dpsABCDpromoter. This work demonstrates that these specificnucleotides play a critical role in maintaining precise tran-scriptional control of antibiotic biosynthetic genes. In addi-tion, it has been revealed that proper spacing between thetandem repeats must be preserved to sustain adequateDNA–DnrI interaction and, consequently, successful tran-scriptional complex formation.

© 2002 Blackwell Science Ltd, Molecular Microbiology, 44, 449–460

450 P. J. Sheldon et al.

Table 1. Strains and plasmids used in this study.

Strain or plasmid Relevant characteristic(s) Source or reference

Bacterial strainsS. peucetius

WMH1445 dnrI::aphII mutant Stutzman-Engwall et al. (1992)

E. coliDH5a F– recA f80 dlacZ DM15 Gibco BRLBL21(DE3) F– ompT hsdS gal dcm (DE3) Novagen

PlasmidspUC19 High-copy E. coli vector; Ampr Sambrook et al. (1989)pT7SC Protein expression vector; Ampr Brown and Campbell (1993)pMalc2X MBP fusion vector – cytoplasmic expression NEBpMalp2X MBP fusion vector – periplasmic expression NEBpWHM1102 N-terminal his-tagged dnrI in pET16b Tang et al. (1996)pWHM1115 N-terminal his-tagged dnrI under control of ermE* promoter This workpWHM1116 N27 10% N-terminal truncation of DnrI This workpWHM1117 N54 20% N-terminal truncation of DnrI This workpWHM1118 N81 30% N-terminal truncation of DnrI This workpWHM1119 N108 40% N-terminal truncation of DnrI This workpWHM1120 C246 10% C-terminal truncation his-tagged DnrI This workpWHM1121 C219 20% C-terminal truncation his-tagged DnrI This workpWHM1122 C191 30% C-terminal truncation his-tagged DnrI This workpWHM1123 MBP–DnrI fusion under control of ermE* promoter This workpWHM1124 1.2 kb XbaI–HindIII fragment from pWHM1102 in pUC19 This workpWHM1125 T64A DnrI This workpWHM1126 R71A DnrI This workpWHM1127 G95A DnrI This workpWHM1128 Y96A DnrI This workpWHM1129 R181A DnrI This workpWHM1130 L202A DnrI This workpWHM1131 Native MBP–DnrI fusion This workpWHM1250 High-copy-number shuttle vector containing ermE* promoter, tsr Madduri et al. (1998)

Amp, ampicillin resistance; tsr, thiostrepton resistance.

Doxorubicin biosynthesis regulatory protein 451

Results

In vivo complementation of S. peucetius strainWMH1445 (dnrI) with truncated and alanine-substitutedforms of DnrI

Based upon recent insight that SARPs may contain anal-ogous structures to those found in the three-dimensionalDNA-binding fold of OmpR (Wietzorrek and Bibb, 1997),we were interested in first mapping the region of DnrIinvolved in DNA recognition and interaction. To narrow thesearch for specific amino acids involved in DNA binding,a broad mutagenesis approach was used initially involv-ing the truncation of varying stretches of polypeptide fromthe N- and C-terminal ends of the protein. After deletionof the corresponding spans of DNA, the truncated plasmidconstructs (see Table 1) were transformed into a dnrImutant strain (WMH1445), which lacks the ability toproduce DNR/DXR (Stutzman-Engwall et al., 1992).When wild-type dnrI is supplied in trans to strainWMH1445, the culture gains the ability to produce theDNR biosynthetic intermediate e-rhodomycinone (RHO)(Stutzman-Engwall et al., 1992) (Table 2). This inter-mediate is readily monitored by thin-layer chromato-graphy, with levels of detection in the nanogram range(the chromophore having a red/orange colour). The pres-ence of dnrI in high copy number within the mutant strainled to the overproduction of RHO, at levels comparablewith those observed previously (Stutzman-Engwall et al.,1992). Perhaps this observation is interesting from theperspective of strain development, where it is known thatone can increase the copy number of DnrI and stimulatethe overexpression of most, if not all, of the biosyntheticpathway genes, including the resistance genes (Madduriand Hutchinson, 1995a).

When the truncated forms of dnrI were introduced intostrain WMH1445, not one maintained the ability to com-plement the dnrI mutation. Indeed, even minimal trunca-tions from the N- or C-terminal ends resulted in the

disruption of DnrI function (Table 2). In an effort to ensurethat the failure to restore DNR intermediate productionwas not the result of a lack of mutant protein expression,Western blot analyses of soluble cell fractions were con-ducted using a histidine tag-specific monoclonal antibody.The wild-type and C-terminal truncates should exist as his-tag fusions of DnrI (see Experimental procedures).It was found that the cultures expressed the expectedwild-type and truncated forms of DnrI (data not shown).Additionally, it was observed that the maltose-bindingprotein (MBP)–DnrI wild-type fusion protein (constructpWHM1123) fails to support RHO production in strainWMH1445 (Table 2) (see Discussion). In contrast to theMBP–DnrI fusion, the N-terminal his-tagged wild-typeDnrI (construct pWHM1115) retains the ability to comple-ment strain WMH1445 (Table 2). It appears that the pres-ence of the smaller, less obtrusive histidine tag does notinterfere with DNA recognition and binding or with inter-actions with other transcriptional factors.

To determine which amino acid residues may be critical for DNA interaction and binding, individual alaninesubstitutions were made at targeted positions. As the pre-dicted secondary structure of the N-terminal half of DnrI,and most SARPs, is highly similar to the winged-helixDNA-binding proteins of the OmpR family, a guide for gen-erating precise changes was readily available (see Fig. 1)(Martinez-Hackert and Stock, 1997a; Wietzorrek andBibb, 1997). It was observed that changes to T64, R71,G95 and Y96 caused a serious defect in the ability of theprotein to stimulate RHO production in strain WMH1445(Table 2). These results support the prediction that theseamino acids make major contributions to protein functionthrough participation in specific DNA interactions. If thewinged-helix DNA-binding motif is located within the N-terminal half of the protein, mutations generated in the C-terminal end of DnrI should not inhibit transcriptionalactivation. To test this idea, alanine mutations werecreated within the C-terminal end of the protein. Changes


Strain Phenotype/mutation % RHO production

WMH1445+pWHM1115 Wild-type his-tagged DnrI 100pWHM1116 10% N-terminal truncate DnrI 0pWHM1117 20% N-terminal truncate DnrI 0pWHM1118 30% N-terminal truncate DnrI 0pWHM1119 40% N-terminal truncate DnrI 0pWHM1120 10% C-terminal truncate his-tagged DnrI 0pWHM1121 20% C-terminal truncate his-tagged DnrI 0pWHM1122 30% C-terminal truncate his-tagged DnrI 0pWHM1123 MBP–DnrI fusion 0pWHM1125 DnrI T64A 0pWHM1126 DnrI R71A 0pWHM1127 DnrI G96A 0pWHM1128 DnrI Y97A 0pWHM1129 DnrI R181A 98pWHM1130 DnrI L202A 71

Table 2. Complementation of dnrI mutantstrain.

to R181 and L202 did not abrogate RHO production, ashad the N-terminal mutations. Interestingly, quantificationrevealed that R181A maintained nearly complete com-plementation of RHO production, whereas L202A resultedin less than complete restoration (Table 2).

MBP–DnrI fusion protein isolated as a stable soluble protein

In an effort to avoid the problems of low protein yield andinstability encountered previously during the purificationand storage of DnrI (Tang et al., 1996), we pursued astrategy to clone and express DnrI as an N-terminal MBPfusion protein. It has been demonstrated that obtaining atarget protein in a soluble, biologically active form can beenhanced by fusing the aggregation-prone polypeptide to a highly soluble partner such as MBP (Kapust andWaugh, 1999). From Escherichia coli BL21(DE3), an N-terminal MBP-tagged DnrI fusion protein was overex-pressed and purified via one-step amylose column affinitychromatography (Fig. 2), as were all the mutant proteinsgenerated in this work. The wild-type DnrI fusion proteinwas found to behave similarly to native DnrI (Tang et al.,1996) in DNA-binding assays. Surprisingly, the fusionprotein exhibited exceptional stability, maintaining DNA-binding activity for a period of up to 6 months uponstorage in numerous buffer compositions at only –20∞C.

Analysis of MBP–DnrI DNA-binding properties

With the stable fusion protein in hand, more detailed DNA-binding assays were performed. The stoichiometry ofDNA binding by the MBP–DnrI fusion was characterizedby conducting gel mobility shift (GMS) assays. The DNAsubstrate was a 90 bp DNA fragment from the intergenic

region separating the dpsEF and dnrG–dpsABCD diver-gent operons. This sequence includes one intact pair ofdirect repeat elements known to be critical for DNA recog-nition and binding (Fig. 3) (see Discussion). After precisedetermination of protein and DNA stock concentrations,varying amounts of protein were reacted with constantconcentrations of labelled DNA substrate. As the ratio of protein to DNA substrate approached 2 : 1, nearly alllabelled DNA shifted into the bound complex (Fig. 4). Thisresult does not preclude the idea that DnrI may bind as amonomer to each of the tandem heptameric repeats.Indeed, at 0.5 : 1 and 1 : 1 ratios of protein to DNA, thereare clearly bandshifts that represent an intermediatecomplex (Fig. 4). Perhaps this complex represents asingle molecule of DnrI binding to a single repeat targetwithin the –35 region of the dnrG–dpsABCD operon (seeDiscussion).

As gel shift assays are readily quantifiable, the resultsfrom the experiment used to determine the stoichiometry



Fig. 1. Alignment of the N-terminal amino acid sequence of DnrI with the C-terminal DNA-binding domain of OmpR. Amino acid residues thathave been exchanged for alanines are highlighted above the DnrI sequence. Regions of OmpR sequence known to make specific contactswith DNA (double underline) and holo-RNAP (dashed underline) are marked. The long intervening sequence separating alpha-helices 2 and 3is denoted.

Fig. 2. Representative SDS–PAGE of MBP–DnrI fusion proteinsisolated from E. coli. Lane 1, MBP–DnrI (wild type); lane 2MBP–DnrI N27; lane 3, MBP–DnrI N54; lane 4, MBP–DnrI N81;lane 5, MBP–DnrI N108; lane 6, MBP–DnrI C246; lane 7,MBP–DnrI C219; lane 8, MBP–DnrI C191. Molecular weight markeris shown on the left.


of protein–DNA binding were also used to estimate the dissociation constant (Kd) for MBP–DnrI dnrG pro-moter affinity. The Kd is defined as the concentration ofMBP–DnrI required to shift 50% of the radiolabelled sub-strate DNA. Densitometry of the autoradiogram revealedthat a shift of 50% was observed with 7.0 ng of protein(Fig. 4), resulting in an apparent Kd of 2.4 ¥ 10–9 M, ameasure that is consistent with DNA-binding proteinswithin the genus Streptomyces (Rother et al., 1999).

Determination of the DNA-binding ability of DnrI mutant proteins

From the complementation studies using both truncated

and site-directed mutant forms of DnrI, it was obvious thatcertain manipulations caused a disruption of protein func-tion. Disruption of either transactivation or DNA binding as a cause for the abolition of complementation remainedas two distinct possibilities. DNA-binding assays werecarried out using purified MBP–DnrI fusion proteins of thevarious truncated and N-terminal alanine-substitutedmutants that failed to complement strain WMH1445. TheDNA substrate was a 203 bp FspI–EagI fragment thatcontained the entire intergenic region separating thedpsEF and dnrG–dpsABCD operons and included twointact sets of tandem heptameric repeat sequences(Fig. 3). In reaction mixtures that contained a protein con-centration at least 10-fold greater than DNA substrate,


Fig. 3. Schematic representation of the intergenic region between the dpsEF and dnrG–dpsABCD operons within the DNR/DXR biosyntheticgene cluster. The bold underlines detail the tandem heptameric ‘target’ sequences within the –35 promoter regions, and the dashed underlinehighlights the spacer nucleotides separating the repeats. The 90 bp XhoI–EagI and 203 bp FspI–EagI fragments used as probes for the GMSassays are shown.

Fig. 4. Stoichiometric analysis of DnrI DNAbinding by gel mobility shift assay. Lane 1,free DNA; lane 2, 3.5 ng of DnrI; lane 3, 7.0 ng of DnrI; lane 4, 10.5 ng of DnrI; lane 5,14.0 ng of DnrI. Free and protein-bound DNAlabel are indicated by arrows. Ratios ofprotein and DNA are indicated underneath.

none of the DnrI truncates maintained the ability to bindDNA (Fig. 5A). In similar experiments, the N-terminalalanine-substituted mutant proteins did not bind DNA(Fig. 5B). These results clearly demonstrate that thefailure of the DnrI mutants to complement strainWMH1445 is directly related to the inability of the proteinsto recognize and bind the specific DNA targets. DnrI con-tains a DNA-binding domain within its N-terminus thatincludes specific residues that may be comparable infunction to those in the OmpR family of winged-helix DNA-binding proteins.

Effect of promoter mutations on the in vitro bindingactivity of DnrI

In the dnrG–dpsABCD promoter region, DNA sequencesbound by DnrI have been located by DNase I footprintingand gel mobility shift assay (Tang et al., 1996). Tandemlyarrayed repeat sequences have been discovered withinthis sequence, which includes the region surrounding the –35 hexamer of the promoter (Tang et al., 1996; Wietzorrek and Bibb, 1997). These sequences are pre-dicted to be critical for DNA recognition and binding byDnrI. To determine the importance of nucleotides withinthe tandem repeats, the DNA sequence of the directrepeats and the spacer region separating the repeatsfound within the dnrG–dpsABCD promoter (Fig. 3) was

subjected to site-directed mutagenesis. Base changes,substituting the alternative purine or pyrimidine in eachposition, were created within the direct repeats. In an effort to characterize the effect of the site-directed muta-tions, the binding efficiency of DnrI to the mutant promoterfragments was tested by GMS assays. Each promoterfragment was excised from the mutant constructs in aprocess that preserved the dnrG–dpsABCD tandemrepeats while disrupting the upstream dpsEF repeatsequences. Base substitutions within the 5¢ repeat boxresulted in a decrease in binding efficiency by DnrI(Fig. 6A), characterized by non-discrete bandshifts andsmearing of label, with the one exception being at position6 (Fig. 6A, lane 8). Substituting A for G (Fig. 3) at that posi-tion does not result in inhibition of binding and mayperhaps enhance DnrI–DNA interaction. Every substitu-tion within the 3¢ repeat led to diminished DnrI binding efficiency (Fig. 6B). However, a quantitative analysis of thegel image revealed that binding was disrupted the leastwhere an A residue replaced G at position 4 (Fig. 6B, lane5). An addition of one nucleotide to the spacer sequenceresulted in nearly complete disruption of DnrI binding(Fig. 6C, lane 3). Diminution of DnrI binding through muta-tion of conserved nucleotides within the putative targetrepeats clearly indicates the importance of thesesequences and is in good agreement with similar studiesperformed with OmpR target sequences (Harlocker et al.,



Fig. 5. Determination of DNA-binding abilityof mutant forms of DnrI by gel mobility shiftassay.A. GMS using truncated forms of DnrI. Lane1, free DNA; lane 2, 500 ng (same amount forall lanes) of wild-type MBP–DnrI; lane 3,MBP–DnrI N27; lane 4, MBP–DnrI N54; lane5, MBP–DnrI N81; lane 6, MBP–DnrI N108;lane 7, wild-type MBP–DnrI; lane 8,MBP–DnrI C246; lane 9, MBP–DnrI C219;lane 10, MBP–DnrI C191.B. GMS using SDM forms of DnrI. Lane 1,free DNA; lane 2, 500 ng of wild-typeMBP–DnrI; lane 3, MBP–DnrI T64A; lane 4,MBP–DnrI R71A; lane 5, wild-type MBP–DnrI;lane 6, MBP–DnrI G95A; lane 7, MBP–DnrIY96A; lane 8, free DNA.


1995). Deleting 1bp from within the linker significantly dis-rupts DnrI binding, but not to the extent observed wherethere was an addition of one nucleotide (Fig. 6C, lane 4).Again, these results are comparable with observationsmade after changing OmpR family target sequences (Prattand Lilhavy, 1995), where the proper spacing between therepeats is crucial for promoter activity.

Discussion

The SARP family of transcriptional activators appears tobe genetically and functionally related to the winged-helix

family of transcriptional regulators, of which the E. coliregulatory protein OmpR is a model member. The DNA-binding domain of the SARPs is located within the N-terminus, whereas the comparable binding motif islocated in the C-terminus of the OmpR family. This particular structural orientation is not unprecedented, asother members of the winged-helix OmpR–PhoB familycontain the DNA-binding domain within the N-terminus(Martinez-Hackert and Stock, 1997b). OmpR belongs toa family of response regulators for which signal trans-duction occurs in combination with a sensor kinasepartner through a phosphorelay mechanism, where the


Fig. 6. Gel mobility shift assays using themutated promoter DNA fragments.A. Lane 1, no protein – free DNA; lane 2,wild-type DNA fragment; lanes 3–9, mutantpromoters G1.1–G1.7; lane 10, double mutantpromoter G1.11/G1.4.B. Lane 1, no protein – free DNA; lane 2,wild-type DNA fragment; lanes 3 and 4,mutant promoters G2.1–G2.2; lanes 5–8,mutant promoters G2.4–G2.7.C. Lane 1, no protein – free DNA; lane 2,wild-type DNA fragment; lane 3, mutantpromoter, one nucleotide addition; lane 4,mutant promoter, one nucleotide deletion.Arrows shown on the left mark the positionsof DnrI-free and -bound DNA.

phosphorylation domain resides in the N-terminus. In con-trast, DnrI is the concluding link of a pathway-specifictranscriptional cascade and does not require phosphory-lation to render it an active transcriptional activator.

The amino acid sequences of the DnrI (Stutzman-Engwall et al., 1992), ActII-ORF4 (Fernandez-Morenoet al., 1991), RedD (Narva and Feitelson, 1990), CcaR(Perez-Llarena et al., 1997) and a handful of otherpathway-specific activators (SARPs) are quite similar.Thus, it is reasonable to predict that the tertiary structureof each protein contributes to a related mechanism ofDNA binding and transcriptional activation. Interestingly,expression of dnrI can complement an actII-ORF4 mutantstrain (Stutzman-Engwall et al., 1992), whereas expres-sion of redD (Stutzman-Engwall et al., 1992) or ccaR(Perez-Llarena et al., 1997) do not. Accordingly, it has been proposed that DnrI and ActII-ORF4 recognizeremarkably similar DNA repeat sequences within the promoter regions of the corresponding DNR and ACTbiosynthetic genes respectively (Wietzorrek and Bibb,1997). Indeed, upon inspection, one can find these directrepeats within the gene promoter regions bound by DnrI(Tang et al., 1996) and ActII-ORF4 (Arias et al., 1999).From the stoichiometry analysis, it is apparent that twomolecules of MBP–DnrI bind to the DNA substrate containing one intact pair of heptameric repeat targetsequences. Although three repeat sequences are presentwithin the DNA fragment (Fig. 3), it is thought that productive binding by DnrI requires co-operation betweenmonomers that recognize intact adjacent direct repeats,similar to the essential OmpR–OmpR co-operative interactions required for binding cognate targetsequences (Harlocker et al., 1995). DnrI binding to allthree repeat target sequences should result in the forma-tion of two intermediate complexes. A single intermediatecomplex is observed in Fig. 4. Recently, the three-dimensional structure of the DNA-binding/transactivationdomain of PhoB, an OmpR family member, bound to itstarget DNA was determined via nuclear magnetic reso-nance (NMR) imaging (Okamura et al., 2000). The datasuggest a structure in which one PhoB monomer is boundto each direct repeat comprising the target sites. Hence,we predict that a DnrI monomer binds to each heptamericrepeat within the promoters of DNR/DXR biosyntheticgenes. Where discrete DNA target sequences have beenclearly defined for transcriptional regulators, such asdirect or inverted repeats, the stoichiometry of bindingprotein to DNA is ordinarily found to be 1:1 (Cicero et al.,1998).

Maltose-binding protein fused to the N-terminus dis-rupts the ability of DnrI to complement strain WMH1445,although it does not inhibit DNA substrate binding in GMSassays. It has been suggested that the alpha-subunit ofRNAP contacts the long loop region separating alpha-

helices 2 and 3 (the turn portion of this HTH variant) ofthe OmpR DNA-binding domain (Kondo et al., 1997).Additionally, evidence has been found suggesting that the long turn region of PhoB acts as a putative interactionsite for the RNAP sigma-subunit (Makino et al., 1996;Okamura et al., 2000). Perhaps the existence of MBP onthe N-terminus of DnrI disrupts potential interactions withother transcriptional factors such as RNAP.

From the structural studies of OmpR and PhoB, it hasbecome apparent that the DNA-binding domain architec-ture of the winged-helix family of transcriptional activators(including the SARPs) consists of a central three-helicalbundle containing the HTH variant separated by a longloop (see Fig. 1). The predicted long loop residues are notwell conserved, and it has been proposed that each indi-vidual OmpR family protein takes on a characteristic con-formation within the HTH based upon the make-up of thelong loop region (Okamura et al., 2000). Thus, the partic-ular loop conformation may contribute to the specificity fortransactivation and DNA binding of each individual SARP.Failure to engineer the Streptomyces clavuligerus SARPCcaR into a dnrI-complementing protein by substitutingresidues within helix three of the HTH variant, and notwithin the long separating loop (P. J. Sheldon and C. R.Hutchinson, unpublished data), supports this argument.The function of the C-terminal half of the protein remainsunknown at this time. It may be involved in intra- or inter-molecular interactions required for oligomerization andassociation with other transcriptional complex proteinsrespectively. These predictions may explain the abroga-tion of protein activity upon deletion of just a few aminoacids or the drop in RHO production when certainresidues within the C-terminus are mutated.

The dnrG–dpsABCD promoter was chosen as thetarget for mutational analysis because it is an early actingand important promoter in the biosynthesis of aklanonicacid, the first detectable intermediate of daunorubicinbiosynthesis. Substitutions within the heptameric repeatsresulted in differential affinity of DnrI to the mutant repeatsequences, which is attested to by the variable binding of the protein in the gel mobility shift assays. In naturalconditions, variations in the intracellular environment maycontribute to variations in DnrI binding. OmpR differen-tially associates with individual promoter elementsdepending on the state of osmolarity surrounding andwithin cells (Harlocker et al., 1995; Bergstrom et al.,1998). The heptameric repeats are separated by a specific number of nucleotides; in the case of the dnrGpromoter, this number is four. As the addition or deletionof the spacing nucleotides had such a dramatic effect on DNA binding, the spacing is critical for DnrI–DNAinteraction. Correct spacing appears to enhance the co-operativity of protein monomers while they are binding tothe target sequence(s). Changing the spacing between




adjacent heptameric repeat target sequences could eitherdisrupt the ability of DnrI monomers to interact with theDNA (induced by steric hindrance) or may adversely affectthe spacing of the –35 and –10 hexamers, in a mannerthat prevents a competent transcription complex fromforming. In support of the former idea, gel mobility shiftassays clearly show that binding efficiency is dramaticallydiminished when an addition or deletion occurs within thesequence that separates the direct repeat binding sites.Increasing the space separating the repeats causedgreater interference, suggesting that co-operativityamong protein monomers is significant.

The results reported here suggest that targetedchanges to the repeat sequences recognized by DnrI maybe useful in the engineering and development of moreeffective DNR/DXR-producing strains. As there are dataindicating that DnrI controls transcriptional activation ofmost, if not all, the DNR/DXR biosynthetic genes (Madduriand Hutchinson, 1995b; Tang et al., 1996), metabolicengineering of DnrI could potentially increase DNR andDXR yield. These predictions might also be extended tothe SARP family as a whole. SARP genes isolated fromvarious organisms or from within different regions of thegenome (Culebras et al., 1999) could be exploited in thedevelopment of valuable production strains. Significantly,this family of regulators has been found to be a key com-ponent within the biosynthetic gene clusters of many com-mercially notable Actinomycete-derived pharmaceuticalssuch as cephamycin (Perez-Llarena et al., 1997), tetra-cycline (McDowall et al., 1999) and tylosin (Bate et al.,1999).

Experimental procedures

Bacterial strains, culture conditions and media

Escherichia coli DH5a used as a host for the generation of double-stranded plasmid DNA was grown at 37∞C onLuria–Bertani (LB) medium (Sambrook et al., 1989). E. coliBL21(DE3) (Novagen), used as host for protein expressionand was grown at 37∞C in LB medium. S. peucetius strainWMH1445 (Stutzman-Engwall et al., 1992) was grown onR2YE medium containing 0.5% glycine at 30∞C for the preparation of protoplasts (Hopwood et al., 1985). StrainWHM1445 transformants were grown on ISP medium 4 agarplates amended with 0.2% yeast extract and 25 mg ofthiostrepton ml–1 medium at 30∞C to produce spores forculture stock preservation.

Biochemicals and other reagents

All commonly used biochemicals were purchased fromSigma. Thiostrepton, used for antibiotic selection of Strepto-myces transformations, was kindly provided as a gift from S. Lucania, Bristol-Myers-Squibb, Princeton, NJ, USA. Allrestriction endonucleases and DNA-modifying enzymes were

purchased from either New England Biolabs (NEB), GibcoBRL or Promega.

Complementation of strain WMH1445

From pWHM1102, a construct that produces a his6x-taggedversion of DnrI (Tang et al., 1996), a 1.2 kb XbaI–HindIIIfragment was extracted and ligated to the same sites invector pWHM1250 (Madduri et al., 1998). This construct(pWHM1115) results in dnrI expression under the control ofthe strongly constitutive Streptomyces promoter ermE*p(Bibb et al., 1994). The dnrI N-terminal truncations were fab-ricated using the polymerase chain reaction (PCR) to syn-thesize DNA fragments that incorporated an NdeI restrictionendonuclease site at positions within the 5¢ coding sequenceof the dnrI gene and a HindIII site downstream of the dnrIstop codon. The DNA fragments were then ligated to thesame sites in plasmid vector pT7SC (Brown and Campbell,1993). From the resulting constructs, XbaI–HindIII fragmentswere isolated and ligated to the same sites in plasmid vector pWHM1250 to form plasmids pWHM1116–1119 (seeTable 1). To construct the dnrI C-terminal truncations, PCRwas used to generate DNA fragments that would incorporatea native XbaI site upstream of the start codon and a TGAstop codon followed by a HindIII site at various positionswithin the C-terminal coding sequence of dnrI. The PCR-derived fragments also contained coding sequence thatwould incorporate a histidine tag on the N-terminal end of theprotein. The DNA fragments were digested with XbaI–HindIIIand ligated to the same sites in vector pWHM1250 creatingplasmids pWHM1120–1122 (see Table 1). An MBP–DnrI(wild-type DnrI) fusion under the control of ermE*p was con-structed using PCR to amplify dnrI containing EcoRI andHindIII sites at the 5¢ and 3¢ ends, respectively, usingpWHM1102 as template. The EcoRI–HindIII fragment wasthen cloned into the same sites in vector pMalp2X (NEB) toform the MBP–DnrI fusion. From the resulting plasmid, anNdeI–HindIII fragment containing the MBP–DnrI fusion wasligated to the same sites in pT7SC to add restriction sites anda ribosome binding site (preceding the MBP start site). AnXbaI–HindIII fragment was excised from this intermediateconstruct and cloned into the same sites in pWHM1250 togive plasmid pWHM1123. The DnrI mutant constructs werethen introduced into S. peucetius strain WMH1445. Trans-formants were selected for resistance to thiostrepton (25 mgof drug ml–1 medium) and kanamycin (10 mg of drug ml–1

medium). To analyse metabolite production, transformantswere grown in APM medium according to previously pub-lished protocols (Stutzman-Engwall et al., 1992) andanalysed by thin-layer chromatography (TLC) for the pres-ence of e-rhodomycinone (RHO), a doxorubicin biosyntheticintermediate. Quantification of RHO was made by extractingnormalized culture volumes with CHCl3, drying the extractedcompound under vacuum and determining the mass. As the expression of PKS genes involved in the construction ofthe molecular core of doxorubicin has been shown to resultin an accurate representation of pathway regulation by DnrI(Stutzman-Engwall et al., 1992), the use of the validatedWMH1445 strain for measurement of RHO was warranted.For the oligonucleotide primer sequences used for the PCRs,see Table 3.




Table 3. Oligonucleotides used for DnrI mutagenesis.

N-terminal truncates pWHM1116-1119N27 (10%) 5¢-GGG GAT TCC ATA TGT TCT CAC TGC TCG CTC TC

NdeIN54 (20%) 5¢-GGG GAT TCC ATA TGC CGG CGA GCG CGC TGA CCN81 (30%) 5¢-GGG GAT TCC ATA TGA ACG GAC CCG CCA AGG ACN108 (40%) 5¢-GGG GAT TCC ATA TGG CCT TCG AGC GTC TCG CC

C-terminal truncates pWHM1120-1122C246 (10%) 5¢-CGC AAG CTT TCA GAG ACG GGC GGA CGG TTC

HindIII DnrI stopC219 (20%) 5¢-CGC AAG CTT TCA GCT CGT GCG GCC CGA GCGC191 (30%) 5¢-CGC AAG CTT TCA CGA CAG CTC GGG CAG CAGForward 5¢-TAA TAC GAC TCA CTA TAG GGG AAT TGT GAG

MBP fusion oligonucleotidesNative DnrI 5¢-ATC GAG GGA AGG ATT TCA ATG CAG ATC AAT ATG TTG GGC CCG CTC

XmnI site DnrI startReverse 5¢-CGC AAG CTT TCA GGC AAG CGC GAC GGA CGC

HindIII DnrI stoppWHM1124 5¢-GTA CCG GAA TTC ATG CAG ATC AAT ATG TTG GGC CCG CTC

EcoRI DnrI start

N-terminal truncatesN27 (10%) 5¢-ATC GAG GGA AGG ATT TCA GTA TTC TCA CTG CTC GCT CTC CAG GCAN54 (20%) 5¢-ATC GAG GGA AGG ATT TCA CCG GCG AGC GCG CTG ACC ACC CTT CAGN81 (30%) 5¢-ATC GAG GGA AGG ATT TCA ACC GGA CCC GCC AAG GAC GTG CTG CGTN108 (40%) 5¢-ATC GAG GGA AGG ATT TCA GCC TTC GAG CGT CTC GCC GAG GAG GGC

MBP C-terminal truncations were constructed using the native MBP–DnrI forward primer and the C246-191 reverse primers listed above

Site-directed mutationsT64A 5¢-ACC ACC CTT CAG GCC TAC ATC CTT CAA GTG ACC Æ GCCR71A 5¢-CTT CAA GTG CGC GCC GGC ATC ACC GTG GCC CGG Æ GCCG95A 5¢-ACC TGC TAC GGC GCC TAC CTG CTC GAC GGC Æ GCCY96A 5¢-TGC TAC GGC GGC GCC CTG CTC GAC GTG TAC Æ GCCR181A 5¢-CTG AGG CTG GGC GCG CAC GCG GGC CTG CGG Æ GCGL202A 5¢-ATG CAC GAG AAC GCG TGG GCC CAA TTC CTG Æ GCG

Site-directed mutagenesis of DnrI

To produce DnrI mutants, the Stratagene Quickchange site-directed mutagenesis (SDM) kit was used according to themanufacturer’s protocol. Specific nucleotide substitutionswere introduced using mutagenic oligonucleotides synthe-sized at the University of Wisconsin–Madison BiotechnologyCenter. For the PCR-based reactions, pWHM1124 was usedas the template DNA. This construct contains the 1.2 kbXbaI–HindIII his-tagged dnrI fragment from pWHM1102cloned into the same sites in pUC19. For the dnrI comple-mentation experiments, an XbaI–HindIII fragment from SDMconstructs was extracted and ligated to the same sites inpWHM1250, resulting in plasmids pWHM1125–1130 (seeTable 1). The SDM DnrI mutants were confirmed to containonly the desired changes by nucleotide sequence analysis.These constructs were transformed into S. peucetius strainWMH1445, and the resulting transformants were analysed as described above. The nucleotide sequences of the site-directed mutagenic oligonucleotides are found in Table 3.

Site-directed mutagenesis of the dnrG–dpsABCDpromoter

Site-directed mutations within the dnrG–dpsABCD promoterwere made using the Quickchange kit as described above.For the PCR-based reactions, pWHM1105 (Tang et al., 1996)

was used as the template DNA. This construct contains thedpsEF–dnrGdpsABCD intergenic region cloned into vectorpUC18 (Sambrook et al., 1989). After determining that the correct mutations had been made by sequence analysis,the constructs were digested with SalI and BamHI, and theresulting 450 bp fragments, containing the mutant dnrG pro-moters, were cloned into the same sites in plasmid pGEM11zf(Promega).

Construction and expression of MBP–DnrI fusions

Fusion of DnrI to the MBP was accomplished using PCR togenerate DNA fragments from template vector pWHM1102containing dnrI wth XmnI and HindIII sites before the startand downstream of the translational stop codons respec-tively. The resulting DNA fragments were ligated to theXmnI–HindIII sites of plasmid vector pMalc2X (NEB). To con-struct the N-terminally truncated forms of MBP–DnrI, PCRwas used to amplify DNA fragments that incorporated anXmnI restriction site at positions within the 5¢ codingsequence of the dnrI gene and a HindIII site downstream ofthe dnrI stop codon. The resulting DNA fragments were thentreated as described above. C-terminal DnrI–MBP truncateswere constructed using PCR to generate DNA fragments thatincluded an XmnI site upstream of the native start codon and a TGA stop codon followed by a HindIII site within the C-terminal coding sequence of dnrI. The resulting DNA


fragments were treated in the same manner as above to givethe final DnrI C-terminally truncated MBP fusion constructs.Using the same oligonucleotide primers as those for the construction of the wild-type DnrI fusion, fragments from the site-directed mutant constructs (pWHM1126–1130) wereamplified and cloned in the same manner as described aboveto form MBP–DnrI SDM fusion plasmids. For expression ofMBP–DnrI fusion proteins, the above constructs were trans-formed into E. coli strain BL21(DE3), grown at 37∞C to anOD600 of ª 0.6 and protein expression induced with the addi-tion of IPTG to a final concentration of 1 mM. After disruptionof cells via sonication, the cytosolic fraction was passed overan amylose–agarose resin (NEB), resulting in the isolation ofMBP-tagged proteins. After release of the MBP–DnrI proteinsfrom the amylose column using maltose, the maltose wasremoved by filtration concentration using an Amicon micro-con filter apparatus and repeated washing with storage buffer(10 mM Tris, pH 7.2, 20 mM NaCl).

Mobility shift DNA-binding assays

Either a 203 bp FspI–EagI or a 90 bp XhoI–EagI DNA frag-ment containing the dpsEF–dnrGdpsABCD intergenic pro-moter region was end labelled with [a-32P]-dCTP (Amersham)using the Klenow large fragment of DNA polymerase. Bindingreactions (40 ml) contained 7.5% glycerol, 1.5 mg of poly-(dI–dC), purified protein of varying concentrations and a con-stant concentration of labelled substrate DNA (10 000–15 000c.p.m.) in a buffer composed of 90 mM Tris-HCl, pH 8.5, 90 mM boric acid and 20 mM EDTA. After incubation atambient temperature for 30 min, protein-bound and free DNAwere separated by electrophoresis at ambient temperature ona 5% native polyacrylamide gel running at ª 25 mAmp. Thegels were developed and analysed using a PhosphorImagerSI (Molecular Dynamics) and the IMAGEQUANT softwarerespectively. Densitometry of the developed phosphorimageswas used in the stoichiometric and kinetic calculations ofDnrI–DNA interactions. The mutant promoter constructs wereexamined as follows. PCR was used to amplify a fragmentfrom the dnrGdpsABCD mutagenic promoter regions. Afterdigestion with XhoI and EagI, the fragments were end labelledas described above. Binding reactions (20 ml) contained 7.5%glycerol, 1.5 mg of poly-(dI–dC), 48 ng of purified MBP–DnrIprotein and a normalized concentration of labelled substrateDNA (10 000–15 000 c.p.m.) in the same buffer describedabove.

Acknowledgement

This research was supported in part by a grant from theNational Institutes of Health (CA64161).

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mapping the dna-binding domain and target sequences of the streptomyces peucetius daunorubicin...

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