J. Mol. BioE. (1996) 212, 669-682

Regulation of Divergent Transcription from the Iron-responsive fepB=eratC Promoter-operator Regions in Escherichia coli Timothy J. Brickman, Bradley A. Ozenbergert and Mark A. McIntoshS Department

of Molecular Microbiology and Immunology School of Medicine University of Missouri-Columbia Columbia, MO 65212, U.S.A.

(Received 6 September 1989; accepted 12 December 1989) Transcriptional linkage of the enterobactin gene cluster entCEBA(P15) was confirmed by ent-1acZ gene fusion analysis. Control sequences directing iron-regulated expression of this polyeistronic message were localized to the fepB-entf’ bidirectional promoter region. Transcriptional initiation sites defined by primer extension analysis were located 103 basepairs apart for the divergent fepB and entC messages. Within this divergent regulatory region, strongly consensus -35 and - 10 promoter determinants and potential Fur repressor-binding sequences were identified. A vector containing divergently oriented indicator gene fusions was constructed to monitor regulatory effects of mutations within this iron-responsive control region. The fepB-entC promoter-operator elements were confirmed by mutation, using the dual gene fusion system in multicopy and low copy number states. Mutations in the - 35 and - 10 regions of the fepB and entC promoters that decreased their similarity to consensus resulted in reduced promoter activity. Mutations in the Furcontrolled operators reduced induction ratios (iron-deficient levels/iron-rich levels) for the respective fusion gene activities by approximately sevenfold. Although operator mutants retained some degree of inducibility, complete relief of repression was observed for double operator mutants, suggesting that only minor regulatory influence is exerted by Fur occupation of the opposing operator site. DNase I footprinting experiments were performed to characterize the sequence-specific Fur interactions at the operator sequences. At the fepB operator, a 31 base-pair Fur-protected region was identified, corresponding to positions - 19 t.o + 12 with respect to the transcriptional start site. Similarly, Fur protected a 31 base-pair region in entC, corresponding to positions + 1 to +31 in the message. A contiguous and sequentially occupied secondary Fur-binding site in entC was protected at higher Fur concentrations, extending the protected region to +49, and sequestering the putative Shine-Dalgarno sequence. Operator positional effects and co-operativity are discussed.

The genes encoding enterobact,in biosynthesis and transport functions span approximately 22 kb$ near minute 13 of the E. coli chromosome, and are co-ordinately regulated at the transcriptional level in response to intracellular iron concentration (Fleming et al., 1983). Diverse molecular strategies have determined the following gene order for the enterobactin system (clockwise on the E. coli chromosome): entl). fepA, fes, entF, KfepE, fepC, fepG, fepD, fepB. ent(Y, entE, entB, ent.l and P15. Evidence compiled from complementation genetic studies. nuclrotide

1. Introduction

typh~m,ur~um.

In enteric bacteria, such as Escherichia coli, iron acquisition is accomplished by highly specific ironchelating systems. Certain low molecular weight, high-affinity iron-binding compounds, termed siderophores, are vital to the assimilation of metabolic iron stores under conditions of iron limitation. Enterobactin (Pollack & Neilands, 1970) or enterochelin (O’Brien & Gibson, 1970) the cyclic triester of 2,3-dihydroxybenzoylserine, is the indigenous siderophore of Escherichia coli and Salmonella t Present address: Yale University, Department of Biology, KBTSlO, P.O. Box 6666, New Haven. CT 0651 l-81 12, U.S.A. $ Author to whom all correspondence should he addressed, 0022-2836/90/080669-14

$03.00/O

9 Abbreviations used: kb, lo3 base-pairs; bp, basepair(s); DHBA, 2,3-dihydroxybenzoate; X-gal, 5-bromo4-chloro-3-indolyl-fl-D-galactoside; Xp, 5-bromo-4-chloro3-indolyl phosphate.

669

0

1990 Academic

Press Limited

670

7’. ,J. Hrickman

sequence data, gene fusion analysis, and t,ranscript, mapping suggests that, the genetic loci specifically related to enterobactin biosynthesis and transport) may be co-ordinately expressed as four major polycistronic transcripts originating from two bidirectional iron-responsive control regions located within the fepA -fes and fepB-entC intercistronic regions, although additional secondary control regions are clearly present within this predicted organization. The fepA -fes control region directs the anticlockwise transcription of fepA and entD, and the clockwise t.ranscription of fes, entF and fepE. From t,he fepHentCl control region, transcription of the transport gene, fepB, and possibly the transport, operon, fepDM’, is directed anticlockwise. and the biosynthesis operon entCEBA(P15) is transcribed clockwise. The enzymatic activities encoded hy the rntCEBrl (P15) op eron, with the possible exception of P15 (whose function remains unknown), are required for the biosynthesis and activation of 2Sdihydroxybenzoate (DHBA), an enterobactin precursor. While early investigations produced inconsistent’ results concerning the t,ranscript,ional organization of the DHRA gene cluster, a recent report provided strong suggestive evidence for t,he existence of an iron-inducible operon (Nahlik rt nl.. 1989). However, the precise location and nat’urr of &acting sequences directing the iron-responsive expression of this cluster remained uncharacterized. Transcriptional regulation of the rnterobactin genes, as well as several other genetic1 loci. is mediated by the protein product’ of the fw locus (Bagg Xr Neilands, 1985: de Lorenzo et nl.. 1987: Hantke. 1981), which acts as aorepressor with ferrous iron vifz sequence-specific prot,ein-DNA interactions at the promoter regions of Furcontrolled genes (Bagg & Neilands. 1987: de Lorenzo rt al.. 1987. 1988). For the iron-regulated aerobactin operon of plasmid pColVK30, spe‘cifir Fur repressor-operator interactions at the promoter region were initially demonstrated by DNase I protection experiments (de Lorenzo it al.. 1987), which identified t#wo cont’iguous and sequentially ockcupied operst.or sites. Analysis of the prot,et*trd sequences. and (*omparison with promoter qions of other iron-regulated genes led to the proposal of a 17 bp consensus Fur repressor-binding sequence. 5’-ATAATGATAATCATTAT-3’ (de Lorenzo ef al.. 1987). Based upon t,he dyad symmetry of’ t,he consensus seyuence, as well as in &ro binding studies (Hagg 8r Neilands, 1987). Fur protein was suggested to function as a dimer. Hydroxyl radical footprinting and methyla.tion protection assays defined t’he topology of protein-DNA cont’a,cts as five groups of contacts symmetrically positioned through three turns of R-DNA helix in the primary binding sit,c. suggesting a wrapping modr of Fur binding (de Lorenzo et nl., 1988). Occupation of the secondary binding site at, higher Fur concentrations produced an extended pattern of protected bases exhibiting the same repetitive motif of 2 protected/ 4 unprotected bases, in phase with the primary binding sit,e contacts.

et al

In t’his study, detinit,ive evidenccb ii)r t,rarl scriptional linkage of the DHBA gene cluster was obtained. Control sequences directing iron-regulated cotranscription of the enterobactin biosynthesis genes entC, entE. entB. entil and a closely-linked gene P15. were localized t!o the ,fqH-ent(’ int,rrcistronic region by la.& gene fusion analysis. The transcriptional initiation sites for this polycistronic message and the divergently transcribed gene fepH were mapped by primer extension analysis. A bdirectional fusion vector was constructed to facilitate mut>ational analysis of this control region. Region-specific chemical mutagenesis and oligonucleot,ide-direct’ed site-specific mutagenesis teachused to introduce niques werr kt*y base substitutions in t.he proposed -35 iUld -- 10 promoter determinants and Fur repressor-binding sequences. I’henotypic analysis of these mut.ations in the dual gene fusion construct c~onfirmrd the involvement of these nucleotidr sequences in thr regulation of divergent, iron-responsive t,ranJ’romoter-operator scription from the fepR-ent(’ regions. DNase I protection assays were used to cha.racterizc the sequence-specific interac+ons of operators. Fur repressor wit,h t,he .fepH-ent(’ 2. Materials

and Methods

The E. eoli strain M(:4160 (Pettis &, McIntosh, 1987) was used as the source of RNA in primer clxtension analyses and as the host st,rain for &-la& fusion plasmids. Recombinant, plasmids pITS303. pITS304. pTTS30S and pITFS306(Nahlik it nl.. 1989). pITS45 (Ozenbergrr ~1 nl., 1987), and pTTS55 (Ozenberger it ni.. 1989) were described previously. Thr ant-lnc2 fusion plasmids pITS603. derivatives

pIT6604. pTTS6Os. pITS606 and pITS607. are of the LacZ fusion vrc,tor yIP31i8K (Myers rf al.. 1986). provided bg Uan Myers ~iccVJohn(hmon. The bifunctional fusion plasmid pITS346 is derived from the promoter probe vector pMC:I 403 (C~asadaban rl (1.1.. 1!)80). kindly provided by Mal(~olm Casatiaban. J3acat,eriophage 1TnphoA-I (b221 ~I857 / plasmid isolat’ion and electrophoresis in aparose g&. (j)

DNase

I footprinting

Protection of operator sequences by thr Fur repressox protein against DNase I nicking was analyzed essentially as described by de Lorenzo et al. (1988). Purified Fur protein was generously provided by Dr ,J. B. Neilands. The 0.5 kb XhoI~HindIII promoter cassette fragments of pITS346 and of mutated operator derivatives were 3’-labeled at the XhoI end with [a-32P)dCTP plus dTTP using the large (Klenow) fragment of DK.L\ polymerase.

672

7’. J. thickman

The end-labeled fragment was diluted to a concentration of approximately 025 nM in 100 ~1 of a buffer consisting of 10 mM-bis(Z-hydroxyethyl)imino-tris(hydroxymethy1) methane (pH 7.0), 2 mM-MgGl,, 1 mM-GaGI,, 100 mM-KGl. 100 pg bovine serum albumin/ml, 2.5 pg sonicated salmon sperm DNA/ml, and lOOpM-MnCl,. Fur protein was added at various concentrations and the reaction mixtures preincubated for 2 min at 37”C, after which 2.0 ng of DNase I (Sigma) were added and the incubation at 37°C continued for 2 min. DNA was precipitated by t’he addition of 20 pg carrier tRNA. 25 ~1 7.5 Mammonium acetate and 2.5 vol. cold absolute ethanol. The pellets were rinsed once with 70%) ethanol, dried, resuspended in 80% formamide sequencing sample buffer. and loaded onto SyO acrylamide sequencing gels. After electrophoresis. gels were exposed to Kodak X-Omat RP5 film at, -70°C. The positions of protected sequences were determined by running sequencing ladders as size markers.

3. Results (a) Transcriptional linkage and in vivo regulation of ent biosynthetic genes: analysis of ent-1acZ gene fusions

A nested series of ent-ZacZ gene fusions was constructed to confirm the polycistronic organization of the enterobactin genes entC, entE, entB, entA and P15, and to localize the &s-acting sequences mediating iron-responsive expression of this enterobactin gene cluster in vivo. The 1acZ fusion vector chosen, yIP358R (Myers et al., 1986), is a modified version of the E. coli promoter probe vector pMC1403 (Casadaban et al., 1980) containing the multiple cloning region of pUCl8 (Norrander et al., 1983) proximal to the 1acZ gene. In addition to the selectable E. coli /?-lactamase gene to confer ampicillin resistance, the fusion vector yIP358R also carries the yeast URA3 gene to allow prototrophic selection in appropriate yeast or E. coli host strains. This vector does not express /3-galactosidase in yeast or E. coli in the absence of inserted promoter and translational signals. In a previous study, individual ent-lad fusions were constructed to include only the immediate upstream region of each respective gene and, with the exception of the entC-1acZ gene fusion, which included the fepB-entC intercistronic region, were not responsive to iron availability (Nahlik et al.. 1989). The overlapping ent-ZacZ fusion constructs from the present study include sequences that are contiguous with the fepB-entC intercistronic region. extending from a HpaI site in the divergently transcribed fepB gene to the fusion junction withm each cistron of the rntCEBA(P75) clust,er, and create in-frame translational fusions between each ent gene and la& (Fig. 1). In this context, each ent-la& fusion displays iron-responsive expression of j?-galactosidase. Furthermore, deletion of nucleotide sequences upstream from the unique HindTTI site within entC abrogated expression of all en-la& fusions, providing conclusive evidence that in vivo iron-regulated expression of the enterobactin gene cluster entCEBA(P15) is mediated solely b.y regula-

et al

Figure 1. Construction of e&la& gene fusion plasmids. Designated restriction fragments of the recombinant plasmid pITS55 were ligated into the plasmid vector yIP358R previously digested with BumHILSmaI t,o produce in-phase protein fusions between Ent proteins and fi-galactosidase. Insert DNA fragments for pITS605, pTTS603, pITS606 and pITS604 were generated from BarnHI-linearized pITS55 DNA by partial digestion with P&I or HpaI. Insert DNA for pITS607 was produced by ClaI digestion of pITS55 DNA isolated from E. co/i strain GM119 (dam) to permit cleavage at the methylated site within P15. followed by end-repair using the Klenow fragment of DNA polymerase I and dNTPs and digestion with BamHI. Deletion derivatives of the &-la& gene fusion plasmids were constructed by cleavage of each fusion plasmid with HindITJ, removing the I.1 kb fragment that extends from the Hind111 site 30 bp upstream from the designated BamHT site to the HindIII site in entC. The derivatives are designated pITS605AHS. pITS603AH3, pTTSGOBAH3, pTTS604AH3 and pITS607AH3. Gene fusion plasmids were analyzed by restriction enzyme mapping and fusion junctions were verified by nucleotide sequencing. Cloned insert, I)NA fragments for t,he previously described w&la& gene fusion plasmids pITS305, pITS303. pTTS306 and pITS304 are shown for comparison. Levels of a-galact,osidase act)ivitv expressed from r&la& fusion plasmids and deletion derivatives in response t,o various iron c~onc,entrations in t,he growt#h medium are presented. Abbreviations for restriction enzyme sites: B, BamHI; RV/H, EcoRVIHpnf junction: A. AvaT; H3. HindIIT; I’. PwLIT: E. EcoRT: H. HpaT: C. ClaT.

tory sequences genes comprise

upstream from entC, and that a polycistronic transcript,ional

t,hese turn.

(b) Mapping of transcriptional initiation sites Jar the fepB-entC bidirectional prow&ok Expression of the entCEBA(P15) cluster and the divergent fepB gene is directed from a limited genetic region defined by the fepB (Elkins & Earhart, 1989) and entC (Ozenberger et al., 1989) coding sequences (Fig. 2). To begin to characterize the regulatory sequences involved, sites of in viva transcriptional initiation from t,he fepB-entC intercistronic region were localized by primer extension analysis using total cellular RNA isolated from strain 1MC4160 grown under iron-rich or irondeficient conditions and in the presence or absence of the recombinant plasmid pIT855. For fepB, extension of oligonucleotide primer PER2 (Fig. 2)

-10

*

PMCl

I

1

Figure 2. Nucleotide sequence and regulatory signals of’the fepB-entC bidirectional promoter-operator region. Major promoter determinants (- 35 and - 10) were defined by site-specific mutagenesis, and primary Fur repressor-binding sites (iron boxes) were identified by mutation and by DNase I footprinting studies. Transcriptional initiation sites as defined by primer extension are designated as + 1. Annealing sites for oligonucleotide primers used in transcript mapping (PEBZ and PECl) and to introduce unique restriction sites flanking the bidirectional regulatory region by oligonucleotide-directed in vitro mutagenesis (PMBl and PMCl) are represented by labeled arrows accompanying the nucleotide sequence. The proposed translation initiation codons for the,fepH and rntC genes are underlined, and a large imperfect palindromic sequence identified within the fepH leader is indicated by the converging arrows, The position and orientation of each copy of a short reiterated sequence. 5’-A/GGGNGAGGG-3’, is represented by an open arrow superimposed upon the arrows designating t.he large palindromic sequence.

TATCATTTTGTGGAGGATGAT~GATACGTCACTGGCTGAGGAAGTACAGCAGACCATGGCAACACTTGCGCCCAATCGCTTTTTCTTTATGTCGCCGTACCGCAGTTTTACGACG~ ATAGTAAAACACCTCCTACTATACCTATGCAGTGACCGACTCCTTCATGTCGTCTGGTACCGTTGTGAACGCGGGTTAGCGAAAAAGAAATACAGCGGCATGGCGTCAAAATGCTGCAGT

EntCl)

CCCCGACTTGCAGATTGCCTTACCTCAAGAGTTGACATAGTGCGCGTTTGCTTTTAGGTTAGCGACC~AAAATATAAATGATAATCATTATTAAAGC~~~ GGGGCTCAACGTCTAACGCAATGGAGTTCTCAACTGTATCACGCGCAAACGAAAATCCAATCGCTGGCTTTTATATTTACTATTAGTAATAATTTCGGAA

-35

420 GGAAGGGAGAGGGGGCAGAACGGCGCAGGACATCACATTGCGCTTATGCGAATCCATCAATAATGCTTCTCATTTTCATTGTAACCACAACCAGATGCAA CCTTCCCTCTCCCCCGTCTTGCCGCGTCCTGTAGTGTAACGCGAATACGCTTAGGTAGTTATTACGAAGAGTAAAAGTAACATTGGTGTTGGTCTACGTT -35 G-l -10 [

PEB2 320 w CAAAGCGCACAATCCGTCCCCTCGCCTTTGGGAGAGGGTTAGGGTGAGGGGAACAGCCAGCACTGGTGCGAACATTAACCCTCACCCCAGCCCTCACCCT GTTTCGCGTGTTAGGCAGGGGAGCGGAAACCCTCTCCCAATCCCACTCCCCTTGTCGGTCGTGACCACGCTTGTAATTGGGAGTGGGGTCGGGAGTGGGA h

.

AAAAGTCCTGTTAATAGAAGGGCGTTGCGGTAGAGCGGGGCGAGTCTCACAAATCAGCTTCCTGTTATTAATAAGGTTAAGGGCGTAATGACAAATT~~~ TTTTCAGGACAATTATCTTCCCGCAACGCCATCTCGCCCCGCTCAGAGTGTTTAGTCGAAGGACAATAATTATTCCAATTCCCGCATTACTGTTTAAGCT -

CAGTCCCACTCCGACCACCTTTGTTATGCGACGCCGACCGAAAGGTCACATACACACGGTGCCGACAGTCATTAGACTGCGCCGGTCAGTCGCCGGACTTGACGCCGATAAGGACTTTCG

PMBl . t GTCAGGGTGACGCTGCTGGAAACAATACGCTCCCCCTGGcTT*cc*GTGT*TGTGTGcc*cGGcTGTc*GT**TcTG*cGcGGccAGTcAGcGGccTG**cTGcGGcT*TTccTGA*~~~

(a)

(b)

1

2

3

4

GATC

5’

1

2

3

4

GATC

AT

\ 2

TA 5’ ’

(c) IRON BOX -10

+l

fspB (425.356)

-35 CGGGGTTGCATCTGGlTGTGGTTACAA

mC

.lO +1 -35 AAGAG~~AGTGCGCGllTGCllTj-j-AGCGACC@AAATATAAATGATAATCA~ATTAAAG

GATGGATTCGCATAAGCGCAATG

IRON BOX (447.516)

to oligonucleotide primers were annraled Figure 3. Primer extension of thefepB and rntC tra,nscripts. 32P-radiolabeled total cellular R&A and ext,ended with dNTPs and reverse transcriptase. Oligonucleotide primer PEH2 corresponds to nucleotide positions 286 to 303 on the upper strand in Figure 2. and PECl corresponds to positions 629 t,o 613 on t,hr lower strand. The accompanying DXA sequence ladders were generated using the same primers and plTS55 I)IvX template. RPu’il was isolated from MC4160 cells grown under iron-rich (lane I ) or iron-deficient (lane 2) conditions. and from MC4160 (pITS55) grown under iron-rich (lane 3) or iron-deficient (lane 4) condit,ions. The major in ~:ir~ initiation sites corresponding to the extension products are indicated by the arrows accompanying the nucleotide sequences shown. (a) Extension products and sequencing ladder generated with oligonucleotide primer PER2 (Fig. 2), showing the @f’~pH transcriptional initiation site at the A residue corresponding to base position 384 on the lower strand in Fig. 2. (b) Extension products and sequencing ladder generated with oligonucleotidr primer l’E(‘1 (Fig. 2). showing, the Pnt(’ transcriptional initiation site at the G residue corresponding to base position 488 on t,he upper strand In Fig. 2. (c) Kuclrotide sequence of the DP;A regions flanking the transcription start sites for j”pp,H and cnt(‘. Putat,ive major promoter determinants and primary Fur repressor-binding sites are indicatrd for the rrspect,ivr antisrnsr strands. Sucleotide positions corresponding to those in Fig. 2 are given in parentheses.

resulted in a product (Fig. 3(a)) corresponding t’o an initiation site at the A residue 214 nucleotides upstream from the putative start of the fepB coding region (Elkins & Earhart, 1989). ThefepR initiation site is flanked by a C at position - 1 and a T at position +2, the preferred flanking nucleotides for fi. coli transcriptional initiation sites (Hawley & McClure. 1983), and is spaced seven nucleotldes from the hexamer GAAAAT, which exhibits two of the three most highly conserved nucleotides for E. coli - 10 promoter elements (Hawley & McClure, 1983). Optimally spaced 17 nucleotides from this Pribnow-like sequence is the sequence TTGCAT, which

itself

contains

the

most

highly

favored

nucleotides among E. coli -35 region sequences (Hawley & McClure, 1983). A sequence. 5’-AAAATGAGAAGCATTAT-3’, strongly resembling the proposed consensus Fur-binding sequence. spans - 12 to + 5 relative to the transcription positions start site (corresponding to base positions 380 to 396 in Fig. 2; designated iron box). A 214 nucleotide fepR leader RNA was found to contain extensive inverted repeat sequences with the potential for stable secondary structure (Fig. 2. base positions

+46 to + 152 relative to the fepH transcription start site). as well as short open reading frames of significance. Similar observations unknown concerning potential structures within t’his leader R,NA (Elkins & Earhart, 1989) were reported while this study was under review. Using oligonucleotide primer PECl (Fig. 2), the initiation site for t’he mtC t,ranscript was localized to the G residue 54 nucleotides upst’ream from t,hr entC’ coding region (Fig. 3(b)). The ent(! transcriptional initiation site is spaced seven nucleotides from the Pribnow-like sequence TAGGTT, which is optimally spaced 17 nucleotides from t’he E. coli consensus -35 region sequence TTGACA (Hawk>) & McClure, 1983). A sequence, 5’-TAAATGATAATCATTAT-3’, displaying strong similarity to the consensus Fur-binding sequence, occupies base positions +X to +24 relative to the transcription start site (corresponding to base positions 495 to 511 in Fig. 2: designated iron box). The alignment, of these two promoters and their strongly consensus regulatory determinants is summarized in Figure 3(c). Extension products of identical lengths were observed from both plasmid and chromosomally

Enterobactin Bidirectional derived mRNA for each transcript originating from this divergent promoter region. Iron-deficient growth conditions resulted in a dramatic increase in steady-state levels of both fepB- and en%-specific mRNA, as compared with iron-rich growth conditions. The elevated levels of primer extension products detected for iron-deficient versus iron-rich growth conditions provide direct evidence that in vivo transcriptional activity of this bidirectional promoter is regulated in response bo iron availability. (c) Construction of the bidirectional fusion eector pITS346 A bidirectional fusion vector containing opposing reporter gene fusions was constructed to monitor effects of regulatory mutations within the fepB-entC intercistronic region upon divergent) gene expression. The fortuitous organization of the opposing fepB and entC coding sequences allows the fusion of the structural phoA gene encoding alkaline phosphatase to the periplasmic transport gene fepB and the 1acZ gene encoding /?-galactosidase to entC. Such a construct was necessary to ascertain the degree of co-operativity, if any, between regulatory elements associated with the closely spaced control regions. CC1 18( pITS45) was infected with ATnphoA-1 and plasmid insertions selected as described in Materials and Methods. A TnphoA insertion, designated pITS45.B5, was isolated that mapped to the 3’ end of the fepB gene and exhibited iron-regulated expression of alkaline phosphatase activity, expressing 29 units of alkaline phosphatase under iron-rich growth conditions and 240 units under iron-deficient condit,ions. Examination of pITS45.B5-encoded proteins by minicell analysis (data not shown) identified the fepB-phoA fusion products as two polypeptides (representing the presumed precursor and mature forms of the hybrid product) migrating with a size of approximately 80,000 iV2,in sodium dodecyl sulfate/polyacrylamide gel electrophoresis. pITS45.B5 retained the ability to complement the fepB mutant strain DK214 (Pierce & Earhart, 1986), as judged by growth on iron-deficient medium and by the ability to utilize purified ferric enterobactin in feeding assays. Nucleotide sequence analysis of pITS45.B5 localized the fepB-phoA fusion junction within the third codon from the 3’ end of the proposed fepR coding region. It is interesting to note that this gene fusion produces only two protein products, whereas wild-type fepB expresses four distinct peptides (Ozenberger it ul., 1987: Pierce & Earhart, 1986). This suggests that the C terminus of FepB plays a role in generating some of these putative alternative forms. a conclusion that was also reached from other recently described fusion proteins (Elkins & Earhart, 1989). A 4.6 kb Hind111 fragment containing the fepBphoA fusion gene, the fepB-entC intercistronic region, and the 5’ end of the entC gene was isolated

675

Prtmoter

H3 E Sm/ti3 Y
-----

plTS346

Figure 4. Construction of the bidirectional fusion vector pITS346. The divergently transcribed genesfepB and entC were fused to phoA and la& to produce the dual expression plasmid pITS346. The recombinant plasmid pITS45 was mutagenized with TnphoA t,o produce the fepR-phoA gene fusion plasmid pITS45.B5. The 4.6 kb Hind111 fragment of pITS45.B5, including the fepB-entC intercistronic region and the 5’-coding region of entC was end-repaired using the Klenow fragment and ligated into the SmaI site of the plasmid vector pMC1403 to create an entC-lac2 gene fusion. The 3.2 kb SphI-BamHI fragment of this dual fusion construct was subcloned into the bacteriophage vector M13mp18, and the recombinant single-stranded phage DNA copied in vitro using the mutagenic primers PMBl and PMCl to introduce the unique HindIII and XhoI sites within the 5’-coding regions of the divergent genes. Primer PMBl , 5’-AATACGCTGCGGCAAGCTTTCCAGTGTATG-3’, corresponding to nucleotide positions 24 to 53 in Fig. 2 with 2 internal mismatches at base positions 37 and 38, created an Hind111 site in the .Y-coding region of fepB. Primer PMCl, 5’-TGGATACGTCACTGCTCGAGGAAGTACAGC-3’, corresponding to nucleotide positions 543 to 572 in Fig. 2 with internal mismatches at positions 557. 558 and 559, introduced an XhoI site in the 5’-coding region of e&C. The mutagenized 3.2 kb #phi-BamHI DN4 frag-

ment with the newly created unique restriction sites was ligated into the dual fusion vector to replace the wild-type

fragment and produce pITS346. Abbreviations for restriction enzyme sites: E, EcoRI; Sm/H3, SmaI/HindIII

tion; Sp, SphI; H3, HindIII; SmaI junction;

junc-

X, XhoI; H3/Sm, Hind1111

B. BamHI.

from pITS45.B5, end-repaired using the Klenow fragment, of DNA polymerase I and deoxynucleotides, and ligated into the SmaI site of the 1acZ fusion vector pMC1403, resulting in fusion of entC at its HindIII site to the eighth codon of 1acZ (Fig. 4). This dual gene fusion plasmid (pITS345) expresses 148 units of /?-galactosidase and 35 units of alkaline phosphatase under iron-rich growth conditions, and 936 units of fi-galactosidase and 230 units of alkaline phosphatase under iron-deficient growth conditions. Oligonucleotide-directed mutagenesis of this dual gene fusion plasmid introduced unique restriction sites flanking the bidirectional control region to create a restriction fragment cassette for regionspecific mutagenesis. Sequences within the coding regions of fepB and entC were chosen for sitedirected mutagenesis in order to include the 5’-untranslated regions of these genes and t.he putative signal sequence of FepB within the cassette. First. an XhoI site downstream from the fepB-phoA fusion gene was eliminated by digestion with XhoI. end-repair using the Klenow fragment and religation. This mutation had no significant effect upon alkaline phosphatase or /?-galactosidase activities of

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7’. J. &-i&man

t,he respective hybrid genes. The 3% kb &&I-RnmHI fragment of this derivative was subcloned into the replicative form DNA of bacteriophage M13mp18, and unique XhoI and HindlIT restriction sites flanking the divergent control region were introduced by oligonucleotidedirected in vitro mutagenesis as described in Materials and Methods, using the mutagenic oligonucleotide primers PMBl and PMCl described in t’he legend to Figure 4. A unique XhoI site was created in the entC coding region, result,ing in an amino acid substitution of alanine to leucine at amino acid position 6 (Fig. 2) of EntC, as deduced from the nucleotide sequence. The introduction of a unique HindIII site at the region corresponding to amino acid position 45 in the deduced protein sequence of FepB generated a glutamine to leucinr substit’ution. The mutagenized 3.2 kb SphI-BamHI fragment, with the newly created unique XhoI and HindIII restriction sites was isolated from the recombinant Ml3 replicative form DNA and ligated into the dual gene fusion plasmid to replace the wild-type fragment and produce pITS346. Alteration of the fepB and entC coding regions by these site-specific mutations resulted in no significant phenotypic effects upon the activities of the FepB-PhoA and EntC-La& hybrid proteins in alkaline phosphatase and fi-galactosidase assays; pTTS346 produced 168 units of j?-galactosidase and 36 units of alkaline phosphatase under iron-rich growth conditions, and 914 units of /I-galactosidase and 220 units of alkaline phosphatase under iron-deficient conditions. (d) Construction and analysis of regulatory mutations in the fepB-entC promoter-operators Primer extension analysis localized the transcriptional initiation sites for the divergent fepB and entC messages, biochemically defining t’he -35 and -10 regions of the fepB and entC promoters. Operator sites (iron boxes) were proposed based solely upon similarity to the consensus Fur-binding sequence. Genetic evidence confirming t,he involvement of these sequences in the iron-regulated expression of the divergent transcripts was obtained by mutational analysis, using a combination of in vitro chemical mutagenesis and oligonucleot,idedirected mutagenesis techniques, followed by assessment of in z~ie,o regulatory effects in the dua,l gene fusion system. Compilation and analysis of E. coli promoters and promoter mutations supports the generalization that, with few exceptions, the most highly conserved base-pairs in a promoter are the primary determinants of promoter strength (Hawley & McClure, 1983). This generalization supplied the rationale for the selection of key promoter sequence positions within the -35 and - 10 regions as targets for oligonucleotide mutagenesis. Mutagenic 25-nucleotide primers containing a single mismatch were used to introduce base substitutions into the - 35 and - 10 regions of the fepB promoter, and the

et al.

- 10 region of the ent(’ promoter. As detailed in .Figure 5(a). the !efepB promoter hexamers TTG(‘A’l’ (-35) and GAAAAT (-10) were changed t,o TTACAT and GGAAAT, respectively, and the ent(’ - 10 sequence TAGGTT was changed to TGGGTT. The ent(’ - 35 consensus TTGAC’A was rnut.ated to TTGATA by in vitro treat*ment) of’ the duplex promoter casset’te fragment with the chtrrnic~al mutagen hydrazine. Similarly, formic at:id t.rratt ment resulted in a base substitut,ion in the A[‘(: translat,ional initiation codon of mt(‘. (*hanging t hta sequence t,o AUA. Proposed fepH and ent(.’ operators were mutated by the introduction of a 5 bp substitjut,ion in each of the symmetry dyads by oligonucleot.ide-dire~tetl mutagenesis. These substitutions eliminat)ed one half of the symmetry dyad and were predicted to result in deregulation of the respective fusion genes in ,V~VO.h similar approach was used for thr well characterized promot(er region of the aerobactin operon after at.tempts to isolate deregulat.ed singkbase mutants were unsuccessful (de Lorc,nzo r:l rrl., 1987). This failure may have been due, in part. to the unusual nature of the Fur-operator intrract~iorr. in which Fur repressor appears to wra,p around the DNA helix such that single base changes rnxv not, affect repressor-- operator interactions to n sfiniticant degree. Our atttmpts to identify dtregulatt~d mut,ant#s in the ,jcpH and entC operat,ors following chemical tnut’agenesis were also unsuccessful. The 5 bp substit,utions int,roduced int.o the j’cJJH am! ~~lk.f.C’ operators a,re detailed in Figure 5(a). In order to control for possible variations in pla,smid c’opy numbers. and to morr accurately monit#or in 7:ivo effects of regulatory mutations ate single copy gene dosage. each mult,icopy pTTS346 derivative was stably integrated into a specialized P factor, and activities of the fepR-phoA ~(1 en.t(‘la& fusion genes assayed under iron-ricxh and iron deficient, growth conditjions. /$(;alactosidase and alkaline phosphatase activities of pITS346 and mutated drrivatJives in response to iron c*oricsentration are presented in Figure 5(b), for both multicopy plasmids as well as the corresponding low CW~?; number F cointegrates. These mutations provided direct genetic evidence for the positions of the fepB and enA” promoter elements and repressor-binding sites, c*onfirming t,he biochemical evidence supplied by transcript analysis and rrucleotjidr mapping. gene fusion sequencing data. The base substitJutions introduced - 35 and - 10 regions abrogated int’o the fepB measurable expression of the j”epH-phoA fusion gene in the low copy number state and, in multicopy., reduced the derepressed levels of alkaline phosphatase activity t,o less than 5(:/b of wild-type. Similarly, t.he mutation in the entC1 - 10 region abolished entC’-ZacZ expression, and the rnt(’ -- 35 region mutation reduced the derepressed levels of fi-galacttosidase activity to less than 2 00 of wild-type at low copy number, and t,o less than 6:: of wild-type at multicopy gene dosage. A mutat.ion in the rnt(’ translational initiation codon reduced the dere-

Enterobactin Bidirectional (a)

677

Promoter

IRON BOX -35 fepB (425.356)

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activity IS0 I

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Figure 5. C’onstruction and analysis of regulatory mutations in the fe@-e&C promoter-operators. Base substitutions in the -35 and - 10 regions of the fe# promoter and t,he - 10 region of the e&C promoter were introduced by oligonucleotide-directed mutagenesis. Mutations in the -35 region of the e&C promoter and the entC translation initiation codon were isolated following in vitro chemical mutagenesis of the 05 kb XhoI-Hind111 promoter cassette fragment of the dual gene fusion vector pITS346. Proposed operator sequences were altered by the introduction of a 5 bp substitution in the respective symmetry dyads by oligonucleotide-directed mutagenesis. Mutated promoter cassette fragments were ligated into pITS346 and expression of the divergent gene fusions was measured in response to various iron concentrations in the growth medium. Results are presented for cells harboring multicopy fusion plasmids as well as the corresponding low copy number F cointegrates. Low copy number F cointegrates were constructed by stable integration of multicopy fusion plasmids into a modified F factor. (a) Nucleotide sequence of the DNA regions flanking the transcription start, sites for fepS and entC. Base substitutions in the major promoter determinants and primary Fur repressor-binding sites are indicated below the corresponding wild-type sequence. Nucleotide positions corresponding to those in Fig. 2 are given in parentheses. (b) Expression of fusion genes from wild-type and mutated promoter-operators in response to various iron concentrations in the growth medium. Shaded bars correspond to enzyme activity levels produced under iron-deficient growth conditions, and open bars represent activities from iron-rich cultures.

pressed levels of /?-galactosidase activity to 13% of wild-type. Presumably translation of this hybrid protein initiates at, the nearest downstream methionine codon at amino acid position 13 in the entC coding region. Preceding this methionine codon by 11 nucleotides is the Shine-Dalgarno-like sequence, 5’-GAGGAAG-S’, which may direct initiation of translation (Fig. 2).

An asymmetric relationship was observed for effects of promoter mutations upon activity of the divergent promoter. Whereas mutations decreasing fepB promoter strength decreased entC promoter activity by approximately 20 %, no such effect was exerted upon fepB-phoA expression by mutations decreasing entC promoter strength. Although interpretat,ion of these results is difficult. they suggest

678

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some degree of co-operativity between the ,fepB and entC promoters. As predicted, mutations in the proposed Fur repressor-binding sequences resulted in deregulated expression of the respective fusion gene activities, implicating these sequences in the Fur-mediated repression of the divergent genes under iron-rich conditions. Tnduction ratios (iron-deficient levels/ iron-rich levels) for the respective fusion gene activities were reduced by approximately sevenfold for both operator mutants. Operator mutations did not result in complet,e relief of repression of the corresponding fusion gene activity unless accompanied by mutation of the operator at the divergent promoter. These data indicate t,hat some minor repressive influence is exerted by Fur occupation of the opposing operator site(s). Operator mutations at each promoter resulted in decreased promoter activity, suggesting some negative influence of these base substitutions upon productive transcript initiation. Mutation of the fepB operator altered the weakly preferred CAT consensus sequence (Hawley & McClure, 1983) at the CATTATT transcription initiation region to (1AGGCGG (Fig. 5(a)), possibly reducing the rate of promoter clearance. The entC operator mutation (Fig. 5(a)) altered the sequence, 5’-TTATT-3’. at nucleotide posit,ions +21 to $25 in the *5’-untranxlated region of the entC message, to the sequence 5’-GGCGG-3’. Such a mutation could feasibly result in a significant pause during translocation of the elongating transcription complex, inhibiting the abilit’y of incoming RNA polymerase to bind; isomerize and initiate a new R,NA chain, and causing an overall reduction in maximal initiation frequency. A second possibility is that this mut’ation in t’he initial transcribed region increases the rate of abortive RNA synthesis, thereby decreasing the overall frequency of productive initiation events. Finally, these base substitutions may affect initiation frequency by perturbing the local DNA conformation or the rate of dissociation of CJsubunit from RNA polymerase holoenzyme. The contribution of promoter clearance rates to promoter strength remains unclear at this time (McClure, 1985), and we have no evidence to support the role of the mechanisms suggested above in the decreased promoter activity observed in our operator mutants. (e) DNase I footprinting DNA regions protected by Fur repressor from DNase I nicking were determined at 100 PM-Mn2+, using the 3’-labeled 0.5 kb XhoI-Hind111 promoter cassette fragment of pITS346 and mutated operator derivatives (Fig. 6). At the wild-type fepB promoter-operator (Fig. 6(a)), a 31 bp protect’ed region was identified, which included the 17 bp core dyad (Fig. 2, iron box) as well as 7 bp of flanking sequence on each side, corresponding to base positions 403 to 373 in Figure 2. This region spans positions - I9 to + 12 with respect to the fepB

et, al.

transcriptional st,art site. Protection of this regiorl was apparent at 10 nM-Fur, with complet,e prote(*tion at 20 nM-Fur. No extension of’ this region. designated fepH 1 in Figure 6(a). occurred at higher Fur concentrations. Similarly. Fur prot.ccted a 31 bp region in ent(’ (Fig. 6(a)), consisting of the 17 bp core dyad indicated in Figure 2 (iron box), with 7 bp of flanking sequence on each side. This region spans base positions 488 to 518 in Figurtb 2, corresponding to nucleotide positions + 1 to +31 in the message. Some prot#ect’ion of t)hix primary binding site, designated entC I in Figure 6(a), was observed at 10 ni%-Fur; with complete prote&ion at, 20 nM-Fur. At 20 nM-Fur, a contiguous downstream region of 18 bp was prot,ected. This secondary binding site, designated entC II (Fig. 6(a)), extends t’he primary protected region to base posit’ion 536 in Figure 2. corresponding to +49 in the ent(’ message, and sequesters the putative Shine---l)algarno sequence. Complete protection of the contiguous entP I and II sit#es occzurred at 40 nlw-Fur. In addit’ion. at 80 nM-Fur. a slight extension of the primar) binding sit’r provided prot,ection of an sddibional upstream region of 3 bp. Fur-binding sequences at include many of the the en,tC and fepB promoters key base-pairs shown to participat,e in repressor operator contact at t,he &A promot,er region (de Lorenzo of d.. 1988). w-ith conservation of tnost of the regularly spaced AT sequences t’hroughout, t’hc dyads. The secondary repressor binding sit,e at v&C’ is less well conserved, yet does contain some of t,hr repeated AT motifs in phase wit,h t’he primary binding site. and so most likely represents an rxtension of the primary site rather that) a distinct repressor binding sequence with lowered affinity for Fur. This conclusion is supported by the fact that mutation of the ent(’ 1 binding sittl removes virtually all the Fur-mediated c*ontrol of wt(‘, although the entC II site remains unchanged by the mutation. The end-labeled fragment carrying tjhe mutated fepB operator (Fig. 6(b)) showed slight prot,ection of the fepB primary site at 80 nM and 160 nM-Fur. indicating a greatly reduced affinity for repressor binding (&fold or greater), compared with wild-type. Protection of the wild-type entl,’ primary and secondary binding sit,es was not affected by thefepl? operat,or mutation (Fig. 6(b)). For the mutat,ed w&J occupation of the operator (Fig. 6(c)), simultaneous primary and secondary binding sites was observed at 80 niv-Fur, indicating an approximately eightfold reduction in Fur affinity compared with wild-type. No effect upon Fur binding at, the wild-type fcyfl operator was observed for the fragment carrying the mutated end’ operator (Fig. 6(c)). These data correlate the in viz~o deregulation of’fepB-phoil and e&t’la& expression with altered in citro Fur-DNA interactions for these mutated operators. Failure of operator mutations to significantly alter repressor binding afinity at the divergent wild-type operator in vitro argues against the involvement of Fur protein-protein interactions in coregulation of t,he opposing genes. However, t,he i,n Vito demonstrat’ion

Enterobactin Bidirectional (0)

679

Promoter (c)

(b)

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Figure 6. DNaYe I footprints of Fur repressor protein on wild-type and mutated operators at the fepS and entC promoters. The 3’-labeled @5 kb XhoI-Hind111 fragments of pITS346 a.nd mutated derivatives were partially digested with DNase I in the presence of various concentrations of purified Fur protein at 100 pm-&*+: Fur (monomer) concentrations used were: lanes 1, no Fur; 2, 10 nM; 3, 20 nix; 4, 40 nM; 5. 80 nM; and 6. 160 nix. Positions of the primary and secondary Fur-binding sites at the wild-type fepB and e&C operators are designated I and II to the right of the protected regions. Relative positions of the operator mutations are indicated accompanying the respective autoradiographs. (a) Wild-type operators; (b) fepR operator mutant: (c) entC operator mutant.

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of slight repression of transcription exerted bv Fur occupancy at the divergent operator site(s) indicates some weak operator co-operativity that is not apparent in the in vitro binding experiments and may reflect effects upon RNA polymerase entry to the promoter regions. The precise mechanisms responsible for this effect are not known.

4. Discussion Early attempts to define the transcriptional organization of the entCEBA gene cluster produced conflicting results. Transcriptional linkage of these genes was initially suggested based upon analysis of Mu d(Ap’ Zac) operon fusions (Fleming et al., 1983), which provided evidence for an iron-inducible operon transcribed in a clockwise direction, although the gene order reported for this region has since been revised (Nahlik et al., 1989: Ozenberger et al., 1989). Seemingly conflicting data supported the assertion that the genes of this ent cluster could be independently transcribed. Individual ent genes present on multicopy plasmid vectors were shown to complement ent mutations (Laird et al., 1980; Laird & Young, 1980; Nahlik et al., 1987; Pickett et al.. 1984), and insertion mutations failed to exert, observable polarity effects in sensitive bioassays for enterobactin production (Laird &, Young, 1980; Nahlik et al., 1987). However, recent studies support the original premise that these genes are transcriptionally linked. Nucleotide sequence analysis indicates a lack nucleotide sequences containing of intergenic canonical promoter elements separating the coding regions for entB, entA and the phenotypically undefined P15 locus (Liu et al., 1988; Nahlik et al., 1989). Construction of la& gene fusions to the individual ent cistrons entC, entE, entB and e,ntA, which places each fusion gene under the control of its immediate upstream region, resulted in the iron-regulated fusion gene (Nahlik expression of only the entC-la& et al., 1989). In addition, an insertion mutat)ion in entC had strong polarity effects upon expression of entB and entA (Ozenberger et al., 1989). These observations, along with the fact that chromosomal la& fusions to entC, entE and entA are all clearly iron-regulated (Fleming et al., 1983), supplies presumptive evidence for transcriptional coupling. The data presented herein provide definit’ive iron-regulated evidence for cotranscriptional of the entCEBA(P15) gene cluster. expression Analysis of newly constructed ent-la& gene fusions and their deletion derivatives has shown that the regulatory sequences mediating the co-ordinated iron-responsive expression of this gene cluster reside exclusively within the fepB-entC intercistronic region. The overlapping ent-la& fusion plasmids pITS605, pITS603, pITS606, pITS604 and pTTS607 described in this paper all exhibit iron-regulat’ed of P-galactosidase, although various expression levels of activity are detected among these gene fusions. These disparities can most likely be ascribed to differences in translational efficiency, or

et al

FepB

Figure 7. Spatial organization of the j’epB-e&C’ bidirecational control region. The genetic region separating the coding sequences for the divergent fepS and e&C genes is depicted. Relative positions of major promoter determinants and iron boxes for the respective genes are shown, with the distance between the proposed -35 elements given in base-pairs. Transcriptional initiation sites are designated + 1, and the distance separating these sites is given. The nucleotide positions corresponding to the start of each coding region are indicated. The approximate position of the region of dyad symmetry within the fepB leader sequence is represented by the large convergent arrows; each small open arrow indicates the position and orientation of a short reiterated sequence, !5’-AjCUG NGAGGG-3’. Two small open reading frames located within the fepB leader sequence are represent)4 by the numbered lines.

to variations in hybrid protein stability or enzymatic activity. The transcriptional linkage of these genes allows the co-ordinated expression (or repression) of the enzymatic activities required for the biosynthesis and activation of 2,3-dihydroxybenzoate by regulation of transcription in response to iron availability. Transcriptional initiation sites have been defined by primer extension analysis, allowing preliminary identification of regulatory sequences including the major promoter elements and Fur repressor-binding sites for the fepB-entC bidirectional control region, and demonstrating iron-regulation at the transcriptional level. A schematic representation of the fepB-entC intercistronic region is shown in Figure 7. Fusion of the divergently transcribed fepB and entC genes to the indicator genes phoA and la&, and the introduction of the unique restriction sites flanking the bidirectional control region provided a highly sensitive reagent for monitoring regulat’ory effects of promoter region-specific mutations, and for assessment of possible cooperative effects between the promoter-operators. Genetic evidence confirming the involvement of the proposed regulatory sequences in the iron-regulated expression of the divergent transcripts was obt,ained by mutational analysis, using the dual gene fusion system in vivo. Mutations in the -35 and - 10 regions of t)he fepB and entC promoters that reduced their simiresulted in dramatically larity to consensus decreased levels of fusion gene expression. Operator mutations led to deregulated expression of fusion genes, implicating these sequences in Fur-mediated interactions were characrepression. Fur-operator terized by DNase I footprinting, identifying a single Fur-binding site at the fepB promoter and a pair of contiguous and sequentially occupied binding sites at the 5’-untranslated region of entC’. In vitro characterization of Fur-DNA interaction at the wild-type and mutated fepK and entC operators

Enterobactin Bidirectional correlates with the in vivo analysis of deregulated operator mutants. Interestingly, although entC repression is mediated by a pair of contiguous Fur-binding sites, with a higher affinity primary binding site than fepB, repression of entC expression is less efficient than repression of fepB. These results are most likely attributable to positional effects of the respective operators. Current models for operator position effects (Elledge & Davis, 1989) would attribute entC repression to Fur interference with the formation of the RNA polymerase-promoter initiation complex and promoter clearance, which is predicted t,o be less efficient than the promoter occlusion effect of Fur binding at the fepB promoter region, resulting in interference with promoter recognition and formation of the RNA polymerase-promoter closed complex. Several notable features of this bidirectional control region suggest the possible involvement of additional regulatory mechanisms. An extensive region of dyad symmetry occurs within the fepB leader sequence, which has the potential to form multiple alternative RNA secondary structures of extremely high stability (Fig. 2). It is possible that this inverted repeat sequence is involved in the post-transcriptional regulation of fepB and, potent’ially, of downstream fep genes. A recent report (Elkins & Earhart, 1989), published during the review of this study. also identifies this potential structure and describes similarities regulatory between its sequence and regions upstream from other I$. coli genes, including pstB and adk. Within the large inverted repeat sequence are six symmet,ritally oriented copies of the sequence, 5’-A/GGGNGAGGC-3’ (Fig. 2), which resemble binding sites for transcriptional regulatory factors. In addition, two small open reading frames lie within the fepB leader region, which may modulate fepB expression by an undefined transcription-translation coupling mechanism. Alternatively, the peptide products, if any, may serve some functional role in the regulation of fepB expression or activity. Finally, secondary structures within the fepB leader may confer increased stability to the message, as was shown for the 5’-untranslated regions of the ompA (Belasco et al., 1986) and T4 gene32 transcripts (Gorski et al., 1985). In a preliminary study (T. J. unpublished results) oligonucleotideBrickman, directed deletion of the fepB leader, which precisely excised the region including nucleotide positions 185 to 350 (Fig. 2), completely abolished fepB-phoA expression at single copy gene dosage. This deletion eliminated the major dyad and two potential openreading frames, yet retains the promoter-operator region as well as 34 nucleotides of the initially transcribed region and the proposed ribosome recognition sequences at the translational start of fepB. Experiments are in progress to ascertain the function of the fepB lea.der in t)he regulation of fepB expression. We thank S. Armstrong, G. Pettis and C. Shea for technical advice and helpful discussions during t,he course

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of these studies. We are indebted to M. Casadaban, A. Myers, ,J. Cannon, C. Manoil, D. Pintel and M. Henry for gifts of strains, phages and plasmids. Special thanks to J. B. Neilands for the generous gift of purified Fur protein. This work was supported by grants DMB 8416017 and DMB 8821464 from the National Science Foundation (to M.A.M.) a,nd by Public Health Service predoctoral traineeship 1 T32 AI07276 (to T.J.B.) from the National Institute of Allergy and Infectious Diseases.

References Arraj, J. A. & Marinus, M. G. (1983). J. Hacteriol. 153. 562-565. Bagg, A. b Neilands, J. B. (1985). J. Hacteriol. 161, 450-453. Bagg, A. & Neilands, J. B. (1987). &ochrmistr?l, 26, 5471-5477. Belasco, J. G., Nilsson, G., von Gabain, A. & Cohen. S. N. (1986). Cell, 46, 245-251. Birnboim, H. C. & Daly. J. (1979). NucZ. Acids J&s. 7, 1513-1523. Brickman, E. & Beckwith, J. (1975). ,I. Nol. Biol. 96, 307-316. Casadaban. M. J.: Chou, J. & Cohen. S. N. (1980). J. Bacterial. 143, 971-980. Cohen, S. N., Chang. A. C. Y. & Hou, L. (1972). l’roc. n;at. Acad. Sci.. U.S.A. 69, 2110-2114. de Lorenzo, V., Wee. S., Herrero, M. & Neilands. ,J. B. (1987). J. Bacterial. 169, 2624-2630. de Lorenzo, V.. Giovannini. F., Herrero, LM. & Neilands, J. B. (1988). J. Mol. BioE. 203: 876-884. Elkins. M. F. & Earhart, C. F. (1989). J. Jhctrrinl. 171. 5443-5451. Elledge, S. J. & Davis, R. W. (1989). Uenes l)re~lop. 3, 185-197. Fleming, T. ,P.; Nahlik, M. S. & McIntosh. M. A. (1983). ,J. Bacterial. 156. 1171-1177. Gorski. K., Roth. ,J.. Prentki, P. & Krisch, H. (1985). (‘ell. 43. 461-469. Gutierrez, C.. Barondess, J., Manoil, (1. dt Hwkwith, J. (1987). J. Mol. Biol. 195, 289-297. Hantke, K. (1981). Mol. Gem Genet. 182, 288-292. Hawley, D. K. & McClure, W. R. (1983). NucZ. A&s Res. 11, 2237-2255. Henry. AM. F. & Cronan, J. E. (1989). J. Bactrriol. 171, 5254-5261. ,Joyce, C. M. & Grindley. N. D. F. (1984). J. Backrid. 158. 636-643. Kunkel, T. A. (1985). Proc. Nat. Aead. Sri.. I ‘.S. A. 82. 488-492. Laird. A. J. & Young. I. G. (1980). Gene, 11, 359-366. Laird. A. J.. Ribbons. 1). W., Woodrow. 0 (‘. & Young, 1. 6. (1980). Gene, 11, 347-357. Liu, J.. Duncan, K. & Walsh, C. T. (1988). .I. Racteriol. 171, 79-798. Manoil. C. & Beckwith, J. (1985). Proc. Nat. Acad. Sci., U.A.A. 82 8129-8133. McClure. W. R: (1985). Ann-u. Rec. Biochem. 54, 171-204. Miller, J. Ii. (1972). Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press Cold Spring Harbor, NY. Myers. A. M., Tzagoloff, A., Kinney, I). M. bz Lusty. C. J. (1986). Gene, 45, 299-310. Myers, R. M., Lerman, L. S. & Maniatis, T. (1985). Science, 229, 242-247. Nahlik. M. S., Fleming, T. P. & McIntosh. M. A. (1987). J. Baderiot. 169, 4163-4170.

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9ahlik. $1. 8.. Brickman. T. J., Ozenberger, R A. & McIntosh. M. A. (1989). J. Hacturiol. 171, 784-790. Xorrander, ,J., Krmpe. T. & Messing. .J. (1983). C&w. 26. 101-106. O’Brien, I. G. B Gibson. F. (1970). Hiochim. Hiophys. Acta, 237. 537-549. Ozenberger, H. A., Sahlik. M. S. & McIntosh. M. A. (1987). .J. Bacterial. 169. 3638.--3646. Ozenberger, R. A.. Brickman, T. .J. & McIntosh, M. A. (1989). J. Ba&riol. 171. 775p78R.

et al. Prttis, (:. S. ?z 1lrlntosh. 51. A. (1987). ./ /~n&rio/. 169. 41.54~416”. Pettis, G. S., Brickman, T. J. & McIntosh, M. A (1988). ,J. Biol. Chem. 263, 18857-18863. Pick&t. C”. I,.. Hayes, L. & Earhart, (‘. F. (1984). FENIWS Microbial.

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Edited by N. L. Sternberg

38, 989-992.

Regulation of divergent transcription from the iron-responsive fepB-entC promoter-operator regions in Escherichia coli.

Transcriptional linkage of the enterobactin gene cluster entCEBA (P15) was confirmed by ent-lacZ gene fusion analysis. Control sequences directing iro...
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