The Bordetella pertussis heme utilization gene cluster hurIR bhuRSTUV encodes regulatory and transport functions required for assimilation of iron from heme and hemoproteins . Expression of the bhu genes is iron regulated and heme inducible . The putative extracytoplasmic function [ECF] factor, HurI, is required forheme-responsive bhu gene expression . In this study, transcriptionalactivation of B . pertussis bhu genes in response to heme compoundswas shown to be dose dependent and specific for heme; protoporphyrinIX and other heme structural analogs did not activate bhu geneexpression . Two promoters controlling expression of the hemeutilization genes were mapped by primer extension analysis.The hurI promoter showed similarity to ⁷⁰-like promoters, andits transcriptional activity was iron regulated and heme independent.A second promoter identified upstream of bhuR exhibited littlesimilarity to previously characterized ECF factor-dependentpromoters . Expression of bhuR was iron regulated, heme responsive,and hurI dependent in B . pertussis, as shown in a previous studywith Bordetella bronchiseptica . Further analyses showed thattranscription originating at a distal upstream site and readingthrough the hurR-bhuR intergenic region contributes to bhuRexpression under iron starvation conditions in the absence ofheme inducer . The pattern of regulation of the readthrough transcriptwas consistent with transcription from the hurI promoter . Thepositions and regulation of the two promoters within the hur-bhugene cluster influence the production of heme transport machineryso that maximal expression of the bhu genes occurs under iron starvation conditions only in the presence of heme iron sources.

 
The innate immune system of the human host defends against invading microorganisms in part by sequestering iron, a nutrient essentialfor virtually all living cells . The majority of host iron ismaintained intracellularly in the form of hemoproteins, whileextracellular iron is bound by the host glycoproteins transferrinand lactoferrin [44, 49] . Successful microbial pathogens haveevolved mechanisms to overcome host iron restriction [21, 32,46], including production and utilization of low-molecular-weightiron chelators termed siderophores [40], utilization of siderophores produced by other organisms, and direct removal of iron fromhost proteins via specific bacterial cell surface receptors[11, 59].

In gram-negative and some gram-positive bacterial species, genes encoding iron transport systems are repressed when intracellulariron levels are high by the Fur protein with ferrous iron asthe corepressor [17, 22] . When bacterial cells encounter aniron-limiting environment such as the human host, their intracellulariron stores are depleted, resulting in derepression of ironacquisition genes . Fur derepression is sufficient for full expressionof the genes in certain iron uptake systems, while in othersystems, positive transcriptional regulation requiring the presenceof the cognate iron source is also necessary for maximal geneexpression [12] . Positive regulators of iron acquisition systemsare of three main classes: AraC-like proteins [4, 9, 18, 24, 43], two-component signal transduction systems [14, 50], andextracytoplasmic function [ECF] factors [1, 13, 30, 31, 57].

ECF factors are members of the ⁷⁰ superfamily of bacterialsigma factors and are utilized by diverse species to regulategenes in response to extracytoplasmic stimuli [37, 45] . ECF factors involved in regulating iron stress responses have beentermed members of the iron starvation subfamily of ECF regulators[58] . These ECF factors and their specific anti- factors areproduced under iron-limiting conditions, but the factors remaininactive until the cognate iron source is sensed in the environment.In the presence of the appropriate iron source, a signalingcascade is initiated at the cell surface by the cognate outermembrane receptor . The signal is transduced to the anti- factor,which then either releases or activates the factor, allowingit to associate with core RNA polymerase and initiate transcriptionof genes encoding iron acquisition functions [6, 58] . Membersof the iron starvation family of ECF factors include FecI [1],PupI [31], and PvdS [13], which regulate a subset of iron uptake genes in Escherichia coli, Pseudomonas putida, and Pseudomonasaeruginosa, respectively . Recently, the putative ECF factorsHurI of Bordetella pertussis and Bordetella bronchiseptica [57]and RhuI of Bordetella avium [30] were shown to regulate expression of heme iron transport genes.

Since greater than 90% of the iron within the human body is associated with heme and hemoproteins [42], bacteria that can access these compounds in vivo and utilize host heme iron have a significant nutritional advantage . Vibrio cholerae [25, 26],pathogenic E . coli [55], Shigella species [36], Yersinia species [53, 54], and P . aeruginosa [41] produce TonB-dependent cellsurface receptors and ATP-binding cassette transporters thatallow utilization of heme, hemoglobin, and other hemoproteins.A second type of heme uptake system, employed by species suchas Serratia marcescens [33], Yersinia pestis [47], and P . aeruginosa [41], involves production and secretion of small heme-bindingproteins termed hemophores that obtain and ferry host heme tospecific bacterial cell surface receptors.

B . pertussis, the causative agent of the human disease whooping cough, and B . bronchiseptica, a closely related mammalian respiratorypathogen, possess multiple systems for iron retrieval underiron-limiting environmental conditions . They produce the siderophorealcaligin [8, 20, 28, 38] and are capable of using siderophoresproduced by other organisms [3] . Both species possess the hemeutilization gene cluster bhuRSTUV, which encodes transport functionsrequired for assimilation of iron from heme and hemoproteins[Fig . 1] [56]. B . avium, a more distantly related pathogen ofturkeys and chickens, has an orthologous gene cluster encodinga functional heme utilization system [39] . Expression of B. pertussis and B . bronchiseptica bhu genes is regulated by iron and the presence of heme via Fur and the ECF regulators encoded by the hurIR genes, located immediately upstream of the bhu gene cluster [56, 57].

 
We showed in a previous study that HurI, a putative ECF factor,is required for heme-activated bhuR transcription and for maximallevels of heme utilization [57] . In the present study, the kineticsof the transcriptional response to heme inducer and the structuralcharacteristics of the inducer were examined . We have identifiedtranscriptional start sites for the iron-regulated hurI andheme-inducible bhuR genes and have demonstrated the hurI dependenceof heme-responsive bhuR expression in B . pertussis . Furthermore, iron-regulated bhuR transcription in the absence of heme was assessed, and it was found that transcription from an upstream promoter, reading through the hurR-bhuR intergenic region, contributes to bhuR expression . These data support a model for transcriptional regulation of heme utilization genes that allows Bordetella cells to sense heme and respond by maximally producing the heme transport machinery.

 
Bacterial strains and plasmids. Bordetella strains and recombinant plasmids used in this studyare listed in Table 1 . E . coli DH5 [Invitrogen, Gaithersburg,Md.] was used as the host strain in routine cloning procedures.Plasmid vectors pGEM3Z [Promega, Madison, Wis.] and pRK415 [29]were used in the construction of recombinant plasmids . A pRK415derivative, plasmid pRK40 [57], carries a promoterless trp'-'lacZ gene and was used to construct all bhuR-lacZ transcriptional fusions [Table 1].

 
Growth media and chemical solutions. Luria-Bertani [LB] [48] broth or agar plates were used to cultureE . coli strains . B . pertussis strains were cultured on Bordet-Gengou[BG] agar [5]; B . bronchiseptica strains were cultured on LBagar . All Bordetella liquid cultures were grown in Stainer-Scholte[SS] minimal medium [51, 52] . SS medium was deferrated by Chelex100[Bio-Rad, Richmond, Calif.] as described previously [2] . Iron-depletedSS medium contained no iron supplements, while iron-repleteSS medium was supplemented with FeSO₄ to a final concentrationof 36 µM . Bovine hemin chloride [Sigma, St . Louis, Mo.]was maintained as a 1 mM stock solution as described previously[56] and added to iron-depleted cultures at a final concentrationof 5 µM unless otherwise indicated . Ethanolic stock solutionsof chlorophyll a [Sigma] were prepared at a concentration of1 mM; aqueous solutions of protoporphyrin IX [PPIX] and cytochromec [both from Sigma] were maintained at concentrations of 400µM and 500 µM, respectively, and zinc-PPIX [Sigma]was dissolved in N,N-dimethyl formamide at a concentration of800 µM . Each porphyrin compound was added to liquid culturesat a final concentration of 5 µM unless otherwise indicated.Tetracycline and ampicillin were used at final concentrationsof 15 µg/ml and 100 µg/ml, respectively.

Bacterial culture conditions. B . pertussis and B . bronchiseptica strains were grown on agarplates and subcultured to iron-replete SS medium . B . bronchisepticacells were grown with shaking at 37°C for 24 h, washed,and inoculated at a dilution of 1:200 to iron-replete and iron-depletedSS medium . After 18 h of growth, hemin was added as appropriateto iron-depleted cultures . All cultures were harvested for ß-galactosidaseassays or RNA isolation 4 h after the addition of hemin [aftera total of 22 h of growth] . A similar procedure was used toculture B . pertussis strains except that iron-replete SS cultureswere grown for 36 h; subcultures were inoculated at an initialoptical density [600 nm] of 0.08 and grown for 24 h prior tohemin addition.

RNA isolation and primer extension analysis. Total RNA was harvested from cultures by a modification [27]of the acid-guanidinium thiocyanate-phenol-chloroform extractionmethod of Chomczynski and Sacchi [10] . Primer extension reactions contained 25 µg of RNA, 1 pmol of ³²P-end-labeled primer, 1X Superscript II buffer [Stratagene, La Jolla, Calif.], 1 mM deoxynucleoside triphosphate mixture, 10 µM dithiothreitol,1 mg of bovine serum albumin per ml, in a total reaction volumeof 20 µl . This mixture was heated to 70°C for 5 minto denature the RNA, hybridized at 45°C for 30 min, andcooled to 37°C for 10 min . Superscript II RNase H^- reversetranscriptase [10 units] [Stratagene] was added to each reaction,which was incubated for an additional 30 min at 37°C . Theprimer extension reaction was stopped, and primer-extended cDNAwas isolated by standard methods [48] . Prior to loading on an8% polyacrylamide gel, the products were denatured by boilingfor 5 min . One-half of the final volume was loaded on the gelnext to a nucleotide sequencing ladder generated by appropriateprimers with plasmid DNA templates.

Reverse transcription-PCR analysis. Reverse transcription reactions with Bordetella RNA as templateswere performed as described for primer extension, except thatnonradiolabeled primer was used . After reverse transcription,the mixture was diluted by addition of an equal volume of distilledwater, and 2 µl was used as the template for PCR . Thefollowing components were used in the PCR: water to a totalvolume of 50 µl, 1X Pfu Turbo buffer [Stratagene], 800µM deoxynucleoside triphosphate mixture, 2 µl of diluted reverse transcription reaction, 8 pmol of each primer,5% dimethyl sulfoxide, 1 unit of Pfu Turbo DNA polymerase [Stratagene]. The thermal cycler was programmed for one cycle of denaturationat 96°C for 5 min, 30 cycles of denaturation at 96°Cfor 1 min, primer annealing at 62°C for 1 min, and extensionat 72°C for 30 s, and one cycle at 72°C for 10 min.

Genetic methods. Bordetella pertussis nucleotide sequence data were producedby the Bordetella Sequencing Group at the Sanger Centre [http://www.sanger.ac.uk/Projects/B_pertussis/]. Other nucleotide sequences were obtained from GenBank at the National Center for Biotechnology Information at the NationalLibrary of Medicine.

Reporter plasmid pRK40 and bhuR-lacZ plasmids pRK41 and pRK42 were described previously [57] . ß-Galactosidase assays of cells carrying reporter plasmids were performed by a modification [7] of the method of Miller [35] . The results reported are representativeof at least two experimental trials . Deletion derivatives ofthe bhuR promoter fragment were generated by PCR with B . pertussiscosmid pCPbhu1 [carrying hurIR bhuRSTUV] [56] as the template. The source of the chloramphenicol [Cm] cassette used to constructthe terminator insertion in plasmid pRK49 was mini-Tn5 Cm [15].

The block substitution and BglII site insertion in plasmids pRK47 and pRK48, respectively, were constructed by whole-plasmidPCR mutagenesis by a method described previously [60] . Briefly, primers that were antisense to one another were designed tobe complementary to the hurR-bhuR intergenic region except for the bases to be substituted . Primers mECF1 and mECF2 containeda 12-nucleotide block substitution in the center of each primer,with 16 nucleotides of complementarity to the template DNA flankingboth sides of the mutation . Primers Bgl1 and Bgl2 containedthree single-nucleotide substitutions to create a BglII restriction site . These primer sequences were as follows: mECF1, 5'-CGTGCCTGCTCTCGATCCCTTTCCTTCTTCATGGTTTACGCTTGC-3';mECF2, 5'-AAGCGTAAACCATGAAGAAGGAAAGGGATCGAGAGCAGGCACGAG-3';Bgl1, 5'-CGGCAAAAAAAATTCCAGATCTCTGTCCGGTTTCGACG-3'; and Bgl2, 5'-CGTCGAAACCGGACAGAGATCTGGAATTTTTTTTGCCG-3'.

For whole-plasmid PCR mutagenesis, the following componentswere mixed in order: water to a total volume of 50 µl,100 ng of p3Z102 plasmid DNA, 50 pmol of each primer, 1 mM deoxynucleoside triphosphate mixture, 1X Pfu Turbo buffer [Stratagene], 5% dimethyl sulfoxide, and 2.5 U of Pfu Turbo DNA polymerase [Stratagene].The thermal cycler was programmed for one cycle of denaturationat 96°C for 5 min, 16 cycles of denaturation at 96°Cfor 1 min, primer annealing at 55°C for 1 min, and extensionat 68°C for 10 min . Following the PCR, DpnI was added todigest the methylated parental template DNA . E . coli DH5 wastransformed with 10 µl of the reaction, and plasmids from several independent transformants were sequenced to identify plasmids containing the desired mutations.

 
Temporal analysis of bhuR induction. The transcriptional response of iron-starved Bordetella cellsto various heme concentrations was monitored to determine thesensitivity and time course of bhu gene activation . Wild-typeB . bronchiseptica B013N cells carrying the hurIR bhuR-lacZ plasmidpRK42 were cultured in parallel in iron-replete SS medium andiron-depleted SS medium with or without hemin . Cells grown iniron-replete medium showed low levels of ß-galactosidaseactivity [400 Miller units] that remained constant for the durationof the experiment [data not shown] . Cells grown in iron-depletedmedium without hemin showed 2-fold-higher levels of reportergene activity compared with iron-replete cells, demonstratingiron-regulated bhu gene expression.

Replicate cultures of iron-starved cells were exposed to concentrations of hemin ranging from 0.32 µM to 20 µM [Fig . 2], and transcription of bhuR was activated in response to all concentrationsof hemin tested . Interestingly, the lowest concentration ofhemin [0.32 µM] did not measurably stimulate the growthof iron-starved cells [data not shown] but did induce bhuR transcription[Fig . 2], indicating that heme responsiveness and bhuR activationare highly sensitive . The induction kinetics of bhuR transcriptionalactivation varied with the concentration of hemin provided.Cells exposed to a low concentration of hemin [0.32 µM]showed a modest induction that slowly increased to a maximumat 8 h after hemin addition . Cells induced by intermediate heme concentrations [1.25 or 5 µM] showed higher peak levelsof transcription that increased rapidly within 2 h and declinedfrom maximum levels after 8 h of heme exposure . Cells giventhe highest dose of heme [20 µM] showed a pattern of rapidbut transient induction followed by a sharper decrease in transcriptionalactivity, consistent with uptake of heme iron resulting in Fur-mediated repression of the fusion gene . The cultures exhibiting the highest sustained levels of bhuR transcription were those exposed to intermediate concentrations of hemin.

 
Analysis of inducer specificity. Initiation of the signaling cascade that results in bhu genetranscriptional activation involves recognition of heme by theBhuR receptor protein [57] . As a means to elucidate the structuralrequirements for BhuR inducer recognition, molecules structurallysimilar to heme were tested for their ability to induce bhuRtranscription . Cytochrome c is a hemoprotein in which the hememoiety is covalently linked to the cytochrome protein . Althoughintact cytochrome c cannot supply nutritional iron to Bordetellacells [data not shown], it was hypothesized that recognitionof heme at the cell surface, independent of transport, couldlead to signaling and transcriptional activation of bhuR . PPIX,the heme biosynthetic precursor lacking a coordinated iron atom,zinc-PPIX, and chlorophyll a, a porphyrin with a coordinatedmagnesium atom, all bear significant structural similarity toheme.

To test bhuR transcriptional responsiveness to these heme analogs, B . bronchiseptica B013N[pRK42] was grown in iron-depleted SS medium and exposed to chlorophyll a, PPIX, zinc-PPIX, cytochrome c, or hemin . In multiple experiments, cells exposed to hemin showed at least a fourfold induction of bhuR transcription over levels exhibited by iron-starved cells . The highest level of induction in response to any other compound tested was a 1.5-fold induction in response to PPIX [data not shown] . To further assess whether PPIX was a weak inducer of bhuR transcription, iron-starved B013N[pRK42] cells were exposed to PPIX concentrations of 5, 10, 25, and 50 µM and bhuR transcriptional activity was monitored . In contrast to the response to hemin [Fig . 2], therewas no dose-dependent transcriptional activation in response to PPIX [data not shown], indicating that BhuR recognition of inducer is highly specific for the porphyrin ring with boundiron.

Mapping of the transcriptional initiation site for hurI. To elucidate the genetic mechanisms mediating inducible expressionof heme utilization genes, the positions and features of promoters within the heme utilization gene cluster were defined . The hurI and hurR genes encode a putative ECF factor and cytoplasmicmembrane regulator, respectively . In previous studies [56, 57],potential ⁷⁰-like promoter elements and Fur binding sites wereidentified upstream of hurI [shown in Fig . 3A], and functionalFur binding activity in this region was demonstrated, suggestingthat hurI transcription was iron repressible.

 
To directly examine hurI expression and identify the hurI transcriptioninitiation site, total RNA isolated from wild-type B . pertussisUT25Sm1 and wild-type B . bronchiseptica B013N cells was analyzedin primer extension experiments . A hurI transcript was undetectablein cells grown under iron-replete conditions but was presentin RNA isolated from iron-starved cells [Fig . 3B], demonstratingiron regulation at the hurI promoter . Addition of heme to iron-starvedcultures resulted in a significant reduction in hurI transcriptlevels, suggesting that the iron requirements of the cells weresatisfied by the added heme and that Fur repression of hurIwas resumed.

A single major hurI transcription initiation site was observed in both B . pertussis and B . bronchiseptica . In B . pertussis,the site corresponded to a T residue that was 27 nucleotidesupstream of the predicted hurI start codon [Fig. 3A], whilein B . bronchiseptica, the major site was the upstream adjacentG residue . Consistent with previous predictions [57], the transcriptioninitiation sites were optimally spaced from ⁷⁰-like -10 and-35 elements: 5'-TAAAAT-3' and 5'-TTGCAT-3', respectively . Theinitiation sites and promoter elements overlap predicted Fur binding sites, consistent with a promoter occlusion mechanism of Fur repression . The lack of canonical Shine-Dalgarno sequences suggests that the translational efficiency of the hurI mRNA may be low.

Genetic and biochemical characterization of bhuR promoter determinants. Other ECF factors regulating a variety of functions in responseto extracytoplasmic signals have been described [34, 37, 45],and these sigma factors recognize promoter sequences distinctfrom those typical of ⁷⁰ promoters [16, 37] . Nucleotide sequence alignments comparing the bhuR upstream region with promoter sequences of other ECF factor-regulated genes identified potential-10 and -35 elements that we previously hypothesized to comprisethe bhuR promoter [Fig. 4] [56] . Based on these predictions, an oligonucleotide primer [PE1, Fig . 4] was designed for mappingof the transcription initiation site by primer extension analysis.However, in multiple experiments, a bhuR-specific extensionproduct was not produced by PE1 [data not shown] . At that time,it was hypothesized that the extremely high G+C content of the predicted bhuR initial transcribed region [93% from positions 370 to 410, Fig . 4] may be causing premature termination ofreverse transcription.

 
To genetically test the prediction that sequences located at positions 335 to 346 constituted a critical part of the HurI-dependent, heme-responsive bhuR promoter, a block substitution mutation in the predicted -35 region was constructed [Fig . 4] and analyzedin the context of a transcriptional hurIR bhuR-lacZ fusion [plasmidpRK47] . B013N[pRK47] showed the same pattern of iron-regulated,heme-responsive bhuR transcription as cells carrying the wild-typefusion gene [pRK42] [data not shown], indicating that the residuesmutated in pRK47 did not constitute part of the heme-responsivebhuR promoter.

Since predictions based on nucleotide sequence alignments with other ECF factor promoters did not allow identification ofthe bhuR promoter, a series of deletions in the bhuR upstreamDNA region was constructed to spatially define the minimal region required for maximal heme-responsive bhuR promoter activity. It was previously shown that a 0.5-kb region encompassing the3' region of hurR, the 0.2-kb hurR-bhuR intergenic region, and 5' bhuR sequences carried all the regulatory determinants necessary to direct hurI-dependent, heme-responsive transcription of a bhuR-lacZ fusion [pRK41] in B . bronchiseptica [57] . Successive5' deletions of this region were obtained by PCR, yielding 0.44-kb,0.38-kb, and 0.3-kb fragments, which were used to construct bhuR-lacZ fusion plasmids pRK51, pRK50, and pRK45, respectively [Fig . 4].

B . bronchiseptica B013N carrying the bhuR-lacZ fusion plasmids were grown in iron-depleted medium with or without hemin and assayed for ß-galactosidase activity [Fig . 5] . B013N[pRK41] showed a ninefold induction of bhuR transcription when iron-starved cells were exposed to hemin . Cells carrying fusion plasmid pRK51 or pRK50 exhibited essentially equivalent levels of transcriptional activity and induction, indicating that bhuR promoter determinants mediating heme responsiveness were contained within the 0.21-kb region upstream of the bhuR start codon carried on plasmid pRK50 [Fig . 4] . However, B013N[pRK45] showed markedly reduced transcriptionalactivity under both growth conditions, and induction in responseto hemin was reduced to only 3-fold [Fig . 5] . This result indicatedthat nucleotide sequences between the pRK50 and pRK45 endpoints[positions 214 and 291, Fig . 4] were required for wild-typelevels of bhuR promoter activity . The residual activity andpartial heme responsiveness of the fusion borne on pRK45 suggestedthat part of the bhuR promoter may be contained on this clonedDNA fragment.

 
Based on these genetic analyses of the bhuR promoter, it was hypothesized that the bhuR +1 position was located further upstream of the bhuR open reading frame than originally predicted and that failure to obtain extension products in previous experiments was perhaps due to the distance between primer PE1 and the transcription initiation site, as well as the G+C composition of sequences upstream of the bhuR coding sequences . Additional primer extension analyses performed with primer PE2 [complementary to a region upstream of the high-G+C tract] [Fig . 4] demonstrated the presenceof a bhuR transcript in RNA samples from iron-starved B . pertussisand B . bronchiseptica cells that were exposed to hemin [Fig.6] . The transcript was undetectable in RNA samples from iron-repletecultures and was present in very low abundance in iron-starvedB . bronchiseptica cells [detectable only after extended exposuresof the autoradiogram].

 
The major bhuR transcription initiation site corresponded to an A residue 116 nucleotides upstream of the predicted bhuR start codon [Fig . 4 and Fig . 6] in both Bordetella species.A minor initiation site mapped to a G residue 2 nucleotidesfurther upstream . A larger primer extension product [open arrow,Fig . 6] was detected in RNA samples from iron-starved B . bronchisepticacells [Fig . 6, inset], and was present in very low abundancein RNA from iron-starved B . bronchiseptica cells induced withhemin . This primer extension product was also detectable inRNA from iron-starved B . pertussis when the autoradiogram wassignificantly overexposed . This larger product maps to a site36 nucleotides upstream of the major bhuR transcription initiationsite and is likely to be derived from a longer iron-regulatedtranscript initiating upstream of bhuR, possibly at the hurI promoter . The greater abundance of this larger product in RNAfrom iron-starved cells in the absence of hemin is similar tothe pattern of expression of the hurI transcript [Fig . 3], suggestingthat this larger primer extension product may be derived froma transcript initiating at the hurI promoter . It is possiblethat termination of reverse transcription may occur at this point on the transcript due to the presence of a secondary structure in the mRNA.

We previously reported that in B . bronchiseptica, heme-inducible transcription of a bhuR-lacZ fusion was dependent on hurI [57]. To determine whether bhuR transcription was also initiated in a hurI-dependent manner in B . pertussis, primer extension experimentswith RNA obtained from wild-type [UT25Sm1] and hurI mutant [PM8]B . pertussis strains were performed [data not shown] . Similarto the results shown in Fig . 6, in wild-type B . pertussis thebhuR transcript was most abundant in iron-starved cells exposedto hemin . In contrast, the B . pertussis hurI mutant showed nodetectable bhuR transcript under any of the conditions tested[data not shown], indicating that production of the heme-induciblebhuR transcript is dependent on the HurI factor in B . pertussis.The larger iron-regulated product seen in primer extension experiments[such as that shown in Fig. 6] was also observed in other experimentswith both wild-type and hurI mutant strains [data not shown],indicating that this transcript is not hurI dependent.

A nucleotide sequence alignment of the bhuR promoter region with other ECF factor- dependent promoters is shown in Fig.7 . Consistent with previous observations of other investigators[16, 37], certain features of the ECF factor promoters of otherorganisms, including the -35 elements and spacing between -35and -10 elements, are fairly well conserved, while the -10 elementsare poorly conserved . It has been proposed that the -10 elementmay provide specificity for promoter recognition by a particularECF factor, since many bacterial genomes appear to encode multipleECF sigma factors [37, 58] . The bhuR promoter shows little sequencesimilarity to other ECF factor promoters, even that of fecA,which is regulated by another member of the iron starvationsubfamily of ECF factors [1] . Interestingly, although determination of the P . putida pupB promoter [regulated by the ECF factorPupI] has not been reported, alignment of the pupB upstreamregion with the bhuR promoter region revealed striking similaritiesin what are predicted to be the -35 and -10 elements in eachof these promoters . In addition, a tract of A residues upstreamfrom the predicted -35 element is present in the promoter regionsof both bhuR and pupB . The functional significance of this sequencefeature, if any, is unknown.

 
Analysis of transcription through the hurR-bhuR intergenic region. The requirement for BhuR in heme-responsive transcriptional activation of the bhu genes was demonstrated previously [57], suggesting a role for the receptor as an environmental heme sensor and signal-transducing protein in addition to its functionas a heme transporter . Results from the present study indicatethat the bhuR promoter is active almost exclusively under iron starvation conditions in the presence of heme; however, in orderto have BhuR displayed on the cell surface to act as a hemesensor, some transcription of bhuR likely occurs under ironstarvation conditions in the absence of heme.

To determine if iron-regulated transcription originating ata distal upstream promoter reads through the hurR-bhuR intergenic region to contribute to iron-regulated bhuR expression, a polar Cm element insertion was constructed in the hurR-bhuR intergenic region . Plasmid-borne transcriptional lacZ fusions with the wild-type parental hurIR bhuR' fragment [pRK42], the fragment containing an engineered BglII site [pRK48], and the fragment containing the Cm insertion [pRK49] [Table 1, Fig . 8A] were analyzed in wild-type B . bronchiseptica.

 
B013N[pRK42] and B013N[pRK48] exhibited equivalent levels of iron-regulated, heme-inducible bhuR expression [Fig . 8B] . Levelsof ß-galactosidase activity were increased by 2-fold in response to iron starvation compared with levels in iron-replete cells, and further activated by approximately 4.5-fold by the addition of heme . In contrast, in B013N[pRK49], the ß-galactosidase activities of iron-replete and iron-depleted cultures were nearly equivalent, indicating that the insertion abolished iron-regulated bhuR expression . However, transcription of bhuR was heme activated 5.5-fold over iron-depleted levels in B013N[pRK49], demonstratingthat the Cm insertion did not disrupt heme-responsive bhuRpromoter function . These results suggest that transcriptionresulting in iron-regulated, heme-independent bhu gene expression originates upstream of the site of the cassette insertion.Transcription from the hurI promoter was shown to be iron regulated,and thus it is likely that transcription from the hurI promoterreads through the hurR-bhuR intergenic region and into bhuRto allow low levels of bhuR transcription under iron-limitingconditions in the absence of heme induction.

Additional evidence indicating that iron-regulated transcription through the hurR-bhuR intergenic region contributes to bhuR expression in the absence of inducer was obtained by reverse transcription-PCR analysis . Total RNA from B . pertussis cells grown under iron-replete conditions and iron-depleted conditionswith and without hemin supplementation was reverse transcribed,and the products were used as the template in PCR . The predicted0.44-kb product, encompassing the 'hurR-bhuR' region, was obtained when cosmid DNA carrying the entire hur-bhu genetic system was used as a control template [Fig . 9, lane 1] . A 'hurR-bhuR' transcriptwas not detected in RNA from cells grown in iron-replete medium[Fig . 9, lane 3], consistent with Fur repression at the hurIpromoter . In contrast, transcripts spanning the hurR-bhuR intergenicregion were detected in RNA samples from iron-starved cellscultured with and without hemin [Fig. 9, lanes 5 and 7] . Theseresults confirm that RNA transcripts initiating upstream ofthe heme-inducible bhuR promoter [likely at the hurI promoter]proceed through the hurR-bhuR intergenic region and into bhuRunder iron-limiting conditions in the absence of inducer, thusallowing BhuR to be produced at a low level for heme sensingand transport.

 
Studies on the B . pertussis heme utilization system to date[56, 57] support the model proposed in Fig . 10 for iron-repressibleand heme-responsive transcriptional regulation of bhu genes.Under iron-replete conditions, Fur and iron repress hurI promoteractivity [Fig . 10A] . Under iron-depleted conditions, Fur derepressionof the hurI promoter allows transcription initiation at hurI,resulting in HurI and HurR protein production . However, in theabsence of heme, HurI remains inactive through its associationwith HurR . Some transcription initiated at the hurI promoterreads through the hurR-bhuR intergenic region and the bhu genes[Fig. 10B], allowing low levels of BhuR to be produced and displayedon the cell surface . When BhuR binds heme, a signal is transducedthrough HurR, and HurI is released and can associate with coreRNA polymerase to direct high levels of transcription at the bhuR promoter [Fig . 10C] . Transcription at the hurI promotermay continue until the cell's intracellular iron stores arereplenished, at which time Fur repression will resume . HurI-dependenttranscription of the bhu genes may diminish and eventually ceaseas the HurI protein turns over and no new protein is produced.

 
The hurI and bhuR promoters were mapped by mutational and primer extension analyses . The transcription initiation site for the hurI gene was consistent with previous predictions of ⁷⁰-like promoter elements . Iron regulation of hurI was observed, consistent with predicted Fur binding sites and previous determinationof functional Fur binding activity in the hurI promoter region[56] . Several lines of evidence suggest that an iron-regulated polycistronic transcript initiating at the hurI promoter and reading through bhuR provides a low level of bhuR expression in the absence of heme inducer . First, the hurI and hurR openreading frames overlap, and no other obvious promoter elements exist within these coding regions, suggesting that they are cotranscribed . Additionally, reverse transcription-PCR experiments identified iron-regulated transcripts encompassing the hurR-bhuR intergenic region, indicating that readthrough transcription occurs . Concordantly, insertion of a terminator downstream ofhurR abolished the wild-type pattern of iron-regulated bhuR expression but did not affect heme-activated expression [Fig. 8], indicating that the transcript encompassing the hurR-bhuRintergenic region was iron regulated and hurI independent, whichis a pattern of expression identical to that of the hurI transcript[Fig . 3] . Iron-regulated, heme-independent bhuR expression ispredicted to be crucial for BhuR production in the absence ofinducer, which would allow B . pertussis cells to sense the presenceof heme in the environment.

The bhuR transcription initiation site was identified in both B . pertussis and B . bronchiseptica . Consistent with our previousstudies examining the activity of bhuR-lacZ reporter fusions[57], the bhuR transcript was found to be iron regulated, hemeinducible, and hurI dependent . A second, larger product wasalso identified with a bhuR-specific primer in primer extensionanalyses . This product was iron regulated but not heme responsiveor hurI dependent; thus, this pattern of expression is verysimilar to that of the hurI transcript . The bhuR promoter shareslittle similarity with characterized ECF factor promoters fromother organisms, including the fecA promoter of E . coli, whichis regulated by the iron starvation ECF sigma factor FecI . Interestingly,the bhuR promoter region shares several features with the predictedP . putida pupB promoter region, including the presence of anadenine-rich region upstream of the predicted -35 elements,suggesting that the regulation of these promoters may also besimilar.

Though the concentrations of heme to which Bordetella cells are exposed in vivo are unknown, the success of another obligate human respiratory pathogen, Haemophilus influenzae, implies that heme may be accessed in this niche by capable organisms.Similar to B . pertussis, nontypeable H . influenzae is a noninvasive organism that colonizes the human nasopharynx . Haemophilus species are incapable of synthesizing protoporphyrin IX, the precursor of heme, and require exogenously supplied heme or porphyrinin order to grow aerobically [23] . Thus, their ability to successfully colonize the nasopharynx and cause upper respiratory diseasein humans indicates that their heme requirements are satisfiedin the host environment . Unlike Haemophilus species, B . pertussis and B . bronchiseptica can synthesize heme precursors and thus do not require heme as a growth factor . However, heme internalized via the Bhu system may be used both as an iron source and asa prosthetic group for direct incorporation into cytochromesand other metabolic enzymes.

The bhu system is the second example of a positively regulated Bordetella iron acquisition system for which the substrate is known . The native alcaligin siderophore system is positively regulated by an AraC-like protein, AlcR, in response to iron starvation and the presence of alcaligin . We hypothesize that positive regulation of iron acquisition systems in Bordetella species allows the organisms to prioritize expression of genesbased on iron source availability . During the course of infection,it is likely that cells may sense multiple iron sources, forexample, heme and ferric alcaligin, simultaneously . Under thosecircumstances, priority might be assigned to expression of genesthat encode utilization functions for the most abundant or mosteasily assimilated iron source in the environment . The abilityto integrate signals received from multiple iron sources andrespond appropriately may be critical for B . pertussis in thecomplex host environment, which changes over the course of infectiondue to the actions of B . pertussis virulence factors and thehost immune responses.

 
We are grateful to Timothy Brickman for critical reading ofthe manuscript, many useful discussions, and advice and assistancewith transcriptional analyses and graphics . We thank MladenTomich for technical advice related to primer extension methods.We acknowledge Jenny Walder for technical assistance.

Support for this study was provided by Public Health Service grants R01 AI-31088 [S.K.A.] and T32 AI-07421 [C.K.V.] fromthe National Institute of Allergy and Infectious Diseases.

 
* Corresponding author . Mailing address: Department of Microbiology, University of Minnesota, MMC 196, 420 Delaware Street S.E., Minneapolis, MN 55455-0312 . Phone: [612] 625-6947 . Fax: [612] 626-0623 . E-mail: sandra@mail.ahc.umn.edu.

Present address: Laboratory of Molecular Biology, National Cancer Institute, Bethesda, MD 20892-4264.

1. Angerer, A., S . Enz, M . Ochs, and V . Braun. 1995 . Transcriptional regulation of ferric citrate transport in Escherichia coli K-12 . Fecl belongs to a new subfamily of sigma 70-type factors that respond to extracytoplasmic stimuli . Mol . Microbiol . 18:163-174.
2. Armstrong, S . K., and M . O . Clements. 1993 . Isolation and characterization of Bordetella bronchiseptica mutants deficient in siderophore activity . J . Bacteriol . 175:1144-1152.
3. Beall, B. 1998 . Two iron-regulated putative ferric siderophore receptor genes in Bordetella bronchiseptica and Bordetella pertussis . Res . Microbiol . 149:189-201.
4. Beaumont, F . C., H . Y . Kang, T . J . Brickman, and S . K . Armstrong. 1998 . Identification and characterization of alcR, a gene encoding an AraC-like regulator of alcaligin siderophore biosynthesis and transport in Bordetella pertussis and Bordetella bronchiseptica . J . Bacteriol . 180:862-870 .
5. Bordet, J., and O . Gengou. 1906 . Le microbe de la coqueluche . Ann . Inst . Pasteur [Paris] 20:731-741.
6. Braun, V. 1997 . Surface signaling: novel transcription initiation mechanism starting from the cell surface . Arch . Microbiol . 167:325-331.
7. Brickman, T . J., and S . K . Armstrong. 2002 . Bordetella interspecies allelic variation in AlcR inducer requirements: identification of a critical determinant of AlcR inducer responsiveness and construction of an alcR[Con] mutant allele . J . Bacteriol . 184</?VOLUMN-NR>:1530-1539.
8. Brickman, T . J., J . G . Hansel, M . J . Miller, and S . K . Armstrong. 1996 . Purification, spectroscopic analysis and biological activity of the macrocyclic dihydroxamate siderophore alcaligin produced by Bordetella pertussis and Bordetella bronchiseptica . Biometals 9:191-203.
9. Brickman, T . J., H . Y . Kang, and S . K . Armstrong. 2001 . Transcriptional activation of Bordetella alcaligin siderophore genes requires the AlcR regulator with alcaligin as inducer . J . Bacteriol . 183:483-489 .
10. Chomczynski, P., and N . Sacchi. 1987 . Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction . Anal . Biochem . 162:156-159.
11. Cornelissen, C . N. 2003 . Transferrin-iron uptake by gram-negative bacteria . Front . Biosci . 8:D836-847.
12. Crosa, J . H. 1997 . Signal transduction and transcriptional and posttranscriptional control of iron-regulated genes in bacteria . Microbiol . Mol . Biol . Rev . 61:319-336.
13. Cunliffe, H . E., T . R . Merriman, and I . L . Lamont. 1995 . Cloning and characterization of pvdS, a gene required for pyoverdine synthesis in Pseudomonas aeruginosa: PvdS is probably an alternative sigma factor . J . Bacteriol . 177:2744-2750.
14. Dean, C . R., and K . Poole. 1993 . Expression of the ferric enterobactin receptor [PfeA] of Pseudomonas aeruginosa: involvement of a two-component regulatory system . Mol . Microbiol . 8:1095-1103.
15. de Lorenzo, V., M . Herrero, U . Jakubzik, and K . N . Timmis. 1990 . Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria . J . Bacteriol . 172:6568-6572.
16. Enz, S., V . Braun, and J . H . Crosa. 1995 . Transcription of the region encoding the ferric dicitrate-transport system in Escherichia coli: similarity between promoters for fecA and for extracytoplasmic function sigma factors . Gene 163:13-18.
17. Escolar, L., J . Perez-Martin, and V . de Lorenzo. 1999 . Opening the iron box: transcriptional metalloregulation by the Fur protein . J . Bacteriol . 181:6223-6229.
18. Fetherston, J . D., S . W . Bearden, and R . D . Perry. 1996 . YbtA, an AraC-type regulator of the Yersinia pestis pesticin/yersiniabactin receptor . Mol . Microbiol . 22:315-325.
19. Field, L . H., and C . D . Parker. 1978 . Differences observed between fresh isolates of Bordetella pertussis and their laboratory passaged derivatives, p . 124-132 . In C . R . Manclark and J . C . Hill [ed.], International symposium on pertussis . U.S . Department of Health, Education, and Welfare, Washington, D.C.
20. Giardina, P . C., L . A . Foster, S . I . Toth, B . A . Roe, and D . W . Dyer. 1995 . Identification of alcA, a Bordetella bronchiseptica gene necessary for alcaligin production . Gene 167:133-136.
21. Guerinot, M . L. 1994 . Microbial iron transport . Annu . Rev . Microbiol . 48:743-772.
22. Hantke, K. 1981 . Regulation of ferric iron transport in Escherichia coli K12: isolation of a constitutive mutant . Mol . Gen . Genet . 182:288-292.
23. Hardy, G . G., S . M . Tudor, and St . J . W . Geme, 3rd. 2003 . The pathogenesis of disease due to nontypeable Haemophilus influenzae . Methods Mol . Med . 71:1-28.
24. Heinrichs, D . E., and K . Poole. 1993 . Cloning and sequence analysis of a gene [pchR] encoding an AraC family activator of pyochelin and ferripyochelin receptor synthesis in Pseudomonas aeruginosa . J . Bacteriol . 175:5882-5889.
25. Henderson, D . P., and S . M . Payne. 1994 . Characterization of the Vibrio cholerae outer membrane heme transport protein HutA: sequence of the gene, regulation of expression, and homology to the family of TonB- dependent proteins . J . Bacteriol . 176:3269-3277.
26. Henderson, D . P., and S . M . Payne. 1994 . Vibrio cholerae iron transport systems: roles of heme and siderophore iron transport in virulence and identification of a gene associated with multiple iron transport systems . Infect . Immun . 62:5120-5125.
27. Kang, H . Y., and S . K . Armstrong. 1998 . Transcriptional analysis of the Bordetella alcaligin siderophore biosynthesis operon . J . Bacteriol . 180:855-861 .
28. Kang, H . Y., T . J . Brickman, F . C . Beaumont, and S . K . Armstrong. 1996 . Identification and characterization of iron-regulated Bordetella pertussis alcaligin siderophore biosynthesis genes . J . Bacteriol . 178:4877-4884.
29. Keen, N . T., S . Tamaki, D . Kobayashi, and D . Trollinger. 1988 . Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria . Gene 70:191-197.
30. Kirby, A . E., D . J . Metzger, E . R . Murphy, and T . D . Connell. 2001 . Heme utilization in Bordetella avium is regulated by RhuI, a heme-responsive extracytoplasmic function sigma factor . Infect . Immun. 69:6951-6961 .
31. Koster, M., W . van Klompenburg, W . Bitter, J . Leong, and P . Weisbeek. 1994 . Role for the outer membrane ferric siderophore receptor PupB in signal transduction across the bacterial cell envelope . EMBO J . 13:2805-2813.
32. Lankford, C . E. 1973 . Bacterial assimilation of iron . Crit . Rev . Microbiol . 2:273-331.
33. Letoffe, S., J . M . Ghigo, and C . Wandersman. 1994 . Iron acquisition from heme and hemoglobin by a Serratia marcescens extracellular protein . Proc . Natl . Acad . Sci . 91:9876-9880 .
34. Lonetto, M . A., K . L . Brown, K . E . Rudd, and M . J . Buttner. 1994 . Analysis of the Streptomyces coelicolor sigE gene reveals the existence of a subfamily of eubacterial RNA polymerase sigma factors involved in the regulation of extracytoplasmic functions . Proc . Natl . Acad . Sci . 91:7573-7577.
35. Miller, J . H. 1972 . Experiments in molecular genetics . Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
36. Mills, M., and S . M . Payne. 1995 . Genetics and regulation of heme iron transport in Shigella dysenteriae and detection of an analogous system in Escherichia coli O157:H7 . J . Bacteriol . 177:3004-3009.
37. Missiakas, D., and S . Raina. 1998 . The extracytoplasmic function sigma factors: role and regulation . Mol . Microbiol . 28:1059-1066.
38. Moore, C . H., L . A . Foster, D . G . Gerbig, D . W . Dyer, and B . W . Gibson. 1995 . Identification of alcaligin as the siderophore produced by Bordetella pertussis and B . bronchiseptica . J . Bacteriol . 177:1116-1118.
39. Murphy, E . R., R . E . Sacco, A . Dickenson, D . J . Metzger, Y . Hu, P . E . Orndorff, and T . D . Connell. 2002 . BhuR, a virulence-associated outer membrane protein of Bordetella avium, is required for the acquisition of iron from heme and hemoproteins . Infect . Immun . 70:5390-5403 .
40. Neilands, J . B. 1995 . Siderophores: structure and function of microbial iron transport compounds . J . Biol . Chem . 270:26723-26726.
41. Ochsner, U . A., Z . Johnson, and M . L . Vasil. 2000 . Genetics and regulation of two distinct haem-uptake systems, phu and has, in Pseudomonas aeruginosa . Microbiology 146:185-198 .
42. Panter, S . S. 1994 . Release of iron from hemoglobin . Methods Enzymol . 231:502-514.
43. Pradel, E., N . Guiso, and C . Locht. 1998 . Identification of AlcR, an AraC-type regulator of alcaligin siderophore synthesis in Bordetella bronchiseptica and Bordetella pertussis . J . Bacteriol. 180:871-880 .
44. Querinjean, P., P . L . Masson, and J . F . Heremans. 1971 . Molecular weight, single-chain structure and amino acid composition of human lactoferrin . Eur . J . Biochem . 20:420-425.
45. Raivio, T . L., and T . J . Silhavy. 2001 . Periplasmic stress and ECF sigma factors . Annu . Rev . Microbiol . 55:591-624.
46. Ratledge, C., and L . G . Dover. 2000 . Iron metabolism in pathogenic bacteria . Annu . Rev . Microbiol . 54:881-941.
47. Rossi, M . S., J . D . Fetherston, S . Letoffe, E . Carniel, R . D . Perry, and J . M . Ghigo. 2001 . Identification and characterization of the hemophore- dependent heme acquisition system of Yersinia pestis . Infect . Immun . 69:6707-6717 .
48. Sambrook, J . E., F . Fritsch, and T . Maniatis. 1989 . Molecular cloning: a laboratory manual . Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
49. Schade, A . L., and L . Caroline. 1946 . An iron binding component of human blood plasma . Science 104:340-341.
50. Schmitt, M . P. 1999 . Identification of a two-component signal transduction system from Corynebacterium diphtheriae that activates gene expression in response to the presence of heme and hemoglobin . J . Bacteriol . 181:5330-5340 .
51. Schneider, D . R., and C . D . Parker. 1982 . Effect of pyridines on phenotypic properties of Bordetella pertussis . Infect . Immun . 38:548-553.
52. Stainer, D . W., and M . J . Scholte. 1970 . A simple chemically defined medium for the production of phase I Bordetella pertussis . J . Gen . Microbiol . 63:211-220.
53. Stojiljkovic, I., and K . Hantke. 1992 . Hemin uptake system of Yersinia enterocolitica: similarities with other TonB- dependent systems in gram-negative bacteria . EMBO J . 11:4359-4367.
54. Thompson, J . M., H . A . Jones, and R . D . Perry. 1999 . Molecular characterization of the hemin uptake locus [hmu] from Yersinia pestis and analysis of hmu mutants for hemin and hemoprotein utilization . Infect . Immun . 67:3879-3892 .
55. Torres, A . G., and S . M . Payne. 1997 . Haem iron-transport system in enterohaemorrhagic Escherichia coli O157:H7 . Mol . Microbiol . 23:825-833.
56. Vanderpool, C . K., and S . K . Armstrong. 2001 . The Bordetella bhu locus is required for heme iron utilization . J . Bacteriol. 183:4278-4287 .
57. Vanderpool, C . K., and S . K . Armstrong. 2003 . Heme-responsive transcriptional activation of Bordetella bhu genes . J . Bacteriol . 185:909-917 .
58. Visca, P., L . Leoni, M . J . Wilson, and I . L . Lamont. 2002 . Iron transport and regulation, cell signalling and genomics: lessons from Escherichia coli and Pseudomonas . Mol . Microbiol . 45:1177-1190.
59. Wandersman, C., and I . Stojiljkovic. 2000 . Bacterial heme sources: the role of heme, hemoprotein receptors and hemophores . Curr . Opin . Microbiol . 3:215-220.
60. Wang, J., and M . F . Wilkinson. 2000 . Site-directed mutagenesis of large [13-kb] plasmids in a single-PCR procedure . BioTechniques 29:976-978.

Free Online Full-text Article

What Is Growth Medium?, What Is Genome?, What Is Staphylococcus Aureus?, What Is Dna?, What Is Environmental Microbiology?, s, Bacterium, s, Microbes, n, Bacteria, a, Microbe, s, Microbiology, n, Microorganisms, c, Bacteria, r, Cell suspensions, c, Citrobacter, c, Citrobacter, n, Escherichia coli, o, Bacteria, n, Pseudomonas, e, Staphylococcus, n, Gram positive, s, Erythromycin, n, Prokaryotes, c, Anaerobes, i,