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Journal of Bacteriology, December 2002, p. 6942-6951, Vol. 184, No. 24
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.24.6942-6951.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Differential Regulation of the Bordetella bipA Gene: Distinct Roles for Different BvgA Binding Sites

Rajendar Deora*

Department of Microbiology, Immunology and Molecular Genetics, David Getten University of California—Los Angeles, School of Medicine, Los Angeles, California 90095-1747

Received 15 January 2002/ Accepted 18 March 2002


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ABSTRACT
 
The BvgAS signal transduction system of Bordetella controls an entire spectrum of gene expression states in response to differences in environmental conditions. In particular, the Bordetella Bvg-intermediate-phase gene bipA displays a complex regulatory pattern in response to various concentrations of modulators. Expression of bipA is low in the absence of modulating signals, maximal at intermediate concentrations of modulators, and near background levels at high concentrations of modulators. bipA is regulated at the transcriptional level, and the bipA promoter contains multiple BvgA binding sites present both upstream and downstream of the transcriptional initiation site. In vivo transcriptional analyses, utilizing several mutant promoter fusions to the reporter enzyme ß-galactosidase, suggest that the upstream binding site IR1 is essential for expression and that the downstream binding sites IR2 and IR3 are involved in transcriptional repression. Mutations of IR2 or IR3 convert the expression profile of bipA from that of a Bvg-intermediate-specific-phase gene to that of a Bvg+-phase gene. To gain insight into the mechanism responsible for differential bipA regulation, DNase I protection studies were conducted with various mutant promoters. These analyses suggest that IR1 and IR2 function as core binding sites and are the primary determinants for the phosphorylation-induced oligomerization of BvgA to the adjacent regions.


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INTRODUCTION
 
The survival and replication of bacteria in a particular niche within a host or in a constantly changing environment outside the host require continuous monitoring of surrounding conditions and the ability to generate an adaptive response. In general, bacteria respond to environmental cues by controlling gene expression, resulting in the upregulation and/or downregulation of appropriate genes. For most bacteria, a major mechanism for eliciting adaptive responses to subtle changes in their environment is a signal transduction cascade, mediated by the so-called two-component systems. In their simplest form these systems are comprised of two regulatory proteins, a sensor kinase and a transcriptional activator/repressor, also known as the response regulator (22, 37). The sensor is an autophosphorylating protein kinase, and the response regulator undergoes a phosphorylation-induced conformational change, thereby eliciting a specific response. Two-component systems are also found in some lower eukaryotes, reflecting the diversity of this method of regulating gene expression (29).

The Bordetella BvgAS locus encodes a two-component system that regulates the expression of nearly all of the known virulence factors synthesized by these gram-negative respiratory pathogens and plays an important role in their survival strategy. The bvgAS loci of Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica are 96% identical at the nucleotide level (6) and are functionally interchangeable during infection (32). BvgA and BvgS are members of a class of two-component systems that communicate via a four-step His-Asp-His-Asp phosphorelay (22, 46). BvgA is a DNA-binding response regulator, and BvgS is a transmembrane sensor protein. Using the {gamma}-phosphoryl group of ATP, BvgS is able to autophosphorylate and then transphosphorylates BvgA (10, 46-47). Phosphorylated BvgA has an increased affinity for Bvg-activated promoters and activates transcription of a variety of genes (11-12, 23-25, 30, 38-40, 43, 49).

In response to environmental signals, Bordetella can switch between distinct phenotypic phases (27). In an extensive analysis, Lacey showed that members of the Bordetella species can alternate between three distinct phenotypic modes, designated X, I, and C, in response to changes in temperature or in the concentrations of specific ions in the culture medium (27). Results from several studies have now clearly demonstrated that the BvgAS signal transduction system controls the transition between at least three distinct phenotypic phases, Bvg+, Bvgi, and Bvg- (for a review, see reference 34). The Bvg+ phase appears to correspond to Lacey's X mode, and the Bvg- phase apparently corresponds to the C mode. When BvgAS is active, Bordetella organisms are in the Bvg+ phase and express a variety of putative adhesins and toxins (34), such as filamentous hemagglutinin, fimbriae (Fim2 and Fim3), pertactin (Prn), adenylate cyclase toxin (CyaA) (28), and dermonecrotic toxin (Dnt) (34). The other potential virulence determinants include secreted factors of the type III secretion system (48), tracheal colonization factor (TcfA) (20), and a serum resistance factor, BrkAB (18). Inactivation of BvgAS by mutation or the presence of modulating signals transforms Bordetella to the Bvg- or avirulent phase. The Bvg- phase is characterized by expression of motility and other coregulated phenotypes in B. bronchiseptica (2, 3). In contrast, B. pertussis and B. parapertussis strains are nonmotile although these strains contain the motility genes. The Bvg- phase in B. pertussis is characterized by the expression of several outer membrane proteins of unknown function encoded by the vrg loci (7, 26, 35, 44). Experiments with phase-locked and ectopic expression mutants demonstrated that Bvg+ phase is necessary and sufficient for respiratory tract colonization by B. pertussis and B. bronchiseptica (4, 13, 31).

Isolation of a mutant displaying phenotypes intermediate between those characteristic of the Bvg+ and Bvg- phases changed the traditional view of the bvg regulon (14). Compared to its Bvg+ phase-locked parent, this mutant (designated Bvgi for Bvg intermediate) displayed increased resistance to nutrient limitation and a decreased ability to colonize the respiratory tract. Molecular analyses indicated that the Bvgi mutant had lost the ability to express a subset of Bvg+ phase factors and expressed factors unique to the Bvgi phase. Interestingly, the phenotype of this particular mutant appears to be similar to that of the I mode, described by Lacey (14, 27). It is now becoming increasingly clear that as opposed to mediating a biphasic transition, the Bvg regulatory system appears to control an entire spectrum of distinct states of gene expression.

The discovery of Bvgi-phase-specific antigens predicted the existence of a class of genes that are maximally expressed in the Bvgi phase. That such a class exists was confirmed by the identification of bipA, a Bvg-regulated gene that is maximally expressed in the intermediate phase, induced by either mutation (bvgS-I1) or growth under semimodulating conditions (16, 45). BipA protein shares sequence similarity with intimin of enteropathogenic and enterohemorrhagic Escherichia coli and invasin of Yersinia spp. Comparison of the wild-type B. bronchiseptica strain and the {Delta}bipA strain did not reveal a requirement for bipA in colonization of the respiratory tract (45). It is possible that BipA performs important functions in the Bordetella infectious cycle other than colonization, such as in aerosol transmission.

Analyses of DNA binding demonstrated that BvgA specifically binds to a number of sites on the bipA promoter with a hierarchy of binding affinities. Three of these sites (IR1, HS1, and HS2) are present upstream of the transcription initiation site, whereas two of the sites (IR2 and IR3) are present downstream (Fig. 1). The binding sites also vary with regard to the requirement for the phosphorylation state of BvgA. Whereas nonphosphorylated BvgA is able to bind to IR1 and HS2, only BvgA-P binds to IR2, IR3, and HS1 (Fig. 1). It was hypothesized that the unique expression pattern of the bipA gene is a consequence of the concentration-dependent differential occupancy of BvgA-P at the various binding sites (16).



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  		FIG. 1. Summary of the occupancy of BvgA and BvgA-P at the different binding sites present on the bipA promoter. The arrangement and boundaries of the BvgA binding sites relative to the transcriptional start site (+1) are depicted. Sequences of the different binding sites are shown. N represents one of the four nucleotides, and the subscript digits denote the number of nucleotides that separate the half-sites of IR2 and IR3. Schematics of the DNase I footprinting patterns of BvgA and BvgA-P as determined previously (16) and in Fig. 4A are indicated by bars. The relative thicknesses of the bars represent the relative affinities of binding to the different sites.

To gain insight into the complex regulation of bipA gene expression, I have examined the nature and relative contributions of the different BvgA-binding sites to the Bvgi-phase-specific expression pattern of the bipA gene. Mutational analyses of the different BvgA binding sites in the bipA promoter show that the upstream binding site IR1 is essential for activation, and the downstream binding sites IR2 and IR3 are involved in efficient repression of transcription. Further, it is shown that the complex signal-dependent response profile of the bipA gene can be altered by changes in the promoter region.


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MATERIALS AND METHODS
 
Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 1.


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  		TABLE 1. Bacterial strains and plasmids

PCR and oligonucleotide primers. The conditions for PCRs were as described previously (16). Pfu DNA polymerase (Stratagene) was used for all PCRs. Sequences of the oligonucleotide primers are listed in Table 2.


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  		TABLE 2. Oligonucleotide primers

Media and growth conditions. All Bordetella strains were grown on BG agar (Becton Dickinson Microbiology Systems) supplemented with 7.5% sheep blood and incubated at 37°C for 48 h. For RNA extraction and ß-galactosidase assays, cells were grown in SS broth (42) and harvested at mid-exponential phase. E. coli strains were grown in Luria-Bertani medium at 37°C with shaking at 250 rpm. As needed, the culture media were supplemented with ampicillin (100 µg/ml), gentamicin (40 µg/ml), kanamycin (40 µg/ml), chloramphenicol (40 µg/ml), and streptomycin (40 µg/ml).

Construction of the promoter deletion strain. The promoter deletion strain RKD 100 was constructed as follows. An XbaI-HindIII fragment containing sequences 5' to the bipA promoter, spanning -296 to -85 relative to the transcription start site, was amplified from the chromosome of RB50 using primers 351-5 and 353-3. A second HindIII-KpnI fragment containing sequences 3' to the promoter, from +125 to +519, was also amplified using primers 352-5 and 352-3. The XbaI-HindIII-digested fragment was ligated with the HindIII-KpnI-digested fragment and the XbaI-KpnI-digested allelic exchange vector pRE118 (Kmr) (17) in a three-way ligation resulting in plasmid pRD588. This plasmid was transformed into DH5{alpha}{lambda}pir (M. Liu, unpublished data). DH5{alpha}{lambda}pir is a derivative of the E. coli DH5{alpha} strain that allows replication of plasmids containing the conditional R6Kori. Plasmid pRD588 was mobilized from DH5{alpha}{lambda}pir into B. bronchiseptica using the plasmid pRK2013 by triparental mating (19). After conjugation, cointegrants were selected on BG blood agar plates containing kanamycin and streptomycin. Colonies arising from second recombination events were selected on Luria-Bertani agar containing 7.5% sucrose as described previously (17). The genotype of RKD100 was confirmed by PCR and primer extension assays.

Construction of {Delta}IR2-3 strain. RKD 101 containing deletion of the binding sites IR2 and IR3 was constructed using the strategy described above. A 5' XbaI-HindIII fragment was amplified using primers 351-5 and 351-3, spanning a region from -296 to +11 of the bipA promoter. The 3' PCR fragment was the same HindIII-KpnI fragment as described above, spanning a region from +125 to +519. The two fragments were cloned as described above in the KpnI-XbaI-digested pRE118, resulting in pRD578. pRD578 was used to delete the region from +12 to +122, which includes IR2 and IR3, from the chromosome of RB50. The protocol for selection of the first and second recombination events was same as described above. The genotype of RKD101 was confirmed by both PCR and primer extension.

Mutagenesis of the bipA promoter. The Quick-change site-directed mutagenesis kit (Stratagene) was utilized to delete IR2, following the manufacturer's protocol. The mutagenic oligonucleotide primers 341-5 and 341-3 were used, resulting in deletion of the region from +17 to +59 of the bipA promoter. The plasmid pRD303 was used as the DNA template.

Construction of the bipA promoter assay vector. The suicide plasmid pEGZ (32) has unique EcoRI and BamHI sites upstream of the promoterless lacZ gene. An MfeI-EcoRI, 177-nucleotide-region, spanning -298 to -121, was amplified from the genome of RB50 using the primers 401-5 and 401-3 and cloned into the EcoRI site of pEGZ. Digestion with MfeI results in cohesive ends that are compatible with EcoRI. The clones were checked by restriction digest, and a clone where the EcoRI-MfeI hybrid site is distal to the BamHI site was selected, resulting in the plasmid pRD590. Since the EcoRI-MfeI hybrid site cannot be recleaved by EcoRI, cloning of EcoRI-BamHI fragments in EcoRI-BamHI-digested pRD590 places the fragments upstream of the promoterless lacZ gene. These plasmid derivatives were then integrated into the Bordetella genome by a single crossover at the bipA locus.

LacZ transcriptional fusions and ß-galactosidase assays. Plasmids pGMT18, pEG112, and pEGZ421 were used for measuring the activities of the prn, frl, and bipA promoters, respectively (16, 32). Different bipA promoter derivatives were amplified using the primers BipA101 and 402-3. These primers amplify a region spanning -120 to +143 of the bipA promoter. For amplification of the wild-type bipA promoter, {Delta}IR2, and {Delta}IR1, the plasmids pRD403, pRD572, and pRD404 were used, respectively. For generation of the {Delta}IR1 derivative of the promoter, the primer 305-5 was used instead of the BipA101 primer, resulting in a fragment spanning -60 to +143 of the bipA promoter. For the amplification of the {Delta}IR3 derivative, the 3' primer 301-11 was used. The different promoter fragments were cloned into the EcoRI-BamHI site of pRD590, resulting in the corresponding lacZ fusion plasmids, pRD593 (wild type), pRD597 ({Delta}IR2), pRD610 ({Delta}IR1), and pRD622 ({Delta}IR3). Delivery of the different suicide plasmids into the wild-type B. bronchiseptica strain RB50 or the bipA promoter deletion strain RKD100 followed by selection for gentamicin resistance resulted in integration of the plasmids in the chromosome. ß-galactosidase assays were performed as described previously (32).

RNA extraction, primer extension, and reverse transcriptase PCR (RT-PCR) analyses. Extraction of RNA from different Bordetella strains, primer extension, and RT-PCR analyses were performed as described previously (15, 16). The oligonucleotide BipAExt was used for primer extension and DNA sequencing (16). The primers 352-5 and 352-3 were used for amplification of the bipA gene. Mock RT reactions (RT absent) did not result in any PCR product (data not shown).

DNase I footprinting assays. The plasmids pRD555 (wild type), pRD408 ({Delta}IR1), pRD572 ({Delta}IR2), and pRD425 ({Delta}IR3) were utilized to generate the various promoter fragments by first digesting them with EcoRI and then end labeling them with T4 polynucleotide kinase followed by digestion with BamHI to release the promoter fragments. DNase I footprinting assays were carried out as described previously (16).


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RESULTS
 
A combination of sequence comparisons and DNA-protein interaction analyses of the bipA promoter region revealed the conservation of three pairs of heptanucleotide inverted repeats (IR1, IR2, and IR3) that interact with BvgA and exhibit similarity to the consensus BvgA binding sequence TTTC(C/T)TA (Fig. 1) (16). This sequence has been previously shown by genetic and biochemical means to be important for promoter activity of a number of Bvg-regulated promoters (8, 11-12, 23, 25). To characterize the role of the multiple BvgA-binding sequences in expression of the bipA promoter, a series of plasmid-borne bipA-lac transcriptional fusions was generated in pRD590, a variant of the suicide plasmid pEGZ (32), which carries a fragment extending from -298 to -121 upstream of the bipA promoter. Different promoter variants were cloned upstream of the lac operon in pRD590 (Fig. 2) and then integrated in the B. bronchiseptica strain RKD100 by single crossover at the bipA locus. RKD100 is a derivative of the B. bronchiseptica strain RB50, containing deletions of the region between -84 and +122 of the bipA promoter from the chromosome.



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  		FIG. 2. Effect of deletions and substitutions in the bipA promoter region on the transcriptional activity. A schematic of the bipA promoter region with the different BvgA binding site is shown at the top. Immediately below is the genetic structure of RKD100. Regions deleted are depicted by gaps. Below the RKD100 diagram on the left are shown the lac fusion constructs and, on the right, the levels of transcriptional activities with standard deviations when the strains were grown under nonmodulating conditions or in the presence of 20 mM MgSO4 (+MgSO4). All transcriptional fusions were cloned into the bipA promoter assay vector pRD590 and integrated into the chromosome of the strain RKD100 at the bipA locus. N.D., not determined. Footnote 1, no conintegrants were obtained. Footnote 2, the corresponding lac fusions were highly unstable under nonmodulating conditions.

The bipA promoter contains both activation and repression sites. Introduction of the wild-type bipA promoter fragment in RKD100, as described above, did not significantly alter transcriptional activity or the expression profile of the bipA-lacZ fusion in response to modulating signals, compared to that of the bipA-lacZ fusion in wild-type B. bronchiseptica strain RB50 (Fig. 2 and 5A and B) (16). Note that the transcriptional activities of the different promoter derivatives in RKD100 were measured in the absence of modulators—conditions that result in high levels of expression from the bipA promoter (16). Deletion of the upstream binding site IR1 resulted in almost complete inhibition of the bipA promoter activity (Fig. 2, {Delta}IR1). These results suggest that IR1 is essential for expression and activation of the bipA promoter. Deletion of the downstream binding sites IR2 and IR3 led to a greater than 20-fold and 4-fold stimulation in ß-galactosidase activity, respectively (Fig. 2, {Delta}IR2 and {Delta}IR3), suggesting that IR2 and IR3 are required for repression of transcription from the bipA promoter. Transcriptional activity of the different bipA promoter derivatives in this context was clearly BvgA dependent, since growth of the different strains in presence of MgSO4, a known modulator of BvgA activity, caused reduction of the ß-galactosidase activity to levels comparable to that for a strain lacking the reporter fusion (Fig. 2). Thus, the above-described results strongly suggest that while IR1 is essential for expression of the bipA promoter, IR2 and IR3 are involved in repression from the promoter.



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  		FIG. 5. Effects of various concentrations of nicotinic acid on the transcriptional activity of different promoter fusions. (A) Expression profiles of bipA-lacZ (bipA), prn-lacZ (prn), and frl-lacZ (frl) fusions. The different promoters were cloned in the vector pEGZ and integrated at the corresponding loci in the chromosome of the wild-type B. bronchiseptica strain RB50. (B) The wild-type bipA promoter and the different promoter variants were cloned in the vector pRD590 and integrated at the bipA locus in strain RKD100. The levels of transcriptional activities (103 ß-galactosidase units) with standard deviation bars are shown. Note that the x axis is not drawn to scale.

Introduction into RKD100 of promoter derivatives lacking both IR2 and IR3 resulted either in a failure to generate cointegrants or in lac fusions that were highly unstable in the absence of MgSO4 (Fig. 2, {Delta}IR2-3). To circumvent this problem, IR2 and IR3 were deleted from the chromosome of RB50 using allelic exchange (RKD101) (Fig. 3A). The level of bipA transcript was then measured using primer extension and RT-PCR assays.



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  		FIG. 3. Measurement of the levels of the bipA transcript as a result of deletion of IR2-3. (A). Structure of the IR2-3 deletion in strain RKD101 is diagrammed. The primer used for primer extension and DNA sequencing is BipAExt, as indicated. (B). Primer extension assays were performed with RNA from wild-type strain RB50 (Bvg+), the phase-locked strains RB53i (Bvgi) and RB54 (Bvg-), and RKD101. The bipA-specific transcript from RB50 and the phase-locked strains are marked with +1 (wt), whereas the corresponding transcript from RKD 101 is marked with +1 ({Delta}IR2-3). G, A, T, and C represent the nucleotide sequencing ladder. The DNA template for sequencing was the plasmid pRD578. Reaction mixtures were electrophoresed on a 6% polyacrylamide gel and exposed to X-ray film for 24 h. (C) A longer exposure (8 days) of the same polyacrylamide gel as in panel B. Relative intensities of the various transcripts were quantitated by densitometric analysis using a Personal Densitometer SI and Image-Quant Software Program (Molecular Dynamics Inc., Sunnyvale, Calif.). (D). RT-PCR analysis was used to quantitate the levels of bipA from different B. bronchiseptica strains. Total RNA from different strains was reverse transcribed using random hexamers and used as a template for PCR. g denotes the genomic DNA as a PCR template. Genomic DNA was isolated from wild-type B. bronchiseptica strain RB50. The volume of the PCR loaded in the RKD101 lane is half of the volumes for the Bvg+, Bvgi, and Bvg- lanes. The kilobase ladder (L) from Gibco BRL was used to determine the sizes of the PCR products.

Deletion of the downstream BvgA binding sites IR2 and IR3 from the B. bronchiseptica chromosome results in a hyperactivated promoter. Primer extension assays with RNA prepared from RKD101 produced a considerably shorter fragment (Fig. 3B), which corresponds to the transcription initiation site of the bipA promoter obtained when RNA was isolated from wild-type B. bronchiseptica strain RB50 or an intermediate phase-locked strain, RB53i (Bvgi) (Fig. 3B). The bipA-specific transcript from RKD101 is smaller because of a deletion of 110 nucleotides from RKD101 of the transcribed region (Fig. 3A). The presence of more than one band for the bipA-specific transcript from RKD101 (Fig 3B) can be explained by the previous identification of an additional start site two nucleotides downstream of the +1 nucleotide by rapid amplification of cDNA ends PCR analysis (16). As determined by primer extension assays, the amount of bipA-specific transcript from RKD101 was approximately 17-fold higher than the corresponding transcript from RB53i (Bvgi). As shown in Fig. 3C, the intensity of the product from RB50 (Bvg+) was considerably lower than that of the transcript from RB53i (Bvgi). This result is in agreement with previous measurements of the relative levels of bipA promoter activity in Bvg+ and Bvgi strains using a number of independent assays (16).

In order to confirm the results of primer extension analysis, the levels of bipA transcript in different strain backgrounds were determined using RT-PCR (Fig. 3D). The level of the bipA transcript from RKD101 was higher than that from RB50 and RB53i. As a control for RNA levels, PCR was performed with primers specific to the Bvg-independent gene, recA. No significant variation in the expression of recA was observed in the different strain backgrounds (data not shown). Therefore, these results show that deletion of the two downstream binding sites IR2 and IR3 leads to very high levels of transcription from the bipA promoter.

Binding of BvgA to different promoter derivatives. To determine the effect of binding sequence alterations on the interaction of BvgA with the bipA promoter, the DNase I footprints of several mutant promoters similar to those used in the in vivo transcriptional assays were compared with those of the wild-type promoter.

Wild type. Similar to previous results (16), both BvgA and BvgA-P protected a region from about -55 to about -75, which includes IR1 (Fig. 4A). The adjacent region from -55 to -35, encompassing the half-site HS2, displayed greater BvgA occupancy with increasingly higher concentrations of nonphosphorylated BvgA and significantly greater protection with BvgA-P. Higher concentrations of BvgA-P resulted in the protection of additional regions spanning from +10 to +85 and from -80 to -105 (Fig. 4A, lanes 6 to 10). This region includes the downstream BvgA binding sites IR2 and IR3 and the upstream half-site HS1.



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  		FIG. 4. DNase I protection analyses. 32P-labeled variants of the bipA promoter region were incubated with unphosphorylated BvgA (BvgA, lanes 2 to 5) or BvgA phosphorylated in vitro with acetyl phosphate (BvgA-P, lanes 6 to 10). Lanes 1 to 5 contain 0, 0.05, 0.2, 0.4, and 0.8 µM concentrations of BvgA, whereas lanes 6 to 10 contain 0.05, 0.1, 0.2, 0.4, and 0.8 µM concentrations of BvgA-P, respectively. (A) Wt, the wild-type DNA fragment from -120 to +197 of the bipA promoter; (B) {Delta}IR1, bipA promoter fragment from -60 to +271; (C) {Delta}IR2, same as Wt fragment except the region from +17 to +59, comprising the IR2 site of the bipA promoter, was deleted; (D) {Delta}IR3, -120 to +66 region of the bipA promoter lacking IR3. The nucleotide positions of the different DNA bands relative to the transcription initiation site are indicated at the left of each panel, and regions corresponding to HS1, IR1, IR2, and IR3 are indicated on the right of each panel, respectively. Asterisks denote the regions that show reduced affinity of BvgA-P binding as a result of deletion of IR1. -, absence of BvgA or BvgA-P.

{Delta}IR1. Deletion of the upstream binding site IR1 led to a reduction in the binding of BvgA-P to a region downstream from -45 to -30, encompassing the binding site HS2 (Fig. 4A and B, compare the intensities of the bands denoted by asterisks). A higher concentration of BvgA-P was required for protection in this region, and even with the highest concentration of BvgA-P used, the protection observed was less complete than with the wild-type promoter (Fig. 4A and B, compare lanes 10). Note that in contrast to BvgA-P, nonphosphorylated BvgA seems to bind more effectively to the region -45 to -30 (Fig. 4B, lanes 3 to 5). The significance of this observation is not clear. No apparent reduction in binding affinity or the extent of the DNase I footprint of BvgA for the region from +5 to +85, encompassing the binding sites IR2 and IR3, was observed as a result of the deletion of IR1. Thus, while efficient binding to regions surrounding HS2 requires the presence of IR1, BvgA occupancy of IR2 and IR3 occur independent of IR1.

{Delta}IR2 and {Delta}IR3. Footprint analysis of the {Delta}IR2 fragment indicated no apparent binding of BvgA-P to the regions contiguous to IR2 (Fig. 4C, lanes 6 to 10). Even at the highest BvgA-P concentration used, there was no observable protection in the region from +5 to +17 and that from +59 to +85 (Fig. 4A and C, compare lanes 10). Thus, these results clearly demonstrate that IR2 is required for the binding of BvgA-P to the adjacent regions, including IR3. In contrast to deletion of IR2, deletion of IR3 caused no significant changes in the binding affinity or the protection pattern of BvgA-P. However, the region from -105 to -95, containing the binding site HS1, was protected at a lower concentration of nonphosphorylated BvgA than with the wild-type promoter (compare lanes 2 to 5, Fig. 4A and D). Note that as shown earlier (Fig. 4B), a similar enhancement in the binding affinity of BvgA to regions surrounding HS2 also occurred as a result of deletion of IR1.

Alterations in the bipA promoter region alter the expression profile of the bipA gene in response to modulators. It was reported previously that the expression of bipA was maximal when wild-type B. bronchiseptica bacteria were grown under semimodulating conditions (16). To compare the expression pattern of the bipA gene in response to modulating signals with that of different B. bronchiseptica genes, the ß-galactosidase activities of different gene fusions in the wild-type strain RB50 were measured in the presence of various concentrations of nicotinic acid, a chemical modulator of bvg activity. The ß-galactosidase activities of lacZ fusions to prn, the gene encoding the outer membrane protein pertactin, and frl, the motility master regulatory locus that is repressed by BvgAS, were measured. Expression of bipA was low in the absence of nicotinic acid, maximal at semimodulating concentrations, and near background levels at high concentrations of the modulator (Fig. 5A). In contrast, prn-lacZ was expressed at very high levels at concentrations of modulator from 0 to 0.4 mM and at a very low level at concentrations higher than 0.4 mM. An expression profile similar to that of prn was also seen for fha, encoding the major Bordetella adhesin, filamentous hemagglutinin (data not shown). The Bvg--phase-specific gene frl was expressed in a manner reciprocal to that of prn and fha, with very little expression in the absence of the modulating signal and maximal expression at high concentrations of modulator (Fig. 5A).

To determine the effect of deletions in the promoter region of bipA on the expression profile in response to modulators, the individual bipA-lacZ fusions integrated in the bipA promoter deletion strain RKD100 were tested at various concentrations of nicotinic acid. Measurement of the transcriptional activity of the wild-type promoter fusion in RKD100 resulted in an expression profile similar to that observed with the wild-type strain RB50 (compare Fig. 5A and B). Deletion of IR2 and IR3 resulted in quite different profiles of expression. In contrast to the wild-type promoter derivative, lac fusions to the {Delta}IR2 or {Delta}IR3 promoter derivatives were highly expressed at nicotinic acid concentrations ranging from 0 to 0.8 mM. Remarkably, as a result of deletion of IR2, both the relative activity and the expression profile of bipA, a Bvg-intermediate-phase-specific gene, were altered to resemble those of the Bvg+-phase-specific genes prn (compare Fig. 5A and B) and fha (data not shown). These observations suggest that the binding sites IR2 and IR3 play a critical role in determining the Bvg-intermediate-phase-specific pattern of the bipA gene in response to modulators.


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DISCUSSION
 
The bipA gene represented the first identified example of a class of genes in Bordetella that are maximally expressed under semimodulating conditions (16, 45). Expression of bipA in response to modulating signals is unusual in that this gene is most highly expressed at a point along the Bvg-regulatory continuum where the activity and the level of BvgA-P are predicted to be intermediate. It was hypothesized that the complicated expression pattern is the result of the differential concentration-dependent occupancy and the spatial location of the multiple BvgA binding sites relative to the start of transcription of the bipA promoter (16). The data presented here, from the genetic analyses of different BvgA binding sites, provide the first experimental evidence supporting the differential roles of BvgA binding sites in mediating the expression pattern of bipA.

Deletion of the upstream binding site IR1 results in an almost complete abolition of the transcriptional activity of the bipA promoter, suggesting that IR1 is essential for activation of the bipA promoter. Analogous to the primary BvgA binding sites of fha, cyaA, ptx, and prn promoters (10-12, 23-25, 49), IR1 is present upstream of the bipA promoter region. Similar to IR1, the primary binding sites for fha, cyaA, ptx, and prn have been shown to be critical for activation of the individual promoters (8, 12, 24-25). Similar locations of the primary BvgA binding sites suggest the existence of a conserved mechanism of transcriptional activation of these promoters.

The results from in vivo transcriptional analyses and quantitation of bipA-specific transcripts reported in this study suggest that the binding sites IR2 and IR3 act in concert to efficiently repress transcription from the bipA promoter. IR2 and IR3 are located in the "exclusive zone of repression," a term coined by Gralla and Collado-Vides for the region downstream of -30 (21). Binding of a regulatory protein to this region almost always interferes with the functioning of RNA polymerase (21). It is reasonable to speculate that phosphorylation-induced oligomerization of BvgA may have a multitude of outcomes for the bipA promoter. The binding of BvgA at various binding sites could lead to the formation of higher-order complexes that may either compete with RNA polymerase for binding or make the RNA polymerase binding region inaccessible. One possible mechanism that could lead to the efficient formation of higher-order complexes involves DNA looping or bending of the intervening DNA (33, 41). Interestingly, the two sites (IR1 and IR2) are separated by 105 bp (distance counted from centers of symmetry), similar to the distance (113 bp) that separates the operators OE and OI of the two gal promoters (1, 5). When GalR binds to the operators OE and OI, the two GalR dimers associate to form a DNA loop of the intervening DNA, leading to repression of the two gal promoters (1). A similar mechanism of repression can be envisioned for the bipA promoter.

It is apparent from the mapping of the BvgA binding sites on a number of Bvg-regulated promoters that there is a great deal of variability in the spacing of the recognition sequences. The half-sites comprising the BvgA binding sites of the fha and prn promoters are directly joined, whereas these are separated by 2 and 10 bp in the cyaA and ptx promoters (11-12, 24). Similar to those of fha and prn, the two half-sites of IR1 are in tandem. In contrast, half-sites of IR2 and IR3 are separated by 27 bp ({approx}2.5 turns) and 37 bp ({approx}3.5 turns of the DNA helix), respectively (Fig. 1) (16). The large physical separation between the two half-sites poses an interesting question as to how BvgA molecules that are bound to two opposite sides of the helix interact to bridge the large gap separating the two half-sites in IR2 and IR3. A clear understanding of BvgA interaction with its recognition sequences at the bipA promoter will require additional detailed experimental analyses.

Studies of the interaction of BvgA with a number of Bvg-regulated promoters have contributed to a model where BvgA initially binds to a relatively high-affinity primary binding site, which then serves as a starting point for progressive binding of BvgA molecules to additional weak binding sites present downstream (9, 11-12). The data presented here support this model. Analyses of BvgA binding with different mutant bipA promoters revealed the presence of two core binding sites, IR1 and IR2. Occupancy of BvgA by IR1 and IR2 is the primary determinant for the resultant binding of BvgA to the neighboring regions. Deletion of IR1 led to a decrease in the binding affinity of BvgA-P to HS2. In this respect, the interaction of BvgA-P with the bipA promoter is similar to that with the fha promoter. For the fha promoter, it has been observed that BvgA-P binds more efficiently to the downstream secondary region in the presence of an intact primary site that is similar to IR1 (9). Similar to IR1, the binding site IR2 is essential for the BvgA occupancy of contiguous regions including the downstream binding site IR3. In each of these cases, phosphorylation-induced binding of BvgA at the primary site contributes to secondary site occupancy.

What is the molecular basis of differential gene expression of bipA in response to modulating signals in Bordetella? It has been proposed that depending on BvgA-P levels inside the cell, BvgA may either preferentially occupy the sites upstream of the promoter (including IR1), leading to maximal activation of transcription in the Bvgi phase, or in addition it may bind the downstream sites (IR2 and IR3), resulting in partial repression in the Bvg+ phase (16). Although intracellular levels of BvgA-P have not been measured, results from ß-galactosidase assays of the different promoter constructs in response to various concentrations of modulators provide indirect evidence for such a mechanism. In particular, the repression of wild-type bipA promoter activity in the absence or presence of low concentrations of modulators (Bvg+-phase conditions) was relieved as a result of either individually or collectively deleting IR2 or IR3. Thus, it seems likely that binding of IR2 and IR3 by BvgA results in repression of transcription from the bipA promoter under Bvg+-phase conditions. The more than twofold increase above the maximal expression levels of the wild-type promoter as a consequence of IR2 deletion suggests that there is a very tight control on the expression levels of bipA promoter even under Bvgi-phase conditions. Thus, although the expression of wild-type bipA promoter in RKD100 at a 0.4 mM concentration of nicotinic acid is maximal, this in fact might still be a partially repressed promoter. In summary, it can be hypothesized that occupation of a combination of low-affinity repression sites (IR2 and IR3) and high-affinity activation sites (IR1) at the bipA promoter leads to a constellation of protein-DNA and protein-protein interaction that ultimately results in the complex regulatory profile displayed by bipA. Note that the bipA promoter has a long untranslated region (Fig. 1). (16), raising the likelihood that mechanisms at the posttranscriptional level could also control bipA expression.

bipA is the first gene in Bordetella that has been shown to undergo such variegated changes in its expression pattern. The differential regulation of bipA gene expression observed in vitro as a result of quantitative differences in the concentrations of modulators reflects the ability of a precise and highly sensitive signal transduction system that is responsive to various microenvironments encountered during the Bordetella infectious cycle in mammalian hosts. Deciphering the complexities of this regulation would unravel new information that would ultimately lead to a better understanding of the mechanisms employed by organisms of the Bordetella species to survive within mammalian hosts.


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ACKNOWLEDGMENTS
 
I thank Jeff F. Miller for guidance and support. I am grateful to Purnima Dubey, Bill Hendrickson, and Tapan Misra for critical reading of the manuscript. I thank Linda Kenney for helpful suggestions. I extend my thanks to Mingshun Liu and Peggy Cotter for providing strains and plasmids. I am also grateful to the two anonymous reviewers whose detailed critiques of the manuscript led to significant improvement.

This study was supported by grants to Jeff F. Miller. R.D. was supported by microbial pathogenesis training grant T32A107323. Parts of this study were performed in collaboration with Purnima Dubey.


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FOOTNOTES
 
* Mailing address: 715 Gayley Ave. 309, Los Angeles, CA 90024. Phone: (310) 208-8205. E-mail: rdeora{at}ucla.edu. Back


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Journal of Bacteriology, December 2002, p. 6942-6951, Vol. 184, No. 24
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.24.6942-6951.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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