BpsR Modulates Bordetella Biofilm Formation by Negatively ...

BpsR Modulates Bordetella Biofilm Formation by Negatively Regulating the Expression of the Bps Polysaccharide Matt S. Conover,a Crystal J. Redfern,b Tridib Ganguly,b Neelima Sukumar,b Gina Sloan,b Meenu Mishra,b and Rajendar Deoraa,b,c Program in Molecular Genetics,a Department of Microbiology and Immunology,b and Wake Forest Institute of Regenerative Medicine,c Wake Forest University Health Sciences, Winston-Salem, North Carolina, USA

Bordetella bacteria are Gram-negative respiratory pathogens of animals, birds, and humans. A hallmark feature of some Bordetella species is their ability to efficiently survive in the respiratory tract even after vaccination. Bordetella bronchiseptica and Bordetella pertussis form biofilms on abiotic surfaces and in the mouse respiratory tract. The Bps exopolysaccharide is one of the critical determinants for biofilm formation and the survival of Bordetella in the murine respiratory tract. In order to gain a better understanding of regulation of biofilm formation, we sought to study the mechanism by which Bps expression is controlled in Bordetella. Expression of bpsABCD (bpsA-D) is elevated in biofilms compared with levels in planktonically grown cells. We found that bpsA-D is expressed independently of BvgAS. Subsequently, we identified an open reading frame (ORF), BB1771 (designated here bpsR), that is located upstream of and in the opposite orientation to the bpsA-D locus. BpsR is homologous to the MarR family of transcriptional regulators. Measurement of bpsA and bpsD transcripts and the Bps polysaccharide levels from the wild-type and the ⌬bpsR strains suggested that BpsR functions as a repressor. Consistent with enhanced production of Bps, the bpsR mutant displayed considerably more structured biofilms. We mapped the bpsA-D promoter region and showed that purified BpsR protein specifically bound to the bpsA-D promoter. Our results provide mechanistic insights into the regulatory strategy employed by Bordetella for control of the production of the Bps polysaccharide and biofilm formation.

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ordetella bacteria are Gram-negative bacteria that colonize the respiratory tracts of humans, mammals, and avian species and are the causative agents of a variety of respiratory diseases (39). The ability of these bacteria to persistently colonize the mammalian nasopharynx allows for efficient spread of the organisms between hosts (27, 42). Bordetella bronchiseptica is characterized by its striking ability to establish long-term persistence in not only experimentally inoculated laboratory animals but also naturally infected and previously vaccinated domesticated or farm animals. Similarly, Bordetella pertussis continues to circulate, and the disease whooping cough or pertussis remains prevalent in humans despite widespread and highly sustained vaccination coverage (19). Studies from our laboratory and others have recently begun to provide an explanation for the continued persistence and circulation of Bordetella. We have shown that both B. bronchiseptica and B. pertussis are capable of forming biofilms in the upper respiratory tract of infected animals (13, 14, 53). These findings, combined with the presence of clusters, microcolonies, and tangles of B. bronchiseptica and B. pertussis bacteria in explant tissues and nasal biopsy specimens of patients, strengthen the hypothesis that Bordetella bacteria utilize biofilms as a means to persist and circulate between their mammalian hosts (22, 47, 54). When in biofilms, microorganisms produce a hydrated extracellular matrix composed of polymeric substances, which may consist of nucleic acids, proteins, lipids, and/or polysaccharides. Previously, we have demonstrated that the Bps polysaccharide is a biofilm matrix component and is essential for in vitro and in vivo biofilm development in B. bronchiseptica and B. pertussis (14, 53). Moreover, Bps is necessary for persistent respiratory tract colonization of B. bronchiseptica and for efficient early colonization of the nose and the trachea for B. pertussis (14, 53). Thus, while Bps is clearly emerging as an important Bordetella virulence factor, the

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mechanisms by which its expression is regulated in Bordetella are not known. The genes of the bpsABCD (bpsA-D) locus share similarity with loci of other species involved in the production of poly-␤-1,6-Nacetylglucosamine ([PNAG] polygalacturonic acid [PGA] and polysaccharide intercellular adhesin [PIA]) polysaccharides. These loci include the ica locus of Staphylococcus and pga locus of Escherichia coli (16, 31, 38, 61, 63). bpsA is predicted to encode an outer membrane protein with four transmembrane domains, suggesting that it might mediate translocation and/or docking of Bps to the cell surface. BpsB contains a polysaccharide N-deacetylase domain and is homologous to IcaB and PgaB, which are involved in the deacetylation of PIA/PNAG and PGA, respectively (31, 60). IcaB has crucial roles in biofilm formation, immune evasion, and virulence (6, 60). BpsC is homologous to the glycosyltransferase 2 family of proteins and is predicted to encode a processive glycosyltransferase (48). IcaC of Staphylococcus epidermidis and PgaC of E. coli are required for the complete synthesis of the longer oligomeric chains of PIA (25, 31). Finally, IcaD and PgaD are required for optimal production of the PIA and PGA polysaccharides, respectively (25, 31). bpsD does not show any significant similarity to either icaD or pgaD (48). In Bordetella, the expression of lipopolysaccharide (LPS) and a putative type II capsular operon is regulated by the BvgAS signal transduction system (34, 44). Many other bacterial species have

Received 16 August 2011 Accepted 21 October 2011 Published ahead of print 4 November 2011 Address correspondence to Rajendar Deora, [email protected]. Supplemental material for this article may be found at http://jb.asm.org/. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/JB.06020-11

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also evolved complex multifactorial mechanisms to regulate various exopolysaccharides, which are often crucial for an organism’s transition from planktonic to biofilm growth and for its pathogenesis (3, 26, 46, 50, 59, 62). Given this common concept concerning the regulatory circuits that control polysaccharide expression, we sought to examine the regulation of the bpsA-D locus. In this report, we have identified an open reading frame (ORF), bpsR, which is homologous to the MarR family of transcriptional regulators (2, 11, 12, 24, 64), and we have examined its role in the regulation of the bpsA-D locus. MATERIALS AND METHODS Strains and growth conditions. The B. bronchiseptica strain RB50 was used as the wild-type strain for all experiments (15). All B. bronchiseptica strains were maintained on Bordet-Gengou (BG) agar supplemented with 7.5% defibrinated sheep’s blood and streptomycin (50 ␮g/ml). E. coli strains were maintained on Luria-Bertani (LB) medium supplemented with the appropriate antibiotics (50 ␮g/ml chloramphenicol [CM], 25 ␮g/ml kanamycin, and 100 ␮g/ml ampicillin). B. bronchiseptica strains were grown in Stainer-Scholte (SS) broth (55), and the E. coli strains were grown in LB medium. For comparing the levels of bpsA-D transcript and the Bps polysaccharide levels, the respective Bordetella strains were grown in LB broth. This growth medium was specifically chosen because we noticed a slight growth defect for the ⌬bpsR strain compared to that of RB50 when it was grown in SS broth. However, there were no differences in the growth of the wild-type and ⌬bpsR strains when they were cultured in LB medium. Construction of the ⌬bpsR strain. An in-frame deletion of the bpsR gene (BB1771) was constructed using a previously published allelic exchange method (57). A 504-bp XbaI-HindIII fragment including the 5= region and first 10 codons of bpsR was amplified from the RB50 chromosome using primers MM1 and MM2 (see Table S1 in the supplemental material). Next, a 504-bp HindIII-KpnI fragment corresponding to the 3= region and last 10 codons of bpsR was amplified using MM3 and MM4 (see Table S1). These fragments were then ligated into the allelic exchange vector pRE112 (23) and transformed into SM10␭pir, resulting in the plasmid pMM12. This plasmid was then mobilized from SM10␭pir into RB50, and exconjugates were selected on BG plates containing streptomycin and chloramphenicol. Sucrose selection was used to identify colonies which had undergone a second recombination event, as described previously (23, 57). The genotype of the ⌬bpsR deletion strain was confirmed by PCR and subsequent DNA sequencing. Serum enrichment. Approximately 5 optical density units at 600 nm (OD600) of overnight cultures of the ⌬bpsA-D strain were harvested by centrifugation and resuspended in serum from a pertussis-positive patient. This suspension was then incubated at 4°C for 2 h with shaking followed by centrifugation to remove the bacterial cells. The supernatant was removed and used to resuspend another 5 OD600 units of the ⌬ bpsA-D strain for an additional round of absorption. The enrichment protocol was repeated three more times, followed by a final absorption overnight at 4°C. Detection of Bps by immunoblotting. Crude exopolysaccharide extracts were prepared using previously described methods (36, 48). Approximately 5 ⫻ 109 cells of various strains grown overnight at 37°C were harvested by centrifugation, resuspended in 100 ␮l of 0.5 M EDTA, and boiled for 5 min at 100°C. Cells were removed by centrifugation, and the supernatant was treated with 1 mg ml⫺1 of pronase for 3 h at 37°C. At the end of the incubation period, samples were heated to 85°C for 15 min and phenol-chloroform extracted to inactivate the pronase and remove residual protein contamination. Five microliters of the extract was spotted on a nitrocellulose membrane and allowed to dry overnight. The membrane was blocked with 5% nonfat milk and probed with a 1:1,000 dilution of the absorbed human serum, which was enriched for anti-Bps antibodies. A secondary goat anti-human IgG antibody conjugated to horseradish peroxidase (Pierce) was used at a concentration of 1:2,000 for detection in

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conjunction with an Amersham ECL (enhanced chemiluminescence) Western blotting system. Detection of Bps by ELISA. EDTA extracts were prepared as above except that 50 ml of culture was harvested and resuspended in approximately 10 ␮l of 0.5 M EDTA per OD600 unit. Fifty microliters of sample was then added into the wells of a 96-well enzyme-linked immunosorbent assay (ELISA) plate (Nunc) with 50 ␮l of phosphate-buffered saline (PBS) for a total volume of 100 ␮l per well. Samples were incubated at 4°C overnight. Next, the samples were aspirated, and the plate was blocked with 5% skim milk for 1 h before three washes with PBS-Tween 20 (PBST). A 1:1,000 dilution of the absorbed human serum was used as the primary antibody diluted in PBST with 5% skim milk for 2 h. The plate was then washed five times with PBST before addition of the goat antihuman horseradish peroxidase detection antibody at a 1:1,000 dilution in PBST for 2 h at room temperature, followed by five washes with PBST. Finally, 100 ␮l of tetramethyl-benzidine (Sigma) was added to the wells and incubated for 15 min in the dark before the addition of 50 ␮l of 1.0 M H2SO4. The plate was then read at 450 nm using a plate reader. RNA extraction. (i) Biofilm and planktonic RNA. Empty sterile petri dishes were aseptically inoculated with mid-logarithmic-phase cultures (A600 of 0.5) of the wild-type strain and incubated statically at 37°C for 48 h. After incubation, the medium containing the bacterial cells in suspension was removed and spun at 4°C, followed by immediate lysing of the pelleted bacteria in RLT buffer (Qiagen). This sample represented the planktonic population. For biofilm samples, the surface-adherent cells were washed three times gently with cold PBS, followed by addition of RLT buffer directly to the petri dishes. RNA was prepared using a Qiagen RNeasy kit per the manufacturer’s instructions. (ii) RNA from shaking cultures. The Bordetella RB50, RB54, and ⌬bpsR strains were grown under shaking conditions to an OD600 of 1.0. RNA was prepared as described above using an Qiagen RNeasy kit. cDNA synthesis. RNA samples were treated with DNase I to remove contaminating genomic DNA. After DNase I digestion, an RNA cleanup was performed using a Qiagen RNeasy kit before proceeding with cDNA synthesis. Approximately 1 ␮g of RNA was used for cDNA synthesis in a 20-␮l volume. A pool of random primers and 2 ␮l of 10 mM deoxynucleoside triphosphates (dNTPs) was added to the mixture before denaturation at 65°C for 5 min. This was then placed immediately on ice, followed by addition of the first-strand buffer (500 mM KCl, 200 mM Tris-HCl [pH 8.4], 5 mM MgCl2), 10 mM dithiothreitol (DTT), and 40 units of RNase Out (Invitrogen). The reaction mixture was then incubated at room temperature for 10 min before the addition of 200 units of SuperScript III reverse transcriptase (Invitrogen), followed by incubation at 50°C for 1 h. The reactions were terminated by incubation at 85°C for 5 min. As a negative control, a mock cDNA synthesis reaction was carried out in parallel by excluding the reverse transcriptase from the mixture. Reverse transcription-PCR (RT-PCR). To confirm that bpsABCD is an operon, primers were designed to overlap the 3= ends and 5= ends of adjacent ORFs leading to the amplification of part of the ORFs and the intergenic regions (see Table S1 in the supplemental material). PCR was then performed with either the RT products or the mock control obtained as above. For positive control, RB50 genomic DNA was used as the template. Aliquots of the amplified products, along with a 1-kb DNA ladder (Bioline), were electrophoresed on 1% agarose gels. Images of the ethidium bromide-stained gels were captured by using the Alpha Innotech Gel Doc system (Alpha Innotech Corporation). Real-time RT-PCR. Specific primers were designed using the GenScript real-time PCR primer design online program to yield similarly sized products for bpsA (see Table S1 in the supplemental material), bpsD, fhaB, and flaA (57). Real-time RT-PCR was then performed using the TaqMan system (Roche). Reactions were performed according to previously published protocols (57). Calculations for comparison between samples were performed using the ⌬⌬CT (where CT is threshold cycle) method, with recA and rpoD being used as the standardization controls, as described previously (57).

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Primer extension. Primer extension was performed as previously described with a few modifications (21). Total RNA was reverse transcribed at 52°C using Thermoscript RNase-H reverse transcriptase (Invitrogen). The primer Bps PE1 (see Table S1 in the supplemental material) was used to map the transcription start site of the bpsA-D promoter. The previously published primer Bvg P1 was used as a positive control to confirm the integrity of the RNA and the reaction (52). The product of the primer extension reaction was run in parallel on a urea-polyacrylamide gel next to a DNA sequencing ladder generated by a Sequenase, version 2.0, DNA sequencing kit (USB) and the Bps PE1 primer. A region of the bpsA-D locus spanning ⫺402 to ⫹108 bp with respect to the translational start site of bpsA was cloned into pcDNA 3.1, and the resulting plasmid was used as the template for DNA sequencing. Rapid amplification of cDNA ends (RACE)-PCR. RNA harvested from log-phase RB50 cells was reverse transcribed as described above. Instead of random primers, the primer bps GSP-1 (see Table S1 in the supplemental material), which hybridizes to a region of the bpsA ORF, was used to direct the synthesis of first-strand cDNA transcript. One microliter of the cDNA product was used in a PCR using an abridged anchor primer and primer bps GSP-2 (see Table S1). The rest of the protocol was essentially the same as described earlier (21, 35). A 1-␮l sample from this reaction product was used for a secondary PCR using an abridged universal primer and the primer bps GSP-3 (see Table S1). This resulted in a product with XhoI and SpeI restriction sites which was then cloned into the XhoI and SpeI sites of pBluescript KS, and several clones were sequenced. Purification of BpsR. The entire bpsR gene was amplified from the RB50 chromosome using primers BpsR1 and BpsR2 (see Table S1), resulting in a 486-bp fragment with flanking BamHI and XhoI restriction enzyme sites. After enzymatic digestion, the PCR product was cloned into BamHI and XhoI sites of pET24a (Novagen), resulting in the BpsR overexpression plasmid pMC1. This cloning strategy will result in the production of BpsR with a T7 affinity tag at the N terminus. pMC1 was transformed into BL21(DE3)/pLysE E. coli cells for overexpression. Cells were grown in 2⫻ TY medium (16 g of Bacto tryptone [Difco], 10 g of yeast extract, 5 g/liter NaCl) containing 0.4% glucose in the presence of kanamycin and chloramphenicol at 37°C. Upon growth to an OD600 of 1.0, the cells were induced with 1 mM isopropyl-␤-D-thiogalactopyranoside (IPTG) for 2 h. The cells were then harvested via centrifugation and resuspended in 30 ml of TGED buffer (10 mM Tris-HCl [pH 7.9], 0.1 mM EDTA [pH 8.0], 0.2 mM DTT, 0.05% sodium deoxycholate, 5% glycerol, and 2 mM phenylmethylsulfonyl fluoride [PMSF]). After a flash freezethaw to aid in cell lysis, the bacterial suspension was lysed by passage through a French pressure cell three times at 14,000 to 16,000 lb/in2. The lysate was centrifuged, the clear supernatant was passed through a T7 tag affinity purification kit (Novagen), and BpsR was purified according to the manufacturer’s instructions. Electrophoretic mobility shift assays (EMSAs). A 205-bp fragment encompassing the bpsA-D promoter was generated by PCR using the primers bpsprom5 and bpsprom3 (see Table S1 in the supplemental material). This product was then end labeled by using T4 polynucleotide kinase (New England BioLabs) and [␥-32P]ATP (Amersham Biosciences). Excess unincorporated radioactivity was removed by passing the sample through a G-50 quick-spin column (GE Healthcare). Reactions were set up in a 20-␮l reaction mixture containing the radiolabeled promoter, purified BpsR protein, and 1⫻ binding buffer [10 mM Tris-HCl (pH 7.8), 2 mM MgCl2 50 mM NaCl, 1 mM dithiothreitol, 0.5 ␮g of poly(dI-dC), 0.01% NP-40, 100 ng of bovine serum albumin, 10% glycerol]. This mixture was then incubated at 37°C for 15 min to allow for the protein to bind to the DNA. The samples were then electrophoresed on a 5% polyacrylamide gel and visualized by autoradiography as previously described (20, 40). Continuous flow confocal microscopy. Three-chambered sterile flow cell systems were purchased from Stovall. A 500-␮l suspension of either the wild-type or the ⌬bpsR strain, containing the pTacGFP plasmid grown

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FIG 1 bpsA-D locus is transcribed as an operon. (A) Location of the bps locus on the B. bronchiseptica chromosome with respect to the genes in the vicinity. The genes of the bps locus and the ORFs in the opposite orientation are depicted by the open arrowheads. The number within each ORF represents the size of the encoded protein product (in amino acids), and the number below is the intergenic distance between the two ORFs. The small line spanning the bpsC and bpsD ORFs indicates that these two ORFs overlap. Primers used for RT-PCR shown in panel B are represented with black arrows. (B) RT-PCR was performed using sense and antisense primers (indicated by black arrows in A) to amplify across the junctions of the different genes. For each primer pair three reaction mixtures were loaded on the agarose gel. cDNA, RT-PCR mixture; gDNA, purified RB50 genomic DNA as the PCR template; and mock, no RT control mixture; L, 1-kb DNA ladder.

to an OD600 of 0.1, was inoculated into the chambers using sterile 25-5/ 8-gauge needles and allowed to adhere for 2 h. After attachment, the chamber was inverted, and medium flow (supplemented LB medium containing 50 ␮g/ml CM) was initiated at a rate of 0.5 ml/min. Biofilm formation was observed every 24 h using a Zeiss LSM 510 confocal scanning laser microscope as described previously (48). Biofilm features such as average and maximal thickness were examined using the COMSTAT software package (28). Predicted secondary structure analysis of BpsR. Sequence comparisons of the BpsR and MarR proteins and promoter alignments were performed using ClustalW2, available online through EMBL-EBI (10). The putative secondary structure of BpsR was constructed using the SCRATCH SSpro8 software available through the University of California, Irvine (9). Statistical analysis. All statistics were performed using a Student’s t test and were determined to be significant at a P value of ⬍0.05.

RESULTS

The bpsA-D locus is transcribed as an operon. The four genes of the bpsA-D locus, bpsA, bpsB, bpsC, and bpsD, are found together in the Bordetella genome orientated in the same direction (Fig. 1A) and appear, by homology, to carry out the functions of polysaccharide synthesis, assembly, and transport (31, 48). These genes are also tightly linked. There are only 6 and 4 nucleotides (nt) separating bpsA from bpsB and bpsB from bpsC ORFs, respectively, whereas the bpsC and bpsD ORFs overlap by 19 bases (Fig. 1A). This analysis led us to hypothesize that these genes constitute

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FIG 2 Analysis of bpsA and bpsD expression levels by real-time RT-PCR. (A) bpsA is expressed differentially in biofilms. RNA was harvested from 48-h biofilms and planktonic cells grown under static conditions. (B) bpsA is regulated in a Bvg-independent manner. RNA was harvested from log phase cultures of RB50 grown under Bvg⫹ phase conditions or from RB54, the Bvg⫺ phase-locked mutant. Specific primers, bpsA rtf and bpsA rtr (see Table S1 in the supplemental material), were used to compare bpsA expression. (C and D) BpsR negatively regulates the expression of bpsA and bpsD. Real-time RT-PCR was performed with RNA harvested either from RB50 or the ⌬bpsR strain. Fold change of bpsA and bpsD between the two strains is represented. The asterisk indicates a P value of ⬍0.05 (Student’s t test). The fold change was determined using the ⌬⌬CT as described previously (57).

an operon and are cotranscribed. To confirm this, RT-PCR was carried out on RNA extracted from exponential phase cultures of the wild-type strain, RB50. A PCR product of the expected size (bpsA-bpsB, 628 bp; bpsB-bpsC, 547 bp; bpsC-bpsD, 581 bp) was obtained for all of the overlapping regions tested (Fig. 1B). These results strongly suggested that all four genes are cotranscribed on the same mRNA transcript. The bpsA-D locus is expressed at higher levels during biofilm growth. We have previously shown that bpsA-D is not required for the early steps of biofilm formation, in particular, the initial attachment to abiotic surfaces (14, 48). Instead, Bps is required at a step postattachment and contributes to the formation of the complex architecture of Bordetella biofilms (14, 48). This led us to hypothesize that there will be elevated expression of the bpsA-D locus in biofilms. In order to investigate differential expression of bps, biofilms of RB50 were grown in SS medium in petri plates (Materials and Methods). Harvested RNA was used in real-time RT-PCR to compare the levels of the bpsA transcripts between the biofilm and the planktonic populations. The constitutively expressed genes, recA or rpoD, were used as an internal control. It is also important to note that neither of these genes is differently expressed in the biofilm state (data not shown). Our results demonstrate ⬇4-fold increase in the expression of bpsA in biofilm cells compared to that in planktonic cells (Fig. 2A). Similar results were obtained by using rpoD as another internal control (data not shown). The bpsA-D operon is regulated in a Bvg-independent manner. Previous reports by us and others have demonstrated that

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biofilm formation in Bordetella species is positively controlled by the BvgAS locus (29, 41). Since Bps is essential for Bordetella biofilm development, we investigated whether bpsA-D is also subject to control by BvgAS. RNA was harvested from log phase cultures of RB50 and the Bvg⫺ phase-locked strain RB54 grown in SS medium without the addition of modulators (Bvg⫹ phase conditions). When BvgAS is active, Bordetella bacteria are in the Bvg⫹ phase. Inactivation of BvgAS by chemical modulation or by mutation results in the transition to the Bvg⫺ phase. The Bvg⫺ phase-locked strain contains a deletion of the gene encoding the BvgS sensor kinase, resulting in a strain that is locked in the Bvg⫺ phase (15). Our results showed only minor changes in the levels of the bpsA transcript between the wild-type and the Bvg⫺ phaselocked strain (Fig. 2B). Although there were slightly higher levels of bpsA transcript in the wild-type strain than in the Bvg⫺ phaselocked strain, this difference was not statistically significant (P ⫽ 0.404). As a control, we measured the expression profiles of the known Bvg-regulated genes, fhaB and flaA. As expected and consistent with previous results (1, 21, 51, 57), fhaB was expressed at very high levels in the wild-type strain, whereas flaA was maximally expressed in the Bvg⫺ phase-locked strain (data not shown). These results corroborate the validity of the real-time RT-PCR assays for accurately measuring Bvg-regulated gene expression patterns. Taken together, this experiment suggests that the bpsA-D operon is expressed in a predominantly Bvg-independent manner when Bordetella bacteria are grown under planktonic growth conditions. Identification of the MarR-like regulator BpsR. The region

Journal of Bacteriology

BpsR Modulates Bordetella Biofilm Formation

upstream of the bpsA-D operon contains a gene (BB1771) encoding a MarR family protein that we predicted may regulate Bps synthesis (Fig. 1). The putative protein product of BB1771 contains a region spanned by amino acids 45 to 138, which shows similarity to the highly conserved winged helix-turn-helix (wHTH) DNA binding motif of MarR. Alignment of BB1771 and MarR revealed 27% identity and 42% similarity between the two proteins at the amino acid level (see Fig. S1 in the supplemental material). Secondary structure prediction software suggests similar alpha-helical structures between MarR and BpsR (see Fig. S1). Based on these similarities and additional experiments conducted below, BB1771 was named as bpsR, for Bordetella polysaccharide regulator. bpsR represses bpsA-D transcription and the production of the Bps polysaccharide. To investigate the role of bpsR in controlling the expression of the bpsA-D locus, we utilized allelic exchange to construct the ⌬bpsR strain that contains an in-frame deletion in the bpsR gene (48, 57). First, we examined the effect of the bpsR deletion on the transcription of the bpsA-D locus. RNA was harvested from the wild-type and ⌬bpsR strains grown to mid-log phase and reverse transcribed; the levels of the bpsA and bpsD transcripts were measured by real-time RT-PCR utilizing recA as the internal reference control. Both bpsA and bpsD were expressed at higher levels in the ⌬bpsR strain than in the wild-type strain (Fig. 2C and D). Next, we addressed the effect of the bpsR deletion on the production of the Bps polysaccharide. To aid in this analysis, we utilized human serum from a pertussis-positive patient. Previously, we have shown that pertussis-positive patients harbor anti-Bps antibodies (14). The human serum was repeatedly absorbed against the ⌬bpsA-D strain which lacks the ability to produce Bps (Materials and Methods) (48). The specificity of this enriched anti-Bps serum was first determined by immunoblotting the polysaccharide extracts from the wild-type and the ⌬bpsA-D strains. As shown in Fig. 3A, immunoblotting revealed that the anti-Bps serum is highly specific since it did not react with extracts from the ⌬bpsA-D strain. Moreover, immunoblotting with approximately 10-fold larger amounts of the polysaccharide extract from the ⌬bpsA-D strain did not result in significant reactivity (data not shown). Bps was detected at higher levels in the ⌬bpsR strain that in the wild-type strain. Complementation of the ⌬bpsR strain with a plasmid containing the bpsR gene (⌬bpsRcomp) resulted in a substantial decrease in the levels of the reactive material. In contrast, the ⌬bpsRvec strain carrying the vector plasmid alone showed similar levels of Bps as those seen in the ⌬bpsR strain (Fig. 3A). As an additional means to confirm the increase in polysaccharide expression, we utilized an ELISA-based assay. These assays revealed that the ⌬bpsR strain produced 5- to 6-fold larger amounts of Bps than the wild-type strain (Fig. 3B). As expected, complementation of the bpsR gene in the ⌬bpsRcomp strain resulted in reduction in Bps levels compared to that observed in the ⌬bpsRvec strain which contains the vector plasmid only (Fig. 3B). While the levels of Bps in the ⌬bpsRcomp strain were reduced compared to levels in the ⌬bpsRvec strain, these levels were still higher than those observed in the wild-type strain. Since BpsR most probably exerts its repressive functions on the bpsA-D promoter in the vicinity of its own gene, it is likely that BpsR provided in trans may not be able to fully compensate for the effects observed

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FIG 3 Role of bpsR in Bps production. (A) Boiled EDTA surface extracts from

equal numbers (⬇5 ⫻ 109) of cells were treated with pronase, followed by extraction with phenol-chloroform. Five microliters of the aqueous fractions was spotted on a nitrocellulose membrane and probed with human serum enriched for Bps and detected using goat anti-human IgG conjugated to horseradish peroxidase. (B) EDTA extracts were used in an ELISA-based assay to measure Bps production. The absorbed human serum was used to probe for Bps, followed by detection using goat anti-human IgG conjugated to horseradish peroxidase. Error bars are representative of the standard deviation. The asterisk indicates a P value of ⬍0.05 (Student t test). Experiments were repeated in triplicate.

from the chromosomally encoded copy that would be transcribed and translated in the vicinity of the region to which it binds. Taken together, these results strongly suggest that the bpsR gene functions to repress the expression of the bpsA-D locus and Bps synthesis. bpsR is expressed at similar levels during biofilm and planktonic growth. Based on the increased expression of bpsA in biofilms and the observed negative regulation of bpsA and bpsD by BpsR, we hypothesized that bpsR will be expressed at lower levels during biofilm growth. To estimate the abundance of bpsR mRNA during the two modes of growth, we utilized real-time RT-PCR assays. Surprisingly, we found that the levels of bpsR did not vary significantly under biofilm and planktonic growth conditions (see Fig. S2 in the supplemental material). Deletion of bpsR leads to an increase in biofilm formation. We next hypothesized that the observed increase in the levels of Bps polysaccharide in the ⌬bpsR mutant will result in an enhancement of biofilm formation by this strain. To quantitatively assess the role of BpsR in biofilm development, equal numbers of the green fluorescent protein (GFP)-tagged wild-type and the ⌬bpsR strains were inoculated in chambered flow cells. The biofilms formed on the glass coverslips were observed over a period of 96 h by confocal microscopy (48). Differences in biofilm architecture were quantitated by COMSTAT software by analyzing the average and maximal thicknesses of the biofilms (28). After 24 h of growth, the biofilms formed by the wild-type and the ⌬bpsR strains dis-

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TABLE 1 COMSTAT biofilm analysis Biofilm thickness (␮m) Time point (h)

Strain

Avg (SD)

Maximum (SD)

24

RB50 ⌬bpsR strain RB50 ⌬bpsR strain RB50 ⌬bpsR strain RB50 ⌬bpsR strain

5.55 (0.925) 6.55 (0.684) 10.807 (1.415) 16.059 (3.48) 13.315 (1.33) 29.93 (5.175) 15.17 (1.77) 28.72 (3.44)

14.67 (2.08) 17 (4) 23.16 (3.523) 41.977 (4.825) 35.383 (2.948) 57.9 (7.589) 36.67 (5.45) 65.30 (5.91)

48 72 96

played similar attachment patterns and existed as uniform monolayers, with only a modest, but not significant, enhancement of biofilm formation by the ⌬bpsR strain (Table 1 and data not shown). At 48 h, the ⌬bpsR strain developed robust biofilms with taller microcolonies and greater thickness than biofilms formed by the wild-type strain. The differences in the architecture of the biofilms formed by the ⌬bpsR strain were even more pronounced at the later time points of 72 and 96 h. At these time points, the ⌬bpsR strain formed biofilms that were considerably thicker, denser, and more structured than those formed by the wild-type strain (Fig. 4 and Table 1). These results strongly suggest that, by regulating the production of the Bps polysaccharide, BpsR plays a key role in the development of the mature three-dimensional architecture of Bordetella biofilms. Identification of the bpsA-D transcription initiation site. To continue our examination of the regulation of bpsA-D gene cluster

FIG 5 Nucleotide sequence of the bps promoter. The transcriptional start site

(bold type and with a ⫹1) and the putative ⫺10 and ⫺35 promoter elements are indicated. Putative inverted (solid arrows), complementary inverted (dashed arrows), and mirror-like repeats (dotted arrows) are shown. The half sites for each of these repeats are indicated by solid-line boxes. The multiply repeated G/C(A/T)4G/C sequence is indicated by dashed-line boxes. Nonconserved nucleotides between B. bronchiseptica, B. parapertussis, and B. pertussis are underlined.

by bpsR, we first mapped the transcriptional start site of the bpsA-D promoter by 5= RACE (21). RACE-PCR products specific to bpsA were cloned and sequenced. Sequencing of two of the five clones revealed the transcription site to be an A nucleotide located 67 bp upstream of the bpsA ORF (Fig. 5). The other three clones resulted in shorter products, with transcription sites corresponding to the A, G, and T nucleotides 13, 23, and 27 bp, respectively, upstream of the bpsA ORF. To resolve the observed discrepancy in the 5= RACE results, primer extension analyses were performed. These experiments yielded a 164-bp fragment, and the transcriptional start site was found to correspond to the A nucleotide located 67 bp upstream of the bpsA ORF, identified in two of the five RACE PCR clones (Fig. 6). As a positive control, primer extension analysis was per-

FIG 4 bpsR regulates biofilm formation in B. bronchiseptica. Confocal scanning laser microscopy of biofilms formed in flow cells by RB50 or the ⌬bpsR strain. Strains were inoculated directly in the flow cell and visualized in situ throughout the time course. For each micrograph, the middle panel represents the x-y plane, and the adjacent top and side panels represent the x-z and y-z planes, respectively. For each strain, images were taken from at least eight areas, and the experiment was repeated three times. A representative image is shown for each sample.

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FIG 7 Electrophoretic mobility shift assays. (A) Radiolabeled bps promoter was incubated with increasing concentrations of purified BpsR. (B) Specific competitive EMSAs were performed by adding the indicated molar excess of a 205-nt fragment of the bps promoter. (C) Nonspecific competitive EMSAs were performed using the indicated molar excess of a 628-bp fragment internal to the bps locus, (bpsAB fragment from the experiment shown in Fig. 1). BpsR was used at a concentration of 1.4 ␮M in the experiments shown in panels B and C.

FIG 6 Mapping of the bps promoter. Primer extension analysis was performed with RNA prepared from RB50 using the bpsA-specific primer Bps PE1 or the bvgAS-specific primer Bvg P1. The arrow indicates a band corresponding to the Bps PE1 primer extension product. A nucleotide sequencing ladder is denoted by the G, A, T, and C lanes.

formed for the bvgAS operon using the previously published Bvg P1 primer (52). In accordance with previously published results, a primer extension product corresponding to the bvg P1 promoter was observed (Fig. 6). Scanning of the DNA sequences upstream of the ORF identified a hexameric sequence, TAAGAT, centered at ⫺10 relative to the A nucleotide and resembling the consensus ⫺10 element for E. coli ␴70 (43) (Fig. 5). A canonical ⫺35 sequence was not apparent upstream of the A67 nucleotide. A similar lack of a recognizable consensus ⫺35 element has been reported for the Bordetella fimbrial subunit promoters and the bipA promoter (8, 21). We were unable to identify hexameric sequences resembling the canonical ␴70 promoter sequences upstream of the other three possible transcription start sites. Although we cannot completely exclude the presence of other transcription initiation sites, based on a combination of results so far, we conclude that the majority of bpsA-D transcription initiates from the A67 nucleotide, and we designate this as ⫹1. BpsR represses bpsA-D transcription by directly binding to the bpsA-D promoter. The presence of the wHTH DNA binding motif in BpsR strongly suggested to us that BpsR will repress bpsA-D transcription by directly binding to the bpsA-D promoter. To confirm this hypothesis, N-terminal T7-tagged BpsR was overexpressed and purified to near homogeneity from E. coli (Materials and Methods). A radiolabeled 205-bp fragment encompassing the bpsA-D promoter regions from ⫺111 to ⫹94 was combined with purified BpsR and electrophoretic mobility shift assays (EMSA) were conducted. Addition of increasing concentrations

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of BpsR resulted in a considerable retardation in the migration of the promoter DNA through the gel, demonstrating that BpsR is capable of binding to the bpsA-D promoter (Fig. 7A). To investigate the specificity of BpsR binding to the bpsA-D promoter, EMSAs were performed with increasing concentrations of nonradioactive specific competitor (the original bpsA-D promoter DNA fragment used above) and nonspecific competitor (a 628-bp bpsAB fragment corresponding to positions ⫹1721 to ⫹2349 of the bpsA-D locus) DNA fragments. The binding of BpsR to the radiolabeled promoter DNA was lost in the presence of the nonradiolabeled specific competitor (Fig. 7B), whereas incubation with concentrations of the nonspecific competitor as high as 100⫻ of the radiolabeled promoter DNA did not result in a significant change in the DNA binding activity of BpsR (Fig. 7C). In combination, these results demonstrate that BpsR binding to the bpsA-D promoter region is DNA sequence specific. Structure of the bpsA-D promoter. Many transcription factors exert their regulatory functions by binding to repeated DNA elements in promoter sequences. Visual scanning of the bpsA-D promoter revealed an extensive series of palindromic sequences in the form of inverted, complementary inverted, mirror-like, and direct repeats. Spanning the region from ⫺10 to ⫹8 lies an inverted repeat element, GATGG (Fig. 5, solid arrows), separated by 8 nucleotides, whereas the complementary inverted repeat (dashed arrows) is separated by 5 nucleotides and spans regions ⫺65 to ⫺79 of the bpsA-D promoter. Spanning regions ⫺91 to ⫺75 and ⫺69 to ⫺51 lie two mirror-like repeats (dotted arrows) which are separated by 9 and 7 nucleotides, respectively. Moreover, a hexameric sequence (broken squares) containing an ATrich stretch flanked by a G or C was repeated six times in the promoter region of bpsA-D. The BpsR protein and nucleotide sequences of the bpsA-D promoter are conserved in the classical Bordetella spp. It has now been clearly established that B. pertussis and Bordetella parapertussis have evolved by genome decay of a B. bronchiseptica ancestor (49). In B. bronchiseptica, the bpsR ORF is located 792 nt upstream of the bpsA coding region. In between bpsR and bpsA lies ORF BB1770, 209 nt upstream of the bpsA coding region, which is annotated as a hypothetical protein. Despite large variations introduced in Bordetella genomes due to loss of a large number of genes, all three species have maintained these genes. Additionally, the genetic arrangement of the bps locus relative to that of the bpsR

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gene and the intergenic distance between the different ORFS are highly similar in the human-adapted species. Multispecies promoter alignment can also be a valuable tool to determine if the function of a particular transcription factor and the cis-acting regulatory elements are conserved across species (58). We carried out an alignment of the bps promoter sequences 11 nt upstream and 94 nt downstream of the annotated translational ATG start site. We found that there were only three nucleotide mismatches in the bps promoter regions of B. bronchiseptica, B. parapertussis, and B. pertussis (Fig. 5). None of these alterations was found to be located in any of the identified DNA repeat elements. We also carried out a sequence alignment of the B. bronchiseptica BpsR protein with that of its homologs from B. pertussis and B. parapertussis. Although the members of the bacterial MarR family display significant diversity at the amino acid level, the BpsR homologs from the three Bordetella species exhibit ⬎99% sequence identity. Taken together, results from these analyses are consistent with an important function of BpsR in the human pathogens. DISCUSSION

Experimental studies and genome sequencing of strains from multiple Bordetella species have revealed that these bacteria encode a repertoire of virulence factors which includes surfaceassociated, outer membrane, and secreted factors (39, 49). A large number of ORFs have also been annotated to encode proteins with predicted functions in gene regulation (49). Thus, it is reasonable to hypothesize that, similar to other bacterial pathogens, Bordetella will also exert coordinated and tight control over the expression of its virulence factors by utilizing a network of regulatory proteins and signaling pathways. However, very few regulatory proteins and signaling systems have been examined in detail in Bordetella. Probably the most well studied are the BvgAS twocomponent system, the regulatory control of the iron responsive proteins, and, to a lesser extent, the RisAS two-component system (5, 17, 18, 32, 45, 56). In order to gain a detailed understanding of Bordetella pathogenesis, it is critical to identify new transcriptional regulators and investigate the mechanisms by which these proteins control virulence factor expression. In this article, we examined the transcriptional control of the four-gene bpsA-D operon, which is involved in the production of the Bps polysaccharide. Bps is essential for colonization and biofilm development in the mouse respiratory tract (14, 48). We have demonstrated that a previously uncharacterized ORF, BB1771, currently annotated as transcriptional regulator of the MarR family, encodes a protein, BpsR. We show that bpsR negatively regulated bpsA-D transcription and the synthesis of the Bps polysaccharide. We have further elucidated the mechanism of BpsR-mediated repression of the bpsA-D locus by demonstrating that recombinant purified BpsR protein is capable of binding to the bpsA-D promoter in a sequence-specific manner. BpsR possesses a motif that displays high conservation with DNA binding domains of MarR family proteins. For some MarR proteins, this recognition motif adopts a winged helix-turn-helix (wHTH) fold (2, 7, 37). Based on this similarity and DNA binding assays conducted herein, we propose that BpsR blocks bpsA-D transcription by directly binding to the promoter, and the regions corresponding to the wHTH motif will be required for DNA binding. Consistent with this hypothesis, visual analysis of the promoter region revealed inverted, complementary inverted, mirror-

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like, and direct repeats interspersed throughout the bpsA-D promoter. The presence of multiple potential regulatory sites on the bpsA-D promoter also suggests that the expression of bpsA-D may also be controlled by regulatory proteins other than BpsR. In particular, BB1770 is predicted to encode a small hypothetical protein. Given the conservation of BB1770 in B. pertussis and B. parapertussis and its location in the vicinity of bpsA-D and bpsR, it is possible that this ORF may play a regulatory role in controlling bpsA-D or bpsR expression. Deletion of bpsR resulted in an enhancement in the biofilmforming capacity of the mutant strain. Given that BpsR represses the synthesis of the Bps polysaccharide, it is likely that increased amounts of Bps in the bpsR mutant results in higher biofilm formation. Interestingly, while there was increased expression of bps mRNA in biofilms, the levels of bpsR were not significantly different between biofilm and planktonic growth conditions. Thus, it appears that the regulation of the bpsA-D biosynthetic gene cluster by bpsR during biofilm growth is quite complex and cannot be simply explained by differential levels of bpsR influencing bpsA-D transcription. Multiple MarR-like regulators bind diverse effector molecules and in many cases binding of these ligands attenuates the DNA binding activity and function of these proteins (64). Interaction with a small-molecule ligand produced during biofilm growth could result in inhibition of the DNA binding activity and the repressive functions of BpsR thereby resulting in increased expression the Bps polysaccharide. It is also likely that observed expression changes of bpsA-D locus in biofilms may not be due to alterations in BpsR activity and are mediated by a different regulator. We have shown here that the expression of the bpsA-D locus is regulated in a predominantly Bvg-independent manner. A previous study has shown that the carbohydrate component of B. bronchiseptica biofilm matrix is regulated independent of BvgAS and is composed mainly of xylose (30). No genetic loci that may encode this polysaccharide have been identified. While at present it is not possible to unequivocally conclude that Bps and the xylosecontaining polysaccharides are different, our previous studies based on immunoreactivity and enzymatic susceptibility assays suggest that Bps is more similar to the poly-␤-1,6-N-GlcNAc family of polysaccharides (14, 48). We are in the process of determining the precise chemical composition of Bps. Irrespective of the potential outcomes, it is clear that control of biofilm development in Bordetella is complex and occurs by a combination of Bvg-regulated protein factors and BpsR-dependent but Bvgindependent control of polysaccharides. We have previously shown that despite a massive reduction in the genomes of the human-adapted strains of B. pertussis and B. parapertussis, the orthologues of the genes of the bpsA-D locus and the production of the Bps polysaccharide have been maintained (14, 48). This, combined with very high levels of nucleotide sequence identity in the bpsA-D promoter regions, the similar arrangement of the bpsR ORF with respect to the bpsA-D locus, and the highly homologous amino acid sequences of the BpsR proteins among these three species, suggests the preservation of a common bacterial strategy to regulate virulence and biofilm development. By analyzing the genomic content of a large collection of B. pertussis strains isolated over the last 60 years from six different countries, a core genome for B. pertussis has been derived (4, 33). In addition, a group of 589 variable genes that were either absent or divergent in one or more of these strains were also iden-

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tified (33). None of the genes of the bpsA-D locus, the intervening gene, BP1945, or the bpsR gene was found to be missing or divergent in these strains, strongly suggesting that these genes are part of the B. pertussis pan-genome. This striking conservation across different species also serves as a starting point for a more thorough investigation of the functions of BpsR in the human-adapted and other Bordetella species. Future studies of the regulatory mechanisms of BpsR will greatly enhance the understanding of Bordetella virulence. ACKNOWLEDGMENTS Research in the laboratory of R.D. is supported by funds from the NIH (grant number 1R01AI075081) and National Research Initiative (grant number 2006-35604-16874) from the USDA National Institute of Food and Agriculture, Microbial Functional Genomics Program. M.C. is supported by an NIH predoctoral training grant (T32 AI07401).

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