Molecular cloning of haemoglobin-binding protein HgbA in the outer membrane of Actinobacillus pleuropneumoniae

Ramakrishnan Srikumar1, Leonie G. Mikael2, Peter D. Pawelek1, Ali Khamessan1, Bernard F. Gibbs3, Mario Jacques2,{dagger} and James W. Coulton1,{dagger}

1 Department of Microbiology and Immunology, McGill University, 3775 University Street, Montreal, QC, Canada H3A 2B4
2 Groupe de Recherche sur les Maladies Infectieuses du Porc, Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, Canada J2S 7C6
3 MDS Pharma Services, St Laurent, QC, Canada H4R 2N6

Correspondence
James W. Coulton
james.coulton{at}mcgill.ca


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
From the porcine pathogen Actinobacillus pleuropneumoniae cultivated in iron-deficient or haem-deficient media, haemoglobin (Hb)-agarose affinity purification was exploited to isolate an outer-membrane protein of ~105 kDa, designated HgbA. Internal peptide sequences of purified HgbA were used to design oligonucleotide primers for PCR amplification, yielding amplicons that showed partial sequences with homology to hgbA of Pasteurella multocida. Upon screening two genomic libraries of A. pleuropneumoniae serotype 1 strain 4074, positive clones were assembled into an ORF of 2838 bp. HgbA (946 aa) includes a signal peptide of 23 aa and the deduced HgbA sequence (104 890 Da) also demonstrated a possible Ton box. The promoter region of hgbA from A. pleuropneumoniae serotype 1 showed consensus for –35 and –10 sequences and a putative Fur-binding site. RT-PCR confirmed that hgbA of A. pleuropneumoniae is upregulated in response to diminished levels of iron in the culture medium. While an internally deleted hgbA mutant was unable to use pig Hb as sole source of iron for growth, flow cytometry confirmed its Hb binding; the internally deleted sequences may not be required for Hb binding, but appear necessary for the iron supply from Hb. HgbA is required for growth of A. pleuropneumoniae in the presence of Hb as sole iron source.


Abbreviations: EDDHA, ethylenediaminedihydroxyphenylacetic acid; Hb, haemoglobin; OM, outer membrane

{dagger}Mario Jacques and James W. Coulton are affiliated to the Canadian Research Network on Bacterial Pathogens of Swine.

The GenBank accession number for the sequence of hgbA from Actinobacillus pleuropneumoniae reported in this paper is AF468020.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Iron, an essential element for many metabolic pathways of bacterial pathogens, plays a critical role in establishing infections (Byers & Arceneaux, 1998; Litwin & Calderwood, 1993). Availability of free iron in vivo is limited by its low solubility at physiological pH as well as by iron-binding proteins that scavenge iron (Otto et al., 1992). In the mammalian host, extracellular iron is bound to iron-binding glycoproteins lactoferrin and transferrin, whereas most intracellular iron is sequestered as haem-containing proteins, including haemoglobin (Hb) (Lee, 1995). To combat inhospitable conditions of iron starvation, bacterial pathogens have evolved different strategies of high affinity iron acquisition: siderophores chelate external iron and bind to cognate outer-membrane (OM) proteins, including FepA (Buchanan et al., 1999) and FhuA (Ferguson et al., 1998; Locher et al., 1998). Following lysis of erythrocytes, Hb may serve as a source of haem (Palmer et al., 1994). Haem-containing iron transport proteins from Haemophilus influenzae (Maciver et al., 1996; Morton et al., 1999), Haemophilus ducreyi (Elkins et al., 1995) and Neisseria meningitidis (Stojiljkovic et al., 1996) utilize a TonB-dependent OM receptor that is highly adapted to specific haem-containing protein sources, including Hb. Other bacterial pathogens assimilate iron from protein-bound or haem-associated iron that circulates in fluids or locates on surfaces of the mammalian host (Wandersman & Stojiljkovic, 2000; Williams & Griffiths, 1992). Mucosal pathogens are able to extract haem by several haem- or haem-haemopexin-binding proteins (Genco & Dixon, 2001; Hanson & Hansen, 1991; Lee, 1992).

Actinobacillus pleuropneumoniae, a member of the Pasteurellaceae, is the aetiological agent of porcine pleuropneumonia, a severe disease that causes worldwide economic loss to the swine industry (Fenwick & Henry, 1994). Different factors contribute to the virulence of A. pleuropneumoniae and a comprehensive review on its pathogenicity was published by Bossé et al. (2002). Potential iron sources for A. pleuropneumoniae include porcine transferrin (Niven et al., 1989) for which A. pleuropneumoniae possesses specific receptors TbpA and TbpB (Gerlach et al., 1992; Gonzalez et al., 1995), and exogenous siderophores (Diarra et al., 1996), including ferrichrome for which A. pleuropneumoniae has a specific receptor, FhuA (Mikael et al., 2002). While both transferrin-binding proteins of A. pleuropneumoniae are upregulated in response to iron restriction (Gerlach et al., 1992; Gonzalez et al., 1995), the fhuA gene is regulated differently because it does not respond to iron restriction (Mikael et al., 2003). A. pleuropneumoniae is also capable of using porcine Hb and haem compounds as sources of iron for growth (Bélanger et al., 1995; Deneer & Potter, 1989; D'Silva et al., 1995). We therefore hypothesized that A. pleuropneumoniae has a specific OM protein that binds Hb and transports haem-associated iron into the cell. In preliminary experiments, OM proteins from A. pleuropneumoniae with both haemin- and Hb-binding activity were detected (Archambault et al., 2003).

Exploiting Hb-agarose affinity interactions, we purified a protein (~105 kDa, henceforth designated HgbA) from detergent-solubilized OM vesicles of A. pleuropneumoniae. Peptide sequences from HgbA were used to design oligonucleotides and to screen genomic libraries of A. pleuropneumoniae, leading to the complete 2838 bp ORF of hgbA. By using RT-PCR, the role of iron restriction on the expression and the transcript level of hgbA was investigated. An internally deleted hgbA mutant of A. pleuropneumoniae was created by a double-step transconjugation system; the mutant was still able to bind Hb as shown by flow cytometry, but was no longer able to use Hb as a source of iron.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and culture conditions.
Reference strains from serotypes 1–12 of A. pleuropneumoniae (Table 1) were maintained on brain heart infusion agar (BHI; Difco) supplemented with 10 µg {beta}-NAD ml–1. Addition of deferrated ethylenediaminedihydroxyphenylacetic acid (EDDHA; Sigma) to BHI medium produced an iron-deficient growth medium. Haem-sufficient or haem-deficient conditions differed by the addition of pig Hb (Sigma) at 20 or 1·0 µg ml–1 to BHI plates deferrated with 150 µM EDDHA. Chloramphenicol (0·5 µg ml–1) was used for the selection and maintenance of plasmid-encoded resistance in A. pleuropneumoniae. Escherichia coli strains were routinely grown on Luria–Bertani (LB) medium (Difco); growth and maintenance of E. coli strain {beta}2155 required 1 mM diaminopimelic acid (Sigma). Antibiotics used for selection and maintenance of plasmid-encoded resistance markers in E. coli were 50 µg kanamycin ml–1, 150 µg ampicillin ml–1 and 30 µg chloramphenicol ml–1.


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Table 1. Strains and plasmids used in this study

 
Isolation of cell envelopes and OM proteins.
Bacterial cultures grown to an OD600 of 1·0 were harvested by centrifugation and stored at –20 °C. After thawing on ice, cells were suspended in PBS (1·9 mM NaH2PO4, 8·0 mM Na2HPO4, 150 mM NaCl, pH 7·4) and sonicated until the cell suspension cleared. Following centrifugation (8000 g, 10 min, ambient temperature) to pellet unlysed cells and debris, the supernatant was centrifuged (100 000 g, 30 min, 4 °C) and the pellet of cell envelopes was resuspended in water. To extract cytoplasmic membrane proteins, cell envelopes were diluted 100-fold in Tris-Sarkosyl buffer [50 mM Tris/HCl, 150 mM NaCl, 1 % (w/v) N-lauryl-sarcosine, pH 7·5] and incubated for 1 h at room temperature with agitation. The Sarkosyl-insoluble fraction containing OM vesicles was recovered as a pellet by ultracentrifugation (100 000 g, 1 h, 4 °C), resuspended in a small volume of water and stored at –20 °C.

Affinity chromatography for purification of Hb-binding protein(s).
Proteins were solubilized from OM vesicles by 1 % (w/v) Zwittergent 3,14 (Calbiochem) in 50 mM Tris/HCl, 150 mM NaCl, 5 mM EDTA, pH 7·5, at 37 °C for 1 h. Centrifugation (12 000 g, 10 min, 4 °C) of this suspension yielded solubilized OM proteins. Following dialysis (2 h, 4 °C) against 50 mM Tris/HCl, pH 7·5, 150 mM NaCl, 0·5 % Zwittergent 3,14, OM proteins were mixed with Hb-agarose (Sigma). To remove non-specifically bound proteins, the protein-loaded gel was repeatedly washed with 50 mM Tris/HCl, pH 7·5, 1 M NaCl, 0·5 % Zwittergent 3,14, followed by boiling of the gel with its retained proteins in Laemmli sample buffer. Aliquots were loaded onto SDS-PAGE and the gels were either stained with Coomassie brilliant blue or transferred to a membrane for peptide sequencing.

N-terminal amino acid sequence determination.
The affinity-purified Hb-binding protein of A. pleuropneumoniae serotype 1, separated by SDS-PAGE, was electroblotted to a polyvinylidene difluoride (PVDF) microporous membrane (Immobilon-P; Millipore). A protein of 105 kDa, located on the membrane by Ponceau-S staining, was subjected to N-terminal amino acid sequencing (Sheldon Biotechnology Center, McGill University) by repetitive cycles of Edman degradation of the PVDF-bound protein followed by detection of the PTH-derived amino acid residue using an on-line C18 HPLC column with gas-phase/pulsed-liquid Procise Automated Sequence System (Applied Biosystems).

Trypsin digestion and peptide analysis.
The band on SDS-PAGE that corresponded to the 105 kDa protein was excised and digested with trypsin (Wilm et al., 1996). Gel particulates were dried by a Speed-Vac concentrator and then washed successively with water, acetonitrile and an ammonium bicarbonate/acetonitrile solution (50 : 50, v/v). Reduction was achieved by incubating gel particulates in a solution of 100 mM ammonium bicarbonate containing 10 mM dithiothreitol at 56 °C for 45 min; alkylation was in 100 mM ammonium bicarbonate containing 55 mM iodoacetamide. The gel particulates were dried completely and then incubated on ice with 0·1 % (w/v) trypsin in 100 mM ammonium bicarbonate for 45 min. Excess trypsin was decanted and gel particulates were incubated at 37 °C in 1 mM HCl for 12 h. Peptides were recovered by three serial extractions with 0·1 % trifluoroacetic acid (TFA), 0·1 % TFA/30 % (v/v) acetonitrile and 0·1 % TFA/60 % acetonitrile, separated by sonications of 30 min. Supernatants from all three steps were combined, freeze-dried and reconstituted in 20 µl 0·1 % TFA in 60 % methanol. The peptides were purified, concentrated and freed from detergent on ZipTip cartridges (Millipore) containing C18 resin, loaded onto a nanospray capillary (Protana) and infused into a QSTAR (MDS Sciex) mass spectrometer. Several double- and triple-charged peptides were selected for tandem mass spectrometry; peptides with triple charge were interpreted with Mascot (Matrix Science).

PCR amplification of hgbA fragments.
Based on amino acid sequences of N-terminal and internal peptides from the affinity-purified 105 kDa protein of A. pleuropneumoniae, oligonucleotide primers were designed for PCR amplification of genomic DNA. PCR products were sequenced and confirmed hgbA fragments were further used for screening the A. pleuropneumoniae genomic library. To establish the distribution of hgbA among the different serotypes, genomic DNA was isolated from reference strains representing serotypes 1–12 of A. pleuropneumoniae for use in PCR amplifications.

Southern blotting.
EcoRI-digested genomic DNA from A. pleuropneumoniae serotype 1 was separated on 0·8 % (w/v) agarose gels in TAE and transferred to a nylon membrane (Amersham Pharmacia Biotech) for DNA hybridization. Based on the N-terminal sequence (Table 2, P-1) and internal peptide sequence (Table 2, P-3) of HgbA, two degenerate oligonucleotides, 5'-GARGARGARATGGARTTRGA-3' and 5'-GATGARCTNTAYTTYACNTTYAA-3', were synthesized and 3'-end labelled with DIG using the DIG Oligonucleotide 3'-End Labelling Kit (Roche) for use as probes in Southern blotting experiments. DNA hybridization was detected with phosphatase-labelled anti-DIG antibodies and NBT-BCIP as substrate.


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Table 2. Peptides from the N terminus and from internal amino acid sequences of the affinity-purified 105 kDa Hb-binding protein from A. pleuropneumoniae

 
Construction and screening of A. pleuropneumoniae phage banks.
Two DNA libraries of A. pleuropneumoniae serotype 1 reference strain 4074 were constructed (Vézina et al., 1997) in {lambda} Zap Express phage vector (Stratagene). One was a Sau3AI/BamHI library; the other was an EcoRI library. Libraries were screened with DIG-labelled PCR products that were obtained with genomic DNA and the degenerate oligonucleotides described above. Positive plaques were also detected with phosphatase-labelled anti-DIG antibodies and NBT-BCIP as substrates. Strongly reacting plaques were purified by three successive rounds of screening and the pBK-CMV phagemid of positive clones was excised using ExAssist Helper Phage (Stratagene). Finally, plasmids were digested with restriction enzymes known to cut once within the multiple cloning site of the vector, thereby establishing the sizes of their inserts. Dideoxynucleotide sequencing reactions with universal primers T3 and T7 determined the entire sequence of the plasmid inserts; internal primers were designed based on confirmed sequences. All DNA sequencing was carried out at the Sheldon Biotechnology Center, McGill University. Nucleotide data and deduced amino acid sequences were analysed using the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov), the European Molecular Biology Laboratory website (www.embl-heidelberg.de) and the Expasy Molecular Biology Server (ca.expasy.org).

Construction of a deletion mutant of hgbA from A. pleuropneumoniae.
To introduce a deletion into A. pleuropneumoniae serotype 1 hgbA, a double-step transconjugation system using a sucrose sensitivity marker (Oswald et al., 1999) was used. The transconjugation vector pEMOC2 (Table 1) carries chloramphenicol resistance and a sucrose sensitivity marker (Baltes et al., 2003). Generating the A. pleuropneumoniae internal {Delta}hgbA strain involved several subcloning steps. Plasmid pRSC01 contained an 8·2 kb insert in pBK-CMV, including the entire 2·8 kb hgbA sequence with additional sequences 2·1 kb upstream and 3·3 kb downstream. The hgbA sequence harboured two BspEI restriction sites; pRSC01 was digested with BspEI thus releasing a 650 bp fragment and then religated to create pRSC04 with an in-frame hgbA deletion. The 4·3 kb NheI fragment from pRSC04 containing the internal deletion was subsequently cloned into the XbaI site of the transconjugation vector pBMK (Oswald et al., 1999) to yield pRSC08. Finally, the 4·3 kb NotI–SalI fragment from pRSC08 was cloned into the NotI/SalI site of the transconjugation vector pEMOC2 to generate pRSC09. Plasmid pRSC09 containing an in-frame deletion of hgbA plus sequences 1·9 kb upstream and 2·4 kb downstream to facilitate allelic exchange was transformed into diaminopimelic-acid-auxotrophic E. coli {beta}2155 cells as the donor for transconjugation into A. pleuropneumoniae. Transconjugation was achieved by mobilizing plasmid pRSC09 from E. coli {beta}2155 to the recipient strain of A. pleuropneumoniae serotype 1 strain 4074 by conjugal transfer using a mating technique (Dehio & Meyer, 1997) adapted by Oswald et al. (1999). Donor and recipient strains were grown on solid medium overnight; an aliquot of 750 µl donor cells was mixed with 250 µl recipient cells. The mixture was centrifuged (3000 g), resuspended in 200 µl TNM buffer (1 mM Tris/HCl, pH 7·2, 100 mM NaCl, 10 mM MgSO4), spotted onto BHI plates supplemented with 10 µg NAD ml–1, 1 mM diaminopimelic acid and 10 mM MgSO4, and incubated at 37 °C for 6 h. Spots of bacterial cells were resuspended in 400 µl BHI supplemented with NAD and serial 10-fold dilutions were plated on BHI-NAD plates containing 2 µg chloramphenicol ml–1. After incubation at 37 °C for 24 h, transconjugants appeared, carrying a copy of pRSC09 in the chromosome. Transconjugants were plated on Mueller–Hinton agar supplemented with 10 µg NAD ml–1 and 10 % (w/v) sucrose and incubated at 30 °C for 48 h. This procedure yielded isolated colonies which had lost pEMOC2 sequences (chloramphenicol-sensitive) and which carried either an unaltered wild-type copy of hgbA or the internal hgbA deletion. Colonies from sucrose-containing Mueller–Hinton agar plates were streaked on BHI-NAD plates; candidates carrying the internal deletion in hgbA were analysed for their PCR profile as well as their capacity to use porcine Hb as a source of iron.

Growth in iron-deficient conditions.
Kinetics of growth of wild-type A. pleuropneumoniae and A. pleuropneumoniae internal {Delta}hgbA mutant were compared in iron-sufficient and iron-deficient conditions. Overnight bacterial cultures were diluted 100-fold and grown until the culture reached exponential phase. A. pleuropneumoniae strains carrying either wild-type hgbA or the internal {Delta}hgbA mutant were then diluted 100-fold by inoculating into (i) NAD-supplemented BHI broth, (ii) NAD-supplemented BHI broth in the presence of 80 µM EDDHA, and (iii) NAD-supplemented BHI broth in the presence of 80 µM EDDHA and 20 µg pig Hb ml–1. Growth kinetics were monitored by measurement of OD600 at time zero and at 1 h intervals; assays for each condition were performed in triplicate. Strains of A. pleuropneumoniae carrying wild-type hgbA, the internal {Delta}hgbA mutant and the internal {Delta}hgbA mutant plus plasmids (complementation, see below) were also tested in growth promotion assays in the presence of various sources of iron as follows. Twofold serial dilutions of EDDHA were made in BHI-NAD broth supplemented with various sources of iron, including Hb from pig, cow, horse, sheep, goat, human and turkey, haemin, ferricrocin and ferric chloride. The highest and lowest concentrations of EDDHA in the assay were 1280 and 20 µM, respectively. Overnight bacterial cultures were used as inocula at a final concentration of 1x106 c.f.u. ml–1, approximately 1000-fold dilution of overnight culture. The total volume of the assay was 2·5 ml and each assay was set up in triplicate. Cultures were grown overnight at 37 °C with agitation and the concentration of EDDHA that inhibited visible growth in each condition was reported.

Expression of recombinant A. pleuropneumoniae proteins in E. coli.
Both wild-type hgbA and internal {Delta}hgbA recombinant proteins of A. pleuropneumoniae were expressed in E. coli and analysed by SDS-PAGE. These strains were also assessed for binding of fluorescein-labelled Hb by flow cytometry. The expression vector pET24b (Novagen) was used for cloning recombinant A. pleuropneumoniae proteins to which were appended a C-terminal His tag, followed by expression in E. coli BL21(DE3) cells, the host for pET vector derivatives. To generate pRSC06, two primers were synthesized: Sal1-Hgb [5'-GAGGTCGACA(159)ACAGTGCATTGGCACAAGAGC-3'; SalI site underlined and number corresponds to the nucleotide in GenBank AF468020] and Xho1-Hgb [5'-GCGCTCGAGG(–4)AAAGTAACCTCTGCGGTTAAC-3'; XhoI site underlined and number corresponds to the nucleotide in GenBank AF468020] specific to the 5' and 3' region of hgbA. Using chromosomal DNA from A. pleuropneumoniae as template and primers Sal1-Hgb and Xho1-Hgb, a 2·8 kb fragment containing hgbA sequence was amplified and cloned into pGEM-T Easy to yield pHGBA-14. This fragment contained hgbA sequences corresponding to the codons of the mature sequence of HgbA, but did not contain codons for the entire signal sequence or the stop codon. The 2·8 kb fragment was recovered from pHGBA-14 as a SalI–XhoI fragment and cloned into pET24b(+) digested with SalI and XhoI to yield pRSC06. To generate pRSC05, pRSC06 was first digested with BamHI (within hgbA) and XhoI, and the 0·9 kb fragment containing the codons for the C-terminal portion of HgbA was ligated to pET24b(+) digested with BamHI and XhoI; the resulting plasmid was named pRSC03. The 1·9 kb NheI–BamHI fragment from pRSC01 containing the codons for the N-terminal portion of HgbA, including its signal sequence and ribosome-binding site, was then cloned into pRSC03 digested with NheI and BamHI to yield pRSC05. To generate pRSC14, pRSC05 was first digested with NheI and XhoI and the 2·8 kb fragment containing hgbA was ligated to pBK-CMV digested with NheI and XhoI; the resulting plasmid was named pRSC12. Plasmid pRSC12 was then digested with BspEI, releasing a 0·65 kb fragment, and religated. This construct, termed pRSC13, contained an in-frame deletion in hgbA. Next, the 2·15 kb NheI–XhoI fragment from pRSC13 was cloned into pET24b(+) to generate pRSC14 which contained sequences of hgbA with an internal in-frame deletion, its signal sequence and a C-terminal His tag.

Plasmids pET24b, pRSC05, pRSC06 and pRSC14 were each transformed into E. coli BL21(DE3) cells. Cultures containing the pET24b-derived recombinant plasmids or vector alone were grown in LB broth plus 50 µg kanamycin ml–1 for 2 h until cells reached an OD600 of 0·4–0·6. Samples were removed for uninduced control; IPTG (final concentration of 0·4 mM) was added to the remainder of the culture and incubated for 2–4 h at 25 °C (Mikael et al., 2002). Bacteria were harvested by centrifugation and whole-cell lysates were analysed by SDS-PAGE and Coomassie blue staining. Cell envelope proteins were also prepared as described above and similarly analysed by SDS-PAGE and Coomassie blue staining.

Complementation.
A 6·7 kb EcoRI fragment from pRSC01 carrying the entire hgbA sequence was cloned into the EcoRI site of the E. coliA. pleuropneumoniae shuttle vector pJF224-XN (Frey, 1992) to yield pRSC15. To assess complementation, this plasmid was introduced into the A. pleuropneumoniae internal {Delta}hgbA strain by electroporation (Oswald et al., 1999). Briefly, a 50 ml culture of A. pleuropneumoniae internal {Delta}hgbA mutant was grown to an OD600 of 0·5 in BHI. Cells were kept on ice for 30 min and then centrifuged for 10 min at 3000 g at 4 °C, and washed thrice in 30 ml ice-cold GYTT medium. Cells were resuspended in 0·5 ml GYTT (1/100) and kept at –70 °C. A 125 µl sample of cell suspension was mixed with 1–2 µg plasmid pRSC15. Electroporation was performed using a Gene Pulser (Bio-Rad) at U=2·5 kV, C=10 µF, R=600 W and a pulse length of 5·0 ms in a 2 mm cuvette. After electroporation, 1 ml NAD-supplemented BHI broth was added and the cells were incubated for 3 h prior to spreading on BHI-NAD plates containing 0·5 µg chloramphenicol ml–1. A. pleuropneumoniae with a chromosomal deletion in {Delta}hgbA and harbouring either vector pJF224-XN or pRSC15 was inoculated into NAD-supplemented BHI broth in the presence of various concentrations of EDDHA and 20 µg pig Hb ml–1, and growth was monitored.

Labelling of pig Hb with fluorescein.
Labelling of pig Hb with fluorescein was achieved using a FLUOS labelling kit (Roche Diagnostics) (Archambault et al., 1999). The molar ratio was set at 1 : 100 after standardization; 1 mol Hb was mixed with 100 mol FLUOS. Briefly, 39·5 µl of a FLUOS solution (20 mg ml–1) was added to 1 ml of a pig Hb solution (1 mg ml–1 in PBS, pH 7·4) and incubated in the dark for 2 h at room temperature. The Hb–FLUOS reaction mixture was then applied to a Sephadex G-25 column and unbound FLUOS was separated by gel filtration.

Flow cytometry and analysis.
Bacterial cultures of both wild-type and A. pleuropneumoniae internal {Delta}hgbA mutant were grown in NAD-supplemented BHI broth (iron-sufficient) and NAD-supplemented BHI broth in the presence of 40 µM EDDHA (iron-deficient). E. coli BL21 cells harbouring pRSC05, pRSC06, pRSC14 or vector pET24b alone were grown with induction by 0·5 mM IPTG for 2 h. Cultures were harvested, washed in 1 ml PBS, centrifuged and resuspended in 90 µl PBS. Ten microlitres of the FLUOS-Hb (final dilution 1/100) was added to the cells and incubated at 37 °C for 30 min with agitation. After washing, E. coli and A. pleuropneumoniae cells were suspended in 1–2 ml fresh PBS or fresh PBS containing 2 % (w/v) paraformaldehyde, respectively. Green fluorescence in the samples was measured by subjecting cells to flow cytometry using a FACStar flow cytometer (Becton Dickinson Immunocytometry Systems) equipped with a water-cooled 2 W argon ion laser operating at 488 nm and with a 200 mW light output. Negative controls included bacterial cells not incubated with FLUOS-Hb and cells incubated with unlabelled pig Hb. Results were expressed as a percentage of gated fluorescent events where a total of 30 000 events were analysed for each sample. All experiments were performed at least twice; samples were always run in duplicate.

RT-PCR.
A. pleuropneumoniae serotype 1 reference strain 4074 was grown on BHI-NAD solid medium overnight. Colonies were inoculated into 25 ml cultures of (i) BHI-NAD broth for iron-sufficient conditions and (ii) BHI-NAD broth supplemented with 50 µM EDDHA for iron-deficient conditions. Growth was monitored by turbidity; organisms were harvested in exponential phase (OD600~0·8), washed immediately with sterile PBS and total bacterial RNA was isolated with the RNeasy Mini Kit (Qiagen). The eluate containing RNA was treated with DNaseI (RNase-free, FPLC purity; Amersham) in a solution of 40 mM Tris, pH 7·5, 6 mM MgCl2, and incubated at 37 °C for 10 min. RNA samples were purified once more with the RNeasy cleanup protocol and concentrations were determined spectrophotometrically at 260 nm. RT-PCR was performed with the One-Step RT-PCR Kit (Qiagen) as recommended by the manufacturer, with 500 ng RNA sample and a primer pair, F2 (5'-GAGTTACGTCATACGAATG-3') and B5 (5'-TGATCGCTACCGGTCGCA-3'), to amplify an internal fragment of A. pleuropneumoniae hgbA. Controls included a primer pair specific for A. pleuropneumoniae fhuA and another primer pair specific to the tbp locus in A. pleuropneumoniae. All primer pairs were chosen to amplify a product of approximately 800 bp. The same primers used with genomic DNA as template served as a positive control for the RT-PCR. Control experiments for DNA contamination were performed in which no reverse transcriptase was added to the RNA samples prior to the PCR step. RT-PCR was performed with an equal amount of template RNA and equal volumes of the amplicon were loaded for all samples.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Affinity purification of Hb-binding proteins from A. pleuropneumoniae
In our search for Hb-binding receptors in A. pleuropneumoniae serotype 1, we isolated OM vesicles from cells grown in iron-sufficient conditions and in iron-deficient conditions. Several OM proteins (47, 55, 75, 80 and 105 kDa) were enhanced in their relative abundances when bacteria were grown in iron-deficient conditions compared to cells grown under iron sufficiency (Fig. 1a, lanes 1 and 2). Cells were cultivated in low-iron medium and proteins from detergent-solubilized OM vesicles were subjected to affinity purification over a column of bovine Hb immobilized on agarose. Affinity-purified proteins from cells grown in low-iron medium showed (Fig. 1a, lanes 3 and 4) a minor band (75 kDa) as well as a major species (105 kDa), suggesting that both proteins may be iron-regulated. The 105 kDa OM protein was the prominent species capable of binding tightly and specifically to Hb-agarose.



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Fig. 1. Iron- and haem-deficient expression of an OM protein in A. pleuropneumoniae serotype 1 reference strain 4074. (a) Vesicles were analysed from cells grown in iron-sufficient medium (lane 1) and from cells grown in medium made deficient for iron by the addition of 40 µM EDDHA (lane 2). After affinity purification on an Hb-agarose column, those proteins that bound to Hb were isolated. Hb-agarose-purified proteins from cells grown in iron-sufficient (lane 3) and iron-deficient media (lane 4) were analysed. (b) OM vesicles were analysed from cells grown in medium made sufficient for haem by the addition of 20 µg pig Hb ml–1 and 150 µM EDDHA (lane 1), and from cells grown in medium made deficient for haem by the addition of 1 µg pig Hb ml–1 and 150 µM EDDHA (lane 2). Hb-agarose-purified proteins from strains grown in haem-sufficient (lane 3) and haem-deficient media (lane 4) were analysed. Detection was by Coomassie blue staining. Broad-range protein molecular mass markers are shown in both panels (lanes M).

 
Parallel experiments varied levels of haem in the culture medium, followed by isolation of OM vesicles and affinity purification on Hb-agarose. Overexpression was observed for some proteins (36, 47, 75 and 105 kDa) under haem-deficient growth conditions (Fig. 1b, lanes 1 and 2). Affinity purification confirmed that the 105 kDa protein bound to immobilized bovine Hb and that the amount of the 105 kDa species was enhanced in cells grown in haem-deficient conditions compared to haem-sufficient conditions (Fig. 1b, lanes 3 and 4). These data implicated the 105 kDa OM protein from A. pleuropneumoniae as an iron-regulated Hb-binding protein, now designated HgbA.

N-terminal and internal peptide sequences of HgbA from A. pleuropneumoniae
By Edman degradation, the N-terminal sequence was determined (Table 2, P-1) from HgbA of A. pleuropneumoniae serotype 1 reference strain 4074. A BLASTP search for this peptide found no homologies with proteins in the NCBI GenBank database. HgbA was then digested with trypsin followed by liquid chromatography and peptide fragments were subjected to de novo sequencing by electrospray ionization mass spectrometry. Two internal peptide sequences (P-2 and P-3) were identical to sequences from a known TonB-dependent Hb-binding OM protein of P. multocida (accession no. AF237932) and from H. influenzae (accession no. U51922). A third internal peptide (P-4) displayed 70 % identity with the same protein from P. multocida. We conclude that HgbA protein from A. pleuropneumoniae is most highly related to the TonB-dependent Hb-binding protein from P. multocida (Bosch et al., 2002).

Cloning and analysis of hgbA from A. pleuropneumoniae
Synthetic oligonucleotides based on the N-terminal (P-1) and internal peptide (P-3) sequences of HgbA were DIG-labelled as probes in Southern hybridization experiments with genomic A. pleuropneumoniae DNA. Both probes hybridized to a 6 kb EcoRI fragment of A. pleuropneumoniae DNA. The positions of the internal peptide sequences of HgbA of A. pleuropneumoniae (Table 2) were compared to homologous peptides in HgbA of P. multocida. Three pairs of degenerate oligonucleotide primers were then used for PCR amplification of genomic A. pleuropneumoniae DNA, yielding three fragments. Their nucleotide sequences were assembled into an incomplete ORF of 2355 bp (785 aa). The predicted protein showed similarities with the TonB-dependent family of Hb-binding OM proteins involved in haem transport: HgbA of P. multocida (69 %), HgbA of H. influenzae (68 %), HupA of H. ducreyi (64 %) and HgbA of Actinobacillus actinomycetemcomitans (75 %). To isolate larger genomic fragments from A. pleuropneumoniae containing the entire hgbA, a Sau3AI-digested genomic DNA library from A. pleuropneumoniae serotype 1 was screened with DIG-labelled PCR products. Following three rounds of purification, sequencing of the insert from clone pC157A showed a partial hgbA sequence, interrupted by a BamHI restriction site that was attributed to the design of the library. Parallel screening of an EcoRI-digested genomic library yielded a positive clone, pA111. These clones contained sequences of hgbA that were overlapping, yet individually neither had the entire hgbA sequence. By ligating a 4·2 kb EcoRV–XhoI fragment from pA111 with a fragment from pC157A digested with EcoRV (within hgbA) and SalI (multiple cloning site), we generated a plasmid (pRSC01) that had pBK-CMV vector as backbone and 8·2 kb from A. pleuropneumoniae. Plasmid pRSC01 contained the full-length 2·8 kb hgbA sequence with additional sequences: 2·1 kb upstream and 3·3 kb downstream (Fig. 2a). The complete ORF (2838 bp) encoded HgbA of 946 aa; the N-terminal sequence and the three internal peptide sequences (P-1, P-2 and P-3) were positioned within the predicted HgbA, confirming its identity. Upstream of hgbA was a complete ORF for a potential haemin-binding protein (174 aa) homologous to HugZ in Plesiomonas shigelloides. The promoter sequences (Fig. 2b) revealed a putative Shine–Dalgarno sequence (AAGGAG) 7 bases upstream of the proposed GTG start codon; –10 (TAGTTT) and –35 (ATAATTAA) boxes, respectively, were located 40 and 60 bases upstream of the translation start codon. A potential Fur-binding sequence (ATAATTAAACATTTAC) was identified 60 bases upstream from the proposed start codon.



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Fig. 2. Features of the coding region of hgbA in A. pleuropneumoniae and flanking sequences. (a) Schematic representation of the DNA sequences containing hgbA and flanking sequences in the chromosome of wild-type A. pleuropneumoniae. The 8·2 kb A. pleuropneumoniae DNA sequence cloned into pRSC01 is shown. Restriction sites useful to this study are identified. The arrow denotes sequences encoding hgbA; the small box identifies the region upstream of the coding sequences that contain regulatory elements. The arrow and small box constitute sequences that have been submitted to GenBank (accession no. AF468020); numbers correspond to the GenBank entry. (b) Sequences showing part of the coding and upstream regions of hgbA. The consensus promoter sequence (–35 and –10), a Fur-binding site (Fur box), the consensus ribosome-binding site (RBS) and the putative start codon are identified. The signal sequence of HgbA is shown in single-letter amino acid code and the beginning of the mature sequence of HgbA is presented in bold type. A region for a Ton box in the protein sequence is also proposed.

 
From the full-length sequence of HgbA, the first 23 aa are proposed as a signal peptide ending with a peptidase recognition sequence, Ala21-Leu-Ala23 (Fig. 2b), yielding mature A. pleuropneumoniae HgbA of 923 residues (104 890 Da). TonB-dependent receptors contain a highly conserved motif at their N termini; a Ton box (LDTVIV) is predicted at the N terminus of HgbA, residues 6–11 (Fig. 2b). An RPS-BLAST search for conserved protein domains identified a motif (NRFTAPGRNFKLTAEVTF) at the C-terminal region of HgbA, residues 906–923. This motif, termed the TonB boxC, is found in the C-terminal region of a pfam protein family (PF00593) of TonB-dependent receptors (Eddy et al., 2000). The conserved NPNL and FRAP motifs of haem/Hb transporters (Simpson et al., 2000; Wandersman & Stojiljkovic, 2000) were found in HgbA: residues 333–337 plus Glu341, the latter motif differing by an extra glutamine residue (NQPNL); residues 655–658 plus His669 for FRAP.

Distribution of hgbA among A. pleuropneumoniae serotype reference strains
To determine whether the hgbA gene was unique to the A. pleuropneumoniae serotype 1 reference strain or widely distributed among different serotypes of this organism, DNA from serotypes 2–12 was tested by PCR and Southern blotting. Two pairs of primers were used to amplify fragments of 750 and 350 bp, corresponding to an internal and a C-terminal region of hgbA, respectively. All 12 serotypes tested positive for both amplicons by PCR and all gave positive hybridization signals (data not shown). These collective results indicate that hgbA is present among all serotypes 1–12 of A. pleuropneumoniae.

Deletion mutant of A. pleuropneumoniae hgbA
To construct a {Delta}hgbA strain of A. pleuropneumoniae serotype 1, a double-step transconjugation system was used to introduce an unmarked in-frame deletion. The mutant strain had an internally deleted fragment (648 bp) extending from Asp592 to Pro807 that maintained the reading frame of hgbA. Using a pair of primers specific to the coding sequences of hgbA, both first and second crossover events were analysed by PCR. The single crossover event, integration of pRSC09, carried both wild-type and the internal {Delta}hgbA; successful counter selection with sucrose yielded the mutant strain carrying the chromosomal deletion in hgbA (Fig. 3). To investigate whether the A. pleuropneumoniae internal {Delta}hgbA strain was compromised for use of Hb as iron source, growth promotion tests were performed with various sources of iron on both wild-type and internal {Delta}hgbA strains under iron-deficient conditions. Wild-type A. pleuropneumoniae and the internal {Delta}hgbA strain required the same concentration of EDDHA (40 µM) to inhibit growth. At this concentration of EDDHA, the internal {Delta}hgbA strain could not grow in NAD-supplemented BHI medium in the presence of any source of Hb tested: pig, cow, horse, sheep, goat, human and turkey, all at 20 µg ml–1. When other sources of iron (haemin at 20 µg ml–1, ferricrocin at 40 µM, ferric chloride at 80 µM) were tested, the wild-type and the internal {Delta}hgbA strain behaved alike; both strains were able to use these alternative sources of iron for growth. Growth characteristics of both wild-type and internal {Delta}hgbA strains were also assessed (Fig. 4). While the internal {Delta}hgbA strain lagged slightly behind the wild-type strain in NAD-supplemented BHI medium, the wild-type strain recovered from 80 µM EDDHA iron deprivation by addition of 20 µg pig Hb ml–1; the mutant strain could not recover.



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Fig. 3. Confirmation of the chromosomal deletion in hgbA. DNA isolated from different strains was subjected to PCR using primer pairs specific to the coding sequences for hgbA. Templates used for the reactions were: pRSC01 (lane 1); pRSC04 (2); pRSC09 (3); chromosomal DNA from wild-type A. pleuropneumoniae (4); chromosomal DNA from A. pleuropneumoniae internal {Delta}hgbA mutant (5); and chromosomal DNA from an A. pleuropneumoniae strain with a co-integration of pRSC09 into its chromosome after a single crossover (6). Genomic DNA from bacteriophage {lambda} digested with HindIII (lane M) was used as a DNA molecular mass marker.

 


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Fig. 4. Growth of A. pleuropneumoniae wild-type and internal {Delta}hgbA mutant. Growth was monitored by measuring OD600. Growth curves are shown of wild-type A. pleuropneumoniae and A. pleuropneumoniae internal {Delta}hgbA mutant with no addition (open and filled diamonds, respectively), wild-type A. pleuropneumoniae and A. pleuropneumoniae internal {Delta}hgbA mutant in the presence of 80 µM EDDHA (open and filled triangles, respectively), and wild-type A. pleuropneumoniae and A. pleuropneumoniae internal {Delta}hgbA mutant in the presence of 80 µM EDDHA and 20 µg pig Hb ml–1 (open and filled circles, respectively).

 
Expression of recombinant A. pleuropneumoniae HgbA in E. coli
Wild-type hgbA genes with and without signal sequence (pRSC05 and pRSC06, respectively) and the internal {Delta}hgbA gene (pRSC14) were cloned into pET24b(+) and expressed in E. coli. After induction with IPTG, recombinant proteins from whole-cell lysates and cell envelope proteins were analysed by 10 % acrylamide SDS-PAGE (Fig. 5). Whereas the wild-type HgbA with and without signal sequence migrated at ~105 kDa, the internally deleted HgbA migrated at ~80 kDa, its position anticipated from the in-frame deletion that produced a mature protein of 707 aa (80 296 Da). The signal sequences preceding the wild-type gene and the internal {Delta}hgbA mutant in pRSC05 and pRSC14, respectively, apparently routed the recombinant proteins to the cell envelope where minor amounts of protein were detected.



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Fig. 5. Expression of recombinant A. pleuropneumoniae proteins in E. coli. Wild-type hgbA and internal {Delta}hgbA A. pleuropneumoniae genes were cloned into pET24b(+) and expressed in E. coli BL21(DE3). Proteins from whole-cell lysates and cell envelopes are shown in pairs: lanes 1 and 5, from E. coli carrying pET24b(+), vector alone (lanes 1 and 5); from E. coli carrying pRSC05, wild-type HgbA (lanes 2 and 6); from E. coli carrying pRSC14, internal {Delta}HgbA protein (lanes 3 and 7); and from BL21(DE3) carrying pRSC06, HgbA minus signal sequence (lanes 4 and 8). Broad-range protein molecular mass markers are shown in lane M. Proteins were stained with Coomassie blue.

 
Hb binding to A. pleuropneumoniae
When grown in the presence of EDDHA to create an iron-deficient environment, A. pleuropneumoniae serotype 1 cells expressing wild-type HgbA reacted with fluorescent pig Hb, as shown by flow cytometry (Fig. 6b). No reactivity was evident when the same cells were grown in rich medium (Fig. 6a). Addition of EDDHA apparently induced expression of HgbA sufficient to cause binding of the fluorescent probe, even though the relative fluorescence intensity did not display a symmetrical profile (Fig. 6b). Some cells may not have fluoresced after incubation (30 min) because they would have removed haemin from Hb and thereby satisfied their iron requirements. Surprisingly, when A. pleuropneumoniae expressing the internally deleted HgbA was grown in the presence of EDDHA, the majority of cells also reacted with fluorescent pig Hb (Fig. 6d). HgbA may form a stable complex with Hb without allowing transport of iron into the cells, thus preventing release of Hb from the receptor. A. pleuropneumoniae internal {Delta}hgbA mutant grown in rich medium did not react with pig Hb (Fig. 6c); expression of internal {Delta}hgbA was likewise downregulated in the presence of iron.



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Fig. 6. Flow cytometry of bacterial strains using fluorescent Hb. Fluorescence is shown for: wild-type A. pleuropneumoniae under iron-sufficient (a) or iron-deficient (b) growth conditions; A. pleuropneumoniae internal {Delta}hgbA mutant under iron-sufficient (c) or iron-deficient (d) growth conditions; E. coli BL21(DE3) strains carrying the vector pET24b(+) alone (e), carrying pRSC05, wild-type HgbA (f), carrying pRSC14, internal {Delta}HgbA protein (g) and carrying pRSC06, HgbA minus signal sequence (h).

 
Although the in-frame deletion of 216 aa in HgbA affected utilization of Hb as an iron source (Fig. 4), we required independent confirmation of how the deletion influenced Hb binding. Wild-type hgbA and the in-frame {Delta}hgbA were each cloned into pET24b(+) and expressed in E. coli BL21(DE3). Whereas an E. coli strain carrying vector alone showed no reactivity by flow cytometry with pig Hb (Fig. 6e), E. coli strains expressing wild-type hgbA (pRSC05) or the internally deleted hgbA (pRSC14) showed comparable profiles of reactivity to fluorescent pig Hb (compare Fig. 6f and 6g). Processing of the native signal sequence of HgbA may have been inefficient in E. coli, consistent with low levels of recombinant receptor in the OM (Fig. 5). This would account for modest but still discernable Hb binding by the two E. coli constructs. An E. coli strain expressing an HgbA protein without its signal sequence (pRSC06) did not react with pig Hb (Fig. 6h); the protein apparently accumulated inside the cell, thus confirming the role of the signal sequence in targeting and assembly of recombinant HgbA in the OM of E. coli.

Complementation
To assess whether utilization of pig Hb could be restored in the internal {Delta}hgbA strain of A. pleuropneumoniae by complementation, a growth promotion assay was performed on this mutant strain that had been transformed with pRSC15 carrying a wild-type hgbA. Results (Table 3) show that the complemented strain could use 20 µg pig Hb ml–1 in the presence of up to 640 µM EDDHA; the internal {Delta}hgbA strain transformed with vector pJF224-XN alone could not use pig Hb. These data confirmed successful complementation of the A. pleuropneumoniae internal {Delta}hgbA strain by a single gene, hgbA.


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Table 3. Complementation of A. pleuropneumoniae serotype 1 internal {Delta}hgbA strain

 
RT-PCR
To investigate the effects of iron levels in the culture medium that influenced expression of hgbA transcripts, RNA was isolated from A. pleuropneumoniae cells grown under iron-sufficient and iron-deficient conditions. RT-PCR was performed with a pair of primers specific to A. pleuropneumoniae hgbA; primers specific to A. pleuropneumoniae fhuA and the tbp locus were used as controls. All primer pairs amplified products of ~800 bp. Levels of hgbA transcript increased in response to a decrease in the level of iron, a result that matched RT-PCR for the tbp locus; transcripts of fhuA were not enhanced in response to low-iron growth conditions (Fig. 7), an observation that matched published data (Mikael et al., 2003). Expression of A. pleuropneumoniae hgbA is therefore upregulated under iron-deficient conditions, consistent with results of the Hb-agarose affinity purification of the 105 kDa OM protein that was purified from A. pleuropneumoniae cells grown under iron-deficient conditions.



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Fig. 7. Iron-deficient expression of hgbA in A. pleuropneumoniae by RT-PCR. RNA templates used in the reaction were from wild-type A. pleuropneumoniae grown under iron-sufficient (lanes 1, 3 and 5) or under iron-deficient (lanes 2, 4 and 6) conditions. One primer pair specific to A. pleuropneumoniae fhuA (1 and 2), one primer pair specific to A. pleuropneumoniae tbp locus (3 and 4) and one primer pair specific to A. pleuropneumoniae hgbA (5 and 6) were used in the reactions.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Virulence of pathogenic bacteria is partly determined by their ability to compete with their infecting host for the supply of essential nutrients, including iron. Because the mammalian host restricts bacterial growth by limiting or withholding iron, bacteria have evolved specific mechanisms for efficient acquisition of iron that may be bound to macromolecules such as haem-containing proteins, transferrin and lactoferrin. Many different genera are capable of using haem; these haem-utilizing systems usually require a surface-located OM receptor that recognizes the iron-donating protein, followed by a transport process that imports haem across the cell envelope. Given the ability of A. pleuropneumoniae to acquire iron from haem, and given preliminary evidence that this organism was capable of utilizing Hb as sole source of iron, we wanted to identify the specific OM receptor that interacts with this haem-donating protein. Our experimental strategies were to isolate OM vesicles from cells grown under iron-deficient conditions, and then by immobilized affinity ligand chromatography to locate a prominent Hb-binding protein (105 kDa). Partial amino acid sequence determinations gave peptides that matched Hb-binding proteins in other bacterial species, proteins identified chiefly by their sequence homologies. Our cloning of the full-length hgbA gene allowed us to designate a specific function for the wild-type protein in A. pleuropneumoniae and to deduce some role for an internally deleted HgbA. We conclude that full-length HgbA is a necessary surface-located OM protein of this bacterial species that interacts with Hb to import haem into the cell. HgbA may be sufficient as sole Hb receptor or HgbA may function in concert with another protein to mediate haem import. This latter mechanism would resemble transferrin-mediated iron uptake in which two OM proteins, TbpA and TbpB, are required for efficient iron uptake.

Our most sensitive assay for recognition and binding of Hb by HgbA employed flow cytometry and confirmed (Fig. 6) that Hb bound to native HgbA in the OM of A. pleuropneumoniae, to the internal {Delta}HgbA protein on intact cells and to the two homologous recombinant HgbA species expressed in E. coli. Hence, A. pleuropneumoniae expressing HgbA with 216 aa, Asp592 to Pro807, deleted remains proficient for Hb binding but unable to use Hb as its iron source. It is noteworthy that this large deletion covers the FRAP motif (HgbA Phe655 to Pro658 plus His669), a region reported (Bracken et al., 1999) to be essential for haem utilization by the HemR receptor of Yersinia enterocolitica. Conversely, these authors showed that the FRAP motif was not essential for binding of HemR to an Hb-agarose affinity column. We now show concordance between the properties of HemR from Y. enterocolitica and the activity of HgbA of A. pleuropneumoniae. Bacterial growth kinetics (Fig. 4) substantiated these observations, leading to the conclusion that, following Hb binding, the sequence Asp592 to Pro807 of HgbA contributes to haem import. The molecular determinants of the release of haem from Hb and its traverse across HgbA are presently under investigation.

What structural elements might be affected by a deletion in HgbA that spans 216 aa? We recently completed analyses of HgbA by comparative modelling and by a Hidden Markov model (P. D. Pawelek & J. W. Coulton, unpublished data), identifying the topological similarities of HgbA to bacterial OM receptors BtuB, FepA, FhuA and FecA of E. coli. Our HgbA model has a globular N-terminal cork domain contained within a 22-stranded {beta} barrel domain, its folds being similar to the structures of OM receptors that have been solved by X-ray crystallography. HgbA residue Asp592 in our model was assigned to surface-exposed loop 6; Pro807 was positioned near the end of {beta} strand 18 as it emerged on the periplasmic face. The net deletion therefore removes {beta} strands 12–17 and most of {beta} strand 18, meaning that an odd number of strands would be missing. Although unusual, such structural reorganizations are not impossible, especially since the mutant protein appeared to be remarkably stable and was not subject to endogenous proteolysis.

Might HgbA merit consideration as a vaccine candidate against A. pleuropneumoniae? From our Southern blotting survey for the presence of hgbA among 12 serotypes of this organism, we determined that the gene is widely distributed in this species. The bacterium shows a clear preference for the source of Hb (swine) over other mammalian sources, suggesting that the determinants for interaction of HgbA with Hb are conserved in surface-located loops of the receptor at the OM. Preliminary investigations into the virulence of the A. pleuropneumoniae internal {Delta}hgbA mutant compared to infection of swine by wild-type A. pleuropneumoniae are in progress. Continuing studies on a recombinant, purified HgbA will establish its utility as a swine vaccine.


   ACKNOWLEDGEMENTS
 
This work was initially supported by a Strategic Grant (224192) from the Natural Sciences and Engineering Research Council (NSERC) of Canada. Recent funding was from the NSERC Canadian Research Network on Bacterial Pathogens of Swine and its industrial partners. We thank G. Gerlach, Institut für Mikrobiologie und Tiersüchen, Hannover, Germany, for E. coli {beta}2155 and vectors pBMK1 and pEMOC2; J. Frey, Institut für Veterinär-Bakteriologie, Universität Bern, Switzerland, for vector pJF224-XN; and M. Sirois, Université de Québec à Trois-Rivières, QC, for the genomic bank of A. pleuropneumoniae. Facilities of Sheldon Biotechnology Center, McGill University, supported by Canadian Institutes of Health Research, provided DNA sequencing, oligonucleotide synthesis and peptide sequencing. C. Ng assisted with affinity purifications and J. Labrie with screenings. J. A. Kashul edited the manuscript.


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Received 21 January 2004; revised 25 February 2004; accepted 5 March 2004.



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