Groupe de Recherche sur les Maladies Infectieuses du Porc, Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, CanadaJ2S 7C61
Department of Microbiology and Immunology, McGill University, Montréal, Québec, CanadaH3A 2B42
Département de Chimie-Biologie, Université du Québec à Trois-Rivières, Trois-Rivières, Québec, CanadaG9A 5H73
Author for correspondence: Mario Jacques. Tel: +1 450 773 8521 ext. 8348. Fax: +1 450 778 8108. e-mail: jacqum{at}medvet.umontreal.ca
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ABSTRACT |
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Keywords: outer-membrane protein, siderophore transport
Abbreviations: DIG, digoxigenin; fhu, ferric hydroxamate uptake; OMP, outer-membrane protein; phoA, alkaline phosphatase; TbpA, transferrin-binding protein A; TbpB, transferrin-binding protein B
The GenBank accession number for the sequence of the fhuCDBA operon of Actinobacillus pleuropneumoniae serotype 1 reference strain 4074 described in this study is AF351135.
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INTRODUCTION |
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A. pleuropneumoniae is capable of using haemoglobin, haemin-containing compounds and porcine transferrin as sources of iron for its growth (Bélanger et al., 1995 ; Deneer & Potter, 1989
). In addition, it can produce haemolysins, toxins that belong to the RTX (repeats-in-toxin) group of proteins (Schaller et al., 1999
). All of these factors may contribute to the virulence of this bacterium. A. pleuropneumoniae also responds to iron-restricted conditions by inducing the synthesis of a specific subset of outer-membrane proteins (OMPs) (Deneer & Potter, 1989
; Niven et al., 1989
; Soltes & MacInnes, 1994
; M. Archambault, personal communication), including two membrane-bound transferrin-specific receptors called TbpA and TbpB (Gerlach et al., 1992a
, b
; Gonzalez et al., 1995
). Although it was tempting to speculate that one of these proteins might serve as a receptor for a siderophore, preliminary bioassays by Deneer & Potter (1989)
did not demonstrate any siderophore production in A. pleuropneumoniae. However, it was suggested that A. pleuropneumoniae might obtain iron in vivo directly from host sources in a manner similar to that of Neisseria species, which apparently also do not produce siderophores (Mickelson et al., 1982
; West & Sparling, 1985
). Niven et al. (1989)
were unable to detect hydroxamate and catecholate siderophores in culture supernatants of A. pleuropneumoniae grown under iron-restricted conditions. By contrast, when Diarra et al. (1996)
tested the ability of all serotypes of A. pleuropneumoniae to use different exogenous sources of iron (specifically catecholates and hydroxamates), growth promotion assays showed that all of the A. pleuropneumoniae strains tested (with the exception of one field strain of serotype 5) were capable of using ferrichrome as a growth-promoting substance under iron-limited conditions. They also demonstrated that A. pleuropneumoniae strain 87-682 of serotype 1 and strain 2245 of serotype 5 secreted an iron chelator into the culture medium in response to iron stress. However, this potential A. pleuropneumoniae siderophore had a structure that did not conform to that defined by the well-characterized assay for catechols established by Arnow (1937)
or the assay of Csaky (1948)
for hydroxamates. It is worth noting that some bacteria are known to use siderophores that are produced by other micro-organisms; hence, these bacteria must have the necessary receptors for the assimilation of different siderophores.
Several fungi, including Ustilago sphaerogena, synthesize ferrichrome, a hydroxamate siderophore. The ferric hydroxamate uptake (fhu) system in Escherichia coli is well recognized as one of the paradigms for siderophore transport (Braun, 1995 ; Coulton et al., 1983
; Locher et al., 1998
). The E. coli fhu system consists of four genes, designated fhuA, fhuC, fhuD and fhuB, which are arranged in one operon at minute 3 of the linkage map (Fecker & Braun, 1984
) and transcribed clockwise in the same order. fhuA encodes the multifunctional OMP FhuA (79 kDa) that acts in E. coli as the ferrichrome-iron receptor as well as the receptor for phages T1, T5,
80 and UC-1, for the bacterial toxin colicin M and for some antibiotics, such as albomycin (a structural analogue of ferrichrome) and rifamycin CGP 4832 (Ferguson et al., 2001a
). FhuA is a key player in the fhu system, as it is specific for Fe3+-ferrichrome and functions as a ligand-specific gated channel (Ferguson et al., 1998a
). The elucidation of the high-resolution X-ray crystallographic structure of FhuA from E. coli (Ferguson et al., 1998a
; Locher et al., 1998
) provided a major advance in the understanding of some of the structurefunction relationships of this protein.
The other proteins of the fhu system, namely FhuD, FhuC and FhuB, are also essential to its function. Periplasmic FhuD (31 kDa) and cytoplasmic-membrane-associated FhuC (29 kDa) and FhuB (41 kDa) are proteins necessary for the transport of ferrichrome and other Fe3+-hydroxamate compounds (Fe3+-aerobactin, Fe3+-coprogen) from the periplasm, across the cytoplasmic membrane into the cytoplasm (Braun et al., 1991 ; Coulton et al., 1987
; Mademidis et al., 1997
). The protein complex TonBExbBExbD (Günter & Braun, 1990
; Postle, 1993
) provides energy for this process.
Here, we report that the genome of A. pleuropneumoniae contains an operon with genes homologous to those of the E. coli fhu system, albeit in a different gene order. We also studied the distribution of these fhu genes among the different serotypes of A. pleuropneumoniae and the expression of the gene encoding the OMP receptor FhuA. The structural similarities between FhuA of E. coli and FhuA of A. pleuropneumoniae were deduced by three-dimensional modelling.
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METHODS |
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Genetic techniques.
Plasmids from the PhoA-positive colonies were isolated using the Plasmid QIAprep Spin Miniprep Kit (Qiagen). The A. pleuropneumoniae DNA insert was sequenced using the oligonucleotide primer foA (Table 2), which hybridizes to the negative strand of phoA. Subsequent primers (F7 and R7; Table 2
) were designed from within the plasmid sequence of the positive clone of relevance to this study. An oligonucleotide primer (FA1; Table 2
) based on a region of promoter sequences for fhuA from E. coli was also designed. PCR was carried out with standard conditions and varying annealing temperatures, depending on the sequence of the primers used. When the expected PCR product was larger than 5·0 kb, the Expand Long Template PCR System 1 (Roche Diagnostics) was used in the reaction, rather than Taq polymerase. DNA sequencing of the PCR product was performed at the DNA Sequencing Core Facility of the University of Maine, by using an ABI model 373A stretch DNA sequencer (Applied Biosystems). Single-stranded synthetic oligonucleotides (Table 2
) were synthesized by BioCorp. Various PCR products were then digoxigenin (DIG)-labelled using the DIG DNA Labelling and Detection Kit (Roche Diagnostics) and used as DNA hybridization probes in Southern blots and plaque-lift assays. Chromosomal DNA was extracted by the method of Pitcher et al. (1989)
. For Southern-blotting experiments, the genomic DNA of the A. pleuropneumoniae serotypes was digested with different enzymes, run on a 0·7% agarose gel and then transferred to positively-charged nylon membranes (Ausubel et al., 1998
). Conditions of high stringency were applied. Hybridization of the DIG-labelled DNA probes was detected by using phosphatase-labelled anti-DIG antibodies and revealed colorimetrically with the use of NBT-BCIP as substrate.
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Analysis of sequence homology and protein localization.
DNA sequence analysis was carried out with the aid of programs from University of Wisconsin Genetics Computer Group Software (Devereux et al., 1984 ) using the National Center for BiotechnoIogy Information database (http://www.ncbi.nlm.nih.gov). The multiple-sequence alignment of proteins employed the CLUSTAL alignment algorithm, provided by the Baylor College of Medicine Search Launcher (http://dot.imgen.bcm.tmc.edu:9331/multi-align/multi-align.htm). Prediction of protein localization and cleavage sites for signal sequences was carried out by applying the method of Nakai & Kanehisa (1991)
.
Expression of recombinant histidine-tagged FhuA.
We elected to amplify the coding sequences (amino acids 1668) of mature FhuA using primers AFor and ARev (Table 2); these primers were designed to maintain the reading frame of the fhuA gene. The PCR product was purified using a PCR Purification Kit (Qiagen), ligated into the pGEM-T Easy Vector (Promega) and then transformed into E. coli XL-1 Blue cells. The recombinant plasmid, pLM201, was digested with NotI, and the resulting NotI fragment was cloned directionally and in-frame into the NotI site of the pET30a+ expression vector (Novagen); this was done to append a hexahistidine tag to the amino terminus of FhuA. Plasmid pLM202, carrying the fhuA sequence for mature FhuA with a hexahistidine tag at its amino terminus, was transformed into E. coli DH5
. It was then extracted from DH5
, purified and the orientation and reading frame of the fhuA gene were verified by DNA sequencing. pLM202 was then transformed into E. coli BL21(DE3) (Novagen), the recommended host background for the expression of recombinant proteins in pET vectors. To express the histidine-tagged fusion protein, and following the manufacturers suggested procedure, cells containing the recombinant plasmid were grown in broth culture containing kanamycin and induced with IPTG (0·4, 1·0, 1·5 and 3·0 mM). Various times (2, 4, 6, 8 and 24 h) and temperatures (37 and 25 °C) were tested for the optimal expression of the fusion protein. Whole-cell protein samples were separated by SDS-PAGE following standard procedures; the proteins within the gel were then either stained with Coomassie blue or transferred to a nitrocellulose membrane for immunoblotting (Harlow & Lane, 1999
).
Western blotting.
After the proteins had been transferred, the nitrocellulose membranes were blocked for 1 h with a solution of 1% BSA and then incubated overnight at 4 °C with a mouse-anti-hexahistidine mAb (Roche), a mouse mAb against E. coli FhuA (mAb Fhu6.1) (Moeck et al., 1995 ) or a convalescent serum obtained from a pig that had been experimentally infected with A. pleuropneumoniae serotype 1 (Jacques et al., 1996
). The secondary antibodies used were either a goat-anti-mouse IgG+IgM (heavy+light) or an anti-swine IgGhorseradish peroxidase conjugate (Jackson ImmunoResearch Laboratories). Reactions were revealed by the addition of 4-chloro-1-naphthol and H2O2 (Sigma) to the membranes (Harlow & Lane, 1999
).
Homology model for FhuA of A. pleuropneumoniae.
Using the sequence data for the gene encoding A. pleuropneumoniae FhuA, the predicted amino-acid sequence (residues 1673 of the mature protein) of this protein was submitted to the JIGSAW 3D Protein Homology Modelling Server (http://www.bmm.icnet.uk/servers/3djigsaw) (Bates & Sternberg, 1999 ). The JIGSAW server returned the E. coli FhuA structure, PDB code 1qfg (Ferguson et al., 2000
), as the only successful structural template within its sample space. Visual inspection of this homology model revealed a number of discontinuities within the extracellular- and periplasmic-loop regions, namely C
pairs in which the distances were too great to form a covalent bond. To better model the loops of A. pleuropneumoniae FhuA, anchor regions flanking these discontinuities were submitted to the CODA server (http://www-cryst.bioc.cam.ac.uk/coda/coda.html) (Deane & Blundell, 2001
). CODA models amino-acid sequences of proposed loops by searching against known structures of loop regions within other proteins. The CODA server was able to model all loop regions within the A. pleuropneumoniae FhuA sequence that the JIGSAW server could not. The acceptable r.m.s. (root mean square) deviation was <1·00
. The loops and anchor regions modelled by the CODA server are as follows, 158166, 183191, 228236, 291299, 301308, 405412, 538546, 564572, 597604, 610617 and 630637. This numbering is based on the amino acid residues of the FhuA sequence of A. pleuropneumoniae.
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RESULTS |
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When our A. pleuropneumoniae signal-sequence library, representing approximately 8250 individual colonies in the phoA-negative E. coli strain CC118, was screened for the blue colony phenotype on medium containing 5-bromo-4-chloro-3-indolyl phosphate, 95 colonies were found to be PhoA-positive. One plasmid (pI-25) from the PhoA-positive colonies contained a 375 bp A. pleuropneumoniae insert that was relevant to our objectives. BLASTX analysis of this fragment showed that it displayed 36% identity with and 57% similarity to the Rhizobium leguminosarum FhuD protein (amino acids 2380, out of a total of 301). The 375 bp fragment also showed 35% identity with and 48% similarity to the E. coli FhuD protein (amino acids 2880, out of a total of 296). To obtain the full fhuD sequence from the chromosome of A. pleuropneumoniae serotype 1, oligonucleotide primers (F7 and R7; Table 2) were designed from the 5' and 3' ends of the sequence in pI-25. These were paired with a primer (FA1; Table 2
) that was based on a region of promoter sequences for fhuA from E. coli. A PCR product of 1063 bp was obtained with the primer pair FA1/R7. The deduced amino acids of this amplicon displayed an uninterrupted ORF for a sequence encoding FhuD; this ORF had 27% identity with the protein and 44% similarity to the gene in E. coli.
To isolate larger genomic fragments from A. pleuropneumoniae that contained fhuD and its neighbouring sequences, we used a genomic library of A. pleuropneumoniae serotype 1 strain 4074 DNA made in the ZAP Express phage; the fragments in this library were approximately 712 kb in size. As a screening tool, we labelled the 1063 bp PCR product (i.e. the fhuD sequence) with DIG. From 10 plaques that gave a positive signal with the fhuD probe, one was subjected to three rounds of purification; the plasmid (pLM101) of this positive clone was then excised. Restriction analysis of pLM101 (Fig. 1
) revealed an insert of 8·0 kb. The entire insert was then sequenced with universal primers T3 and T7, which annealed with the vector, and then with internal primers designed from the sequence walking. The total sequence information displayed four different ORFs corresponding to the genes fhuC, fhuD, fhuB and fhuA which appear in the fhu operons of several bacteria, including that of E. coli (Table 3
).
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Sequence of the fhuD region
In A. pleuropneumoniae, the last codon (TGA) of fhuC overlaps with the initiation codon (ATG) of the following gene fhuD, which extends from nucleotide 1656 to nucleotide 2609 (Fig. 1 and Table 3
) this genetic organization matches the overlap of the fhuC and fhuD genes in E. coli (Coulton et al., 1987
). This stretch of nucleotide sequence displays an uninterrupted ORF that terminates with TAA and which encodes a protein whose deduced amino-acid sequence reveals a protein with identity to the FhuD proteins of several bacteria (Table 3
). FhuD is a periplasmic protein that is responsible for transporting ferrichrome from the FhuA receptor in the outer membrane to the FhuB protein in the cytoplasmic membrane (Köster & Braun, 1989
; Moeck et al., 1996
). Using software (Nakai & Kanehisa, 1991
) for the prediction of signal-sequence-cleavage sites, a possible cleavage site was identified and is proposed to be situated at amino acid 47 for FhuD of A. pleuropneumoniae; however, the program failed to characterize this region as an N-terminal signal sequence that is usually displayed by proteins destined for export into the periplasm or outer membrane.
Sequence of the fhuB region
The initiation codon (ATG) for fhuB is situated 8 bp upstream of the termination codon TAA of the preceding gene fhuD in the fhu operon of A. pleuropneumoniae (Fig. 1). The ORF for FhuB extends from nucleotide 2603 to nucleotide 4555 (Fig. 1
and Table 3
) and terminates with a TAA stop codon. The deduced amino-acid sequence of fhuB encodes a protein that displays identity with the FhuB protein of several bacteria (Table 3
). Software for the prediction of protein localization (Nakai & Kanehisa, 1991
) identified the FhuB homologue in A. pleuropneumoniae as being a cytoplasmic-membrane protein with 19 membrane-spanning regions, as compared to the 16 membrane-spanning regions that were predicted for E. coli. The FhuB homologue was also characterized as having an ABC-transporter-family signature sequence (IASGDPRANQLITWT, amino acids 489503).
Sequence of the fhuA region
The last gene in the A. pleuropneumoniae fhu operon is an ORF that commences 46 bp downstream of fhuB, stretching from nucleotide 4602 to nucleotide 6689 (Fig. 1) and terminating with a TAA stop codon. The deduced amino-acid sequence of this ORF encodes a protein which displays identity with the FhuA proteins of several bacteria (Table 3
). Software analysis for the prediction of protein localization (Nakai & Kanehisa, 1991
) identified the putative A. pleuropneumoniae FhuA homologue as an OMP with 11 extracellular loops and 10 periplasmic turns, as well as a cleavable N-terminal signal sequence characteristic of proteins destined for export to the outer membrane. A potential cleavage site for the signal sequence was detected between amino-acid residues 23 and 24. The predicted molecular mass of the mature protein is 74750 Da. A multiple-sequence alignment generated using CLUSTAL W allowed a comparison of the amino termini of three known OMPs of A. pleuropneumoniae, TbpA (Gonzalez et al., 1995
), FhuA (this study) and haemoglobin-binding protein, HgbA (A. Khamessan, personal communication). A stretch of 6 aa residues (shown in bold) that may act as a TonB box is located within the first 13 aa of these three target sequences (TbpA, EQAVQLNDVYVTG; FhuA, QETAVLDEVSVVS; HgbA, QEQMQLDTVIVKD). In other Gram-negative bacteria, the TonB box serves as a site of physical interaction between some outer-membrane receptors and TonB, a protein that delivers the proton motive force of the cytoplasmic membrane to the outer membrane (Moeck & Coulton, 1998
; Postle, 1993
).
Characterization of the fhu chromosomal locus in A. pleuropneumoniae serotype 1
In contrast to the fhu operon in E. coli in which fhuA is at the 5' end, fhuA in A. pleuropneumoniae is preceded by the genes fhuC, fhuD and fhuB. Upstream of fhuC in A. pleuropneumoniae is an incomplete ORF in the opposite direction, separated from the 5' start of fhuC by a 244 bp intergenic region and encoding a protein homologous to E. coli YaaH. This region contains promoter sites for the fhu genes, as well as a potential Fur-binding site for their regulation. A putative TAATTA box at -10 (nucleotides 854859, Fig. 1) and a ShineDalgarno sequence (GGAG; nucleotides 883886, Fig. 1
) were also found in this stretch, along with a consensus sequence (TTTAA) for the -35 region (nucleotides 835839, Fig. 1
). Both the -35 and ShineDalgarno sequences match the consensus sequence proposed for promoter regions in A. pleuropneumoniae (Doree & Mulks, 2001
). The spacing between the -10 and -35 regions was 14 bp, a value which falls within the range of 13 and 16 bp proposed for this region in A. pleuropneumoniae (Doree & Mulks, 2001
). In E. coli, the fur gene product is a negative regulator of iron-dependent genes and the consensus sequence for the Fur box is GATAATGATAATCATTATC. In A. pleuropneumoniae the region upstream of fhuC (nucleotides 833851) and the stretch of 46 non-coding base-pairs that falls between fhuB and fhuA (nucleotides 45984616) both demonstrated putative Fur boxes nine out of 19 of the nucleotides were conserved upstream of fhuC, whereas 10 out of 19 of the nucleotides were conserved upstream of fhuA.
Analysis of the DNA sequence immediately downstream of the A. pleuropneumoniae fhuA gene shows that this region displays similarity with a hypothetical protein from Neisseria meningitidis Z2491 (NMA0986; Parkhill et al., 2000 ), which is transcribed in the same direction as the fhu genes. Four-hundred base-pairs downstream of fhuA a homologue to a Haemophilus influenzae protein is encoded (phospho-ribosyl-aminoamidazole-succinocarboxamide synthase), which is divergently transcribed.
Distribution of the fhu genes among A. pleuropneumoniae serotype reference strains
To determine if the fhu operon was unique to the A. pleuropneumoniae serotype 1 reference strain or if it was widely distributed among the different serotypes of this organism, samples of DNA from reference strains representing serotypes 212 of A. pleuropneumoniae were investigated by PCR. PCR amplification was carried out with primers 5T3W1 and 5T7W2 (Table 2), which flank the fhu region in A. pleuropneumoniae serotype 1, and the Expand Long Template PCR System 1. Reference strains from serotypes 2, 6, 8, 9, 10, 11 and 12 all showed (Fig. 2
) the expected 6·0 kb PCR product, identical to the one observed for serotype 1. Using primers 5T3W1 and 5T7W2 for PCR, some serotype strains yielded a negative PCR result for the entire fhu operon. To investigate these serotype strains further, amplification of the fhu operon was attempted with primers internal to this operon (CFor and ARev; Table 2
). Using primers CFor and ARev, the expected PCR product of 5·7 kb in size was obtained for the serotype 3 strain, confirming that the fhu operon is present in this serotype and with an arrangement of fhu genes similar to that seen in the serotype 1 strain. As the reference strains from serotypes 4, 5 and 7 produced a negative PCR result when CFor and ARev were used as primers, these serotypes were investigated with respect to the individual genes within the fhu operon both by PCR and Southern blotting. Pairs of primers internal to each gene of the fhu operon were used for PCR (AFor/ARev for fhuA, BFor/BRev for fhuB, CFor/CRev for fhuC and DFor/DRev for fhuD; Table 2
). To elucidate the arrangement of the fhu genes in serotypes 4, 5 and 7, we performed PCR amplifications using various combinations of primers. For the Southern-blot analyses, EcoRI-digested genomic DNA from these serotypes was tested for hybridization to a DIG-labelled PCR product for each gene. Reference strains from serotypes 4, 5 and 7 tested positive for the presence of the fhuA, fhuB, fhuC and fhuD genes both in PCR and Southern-blot analyses, although the conditions of stringency had to be lowered to obtain a PCR amplicon for the fhuA and fhuD genes and a positive hybridization signal for fhuD this may be due to some dissimilarity in the fhuD and fhuA gene sequences between serotype 1 and serotypes 4, 5 and 7.
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Protein expression and Western blotting
The fhuA gene of the A. pleuropneumoniae serotype 1 strain was cloned into the expression vector pET30a+ to yield pLM202 (Table 1). pLM202 was then transformed into E. coli BL21 cells. The E. coli BL21(pLM202) cells were grown under various conditions to optimize the expression of recombinant FhuA with the amino-terminal histidine tag, i.e. we tested different concentrations of IPTG and varied the parameters of time and temperature. Whole-cell samples of E. coli BL21(pLM202) were run on 8 or 12·5% SDS-polyacrylamide gels and the gels were subsequently stained with Coomassie blue. A protein of approximately 79 kDa in size showed the highest level of expression at 25 °C (Fig. 3a
). Induction with IPTG concentrations higher than 1·0 mM and induction periods longer than 6 h did not further enhance the expression of recombinant FhuA. The proteins from the gels were then transferred to a nitrocellulose membrane and blotted with an anti-hexahistidine mAb which reacted specifically with the 79 kDa protein, namely A. pleuropneumoniae FhuA with an N-terminal histidine tag (Fig. 3b
). We then tested mAb Fhu6.1 (Moeck et al., 1995
) (Fig. 3c
), which recognizes a linear epitope between amino acids 241 and 281 of E. coli FhuA, against the 79 kDa protein. For both Western blots, a purified FhuAhexahistidine of E. coli (Moeck et al., 1996
) served as a positive control and uninduced E. coli BL21(pLM202) cells served as a negative control. We also tested a polyclonal antiserum taken from a pig infected experimentally with A. pleuropneumoniae serotype 1 and were able to show that the 79 kDa protein also reacted with this immune serum (Fig. 3d
).
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DISCUSSION |
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A. pleuropneumoniae, a Gram-negative bacterium that is an important swine pathogen, is capable of using transferrin, haemoglobin, haemin and exogenous siderophores, including ferric hydroxamates, as sources of iron for growth. The transferrin receptor complex includes TbpA and TbpB in the outer membrane of A. pleuropneumoniae (Deneer & Potter, 1989 ) as well as the exbB and exbD genes upstream of tbpA and tbpB in the same operon (Tonpitak et al., 2000
). The objectives of the present study were to elucidate the genes and products involved in the uptake of ferric hydroxamates in A. pleuropneumoniae. We successfully cloned the homologues of the E. coli fhuACDB operon from A. pleuropneumoniae. In A. pleuropneumoniae the proteins encoded by this operon are: FhuA, a 77 kDa OMP that acts as the receptor for ferric hydroxamate; FhuD, the periplasmic protein responsible for the translocation of ferric hydroxamate from the outer to the inner membrane; and FhuC and FhuB, cytoplasmic-membrane-associated proteins that are components of an ABC transporter which internalizes ferric hydroxamate. An ABC-transporter-family signature sequence was identified for the FhuC and FhuB proteins of A. pleuropneumoniae. In other Gram-negative bacteria FhuB is a hydrophobic protein that is embedded in the cytoplasmic membrane. It is twice the size of hydrophobic proteins usually found in periplasmic binding transport systems (Linton & Higgins, 1998
) and displays an internal amino-acid-sequence homology between its N-terminal and C-terminal halves. This internal homology led Köster & Braun (1989)
to suggest that fhuB originated from the duplication of an ancestral gene, with the two DNA fragments fusing to form a single gene. The A. pleuropneumoniae FhuB homologue is the same size as its E. coli counterpart, and comparison of the two halves of the fhuB sequence of A. pleuropneumoniae revealed 64% sequence identity, hence suggesting similar ancestry. Southern blot and PCR analyses showed that all of the genes of the fhu operon are present in all of the reference strains tested here, which represented serotypes 112 of A. pleuropneumoniae. However, testing for the individual fhuC, fhuD, fhuB and fhuA genes revealed that although they are present individually in the reference strains of serotypes 4, 5 and 7, the sequences of fhuA and fhuD are somewhat different in these serotypes compared to the sequences of these genes in the other nine serotypes studied. Nevertheless, it appears that in all of the serotypes of A. pleuropneumoniae studied, the fhuC, fhuD, fhuB and fhuA genes are arranged in the same order as seen in the serotype 1 reference strain 4074.
The genes involved in the uptake of ferrichrome in A. pleuropneumoniae are arranged differently than their corresponding genes in E. coli. In the latter, the fhu receptor gene fhuA is located upstream of fhuCDB, whereas in A. pleuropneumoniae it is the last gene transcribed in the fhu operon. This difference in gene organization between the two species may reflect differences in the regulation of their iron-transport systems. To explore this possibility, further analyses are needed on the sequences upstream of fhuC and fhuA in A. pleuropneumoniae. The G+C content of the fhu operon in A. pleuropneumoniae is 44 mol%, a value that correlates well with the estimated 43·2 mol% reported (Pohl et al., 1983 ) for the genome of A. pleuropneumoniae. Interestingly, Galindo et al. (2001)
have reported that the fhuA gene in the fhuABD operon of Campylobacter jejuni is strikingly GC-rich (65 mol%) compared to the C. jejuni genome (35 mol%).
The predicted primary sequence of FhuA from A. pleuropneumoniae has allowed us to propose a homology-based three-dimensional model for this protein (Fig. 5b). The overall fold of the A. pleuropneumoniae FhuA model resembles that of E. coli FhuA, for which the crystal structure is known (Ferguson et al., 1998a
; Locher et al., 1998
), with the most significant deviations from the known structure occurring in the extracellular- and periplasmic-loop regions. Overall, the sizes of the extracellular loops of the FhuA model are similar to the corresponding loops in the E. coli structure. However, significant differences between the lengths of two key extracellular-loop regions were observed. Loop L3 in the E. coli structure is 31 residues long compared to 35 residues for the same loop in the A. pleuropneumoniae FhuA model, a difference due to an extension of the loop at its N-terminal end. Furthermore, L4 in the A. pleuropneumoniae FhuA model is considerably longer than the E. coli L4 region (28 residues compared to 20 residues) and does not contain the short ß-strands that are seen in the E. coli FhuA structure. Given that loops L3 and L4 are involved in ligand recognition and uptake (Ferguson et al., 1998a
), it remains to be determined whether these structural variations correspond to differences in function of FhuA from A. pleuropneumoniae relative to the E. coli protein. These differences could also prove to be responsible for the lack of susceptibility of A. pleuropneumoniae to the antibiotics albomycin and rifamycin CGP 4832 and to the bacterial toxin colicin M, all of which use FhuA as a docking site for entry into E. coli bacterial cells.
The X-ray crystallographic structure of E. coli FhuA complexed with ferricrocin indicates that 10 residues of this protein are within 4 of the bound ligand atoms (Ferguson et al., 2001b
). Inspection of the structure-based alignment of A. pleuropneumoniae FhuA with the primary sequence of the E. coli protein (Fig. 4
) shows that six out of 10 positions are highly homologous. Absolute conservation is observed for residues Y292 and Y294 of the A. pleuropneumoniae protein, which align with E. coli ligand-binding residues Y313 and Y315. Residues F92 and F224 share the aromatic character of the aligned E. coli residues Y116 and W246. A position of hydrophobicity is maintained at position L364, which aligns with F391 in the E. coli sequence. Finally, S222, which has the potential to hydrogen bond to the siderophore ligand, is aligned with E. coli residue Y244. Interestingly, two of these six residues retain an aromatic character relative to their homologous E. coli ligand-binding residues, yet they do not have the ability to form hydrogen bonds with a siderophore ligand. This suggests either that the mode of ligand binding for the A. pleuropneumoniae protein may have hydrophobic-stacking interactions as a more predominant component or that the hydrophobic component of these residues in the E. coli protein may be more critical to ligand binding than to their ability to form hydrogen bonds. Functional analysis of the ligand-binding ability of a mutant E. coli FhuA protein in which the two aromatic residues have been modified to resemble those in the A. pleuropneumoniae sequence (Y116F, W246F) may resolve this issue.
The structure of E. coli FhuA also possesses a stretch of highly conserved residues along the longitudinal axis of the inner wall of the ß-barrel that is thought to form a staircase, potentially to facilitate siderophore transfer from the ligand-binding site to the periplasm (Ferguson et al., 1998a , 2001a
). Of these eight staircase residues, four homologous positions are conserved in the A. pleuropneumoniae primary sequence, as indicated by the structure-based alignment in Fig. 4
. With a two-residue shift towards the C terminus, R274 and N276 of the A. pleuropneumoniae protein align with E. coli residues R297 and N299, respectively. Absolute conservation is seen with two charged residues, D331 and D352, which align with D358 and D379, respectively, in the E. coli sequence. Position Q397 in the A. pleuropneumoniae protein aligns perfectly with E. coli staircase residue Q431. Position R333 aligns with E. coli staircase residue Q360. Although at this position there is a charge difference between the two proteins, both side-chains are of approximately the same length, suggesting that the role of this staircase residue may be based more on the steric character of the side-chain than on its charge. It is interesting to note that with the exception of the Q397/Q431 pair, the highest degree of homology in the staircase is clustered at its N-terminal end, proximal to the periplasmic end of the ß-barrel domain.
This is, to the best of our knowledge, the first description of a siderophore receptor in A. pleuropneumoniae. In view of the fact that we now have genetic evidence as well as a three-dimensional model for the OMP FhuA of A. pleuropneumoniae, we can now distinguish different molecular mechanisms of siderophore receptors from different bacterial species and compare structural information between these species.
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ACKNOWLEDGEMENTS |
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Received 14 January 2002;
revised 15 April 2002;
accepted 21 May 2002.