May 11, 1992 - Cells were pelleted,resuspended in sample buffer, ..... V N G. K L F D. K G G. 1531 GTAAATCCTG TATTTACCGT AGATGCGACA ATTAATGGTA ATGGCTTTAT .... in agreement with several recent reports showing the lipid-. 45.
Vol. 60, No. 8
INFECTION AND IMMUNITY, Aug. 1992, p. 3253-3261
0019-9567/92/083253-09$02.00/0 Copyright X 1992, American Society for Microbiology
Characterization of Two Genes Encoding Distinct TransferrinBinding Proteins in Different Actinobacillus pleuropneumoniae Isolatest GERALD-F. GERLACH,* SANDY KLASHINSKY, CAROL ANDERSON, ANDREW A. POTTlER, AND PHILIP J. WILLSON Vetennary Infectious Disease Organization, University of Saskatchewan, 124 Veterinary Road, Saskatoon, Saskatchewan, Canada S7N OWO Received 29 January 1992/Accepted 11 May 1992
The gene encoding the ActinobaciUus pleuropneumoniae serotype 1 transferrin-binding protein (tfbA) was cloned, and the carboxy-terminal 70%o of the protein was expressed as an aggregate protein in Escherichia coli. The nucleotide sequences of the aJbA genes from A. pleuropneumoniae serotypes 7 (G.-F. Gerlach, C. Anderson, A. A. Potter, S. Klashinsky, and P. J. Willson, Infect. Immun. 60:892-898, 1992) and 1 were determined, and a comparison revealed that they had 65% sequence identity. The deduced amino acid sequences showed a sequence agreement of 55%, and both proteins possessed a lipoprotein-like signal sequence. The serotype 1 TfbA protein had a predicted molecular mass of 65 kDa, compared with 60 kDa for the serotype 7 TfbA protein, and both proteins were immunologically distinct as assessed in a competitive enzyme-linked immunosorbent assay. Southern hybridization and Western blot (immunoblot) analysis of the 13 A. pleuropneumoniae type strains revealed that serotypes 2, 3, 4, 8, 9, 10, and 11 encode and express a TfbA protein highly homologous to that ofA. pleuropneumoniae serotype 7 whereas the TfbA proteins and the encoding genes of serotypes 6 and 12 were highly homologous to that found in A. pleuropneumoniae serotype 1. The aJbA genes of A. pleuropneumoniae serotypes 5A and 5B were recognized, under medium-stringency hybridization conditions, by the A. pleuropneumoniae serotype 1-derived tJbA probe, and the respective proteins were weakly reactive with the antibody raised against the A. pleuropneumoniae serotype 7 TfbA protein.
B-lymphocyte mitogens (22), and their potential use as adjuvants has been investigated (5, 31). Also, an increased number of bacterial lipoproteins have been identified in recent years, and several of them have been shown to possess binding and/or metabolic functions (4, 11, 12). Several bacterial species, among them A. pleuropneumoniae, can use transferrin of their natural host as the only source of iron (9, 15, 30, 35). This ability correlates with the binding of transferrin by iron-starved cells (40), and transferrin-binding proteins from Neissera spp., Haemophilus influenzae, Pasteurella haemolytica, and A. pleuropneumoniae have been identified by affinity chromatography or a Western blot (immunoblot)-like assay (9, 23, 30, 35). We have recently described the cloning of a gene (tfbA) encoding a transferrin-binding protein (TfbA) from A. pleuropneumoniae serotype 7 (8). We showed, by Southern blotting, that a possible analogous gene from A. pleuropneumoniae serotype 1 is substantially different. In order to further investigate these differences, we have cloned the tfbA gene from an A. pleuropneumoniae serotype 1 isolate. We have determined and compared the nucleotide sequences of both genes as well as the deduced amino acid sequences. Also, we have determined, by Southern and Western blotting, which type of transferrin-binding protein occurs in the A. pleuropneumoniae type strains.
Actinobacillus pleuropneumoniae is the etiologic agent of porcine pleuropneumonia (36). It causes disease ranging from peracute to chronic, with infected pigs typically showing a hemorrhagic, necrotizing pneumonia often associated with fibrinous pleuritis. Pigs which survive the infection develop a protective immunity but may suffer from chronic lesions and become subclinical carriers of the pathogen (37). A. pleuropneumoniae is host specific and transmitted among pigs in close contact or by aerosol. The pathogen is encountered worldwide, and its prevalence has increased over the last decade, reflecting, in part, intensified production conditions. To date, 12 serotypes of A. pleuropneumoniae have been identified (10, 25-29). Multilocus enzyme electrophoresis has been performed on multiple isolates and indicates a clonal origin for the different serotypes (24). Also, immunization studies have shown that the vaccination of pigs with a bacterin obtained from one serotype will protect against infection with strains from the same serotype but not from other serotypes (16). Lipoproteins are commonly found in association with bacterial cell membranes (17, 32). Characteristic for lipoproteins is the sequential modification and processing by glyceryl and O-acyltransferase, signal peptidase II, and N-acyltransferase resulting in the mature protein with a triple-acylchain modification of its amino-terminal cysteine residue (13). This processing of lipoproteins is prevented by globomycin, a specific inhibitor of signal peptidase II (6). Lipoproteins have been shown to act as potent antigens and *
MATERIALS AND METHODS Bacterial strains, plasmids, and media. The bacterial strains and recombinant plasmid constructs used in this study are listed in Table 1. The expression vectors pGH432 and pGH433 contain a tac promoter, a translational start site with restriction enzyme sites allowing ligation in all three
Corresponding author.
consent of the director of the Veterinary Infectious Disease Organization as Journal Series no. 142.
t Published with the
3253
3254
GERLACH ET AL.
INFECT. IMMUN. TABLE 1. Bacterial strains and plasmids used in this study
Strain or plasmid
Genotype and characteristics
E. coli
HB1O1 ...................... JM105 .......................
hsdS20(rB- MB-) supE44 recA13 hsdR4 A(lac-pro) [F' lacIqZAM15]
A. pleuropneumoniae
AP205 ......................
Serotype 7 porcine lung isolate provided by M. L. Chepok, Modern Veterinary Products, Omaha, Nebr. Serotype 1 porcine lung isolate obtained from the Western College of Veterinary
AP37 ......................
Medicine, Saskatoon, Saskatchwan, Canada
ATCC 27088 ...................... ATCC 27089 ...................... ATCC 27090 ...................... ATCC 33378 ...................... ATCC 33377 ...................... L20 ...................... ATCC 33590 ......................
WF83 ...................... 405 ...................... CVJ13261 ...................... D13039 ...................... 56153 ...................... 8329 ......................
Type strain, serotype 1 Type strain, serotype 2 Type strain, serotype 3 Type strain, serotype 4 Type strain, serotype 5A Type strain, serotype SB, provided by R. Nielsen, State Veterinary Serumlaboratory, Copenhagen, Denmark Type strain, serotype 6 Type strain, serotype 7, provided by S. Rosendahl, University of Guelph, Guelph, Ontario, Canada Type strain, serotype 8, provided by R. Nielsen Type strain, serotype 9, provided by R. Nielsen Type strain, serotype 10, provided by R. Nielsen Type strain, serotype 11, provided by R. Nielsen Type strain, serotype 12, provided by R. Nielsen
Plasmids
pTF205/E1 ...................... pTF205/E2
Encoding the TfbA protein from A. pleuropneumoniae AP205 ..BgII-BamHI deletion derivative of pTF205/E1 encoding the full-length TfbA protein from A. pleuropneumoniae AP205
Initial plasmid clone encoding the TfbA protein from A. pleuropneumoniae AP37 pTF37 ...................... pTF37/E1 .......................NsiI-SspI-fragment of pTF37 cloned into NsiI-SmaI-restricted pTF205/E1 Styl fragment of pTF37/El, blunt ended with the Klenow fragment and ligated into pTF37/E13 ......................
SmaI-restricted pGH433
reading frames followed by stop codons in all three reading frames (1). A. pleuropneumoniae strains were grown on PPLO medium (Difco Laboratories, Detroit, Mich.) supplemented with 3-NAD (10 mg/liter; Sigma Chemical Co., St. Louis, Mo.). Iron restriction was obtained by adding 2,2'-dipyridyl (Sigma Chemical Co.) to a final concentration of 100 ,uM. Escherichia coli transformants were grown in Luria medium (20) supplemented with ampicillin (100 ,ug/ml). Transcription from the tac promoter was induced by the addition of isopropylthiogalactopyranoside (IPTG; 1 mM final concentration). Preparation of DNA and Southern blotting. Genomic DNA was prepared by sodium dodecyl sulfate (SDS)-facilitated freeze-thawing-induced lysis as described previously (38). Plasmid DNA was prepared from chloramphenicol (100 ,ug/liter)-amplified cultures by alkaline lysis and cesium chloride-ethidium bromide gradient centrifugation (20). All restriction endonuclease digests were done in T4 DNA polymerase buffer (20) supplemented with 1 mM dithiothreitol and 3 mM spermidine. Digested DNA was separated on 0.7% agarose gels and transferred onto nitrocellulose by capillary blotting. 32P-labelled probes were prepared by random priming (7), and unincorporated nucleotides were removed by passage through a Sephadex G-50 column. Filters were prehybridized in 5x Denhardt's solution-6x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate [pH 8])-0.5% SDS) at 65°C. Filters were hybridized in the same solution at 55°C and washed at 550C in 3x SSC-0.5% SDS (low stringency), at 65°C in lx SSC-0.5% SDS (medi-
um stringency), or at 650C in O.lx SSC-0.5% SDS (high
stringency). Preparation of aggregate protein and determination of protein concentrations. Aggregate protein was prepared by detergent lysis from IPTG-induced E. coli transformants as previously described (8). The concentration of the protein preparations was determined by separating serial dilutions of the protein by SDS-polyacrylamide gel electrophoresis (PAGE) and staining with Coomassie blue. The intensity of the bands was compared with those of a bovine serum albumin standard (Pierce Chemical Co., Rockford, Ill.). Preparation of antisera. Porcine serum against A. pleuropneumoniae serotype 1 was raised by immunizing pigs with the anionic fraction of a sodium chloride extract (19). The extract was emulsified with Emulsigen (MDP Laboratories, Ralston, Nebr.) as an adjuvant and injected twice, with a 3-week interval, intramuscularly. Porcine convalescent sera were obtained from pigs experimentally infected by aerosol with A. pleuropneumoniae AP37 or AP205. Sera against the recombinant A. pleuropneumoniae TfbA proteins were raised in mice by intraperitoneal injection of 30 ,ug of guanidine hydrochloride-dissolved protein in complete Freund's adjuvant and two subcutaneous boosts with 30 ,ug of protein in incomplete Freund's adjuvant. Enzyme-linked immunosorbent assay (ELISA) and competitive ELISA. To show transferrin binding of the E. coli pTF37/E1 transformants, plates were coated overnight at 4°C with 10 pLg of porcine transferrin per well, blocked with 0.5% gelatin in washing buffer (150 mM NaCl, 10 mM Tris-HCl [pH 8.0], 0.05% Tween 20), and washed. E. coli
VOL. 60, 1992
transformants were grown overnight in the presence of 10 ,umol of IPTG, collected by centrifugation, resuspended in 1/20 volume of washing buffer, exposed to one freeze-thaw cycle, and sonicated for 40 s. A serial dilution of this lysate was incubated for 1 h at room temperature in the transferrincoated plates. Subsequently, the plates were washed and developed with serum raised against the TfbA aggregate protein prepared from E. coli pTF37/E13 transformants, alkaline phosphatase-labelled conjugate (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Md.), and p-nitrophenyl phosphate (15 mM in 1 M diethanolamine [pH 9.8]0.5 mM MgCl2) as a substrate. The color development was measured at 405 nm. For the competitive ELISA, plates were coated overnight at 4°C with guanidine hydrochloride-solubilized recombinant TfbA protein at a concentration of 1 p,g/ml, blocked with 0.5% gelatin in washing buffer, and washed. Mouse serum raised against the recombinant proteins and porcine convalescent-phase serum were titrated, and the highest dilution still resulting in the maximum optical density at 405 nm after an incubation at 37°C for 30 min was incubated with various dilutions of the recombinant TfbA proteins for 1 h at room temperature in washing buffer. This mixture was transferred onto the coated plates, incubated for 1 h at room temperature, and developed as described above. Plates were read at 405 nm, and the concentrations of homologous and heterologous TfbA protein causing 50% inhibition were determined. Western blotting. Whole-cell lysates of A. pleuropneumoniae grown in broth under iron-restricted conditions were separated by SDS-PAGE (18) and electroblotted onto nitrocellulose (39). Unspecific binding was blocked by incubation in 0.5% gelatin in washing buffer. Subsequently, mouse serum raised against the recombinant TfbA proteins and alkaline phosphatase-labelled goat anti-mouse conjugate, both in washing buffer, were added, and each was incubated for 1 h at room temperature. The blots were developed with a substrate containing Nitro Blue Tetrazolium and 5-bromo4-chloro-3-indolyl phosphate. Intrinsic radiolabelling with 3H-palmitic acid and globomycin treatment. Labelling was done essentially as described by Ichihara et al. (17). Briefly, 9,10-3H-palmitic acid (55 Ci/mM) in toluene (Amersham Corp., Arlington Heights, Ill.) was lyophilyzed and dissolved in isopropanol to a concentration of5 mCi/ml. A. pleuropneumoniae AP205 (in PPLO broth) and E. coli transformants (in Luria broth containing 1 FM IPTG) were grown with shaking to an optical density at 660 nm of 0.4, palmitic acid was added (10 [LI/ml), and growth was continued for 1 h. The cultures were precipitated with 10% (vol/vol) trichloroacetic acid, and the pellets were washed with methanol and resuspended in SDS-PAGE sample buffer. After SDS-PAGE, gels were fixed, treated with Amplify (Amersham Corp.), dried, and exposed to X-ray film. Globomycin (a gift from M. Arai, Sankyo Co., Tokyo, Japan) was dissolved in 50% dimethyl sulfoxide at a concentration of 10 mg/ml. It was added to an A. pleuropneumoniae AP205 culture grown under ironlimiting conditions to an optical density at 660 nm of 0.6 to a final concentration of 100,ug/ml, and growth was continued for 1 h. Cells were pelleted, resuspended in sample buffer, and analyzed by SDS-PAGE and Western blot using a TfbA-specific serum. Preparation and screening of the A. pleuropneumonwae
serotype
1 expression library. Genomic DNA from A. pleu-
ropneumoniae AP37 was partially digested with the restriction endonuclease Sau3AI. Fragments of 3 to 8 kb were isolated by sucrose gradient centrifugation (20) and ligated
TRANSFERRIN-BINDING PROTEIN
3255
C1)> -
pTF205/E1
z
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FIG. 1. Physical map of plasmids pTF205/El, pTF205/E2, pTF37/E1, and pTF37/E13. Vector sequences are represented by the thick lines, A. pleuropneumoniae AP205-derived sequences are shown by the thin lines, and A. pleuropneumoniae AP37 sequences are indicated by the hatched bars. The vertical arrows labelled P,ac indicate the position of the tac promoter, and the asterisk-labelled arrows indicate the location of stop codons in all three reading frames. into the BamHI and BglII sites of the expression vectors pGH432 and pGH433, thus allowing for fusions in all three reading frames. E. coli JM105 was transformed and plated at a density of approximately 400 colonies per plate. Colonies were replica plated onto nitrocellulose disks, induced for 2 h with 1 mM IPTG, and lysed in chloroform vapor. Unspecific binding was blocked with 0.5% gelatin in washing buffer, and after removal of the cellular debris, the membranes were incubated with porcine serum raised against the anionic fraction of a sodium chloride extract and developed with goat anti-porcine conjugate and substrate as described above. Transposon mutagenesis. The transposon TnphoA, carried by a lambda phage, and the phosphatase-negative E. coli strain CC118 were kindly provided by J. Beckwith, Harvard Medical School, Boston, Mass. The mutagenesis was performed as described by Manoil and Beckwith (21), and the nucleotide sequence at the insertion site was determined by using an oligonucleotide primer complementary to the first 20 bases of the phoA gene in TnphoA (3, 21).
3256
GERLACH ET AL.
INFECT. IMMUN.
Sau3AI 1 GATCACAATG CCAATATTAA CCCAATCTAT TCCACTTGAA TTACCAACCT CCAGTATTGA GAAAAAAGAT GAGCCAAAAG ATATCTTCAG
91 AGTGGCGATT AATCCTACGG GCATTTATTT AGGCGAGAAG CTAGTGAATG AAGAAGAATT AAAACAATCT TTTCTGACAA AATTTCAGGA 181 AAATAAAAAT ACCGTTATTG CTATTTCTGC GGATATTTCC GTGGAATATC AACATATCGT GAAAGTCCTT GAATTAGCTC AAAACGTCGG
NsiI
271 GCTAACGAAA ATAGGCTTTG TGACTCACCT AGTAAATAAA AGCAGAAATT TTATATTGGA GGCAATATGC ATTTTAAACT TAATCCCTAT M SD HF K L N P Y Y
361 GCGTTAGCGT TTACTTCGCT GTTTCTTGTC GCTTGTTCTG GCGGAAAAGG AAGTTTTGAT TTAGAAGATG TCCGGCCTAA TAAGACAACA A L A F T S L F L V A C S G G K G S F D L E D V R P N K T T Y
451 GGCGTGTCTA AAGAGGAGTA CAAGGATGTA GAAACAGCCA AGAAAGAAAA AGAACAGTTA GGGGAATTAA TGGAACCTGC TTTGGGGTAT G V S K E E Y K D V E T A K K E K E Q L G E L M E P A L G Y 541 GTTGTAAAAG TTCCGGTGAG TTCTTTTGAA AATAAGAAAG TTGATATTTC AGATATAGAA GTGATTACGA ACGGAAATTT AGACGATGTG V V K V P V S S F E N K K V D I S D I E V I T N G N L D D V 631 CCGTACAAGG CAAATTCATC TAAATATAAC TATCCAGATA TAAAAACAAA AGATTCTTCT CTTCAGTACG TTCGCTCAGG ATATGTTATT P Y K A N S S K Y N Y P D I K T K D S S L Q Y V R S G Y V I V
721 GAGTGGGAAC ACTCTGGTTC TAATGAAAAG GGATATGTGT ATTATAAAGG TAATTCACCT GCAAAAGAAT TACCCGTTAA TCAGCTTTTA H S G S D G E N E K G Y V Y Y K G N S P A K E L P V N Q L L 811 ACTTATACAG GAAGTTGGGA TTTTACTTCC AATGCGAATT TAAATAATGA AGAGGGAAGA CCTAATTATT TAAACGACGA TTATTATACT T Y T G S W D F T S N A N L N N E E G R P N Y L N D D Y Y T 901 AAATTTATAG GTAAACGGGT GGGCTTGGTT TCGGGAGATG CGAAACCTGC AAAGCATAAA TACACTAGCC AGTTTGAAGT TGATTTTGCA K F I G K R V G L V S G D A K P A K H K Y T S Q F E V D F A 991 ACTAAAAAAA TGACAGGTAA ATTATCCGAT AAAGAGAAAA CGATTTATAC AGTCAATGCT GATATTAGAG GCAATCGTTT TACGGGGGCT T K K M T G K L S D K E K T I Y T V N A D I R G N R F T G A 1081 GCTACAGCGA GTGATAAAAA TAAAGGGAAA GGCGAATCAT ATAACTTCTT TAGTGCCGAT TCTCAGTCTT TAGAAGGCGG CTTCTATGGT A T A S D K N K G K G E S Y N F F S A D S Q S L E G G F Y G V
1171 CCAAAAGCAG AAGAAATGGC AGGGAAATTT GTAGCTAACG ACAAATCTCT TTTTGCCGTT TTTTCAGCAA AACACAATGG CTCTAATGTT P K A E E M A G K F V A N D K S L F A V F S A K H N G S NV
HpaI
1261 AACACCGTTC GGATTATTGA TGCCTCAAAA ATTGATTTAA CTAATTTCAG CATTTCAGAA CTTAACAATT TTGGTGATGC TTCCGTTTTA N T V R I I D A S K I D L T N F S I S E L N N F G D A S V L 1351 ATTATTGATG GGAAAAAAAT AAAGCTAGCT GGTAGCGGGT TTACAAATAA GCACACTATT GAAATCAATG GCAAAACAAT GGTAGCCGTA I I D G K K I K L A G S G F T N K H T I E I N G K T M V A V 1441 GCCTGCTGTA GTAATCTGGA ATATATGAAG TTTGGTCAAT TATGGCAACA AGCAGAGGGC GGAAAACCCG AGAATAATAG TTTATTCCTA A C C S N L E Y M K F G Q L W Q Q A E G G K P E N N S L F L 1531 CAAGGCGAA CGTACCGCAAC AGATAAGATG CCAAAAGGCG GAAACTATAA ATATATTGGT ACTTGGGATG CTCAGGTTTC AAAAGAAAAT Q G E R T A T D K M P K G G N Y K Y I G T W D A Q V S K E N 1621 AACTGGGTTG CTACGGCAGA TGATGATAGA AAAGCTGGCT ATCGGACAGA ATTTGATGTT GATTTTGGCA ACAAAAATTT AAGTGGTAAG N W V A T A D D D R K A G Y R T E F D V D F G N K N L S G K 1711 TTATTTGATA AAAACGGTGT AAATCCTGTG TTTACCGTAG ATGCAAAAAT TGATGGTAAT GGTTTTACTG GCAAAGCTAA AACCTCAGAT L F D K N G V N P V F T V D A K I D G N G F T G K A K T S D
XbaI
1801 GAAGGCTTCG CTCTAGATTC AGGTAGTTCA CGTTATGAGA ATGTGAAATT TAACGATGTA GCAGTTAGTG GTGGCTTCTA TGGTCCAACG E G F A L D S G S S R Y E N V K F N D V A V S G G F Y G P T 1891 GCAGCAGAGC TTGGCGGACA ATTCCACCAT AAATCAGAAA ATGGCAGTGT AGGTGCTGTC TTTGGTGCAA AACAACAAGT AAAAAAATAA A A E L G G Q F H H K S E N G S V G A V F G A K Q Q V K K * 1981 TAAGGAATTT GCAATGAAAA ATAAATTAAA TCTGATTAGC CTTGCTCTGC TTAGCCTCTT TGCCGTACAA AGCTATGCAG AACAAGCGGT
KpnI
2071 GCAATTGAAC GATGTTTAT GTCACAGGTAC CAAAAAGAAA GCACATAAAA AAGAGAACGA AGTGACAGGC TTAGGGAAAG TAGTGAAAAC 2161 ACCAGATTCT CTTAGTAAGG AGCAAGTGTT AGGAATGCGA GATCTGACTC GCTACGATCC GGGTATTTCT GTAGTAGAGC AAGGACGAGG 2251 TGCAACGACA GGCTACTCAA TTCGTGGGGT AGATCGTAAT CGTGTGGGCT TGGCATTAGA CGGTTTGCCA CAGATTCAAT CCTATGTAAG 2341 TCAATATTCA CGTTCCTCAA GCGGTGCCAT TAATGAAATA GAATACGAAA ATCTGCGTTC GATCCAAATT AGTAAAGGAG CTAGTTCTTC 2431 TGAGTTTGGC AGTGGCTCGC TAGGCGGTTC GGTGCAATTC CGTACCAAAG AGGTAAGCGA CATTATTAAG CCAGGGCAAT CTTGGGGACT 2521 AGATACCAAA AGTGCCTACA GCAGCAAAAA TCAACAATGG TTAAACTCAC TTGCTTTTGC GGGTACTCAC AATGGCTTTG AGTCTCTTGT Sau3AI 2611 GATTTACACT CACCGTGATG GTAAGGAAAC GAAAGCTCAT AAGGATGCAG AAAGCCGTTC TAAGAGTATT CAGAGAGTGG ATC
FIG. 2. Nucleotide sequence of the A. pleuropneumoniae AP205-derived DNA in pTF205/E1 and the deduced amino acid sequence of the TfbA protein. The Sau3AI sites correspond to the insertion sites of the A. pleuropneumoniae-derived sequence in pTF205/El. SD indicates the location of a Shine-Dalgarno consensus sequence. The arrowheads mark the locations of alkaline phosphatase-positive TnphoA fusions. The underlined amino acids are encoded by rare codons.
Nucleotide sequence analysis. DNA sequencing was performed by using M13 vectors and the dideoxy-chain termination method essentially as described by Sanger et al. (34). Nested deletions were prepared by exonuclease III treat-
ment (14), and specific primers were synthesized by using the Pharmacia Gene Assembler. Both strands were sequenced in their entirety. The open reading frame (ORF) of the tfbA gene from A. pleuropneumoniae AP205 was con-
TRANSFERRIN-BINDING PROTEIN
VOL. 60, 1992
b
a 1
2
3 4
3257
1
5
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. .
_:.
-97 -97
i.......
-66
i-45
It
-66
-31 FIG. 3. The TfbA protein from A. pleuropneumoniae AP205 is a lipopeptide. (a) SDS-PAGE showing whole cell lysates from
14C-palmitate-labelled A. pleuropneumoniae AP205 grown under standard (lane 1) and iron-limiting (lane 2) conditions and from E. coli transformants containing pTF205/El (lane 3), pTF205/E2 (lane 4), or pGH433 (lane 5). (b) Western blot, using TfbA-specific serum, of A.
pleuropneumoniae AP205 whole-cell lysates after growth under iron-limiting conditions before (lane 1) and after (lane 2) globomycin treatment. The arrowhead indicates the position of the TfbA precursor appearing after globomycin treatment. The numbers to the right indicate the positions of marker proteins in kilodaltons.
firmed by TnphoA insertion mutagenesis; the ORF of the A. pleuropneumoniae AP37-derived tfbA gene was confirmed by construction of the plasmid pTF37/E13 containing an in-frame fusion to a vector-derived translational start site. Sequences were analyzed by using the IBI/Pustell program and the GenBank data base. Nucleotide sequence accession number. The GenBank nucleotide sequence data base accession numbers for the A. pleuropneumoniae tibA genes are M85275 (derived from AP205) and M85274 (derived from AP37). RESULTS
Cloning and expression of the A. pleuropneumoniae AP37 gene. Immunoreactive colonies were subcultured and screened in a Southern blot, under low-stringency conditions, using the EcoRV-KpnI fragment of pTF205/E2 as a probe (8). One transformant which also reacted with convalescent-phase serum and contained a plasmid of approximately 11 kb hybridizing to the pTF205/E2-derived probe was identified, and the encoding plasmid was designated pTF37. Physical mapping showed that this plasmid contained an NsiI and a Kpnl site, and initial nucleotide sequence analysis revealed that the adjacent sequences were homologous to those in pTF205/E1. Therefore, in order to express the A. pleuropneumoniae AP37-derived TfbA protein, an NsiI-SspI fragment from pTF37 was cloned into NsiI-SmaI-cut pTF205/E1, and the resulting clone was designated pTF37/E1 (Fig. 1). Upon IPTG induction, pTF37/E1 transformants produced TfbA protein as assessed by Western blot but did not form inclusion bodies like pTF205/E1 transformants. Therefore, on the basis of the nucleotide
(bA
sequence analysis (see below), a StyI fragment which encodes for the carboxy-terminal 70% of the protein was cloned in frame into pGH433, thus fusing it to a 5-amino-acid leader peptide provided by the vector. This construct was designated pTF37/E13 (Fig. 1), and E. coli HB101 containing this plasmid produced protein aggregates upon IPTG induction. However, the yield from cells induced at an optical density at 660 nm of 0.5 was only 5 mg/liter, compared with 20 mg/liter obtained from pTF205/E1 transformants under the same conditions. Subsequently, by using serum raised against this aggregate protein, the transferrin-binding ability of the protein encoded by pTF37/E1 was confirmed on whole-cell lysates of E. coli pTF37/E1 transformants in the ELISA described above (data not shown). Analysis of the A. pleuropneumoniae AP205 and AP37derived (fbA genes. The nucleotide sequence analysis of pTF205/E1 revealed one ORF of 1,641 bp coding for a protein with a predicted molecular mass of 59,844 Da (Fig. 2). It is preceded by a Shine-Dalgarno consensus sequence (GGAGG) 4 bp upstream of the methionine codon. The first 19 amino acids of the polypeptide have the characteristics of a lipoprotein signal peptide with a predicted cleavage site in front of the cysteine residue at position 20. The ORF and the presence of a signal peptide were confirmed by four independent TnphoA insertions 85, 148, 409, and 922 bp downstream from the methionine codon which, upon transformation of thephoA-negative E. coli CC118, gave rise to alkaline phosphatase-positive transformants. The predicted signal peptide cleavage site resulting in an amino-terminal cysteine residue of the mature protein was confirmed by labelling of the E. coli transformants with 14C-palmitate in the presence of 1 ,uM IPTG, resulting in a
3258
INFECT. IMMUN.
GERLACH ET AL.
NsiI 1 ATGCATTTTA AACTTAATCC CTATGCGTTA GCGTTTACTT CGCTGTTTCT TGTCGCTTGT TCTGGCGGAA AAGGAAGTTT TGATTTAGAA K G S F D L E S G G S L F L V A C A F T Y A L K L N P W7 F 91 GATGTCCGGC CAAATCAAAC TGCAAAAGCA GAAAAAGCAA CAACCTCTTA TCAAGATGAG GAAACGAAGA AAAAGACAAA GGAAGAATTA E E L E T K K K T K T T S Y A K A E K A P N Q T D V R Q D E
181 GATAAGTTGA TGGAGCCTGC TTTGGGGTAT GAAACTCAAA TTTTACGGCG AAATAAGGCT CCTAAAACAG AAACAGGAGA GAAAAGGAAT K R N E T G E N K A P K T E T Q I L R R L G Y M E P A D K L 271 GAGAGAGTTG TTGAGTTATC CGAAGATAAA ATTACGAAAT TATACCAAGA GAGTGTAGAA ATAATCCCTC ATTTAGATGA GCTAAATGGA H L D E L N G I I P S V E L Y Q E E D K I T K E R V V E L S
B?=TI
361 AAAACAACGA GCAATGATGT TTATCATTCT CACGATAGTA AAAGGCTTGA TAAGAATAGA GATCTCAAAT ATGTTCGTTC AGGTTATGTT G Y V Y V R S K N R'TDL K K R L D H D S Y H S K T T S N D V
StyI
451 TATGATGGGT CTTTCAATGA AATACGACGA AATGACTCAG GATTCCATGT TTTTAAACAG GGTATAGATG GCTATGTCTA TTACCTTGGA G Y V Y G I D F K Q G F H V N D S Y L G I R R S F N E Y D G 541 GTTACTCCAT CAAAAGAGTT ACCAAAAGGA AAAGTCATAA GTTATAAAGG TACTTGGGAT TTTGTAAGTA ACATCAATTT AGAGCGTGAA E R E N I N L F V S T W D S Y K G K V I P K G S K E L V T P 631 ATAGATGGAT TCGACACTTC AGGTGATGGT AAAAATGTAT CTGCAACATC TATTACAGAA ACTGTCAATC GAGATCATAA AGTTGGTGAA V G E T V N R D H K I T E S A T S K N V G D G F D T S I D G 721 AAACTAGGTG ATAATGAAGT TAAAGGGGTA GCTCATTCTA GTGAATTTGC AGTAGATTTT GATAACAAAA AATTGACAGG TAGTTTATAT S L Y D N K K L T G V D F S E F A K G V A H S D N E V K L G 811 CGTAATGGTT ATATCAACAG AAATAAAGCG CAAGAAGTAA CGAAACGCTA TAGCATTGAA GCTGATATTG CAGGCAACCG TTTTAGGGGA F R G A G N R S I E A D I T K R Y N K A Y I N R Q E V R N G 901 AAAGCCAAAG CAGAAAAAGC AGGTGATCCG ATCTTTACTG ATTCAAATTA TCTTGAAGGG GGATTCTATG GTCCTAAAGC TGAAGAAATG E E M G P K A G F Y L E G D S N Y I F T A E K A G D P K A K 991 GCAGGGAAGT TTTTCACAAA TAATAAATCT CTCTTTGCAG TATTTGCAGC TAAAAGTGAA AACGGCGAGA CGACCACAGA ACGAATCATT R I I N G E T T T E K S E V F A A L F A N K S F F T N A G K
1081 GATGCAACTA D A T Bql II 1171 ATAGATCTAG I D L
AAATTGATTT AACCCAATTT AATGCTAAAG AACTCAACAA TTTTGGTGAT GCCTCTGTTT TAATTATTGA TGGACAAAAA G Q K A S V L I I D F G D E L N N N A K T Q F K I D L CAGGTGTCAA TTTTAAAAAT AGTAAAACGG TTGAAATCAA CGGCAAAACA ATGGTAGCCG TAGCTTGCTG TAGTAATCTG S N L V A C C M V A G K T V E I N S K T F K N A G V N
1261 GAATATATGA AATTTGGTCA ATTGTGGCAA AAAGAGGGCA AACAACAAGT TAAAGATAAT AGTTTATTCC TACAAGGTGA ACGTACTGCA R T A S L F L Q G E K D N K Q Q V K E G L W Q K F G Q E Y M 1351 ACGGATAAAA TGCCCGCAGG AGGTAACTAT AAGTATGTTG GAACTTGGGA TGCACTCGTA TCTAAAGGGA CGAACTGGAT AGCGGAAGCA A E A T N W I A L V S K G G T W D K Y V G N Y M P A G T D K 1441 GATAATAATC GAGAATCGGG CTATCGCACT GAATTTGATG TTAATTTTAG TGATAAAAAA GTAAACGGTA AGTTATTTGA TAAAGGCGGT K G G K L F D V N G D K K V N F S Y R T E F D D N N R E S G 1531 GTAAATCCTG TATTTACCGT AGATGCGACA ATTAATGGTA ATGGCTTTAT CGGCAGTGCG AAAACCTCTG ATAGTGGCTT TGCTTTAGAT A L D D S G F K T S G S A N G F I I N G D A T V F T V V N P 1621 GCAGGCTCTA GCCAACACGG AAATGCGGTA TTTAGTGATA TAAAAGTCAA TGGTGGCTTC TATGGTCCAA CCGCTGGAGA ACTTGGCGGA L G G T A G E G G F Y G P I K V N F S D N A V S Q H G A G S 1711 CAATTCCATC ATAAATCAGA CAATGGCAGT GTTGGCGCTG TCTTTGGTGC AAAACGACAA ATAGAAAAAT AATAAGGAAT TTGCTATGAA I E K K R Q V F G A N G S V G A * H K S D Q F H 1801 AAATAAATTA AATCTGATTA GCCTTGCTCT TCTTAGCCTA TTTGCCGTAC AAAGCTATGC AGAACAAGCG GTACAATTAA ATGATGTTTA KpnI 1891 TGTCACAGGT ACC
FIG. 4. Nucleotide sequence of the A. pleuropneumoniae AP37-derived NsiI-KpnI fragment in pTF37/El and the deduced amino acid sequence of the TfbA protein. The underlined amino acids are encoded by rare codons.
labelled 60K protein (Fig. 3a). In addition, it was shown that growth of A. pleuropneumoniae AP205 in the presence of globomycin inhibited processing of the TfbA precursor protein (Fig. 3b). The nucleotide sequence analysis of pTF37/El revealed one ORF of 1,779 bp. It encodes a protein (TfbA37) with a predicted molecular mass of 65,523 Da (Fig. 4). The aminoterminal 35 amino acids are identical to those found in TfbA205. A further comparison with the tfbA205 sequence showed 65% nucleotide sequence identity. A comparative analysis of the codon usage revealed that the tfbA37 gene contained several rare codons (2) such as seven CGA and three AGG codons for arginine and nine ATA codons for isoleucine (Fig. 4). Seven of these codons, among them a block of three rare codons (ATA CGA CGA), were located upstream of the StyI site. In contrast, the tJbA205 gene
contained no CGA or AGG codons and only four ATA codons. The primary amino acid sequences had an overall identity of 55%, with the amino-terminal half of the TfbA205 protein being only 36% identical to its A. pleuropneumoniae serotype 1 counterpart (Fig. 5). These differences in primary amino acid sequence were reflected in differences in antigenicity as assessed in a competitive ELISA. Thus, with recombinant TfbA protein as the surface-bound antigen, approximately 60 times more heterologous than homologous recombinant TfbA protein was required to cause 50% inhibition (Table 2). This degree of inhibition was obtained with both mouse serum raised against the recombinant proteins and porcine convalescent serum. A GenBank data base search with the two predicted amino acid sequences did not reveal any likely similarities (>35%) to known proteins.
3259
TRANSFERRIN-BINDING PROTEIN
VOL. 60, 1992 MHFKLNPYAL AFTSLFLVAC SGGKGSFDLE DVRPNKTTGV SKEEYKDVET AKKEKEQL--
1
2 3
4 5 6 7
8 9 10
11
12 13 14 15
MHFKLNPYAL AFTSLFLVAC SGGKGSFDLE DVRPNQTAKA EKATTSYQDE ETKKKTKEEL GELMEPALGY
-4.0
------ VVKV PVSSFENKKV ----DISDIE VITN------ -----GNLDD
11111111
I I
-I
DKLMEPALGY ETQILRRNKA
PKTETGEKRN
-2.2
ERVVELSEDK ITKLYQESVE IIPHLDELNG
VPYKANSSKY NYPDIKTKDS SLQYVRSGYV IDG------- -----EHSGS NEKGYVYYKG 1111111
I
H
1I
ill1
KTTSNDVYHS HDSKRLDKNR DLKYVRSGYV YDGSFNEIRR NDSGFHVFKQ GIDGYVYYLG
NSPAKELPVN QLLTYTGSWD FTSNANLNNE EGRPNYLNDD ---------- YYTKFIGKRV VTPSKELPKG KVISYKGTWD FVSNINLERE IDGFDTSGDG KNVSATSITE TVNRDHKVGE
-0.6
GLVSGDAKPA KHKYTSQFEV DFATKKMTGK LSDKEKTIYT VNADIRGNRF TGAATASDKN
KLGDNEVKGV AHSSEFAVDF DNKKLTGSLY RNGYINRNKA QEVTKRYSIE ADIAGNRFRG
KGKGE-SYNF FSADSQSLEG GFYGPKAEEM AGKFVANDKS LFAVFSAKH- NGSNVNTVRI
11111111111
lIl l
1111
1
1111111111
11
1 11
KAKAEKAGDP IFTDSNYLEG GFYGPKAEEM AGKFFTNNKS LFAVFAAKSE NGE-TTTERI
-4.0
IDASKIDLTN FSISELNNFG DASVLIIDGK KIKLAGSGFT NKHTIEINGK TMVAVACCSN 11111111
111111
111111111
111111111111111
111111
ATDKMPKGG NIYKYIGTWDA QVSKENNWVA I ER III 11111 11 1111111 11 III LEYMKFGQLW -QKEGKQQVK DNSLFLQGER TATDKMPAGG NYKYVGTWDA LVSKGTNWIA F LEYMKIFG IL QfAEGGK-PE NINSLF 1111 11 -1 11 H IM
TADDDRKAGY RTEFDVDFGN KNLSGKLFDK NGVNPVFTVD AKIDGNGFTG
11 1
I
111111
111111 1111111111
I
I I
-2.2
#
IDATKIDLTQ FNAKELNNFG DASVLIIDGQ KIDLAGVNFK NSKTVEINGK TMVAVACCSN
_db~
rs
_di
KAKTSDEGFA
11 11 11111
III
EADNNRESGY RTEFDVNFSD KKVNGKLFDK GGVNPVFTVD ATINGNGFIG SAKTSDSGFA
~-0.6
LDSGSSRYEN VKFNDVAVSG GFYGPTAAEL GFHHKSEN GSVGAVFGAK OQVKK 111111 111111 1111111 11 I I 11 LDAGSSQHGN AVFSDIKVNG GFYGPTAGEL GGQFHHKSDN GSVGAVFGAK RQIEK
FIG. 5. Comparison of the amino acid sequences of the A. pleuropneumoniae AP205 (serotype 7; top) and AP37 (serotype 1; bottom) TfbA proteins. The horizontal dashes indicate gaps introduced in order to obtain maximal alignment. The vertical lines indicate identical residues.
Distribution of different sfbA genes and TfbA proteins in the A. pleuropneumonuae type strains. Genomic DNA from all A. pleuropneumoniae type strains was analyzed in a Southern blot using the A. pleuropneumoniae AP205- and AP37derived tfbA genes as probes. The NsiI-BglII-restricted DNA from the A. pleuropneumoniae type strains for serotypes 2, 3, 4, 7, 8, 9, 10, and 11 reacted with the pTF205/ El-derived probe under high-stringency conditions (Fig. 6). The DNA from serotypes 1, 6, and 12 reacted with the pTF37/El-derived probe under high-stringency conditions, and the DNA from serotypes 5A and SB was weakly reactive with this probe under medium-stringency washing conditions (Fig. 6). When the NsiI-BglII-restricted DNA from A. pleuropneumoniae serotype 1, 6, and 12 type strains was investigated by Southern blotting after electrophoresis on a 1.2% agarose gel, the single hybridizing band seen after electrophoresis on a 0.7% agarose gel was resolved into two bands corresponding in size to the NsiI-BglII and BgIII fragments of pTF37/El (data not shown). Whole-cell lysates from all A. pleuropneumoniae type strains, grown under iron-restricted conditions, were ana-
FIG. 6. Southern blot analysis of the A. pleuropneumoniae type strains, using NsiI-BglII-restricted DNA. (A) Probed with the NsiIXbaI fragment from pTF205/El; (B) identical to the blot in panel A but hybridized with both the pTF205/E1-derived probe and the NsiI-KpnI fragment from pTF37/E1. Lanes 1 to 13 indicate the positions of the type strains for serotypes 1 (lane 1), 2 (lane 2), 3 (lane 3), 4 (lane 4), 5A (lane 5), SB (lane 6), 6 (lane 7), 7 (lane 8), 8 (lane 9), 9 (lane 10), 10 (lane 11), 11 (lane 12), and 12 (lane 13). Lane 14 contains A. pleuropneumoniae AP37 DNA; lane 15 contains DNA from A. pleuropneumoniae AP205. The numbers to the right indicate the relative positions of size markers in kilobases.
lyzed in a Western blot using the mouse sera raised against the recombinant TfbA proteins from A. pleuropneumoniae AP205 and AP37 (Fig. 7). The binding pattern of both sera reflected that of the respective DNA probes (Fig. 7B and C), and the TfbA proteins from A. pleuropneumoniae serotypes 1 and 7 were shown to have different electrophoretic mobilities consistent with their predicted molecular masses (Fig. 7D). The TfbA proteins from serotypes 5A and 5B were weakly detected by the serum raised against the A. pleuropneumoniae AP205 TfbA protein, and their molecular mass, as assessed by their electrophoretic mobility, was 62 kDa. DISCUSSION Recently, we have described the cloning and expression of a transferrin-binding protein (TfbA) from A. pleuropneumo-
TABLE 2. Results of competitive ELISA showing antigenic differences between the TfbA proteins of A. pleuropneumoniae AP205 (serotype 7) and AP37 (serotype 1)
Origin of ELISA solid-phase TfbA protein
Serum
pTF205/E2 transformants
Pig convalescent (AP205 infection) Mouse anti-TfbA (from pTF205/E2 transformants)
pTF37/E13 transformants
Pig convalescent (AP37 infection) Mouse anti-TfbA (from pTF37/E13 transformants)
Competing antigena (,ug/ml) isolated from E. coli transformants containing:
pTF2O5/E2 1 0.7
>128 64
a Inhibition values are stated as the quantity of antigen required to decrease the reaction of antiserum with solid-phase antigen by 50%.
pTF37/E13 >128 100
1 0.7
3260
,*0-$t =* GERLACH ET AL.
1 2 3 4 5 6 7 8 9 1011 12131415 ......-
-96
T,'>4
*:-6 45 -31
-22
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- -66
- 66
dWesterneblotsn(B,
FIG. 7. Coomassie blue-stained gel (A) an C, and D) of whole-cell lysates from the A. p leuropneumoniae type strains grown under iron-limiting conditions. the positions of the type strains for serotype s 1 (lane 1), 2 (lane 2) 3 (lane 3), 4 (lane 4), 5A (lane 5), 5B (lane 6)), 6 (lane 7), 7 (lane 8), 8 (lane 9), 9 (lane 10), 10 (lane 11), 11 (lane 12), and 12 (lane 13). Lanes 14 and 15 contain A. pleuropneumoi,niae AP37 and AP205 whole-cell lysates. The numbers to the rigl ht indicate the relative positions of marker proteins in kilodaltons. The sera used in the Western blots were specific for the A. pleiuropneumoniae AP205 (serotype 7) TfbA protein (B), specific for th(e A. pleuropneumoniae AP37 (serotype 1) TfbA-protein (B), and a c,ombination of the sera used in panels B and C (D).
niae serotype 7 (8). We showed by Soouthern blot analysis that the A. pleuropneumoniae serotype '7-derived tfbA probe did not hybridize to the genomic DN{A from several A. pleuropneumoniae serotype 1 isolates u nder high-stringency conditions. In order to more closely in,ivestigate this observation, we have now cloned the tff.,,4 gene from an A. pleuropneumoniae serotype 1 clinical is ;olate and expressed its carboxy-terminal portion. To furthi er define the differences between these A. pleuropneumorniae serotype 1- and 7-derived genes, we performed a comr plete nucleotide sequence analysis. We observed that both genes were identical over the first 100 bp (Fig. 5). In both casses the first 19 amino acids encode a sequence with the char acteristics of a lipoprotein signal peptide, and this observ,ation was confirmed by palmitate labelling of E. coli pTF2'05/El transformants and by globomycin treatment of A. pleuropneumoniae AP205 (Fig. 3). The sequence has arn uncommon amino terminus with the residues M-H-F-K ( 13), and it does not possess a marked hydrophobic core. AIso, the sequence at the cleavage point, L-V-A-C-S, shows a valine residue at position 2 which is not commonly fou nd in the consensus sequence for this region (13). The secon Ld residue behind the cleavage point has been shown to be in nportant for the final localization of the protein (41), and tthis residue being a serine is consistent with its outer mermbrane location (8). Also, the lipid-modified nature of the TfbA protein is in
INFECT. IMMUN.
agreement with our previous observation that it could be removed from the outer membrane by detergent treatment (8). In addition, the lipid modification of the TfbA protein is in agreement with several recent reports showing the lipidmodified nature of proteins involved in substrate binding and/or uptake (4, 11, 12). We observed that expression of the entire A. pleuropneumoniae serotype 1 TfbA protein, in the form of inclusion bodies, did not occur in transformants containing the plasmid pTF37/El. This result was unexpected because pTF37/El is a construct in which the A. pleuropneumoniae AP205-derived sequence downstream from the NsiI site of the aggregate-producing plasmid pTF205/El was replaced
with an analogous A. pleuropneumoniae AP37 sequence as determined by restriction enzyme mapping and partial nucleotide sequence analysis (Fig. 1, 2, and 4). However, the complete nucleotide sequence analysis revealed the presence of several rare codons (2) within the ORF of the A. pleuropneumoniae AP37-derived sequence with the triplet ATA CGA CGA at position 472 (Fig. 4). Since this sequence might inhibit high-level translation, a deletion derivative was constructed by using the StyI site at position 534. The downstream sequence was ligated in frame to a leader sequence provided by the vector pGH433. E. coli HB101 transformed with this construct produced inclusion bodies upon IPTG induction with a comparatively low efficacy which may be due to the presence of 11 additional rare codons downstream from the fusion site (Fig. 4).
A comparison of the TfbA sequences fromA. pleuropneumoniae serotype 7 (AP205) and serotype 1 (AP37) revealed that they strongly diverged downstream from the first 35 amino acids. The A. pleuropneumoniae serotype 1-derived TfbA protein possessed several insertions in this region, thus accounting for the differences in molecular mass. Further downstream, starting approximately at the middle of the molecule, both proteins again showed a high degree of sequence agreement. These differences in primary amino acid sequence were substantiated by the competitive ELISA which indicated that both proteins are immunologically distinct (Table 2). The Southern and Western blots of the A. pleuropneumoniae type strains further confirmed these results and indicated that a third distinct TfbA protein is present in the A. pleuropneumoniae type strains SA and SB (Fig. 6 and 7). Since these serological distinctions are also made by convalescent-phase serum (Table 2), it could be that the variation among the different TfbA proteins allows the different A. pleuropneumoniae serotypes to avoid the host immune response against heterologous strains. This would be particularly advantageous since the TfbA protein is the predominantly recognized antigen in a Western blot with convalescent-phase serum (33). The alternative possibility that the different proteins facilitate the binding of different epitopes of the transferrin molecule remains to be investigated. ACKNOWLEDGMENTS This work was supported by operating grant MA-11747 from the Medical Research Council of Canada and by Agri-Food Project 12104-1 of the Canada-Manitoba Economic and Regional Development Agreement. We thank Iris Rugg and Sandra Calver for editorial assistance. REFERENCES 1. Anderson, C., A. A. Potter, and G.-F. Gerlach. 1991. Isolation and molecular characterization of spontaneously occurring cytolysin-negative mutants of Actinobacillus pleuropneumoniae serotype 7. Infect. Immun. 59:4110-4116.
VOL. 60, 1992 2. Cerretti, D. P., D. Dean, G. R. Davis, D. M. Bedwell, and M. Noumura. 1983. The spc ribosomal operon of Escherichia coli: sequence and cotranscription of the ribosomal protein genes and a protein export gene. Nucleic Acids Res. 11:2599-2616. 3. Chang, C. N., W.-J. Kuang, and E. Y. Chen. 1986. Nucleotide sequence of the alkaline phosphatase gene of Escherichia coli. Gene 44:121-125. 4. Chapon, C., and 0. Raibaud. 1985. Structure of two divergent promoters located in front of the gene encoding pullulanase in Kiebsiella pneumoniae and positively regulated by the malT product. J. Bacteriol. 164:639-645. 5. Croft, S., J. Walsh, W. Lloyd, and G. J. Russell-Jones. 1991. TraT: a powerful carrier molecule for the stimulation of immune responses to protein and peptide antigens. J. Immunol. 146:793798. 6. Dev, I. K., R. J. Harvey, and P. H. Ray. 1985. Inhibition of prolipoprotein signal peptidase by globomycin. J. Biol. Chem. 260:5891-5894. 7. Feinberg, A. P., and B. Vogelstein. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6-13. 8. Gerlach, G.-F., C. Anderson, A. A. Potter, S. Klashinsky, and P. J. Willson. 1992. Cloning and expression of a transferrinbinding protein from Actinobacillus pleuropneumoniae. Infect. Immun. 60:892-898. 9. Gonzalez, G. C., D. L. Caamano, and A. B. Schryvers. 1990. Identification and characterization of a porcine-specific transferrin receptor in Actinobacillus pleuropneumoniae. Mol. Microbiol. 4:1173-1179. 10. Gunnarsson, A. 1979. Serologic studies on porcine strains of Haemophilus parahaemolyticus (pleuropneumoniae): extraction of type-specific antigens. Am. J. Vet. Res. 40:407-413. 11. Hanson, M. S., and E. J. Hansen. 1991. Molecular cloning, partial purification, and characterization of a haemin-binding lipoprotein from Haemophilus influenzae type b. Mol. Microbiol. 5:267-278. 12. Hayashi, S., H. Hara, H. Suzuki, and Y. Hirota. 1988. Lipid modification of Escherichia coli penicillin-binding protein 3. J. Bacteriol. 170:5392-5395. 13. Hayashi, S., and H. C. Wu. 1990. Lipoproteins in bacteria. J. Bioenerg. Biomembr. 22:451-471. 14. Henikoff, S. 1987. Unidirectional digestion with exonuclease III in DNA sequence analysis. Methods Enzymol. 155:156-165. 15. Herrington, D. A., and F. P. Sparling. 1985. Haemophilus influenzae can use human transferrin as a sole source for required iron. Infect. Immun. 48:248-251. 16. Higgins, R., S. Lariviere, K. R. Mittal, G. P. Martineau, P. Rousseau, and J. Cameron. 1985. Evaluation of a killed vaccine against porcine pleuropneumoniae due to Haemophilus pleuropneumoniae. Can. Vet. J. 26:86-89. 17. Ichihara, S., M. Hussain, and S. Mizushima. 1981. Characterization of new membrane lipoproteins and their precursors of Escherichia coli. J. Biol. Chem. 256:3125-3129. 18. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 19. Maheswaran, S. K., G. H. Johnson, and B. S. Pomeroy. 1973. Studies on Pasteurella multocida. II. The capsular polysaccharides from turkey isolates. Avian Dis. 17:P705-P716. 20. Maniatis, T., E. F. Fritsch, and J. Sambroolk 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 21. Manoil, C., and J. Beckwith. 1985. TnphoA: a transposon probe for protein export signals. Proc. Natl. Acad. Sci. USA 82:81298133. 22. Melchers, F., V. Braun, and C. Galanos. 1975. The lipoprotein of the outer membrane of Eschenichia coli: a B-lymphocyte mito-
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gen. J. Exp. Med. 142:473-482. 23. Morton, D. J., and P. Williams. 1990. Siderophore-independent acquisition of transferrin-bound iron by Haemophilus influenzae type b. J. Gen. Microbiol. 136:927-933. 24. Musser, J. M., V. J. Rapp, and R. K. Selander. 1987. Clonal diversity in Haemophilus pleuropneumoniae. Infect. Immun. 55:1207-1215. 25. Nielsen, R. 1985. Serological characterization of Haemophilus pleuropneumoniae (Actinobacillus pleuropneumoniae) strains and proposal of a new serotype: serotype 9. Acta Vet. Scand. 26:501-512. 26. Nielsen, R. 1985. Serological characterization of Haemophilus pleuropneumoniae (Actinobacillus pleuropneumoniae) strains and proposal of a new serotype: serotype 10. Acta Vet. Scand. 26:581-586. 27. Nielsen, R. 1986. Serology of Haemophilus (Actinobacillus) pleuropneumoniae serotype 5 strains: establishment of subtypes A and B. Acta Vet. Scand. 27:49-58. 28. Nielsen, R. 1986. Serological characterization of Actinobacillus pleuropneumoniae strains and proposal of a new serotype: serotype 12. Acta Vet. Scand. 26:453-455. 29. Nielsen, R., and P. J. O'Connor. 1984. Serological characterization of 8 Haemophilus pleuropneumoniae strains and proposal of a new serotype: serotype 8. Acta Vet. Scand. 25:96-106. 30. Ogunnariwo, J. A., and A. B. Schryvers. 1990. Iron acquisition in Pasteurella haemolytica: expression and identification of a bovine-specific transferrin receptor. Infect. Immun. 58:20912097. 31. Prass, W., H. Ringsdorf, W. Bessler, K.-H. Wiesmuller, and G. Jung. 1987. Lipopeptides of the N-terminus of Escherichia coli lipoprotein: synthesis, mitogenicity and properties in monolayer experiments. Biochim. Biophys. Acta 900:116-128. 32. Regue, M., and H. C. Wu. 1988. Synthesis and export of lipoproteins in bacteria, p. 587-606. In R. C. Gas and P. W. Robbins (ed.), Protein transfer and organelle biogenesis. Academic Press, Inc., San Diego, Calif. 33. Rossi-Campos, A., C. Anderson, G.-F. Gerlach, S. Klashinsky, A. A. Potter, and P. J. Willson. Immunization of pigs against Actinobacilluspleuropneumoniae with two recombinant protein preparations. Vaccine, in press. 34. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467. 35. Schryvers, A. B., and L. J. Morris. 1988. Identification and characterization of the transferrin receptor from Neisseria meningitidis. Mol. Microbiol. 2:281-288. 36. Sebunya, T. N. L., and K. R. Saunders. 1983. Haemophilus pleuropneumoniae infection in swine: a review. J. Am. Vet. Med. Assoc. 182:1331-1337. 37. Shope, R. E. 1968. Porcine contagious pleuropneumonia. I. Experimental transmission, etiology and pathology. J. Exp. Med. 119:357-368. 38. Stauffer, G. V., M. D. Plamann, and C. T. Stauffer. 1981. Construction and expression of hybrid plasmids containing the Escherichia coli glyA gene. Gene 14:63-72. 39. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354. 40. Tsai, J., D. W. Dyer, and P. F. Sparling. 1988. Loss of transferrin receptor activity in Neisseria meningitidis correlates with inability to use transferrin as an iron source. Infect. Immun. 56:3132-3138. 41. Yamaguchi, K., F. Yu, and M. Inouye. 1988. A single amino acid determinant of the membrane localization of lipoproteins in E. coli. Cell 53:423-432.
