DeSmidt_139-152.qxd INTRODUCTION Haemophilus paragallinarum is a gram-negative, polar staining, non-motile bacterium. In 24-h cul- tures it appears as short rods, or coccobacilli, 1–3 µm in length and 0.4–0.8 µm in width, with a ten- dency for filament formation. This organism causes an acute respiratory disease of chickens known as infectious coryza (IC), a disease first recognised as a distinct entity in the late 1920s. Since the disease proved to be infectious and primarily affected the nasal passages, the name “infectious coryza” was adopted (Blackall 1989). The major economic effect of the disease is an increased culling rate in meat chickens and a reduction in egg production (10– 40 %) in laying and breeding hens. The disease is limited primarily to chickens and has no public health significance (Yamamoto 1991). All the com- mercially available bacterins against IC consist of inactivated broth cultures of a combination of two or three different serotypes. Although vaccines against IC have been used in South Africa since 1975, it became apparent in the 1980s that the vaccines were becoming less effective in controlling the dis- ease (Bragg, Coetzee & Verschoor 1996). This may be due to the emergence of previously unknown serovars, serogroups or changes in the population dynamics. Vaccine efficiency is therefore a problem and an alternative to available vaccines is needed. 139 Onderstepoort Journal of Veterinary Research, 71:139–152 (2004) Genetic organisation of the capsule transport gene region from Haemophilus pparagallinarum O. DE SMIDT, J. ALBERTYN*, R.R. BRAGG and E. VAN HEERDEN Department of Microbial, Biochemical and Food Biotechnology, University of the Free State P.O. Box 339, Bloemfontein, 9300 South Africa ABSTRACT DE SMIDT, O., ALBERTYN, J., BRAGG, R.R. & VAN HEERDEN, E. 2004. Genetic organisation of the capsule transport gene region from Haemophilus paragallinarum Onderstepoort Journal of Vet- erinary Research, 71:139–152 The region involved in export of the capsule polysaccharides to the cell surface of Haemophilus paragallinarum was cloned and the genetic organisation determined. Degenerate primers designed from sequence alignment of the capsule transport genes of Haemophilus influenzae, Pasteurella multocida and Actinobacillus pleuropneumoniae were used to amplify a 2.6 kb fragment containing a segment of the H. paragallinarum capsule transport gene locus. This fragment was used as a digoxigenin labelled probe to isolate the complete H. paragallinarum capsule transport gene locus from genomic DNA. The sequence of the cloned DNA was determined and analysis revealed the presence of four genes, each showing high homology with known capsule transport genes. The four genes were designated hctA, B, C and D (for H. paragallinarum capsule transport genes) and the predicted products of these genes likely encode an ATP-dependent export system responsible for transport of the capsule polysaccharides to the cell surface, possibly a member of a super family designated ABC (ATP-binding cassette) transporters. Keywords: Capsular transport genes, Haemophilus paragallinarum, infectious coryza * Author to whom correspondence is to be directed. E-mail: AlbertynJ.sci@mail.uovs.ac.za Accepted for publication 11 November 2003—Editor Capsules are found on the surface of a wide range of bacteria and are often important for virulence. These polysaccharide structures have been the subject of intensive investigation because of their usefulness as vaccines for prevention of bacterial infections (Lee 1987; Boulnois & Roberts 1990). Many researchers sought to understand the role of the capsule in virulence by identifying the genes involved in capsular polysaccharide export and bio- synthesis. The genetic organization of the group II capsule gene loci of Haemophilus influenzae type b (Kroll, Zamze, Loynd & Moxon 1989; Kroll 1992), Escherichia coli K1 and K5 (Boulnois, Roberts, Hodge, Hardy, Jann & Timmis 1987; Jann & Jann 1990), Pasteurella multocida M1404 (B:2) (Boyce, Chung & Adler 2000) and Actinobacillus pleuro- pneumoniae serotype 5a (Ward & Inzana 1997) have been determined and are very similar. In each of these species, a central DNA segment necessary for capsular polysaccharide biosynthesis is flanked by DNA encoding proteins for capsule export. Sub- stantial homology exists in the genes required for capsular polysaccharide export among these spe- cies, suggesting a common evolutionary origin (Frosch, Edwards, Bousset, Kraube & Weisgerber 1991). Genetically defined acapsular mutants have been shown to have reduced virulence in a number of organisms (Boyce et al. 2000). A mutant defective in the export of the P. multocida capsule was con- structed by allelic exchange and virulence assays showed the acapsular P. multocida to be 106 fold less virulent than their encapsulated counterparts (Boyce & Adler 2000). Similar studies have been conducted on the bexA gene of H. influenzae (Kroll, Hopkins & Moxon 1988). A frame shift mutation engineered at a restriction site within the open read- ing frame resulted, when introduced into the cap locus in the chromosome, in the expression of a mutant phenotype. The noncapsulated mutants of A. pleuropneumoniae reported by Inzana, Todd & Veit (1993) showed extreme stability and induced a protective immune response without any symptoms of disease. This not only proves the capsule’s involvement in virulence but also offers the oppor- tunity to investigate the possibility of producing live vaccines. In an attempt to understand the genetic organiza- tion of the capsular genes of H. paragallinarum de- generate PCR primers, based on the capsule loci of H. influenzae, A. pleuropneumoniae and P. multo- cida, were used to amplify a section of the capsule transport genes of H. paragallinarum. This section was employed as a probe to clone the full-length transport region. MATERIALS AND METHODS Bacterial strains Haemophilus paragallinarum strain 1742, obtained from the Department of Poultry Health, University of Pretoria, South Africa, was grown in TM/SN medi- um (1 % biosate peptone, 1 % NaCl, 0.5 % glucose, 0.1 % starch and 0.0005 % thiamine solution, oleic acid-albumin complex and chicken serum as sup- plements) as described by Blackall & Yamamoto (1990), in which 1.5 % agar was used to solidify the medium if required. In liquid culture the organisms were grown without aeration and on solid media in a candle jar at 37 °C. Escherichia coli strain Sure2 (Stratagene) was grown with aeration in Luria-Ber- tani (LB) broth (Sambrook, Fritsch & Maniatis 1989) under selective pressure with 60 µg/ml ampicillin in liquid and solid media when required. Preparation and analysis of genomic and plasmid DNA Genomic DNA was prepared from 20, 5 ml liquid cultures of H. paragallinarum grown for 16 h (Towner 1991). The cells were harvested by centrifugation at 3 000 g for 10 min at 4 °C and the mass of the pel- let was determined. The pellet was washed in TE- buffer (10 mM Tris-HCl, 1 mM EDTA) pH 8 and cen- trifuged again at 3 000 g for 5 min at 4 °C. The pel- let was re-suspended in 40 ml/0.5 g cells buffer (50 mM Tris-HCl, pH 8, 0.7 mM sucrose) and lysozyme (20 mg/ml) was added before the suspension was incubated on ice for 5 min. Six hundred microlitres EDTA (0.5 M, pH 8) and 500 µl 10 % SDS were added for each 0.5 g cells, gently mixed and placed on ice for 5 min. After the addition of 10 ml/0.5 g cells digestion buffer (1 % SDS, 50 mM Tris-HCl pH 8, 0.1 M EDTA, 0.2 M NaCl, 0.5 mg/ml proteinase K), the suspension was incubated at 55 °C for 3–16 h with mild shaking. One time the volume of pH cal- ibrated phenol (pH 7.8) was added to the lysate and incubated a further 3 h at 25 °C with constant inver- sion. Cell debris was removed by centrifugation at 4 000 g for 10 min and the supernatant mixed with 0.1x the volume 5 M NaCl and placed on ice for 5 min. Genomic DNA was precipitated with 10 ml 100 % ethanol, spooled and washed in 1 ml 70 % ethanol. After drying, the pellet was suspended in 500 µl/ 0.5 g cells TE-buffer and incubated at 50 °C for 1 h or kept at 4 °C overnight before use. 140 Genetic organisation of capsule transport gene region from Haemophilus paragallinarum Plasmid DNA was isolated by a rapid alkaline lysis method described by Sambrook et al. (1989) and suspended in TE-buffer containing 10 µg/ml RNase. Genomic and plasmid DNA were analysed by re- striction enzyme digestion. Plasmid DNA was digest- ed with EcoRI or HindIII for 1 h, while genomic DNA was digested using BamHI, EcoRI, HindIII, PstI or XbaI for 3–16 h. All the enzymes used in these di- gestions were obtained from Roche Molecular Bio- chemicals. PCR analysis and cloning techniques PCR analysis was performed in a Perkin-Elmer Geneamp 2400 thermocycler. Haemophilus para- gallinarum genomic DNA (60 ng) was used as tem- plate and PCR reactions were carried out in 50 µl volumes. The reaction mixtures consisted of a 10x dilution of reaction buffer (100 mM Tris-HCl, 15 mM MgCl2, 500 mM KCl, pH 8.3), 2 pmol of each de- generate primer (Table 1) in different combinations, 0.2 mM dNTP mixture and 5 U of Taq polymerase (Roche). The reaction conditions consisted of an initial denaturation cycle of 94 °C for 5 min followed by 25 cycles of 94 °C for 30 s, 45 °C for 30 s, 72 °C for 2 min and a final elongation cycle of 72 °C for 5 min. The same reaction constituents and conditions were used for amplification of the partial H. para- gallinarum capsule transport gene locus and for production of a DNA probe for screening. PCR products were purified and DNA fragments were recovered from agarose gels with the GFXä- PCR DNA and gel band purification kit (Amersham Pharmacia Biotech). Purified fragments were cloned into either pGEM-T Easy or pGEM-3Z (Promega). Escherichia coli strain Sure2 was grown to early log phase at 18 °C in SOB-media as described by Hanahan (1983). Competent E. coli cells were pre- pared according to the method of Inoue, Nojima & Okayama (1990). Blotting techniques Southern hybridisation was used as a method to identify fragments in digested genomic DNA that encode the capsule transport gene locus and col- ony hybridisation to identify positive clones contain- ing the recombinant plasmids. Genomic DNA was digested with BamHI, EcoRI, HindIII, PstI or XbaI and the fragments separated by agarose gel electrophoresis. The DNA was trans- ferred to a Magnacharge nylon membrane (Micron separations, Inc.) by 1 h downward capillary trans- fer as described by Chomczynski (1992). DNA was linked to the membrane with the GS gene linker™ (BIO-RAD) prior to hybridisation. Colony blotting to screen for the presence of clones containing the transport gene locus was performed on transformants grown for 16 h on LB plates con- taining ampicilin. Blotting proceeded as described by the DIG system users’ guide for filter hybridisa- tion (Roche Molecular Biochemicals). Colonies were lifted from the growth media and fixed on a magna- charge nylon membrane. The membrane was sub- jected to lysis in 10 % SDS and denaturation solu- tion (0.5 M NaOH, 1.5 M NaCl) followed by neutral- isation (1 M Tris-HCl pH 7.5, 1.5 M NaCl) and wash- ing twice in SSC until all the cell debris was re- moved. Hybridisation and colorimetric detection were per- formed as described in the DIG Nucleic Acid De- tection Kit (Roche Molecular Biochemicals). Probe labelling and screening methods The 2.6 kb fragment used as a hybridisation probe was amplified from H. paragallinarum genomic DNA with primers HctD-1F and HctA-1R (Table 1). This fragment was prepared as the Hct-probe by random prime labelling with digoxigenin using the DIG label- ling and detection kit. 141 O. DE SMIDT et al. TABLE 1 Degenerate and sequence specific oligonucleotides used for the amplifi- cation of the capsule transport gene locus from H. paragallinarum Degenerate primers HctD-1F 5’- GAT AAA GAT WTW GTH TAT GTR TCR AAT GCA CC -3’ HctC-1F 5’- GCB TCY GAT ATT TAT RTT TCD SAA TCD AG -3’ HctC-1R 5’- CYA AAT AMA RTT GYT GGC GAT C -3’ HctB-1F 5’- ATG ATG TGG CGH AAT GCD TC -3’ HctB-1R 5’- AAC ATT TCY GWR CCR TGA ATC ATY GG -3’ HctA-1R 5’- ATY TTR GTT TCW CAT AGC CCG WVT -3’ G Sequence specific primers HctD-1R 5’- GGT GCA TTC GAC ACA TAT AC -3’ HctA-1F 5’- ATT TTA GTT TCT CAT AGT CCA ACC G -3’ The amount of labelled DNA was determined by comparison of the intensity of the spots of a serial dilution of the Hct-probe to that of a labelled control (supplied by the manufacturer). Sequencing and analysis Plasmid construct pHctA-D was used as a template for sequencing. Sequencing reactions were per- formed with the ABI Prism Big Dye terminator V3.0 cycle sequencing ready reaction kit and data collected on an ABI Prism 377 DNA sequencer (Per- kin-Elmer biosystems). Data was analysed using Sequencing analysis V3.3. Sequences were reverse complemented and compared by using Sequence Navigator V 1.0.1 and assembled using Auto-assem- bler V1.4.0 and DNAssist V2.0. Sequence submission Sequence of the transport gene locus was submit- ted to GenBank, accession number AY116594. RESULTS Partial amplification of the H. pparagallinarum capsule transport gene region Genomic DNA was isolated from H. paragallinarum and used as a template for PCR amplification of the H. paragallinarum capsule transport genes. The capsule transport gene sequences of H. influenzae (bexA-D genes), A. pleuropneumoniae (cpxA-D genes) and P. multocida (hexA-D genes) were obtained from GenBank (accession no. X54987, U36397 & AF067175) and submitted to a multiple sequence alignment using DNAssist V 1.0.2. Six degenerate primers were designed (Table 1) from areas in these aligned gene sequences where the sequence was highly conserved. The PCR performed with different oligonucleotide combinations (Fig. 1A), showed amplification of fragments of expected sizes in lanes 1 (~2.6 kb), 2 (~2.3 kb), 3 (~1.9 kb), 4 (~1.6 kb) and 6 (~1.1 kb). The relative position of each of these fragments in the proposed H. paragallinarum transport gene re- gion is indicated in Fig. 1B. Lanes 5 and 7 showed either non-specific or no amplification. More than one band was visible in some lanes due to non-spe- cific priming and a low annealing temperature of 45 °C. The estimated ~2.6 kb fragment amplified by the oligonucleotides HctD-1F and HctA-1R (Fig. 1A, lane 1), was cloned into pGEM-T Easy and desig- nated pHct. The nucleotide sequence of this frag- ment was determined and analysis revealed con- siderable homology with the capsule transport genes of related organisms (H. influenzae, A. pleuropneu- moniae and P. multocida). This high degree of ho- mology among the four species indicated that the sequenced 2638 bp insert in pHct represented part of the capsule transport gene region of H. paragal- linarum. By comparison with the capsular transport genes of P. multocida, this fragment contained ho- mologues of hexC and hexB as well as small regions of the 3’ end of hexD and the 5’ region of hexA. Construction of a mini-library to isolate the entire capsule transport gene region To facilitate the cloning of the full-length capsular transport region, the pHct insert was used as a probe (designated Hct) in southern blotting followed by colony hybridisation. Genomic DNA of H. para- gallinarum was digested with five different restric- tion enzymes, transferred to a nylon membrane and hybridised with a digoxigenin labelled Hct-probe under stringent conditions. Southern blotting and hybridisation indicated that a HindIII fragment of ~6.15 kb (Fig. 2, lane 3) hybridised to the Hct- probe. Hybridisation products visible in lanes 1, 2, 4 and 5 at a position of ~21 kb correspond to the rel- ative position of undigested genomic DNA or unre- solved large restriction fragments when using restriction enzymes BamHI, EcoRI, PstI and XbaI. The HindIII fragments resolved between 6 kb and 6.5 kb were excised from the gel, purified and cloned into vector pGEM-3Z to construct a mini- library. Colony hybridisation was used as a screen- ing method to identify positive clones containing the transport genes. A total of 93 colonies were visible within 1 day of transformation and two colonies showed hybridisation with the Hct-probe after screening under stringent conditions. Plasmid DNA, extracted from the above-mentioned colonies and digested with HindIII, revealed the presence of a ~6.15 kb insert. To confirm that these plasmid constructs did contain the capsule trans- port region, the 5’ and 3’ terminal regions were sequenced. Sequencing confirmed that both clones were identical and also gave an indication of the ori- entation in which the ~6.15 kb fragment was ligated into the vector. Sequence alignment to known cap- sular genes, using the above-mentioned sequences, indicated that the ~6.15 kb fragment did in fact con- tain the relevant capsule region. PCR reactions were performed to determine which part or parts of the Hct-probe features in the 142 Genetic organisation of capsule transport gene region from Haemophilus paragallinarum 143 O. DE SMIDT et al. M 1 2 3 4 5 6 7 5.1 2.0 1.6 1.4 21 kb 0.83 2 600 bp 2 300 bp 1 900 bp 1 600 bp 1 100 bp 860 bp 1 kb HctD-1F HctC-1F HctB-1F HctA-1RHctC-1R HctB-1R hctA hctB hctC hctD 1 100 bp A B FIG. 1 A Amplification of segments of the H. paragallinarum capsule transport gene region The different degenerate oligonucleotides were used in the following combinations: HctD-1F & HctA-1R (lane 1), HctC-1F & HctA-1R (lane 2), HctD-1F & HctB-1R (lane 3), HctC-1F & HctB-1R (lane 4), HctD-1F & HctC-1R (lane 5), HctB-1F & HctA-1R (lane 6) and HctC-1F & HctC-1R (lane 7) B Schematic representation of the proposed H. paragallinarum transport gene region indicating the relative positions of the degenerate oligonucleotides used in Fig. 1A The PCR fragments expected were as follows: 2 600 bp (lane 1, Fig. 1A), 2 300 bp (lane 2, Fig. 1A), 1 900 bp (lane 3, Fig. 1A), 1 600 bp (lane 4, Fig. 1A), 1 100 bp (lane 5, Fig. 1A), 1 100 bp (lane 6, Fig. 1A), 860 bp (lane 7, Fig. 1A) ~6.15 kb fragment. These PCR reactions were per- formed using sequence specific oligonucleotides designed according to the sequence obtained from the 2.6 kb pHct fragment. Sequence specific oligo- nucleotide HctD-1R was used in combination with T7 (Fig. 3, lane 1) and HctA-1F in combination with SP6 (Fig. 3, lane 2) (SP6 and T7 have binding sites on opposite sides of the multiple cloning site of pGEM-3Z). Amplification of two bands were visible, a ~1.5 kb band in lane 1 representing the segment upstream and a ~2.1 kb band in lane 2 indicating the segment downstream from the Hct-probe sequence. These results and the high degree of sequence homology with the transport genes of related organ- isms, verified that the ~6.15 kb fragment represents the entire H. paragallinarum capsule transport region and was designated pHctA-D. The nucleotide sequence of the full-length capsular transport region was determined through primer walking using the ~6.15 kb HindIII restriction frag- ment of pHctA-D. Analysis of the complete sequence revealed that the H. paragallinarum capsule trans- port gene region is 3 792 bp in length with a GC con- tent of 37 %, comprising four open reading frames representing the four capsule transport genes des- ignated hctDCBA (Fig. 4 and 5). hctD contains 1 188 nucleotides and terminates at a TGA stop codon, encoding a putative protein of 395 amino acids. The next open reading frame, hctC, starts at the third base of the hctD stop codon and encodes a puta- tive protein of 387 amino acids. The third base of the stop codon at the 3’- end of hctC is the first base of the ATG at the start of hctB, 798 nucleotides in length and coding for a putative protein of 265 amino 144 Genetic organisation of capsule transport gene region from Haemophilus paragallinarum ~6.15 kb M 1 2 3 4 5 5.1 2.0 1.6 1.4 21 kb ~2.1 kb ~1.5 kb M 1 2 5.1 2.0 1.6 1.4 21 kb FIG. 2 Southern blot analysis of digested genomic DNA hybridised with the Hct-probe under stringent condi- tions. Genomic DNA was digested with BamHI (lane 1), EcoRI (lane 2), HindIII (lane 3), PstI (lane 4) and XbaI (lane 5) for 3 h. Arrow indicates positive hybridi- sation with a fragment ~6.15 kb in size in the HindIII digestion FIG. 3 Amplification of the regions up- and downstream from the Hct-probe sequence within the ~6.15 kb clone. A ~1.5 kb fragment was amplified when oligonucleotide HctD-1R was used in combination with T7 (lane 1). A ~2.1 kb fragment was amplified by oligonucleotides HctA-1F and SP6 (lane 2) 0 3.79 kb 2 kb 3 kb1 kb hctD 1 188 bp hctB 798 bp hctA 648 bp hctC 1 164 bp Hct-probe FIG. 4 Genetic map of the capsule transport gene region of H. paragallinarum. The locations and directions of transcription of the four open reading frames hctDCBA are indicated. The 2.6 kb fragment (Hct) used as the DNA probe in Fig. 2 is also indicated FIG. 5 Nucleotide sequence of the capsule transport region. Three thousand nine hundred and twenty three nucleotides of the sequence are shown, from arbitrary points 167 bp upstream of hctD to 131 bp downstream of hctA. The four open reading frames are indicated as hctD, hctC, hctB and hctA, in each case from the first ATG, with the translated peptide sequence beneath. The underlined regions are referred to in the text 145 O. DE SMIDT et al. –167 …GA TAA GTG TTG ATA TAA ATA AAA TTT CCC GAG TCT TTA –130 –129 AAA AAT TGG AAT TAT TTT TAT AAA AAA GTT TTC TAC AGG AAA TTG –85 –84 AGC AAA AAT TAA TAA TTA TCT ATG ATA ATT ACT CAC TTT TAA TAG –40 hctD –39 AAA AAT CAT GAT CAA AAA CAA AAT AAT TAA GGT AAA ACT ATG CGT 6 Met Arg 2 7 AAA TCG CTG ATT GCA GTA AGT TAC TGC TTA TTA TTA ATG TCT TGG 51 3 Lys Ser Leu Ile Ala Val Ser Tyr Cys Leu Leu Leu Met Ser Trp 17 52 TCT TAT TTG CCA AAT TCA GGA CCG AGC AAA GGC AAT ATT GAG GTA 96 18 Ser Tyr Leu Pro Asn Ser Gly Pro Ser Lys Gly Asn Ile Glu Val 32 97 GTC AAT AAA CAG AAA TCC AAT GAG GAT TTG CTT GCA GTA CAG TTG 141 33 Val Asn Lys Gln Lys Ser Asn Glu Asp Leu Leu Ala Val Gln Leu 47 142 ATC GAG GTG AAT AAT AAA GTT GCG GAA AGT ATG TTT AAT CAA CAA 186 48 Ile Glu Val Asn Asn Lys Val Ala Glu Ser Met Phe Asn Gln Gln 62 187 CAC CCT CAA TCA TTT TTG CAG TTT CCT TCA TCA AAA GCA CAT TAT 231 63 His Pro Gln Ser Phe Leu Gln Phe Pro Ser Ser Lys Ala His Tyr 77 232 CAT GGG GTA GTT AAA TGC TGG TGT TTA CTT GAT ATT ACT CTC TGG 276 78 His Gly Val Val Lys Cys Trp Cys Leu Leu Asp Ile Thr Leu Trp 92 277 GAA GCA CCC GCC AGC AAC TTT GTT TGG CAG TGT GTT GAA TCA AGC 321 93 Glu Ala Pro Ala Ser Asn Phe Val Trp Gln Cys Val Glu Ser Ser 107 322 CGG TGT GTC GGG CGG ACA AAG CAC TCA CTT ACC GGA ACA GGT GGT 366 108 Arg Cys Val Gly Arg Thr Lys His Ser Leu Thr Gly Thr Gly Gly 122 367 TAT AGC AAT GGA AGA ATA ACC ATT CCT TTT GTT GGT GCA TTA AAA 411 123 Tyr Ser Asn Gly Arg Ile Thr Ile Pro Phe Val Gly Ala Leu Lys 137 412 GTA GCA GGG AAA ACA CCG GAG CAG ATC CAA TCT GAA ATT GTT GGA 456 138 Val Ala Gly Lys Thr Pro Glu Gln Ile Gln Ser Glu Ile Val Gly 152 457 CGT TTA CAA GCA ATT GCC AAT CAA CCA CAA GCA GTG GTG CGA ATT 501 153 Arg Leu Gln Ala Ile Ala Asn Gln Pro Gln Ala Val Val Arg Ile 167 502 GTG AAG AAT AAT TCT GCT AAT GTG ACG GTT TTA ACT AAA TCG ACT 546 168 Val Lys Asn Asn Ser Ala Asn Val Thr Val Leu Thr Lys Ser Thr 182 547 ACT ATT CGA ATG GCT TTA ACT GCT TAC GGT GAA CGA AGT GTT AGA 591 183 Thr Ile Arg Met Ala Leu Thr Ala Tyr Gly Glu Arg Ser Val Arg 197 592 TGC TAT TGC GGC AGC AGG TGG AGC CGG TGG TAT GTG CAA AGA TGT 636 198 Cys Tyr Cys Gly Ser Arg Trp Ser Arg Trp Tyr Val Gln Tyr Cys 212 637 TTC AGT GCG ACT GAC TCG TGG GAA ATC AGG GTG CAA ACG ATT TCT 681 213 Phe Ser Ala Thr Asp Ser Trp Glu Ile Arg Val Gln Thr Ile Ser 227 682 TTA GCC AGG ATT AAC GGA GGG AGC CAC AGG CAA AAT ATC CTA TTA 726 228 Leu Ala Arg Ile Asn Gly Gly Ser His Arg Gln Asn Ile Leu Leu 242 727 CGT TCC GGC GAT GTA GTA ACG TTA TTA AAT AAT CCA CTT TCT TTC 771 243 Arg Ser Gly Asp Val Val Thr Leu Leu Asn Asn Pro Leu Ser Phe 257 772 ACT GCA ATG GGT GCG GTA GGA AAT AGT AAA GAA ATT CGT TTT TCG 816 258 Thr Ala Met Gly Ala Val Gly Asn Ser Lys Glu Ile Arg Phe Ser 272 817 GCA GAA GGT TTA ACT TTA GCA GAA GCA ATC GGT CGT TTA GGT GGA 861 273 Ala Glu Gly Leu Thr Leu Ala Glu Ala Ile Gly Arg Leu Gly Gly 287 FIG. 5 (Continued) 862 TTG AAT GAT GAT CGT GCA GAT CCA AGA GGA GTA TTT ATC TTT CGT 906 288 Leu Asn Asp Asp Arg Ala Asp Pro Arg Gly Val Phe Ile Phe Arg 302 907 TAT GTT CCA TTT GAA GAA ATG CCC TTA AGT AAA CAA AAT GAA TGG 951 303 Tyr Val Pro Phe Glu Glu Met Pro Leu Ser Lys Gln Asn Glu Trp 317 952 CAA GCC AAG GGG TAT CAC AAC GGA ATG AAA ATT CCA ACA GTA TAT 996 318 Gln Ala Lys Gly Tyr His Asn Gly Met Lys Ile Pro Thr Val Tyr 332 997 CAA GCG AAT TTA CTT GAA CCT CAA TCA ATG TTT TGG ATT CAA CAA 1041 333 Gln Ala Asn Leu Leu Glu Pro Gln Ser Met Phe Trp Ile Gln Gln 347 1042 TTT CCA ATT AAA GAT AAA GAT ATT GTT TAT GTA TCT AAT GCA CCA 1086 348 Phe Pro Ile Lys Asp Lys Asp Ile Val Tyr Val Ser Asn Ala Pro 362 1087 TTG GCT GAA TAC CAA ATT TAT TCG TAT GAT TTA CGC CAC CGT TGC 1131 363 Leu Ala Glu Tyr Gln Ile Tyr Ser Tyr Asp Leu Arg His Arg Cys 377 1132 AAC TAC ACC GCC GGT TTC AAC TGT AAA CAA GTG TTA ATA ATC TGT 1176 378 Asn Tyr Thr Ala Gly Phe Asn Cys Lys Gln Val Leu Ile Ile Cys 392 hctC 1177 AGG GGG AGA TGA TG GAA CAA AAT GTA GTA GTT CAA TCG AAA GAA 1220 393 Arg Gly Arg *** Met Glu Gln Asn Val Val Val Gln Ser Lys Glu 406 1221 CAA CTG AGA AAG TTA AAA CAG TGG TTG CGA AAA ATT AAT CTG TTA 1265 407 Gln Leu Arg Lys Leu Lys Gln Trp Leu Arg Lys Ile Asn Leu Leu 421 1266 TTT TTA CTG ACG GTG ATT ATT CCG ACT TTT TGT TCG TTA TTT TAT 1310 422 Phe Leu Leu Thr Val Ile Ile Pro Thr Phe Cys Ser Leu Phe Tyr 436 Region A1 (421–442) 1311 TTT TCT ATT TGG GCT TCC GAT GTT TAT ATT TCG GAG TCC AGT TTT 1355 437 Phe Ser Ile Trp Ala Ser Asp Val Tyr Ile Ser Glu Ser Ser Phe 451 1356 ATT GTG CGT TCT TCT CGT GCT CAG GCA TCG CTC GGA GGT ATG GGG 1400 452 Ile Val Arg Ser Ser Arg Ala Gln Ala Ser Leu Gly Gly Met Gly 466 Region A2 (458–475) 1401 GCT TTA TTG CAG AGT ATC GGT TTT GCT CGT TCG CAA GAT GAT ACT 1445 467 Ala Leu Leu Gln Ser Ile Gly Phe Ala Arg Ser Gln Asp Asp Thr 481 1446 TTT ACG GTG CAA GAA TTT ATG CGT TCG CGT AAT GCG TTG ACA ACA 1490 482 Phe Thr Val Gln Glu Phe Met Arg Ser Arg Asn Ala Leu Thr Thr 496 1491 TTG GAA AGT GAG TTA CCG GTG AGA AAA TTT TAT GAA GAT GAA GGG 1535 497 Leu Glu Ser Glu Leu Pro Val Arg Lys Phe Tyr Glu Asp Glu Gly 511 1536 GAT TTT TTC AGC CCG TTT AAT CCG TTA GGT TTT TTT AAT GAA CAG 1580 512 Asp Phe Phe Ser Pro Phe Asn Pro Leu Gly Phe Phe Asn Glu Gln 526 1581 GAA TTG TTT TAT CAA TAT TTT CGT AAA CAT TTG ATG ATT AAT ATC 1625 527 Glu Leu Phe Tyr Gln Tyr Phe Arg Lys His Leu Met Ile Asn Ile 541 1626 GAT TCT TTA TCT GGG TAT TGC TAC TTT ACA GGT TCC GTG GGT TTA 1670 542 Asp Ser Leu Ser Gly Tyr Cys Tyr Phe Thr Gly Ser Val Gly Leu 556 1671 ATG GCT GAC CTC CGG CAC CAA CAA GAA TTA AAT GGA AGC CAT TAT 1715 557 Met Ala Asp Leu Arg His Gln Gln Glu Leu Asn Gly Ser His Tyr 571 1716 TGC CAT TTT GGC GGG AAA CCA TTT AGT GGA ATA AAC TCA ATG ATC 1760 572 Cys His Phe Gly Gly Lys Pro Phe Ser Gly Ile Asn Ser Met Ile 586 1761 GTG CAC GTA AAG ATA CAA TTA CTT TGC GGA ACA ATC GGT AAT GAA 1805 587 Val His Val Lys Ile Gln Leu Leu Cys Gly Thr Ile Gly Asn Glu 601 1806 GCA GAA AAA TAT TTG TCT GAA ACC TCG ACA GCC TTA AGC CAA TAT 1850 602 Ala Glu Lys Tyr Leu Ser Glu Thr Ser Thr Ala Leu Ser Gln Tyr 616 146 Genetic organisation of capsule transport gene region from Haemophilus paragallinarum FIG. 5 (Continued) 1851 CGT GTA AAA AAT GGG ATA TTT GAT ATT GGG GCA CAA TCT GAA TCG 1895 617 Arg Val Lys Asn Gly Ile Phe Asp Ile Gly Ala Gln Ser Glu Ser 631 1896 ATT TTA ACT TTA GTG CAG AAG TTG CAG GAT GAA CTG ATT GCC ATT 1940 632 Ile Leu Thr Leu Val Gln Lys Leu Gln Asp Glu Leu Ile Ala Ile 646 1941 CAG ACG CAA CTT GAT CAG GTG AGG GGC GTT ATC TCC GGA TAC CCT 1985 647 Gln Thr Gln Leu Asp Gln Val Arg Gly Val Ile Ser Gly Tyr Pro 661 1986 CAG GTT AAA GTG TTA AAG GCA AGG CAA TTT GAA AGT ATT CGT GAA 2030 662 Gln Val Lys Val Leu Lys Ala Arg Gln Phe Glu Ser Ile Arg Glu 676 2031 AGA AGT GGC ACA ACA ATT GAA TCC GGG GTT TTT GAG GGG AAA CCA 2075 677 Arg Ser Gly Thr Thr Ile Glu Ser Gly Val Phe Glu Gly Lys Pro 691 2076 TTC TTT AAC AAC ACA ATC AGC AGA GTA CCA GCC GTT AAT TTA GAT 2120 692 Phe Phe Asn Asn Thr Ile Ser Arg Val Pro Ala Val Asn Leu Asp 706 2121 GAA ACC TTG GCA AAA CAG CAA TTA ACA GCT GCA ATG TCT TGC GTT 2165 707 Glu Thr Leu Ala Lys Gln Gln Leu Thr Ala Ala Met Ser Cys Val 721 2166 ACA AGT GGC AAA GAA GAA GCT GGA AGA CAA CAG CTT TAT CTG GAA 2210 722 Thr Ser Gly Lys Glu Glu Ala Gly Arg Gln Gln Leu Tyr Leu Glu 731 2211 ATT ATT GCT AAA CCT AGC CAT CCA GAT TTA GCA TTG GAA CCG CAC 2255 737 Ile Ile Ala Lys Pro Ser His Pro Asp Leu Ala Leu Glu Pro His 751 2256 CGT TTG TAC AAT ATT TTG GCA ACT TTG ATT CTT GGA TTA GTT ATT 2300 752 Arg Leu Tyr Asn Ile Leu Ala Thr Leu Ile Leu Gly Leu Val Ile 766 Region A3 (753–777) 2301 TAT GGC GTT TCA ACT TTA TTA TTA GCC GGT GTG AGA GAG CAT AAG 2345 767 Tyr Gly Val Ser Thr Leu Leu Leu Ala Gly Val Arg Glu His Lys 781 hctB 2346 AAC TGA TG CAG TAT GGT GAA CAA ACT TCG TTA AAA GAT TCA TTT 2389 782 Asn *** Met Gln Tyr Gly Glu Gln Thr Ser Leu Lys Asp Ser Phe 795 2390 ACT ATC CAA GGA CGG GTG TTG AAA GCG TTG TTG TTG CGT GAA ATT 2434 796 Thr Ile Gln Gly Arg Val Leu Lys Ala Leu Leu Leu Arg Glu Ile 810 2435 ATC ACT CGT TAT GGT CGT AAA AAT TTA GGC TTT TTG TGG GTT GTT 2479 811 Ile Thr Arg Tyr Gly Arg Lys Asn Leu Gly Phe Leu Trp Val Val 825 2480 CGT GAG CCA TTT TTG ATG AGC CTA GTT ATT GTG GTA ATG TGG CAT 2524 826 Arg Glu Pro Phe Leu Met Ser Leu Val Ile Val Val Met Trp His 840 2525 TTT TTT CGT GCT GAT CGC TTT TCA ACA TTA AAC ATT GTT GCT TTT 2569 841 Phe Phe Arg Ala Asp Arg Phe Ser Thr Leu Asn Ile Val Ala Phe 855 2570 GCA ATG ACG GTT ATC CAT TAT TAT GGA TGT GGC GTA ATG CTT CTA 2614 856 Ala Met Thr Val Ile His Tyr Tyr Gly Cys Gly Val Met Leu Leu 870 2615 ACC GTG CAA TTA GCG GGA ATG GAT TCC AAT ATC CCA TTA CTT TTA 2659 871 Thr Val Gln Leu Ala Gly Met Asp Ser Asn Ile Pro Leu Leu Leu 885 2660 TCA CGT AAT GTA CGT CCT CTT GAT ACG CTT TTT TCT CGT ATG ATT 2704 886 Ser Arg Asn Val Arg Pro Leu Asp Thr Leu Phe Ser Arg Met Ile 900 2705 TTG GAG ATT GCT GGT GCG ACT GTA GCA CAA ATT GTG ATG TTA GTG 2749 901 Leu Glu Ile Ala Gly Ala Thr Val Ala Gln Ile Val Met Leu Val 915 2750 ATT TTA ATT GCT ATT GAT TGG ATC GGC TTG CCA AAT GAT GTG TTG 2794 916 Ile Leu Ile Ala Ile Asp Trp Ile Gly Leu Pro Asn Asp Val Leu 930 2795 TAT ATG CTT TTT GCT TGG TTC TTA ATG GCA CTG TTT GCC ATT GGT 2839 931 Tyr Met Leu Phe Ala Trp Phe Leu Met Ala Leu Phe Ala Ile Gly 945 2840 TTA GGT TTA ATT ATT TGT GCT ATT TCT TAT TAT TTA GAG TTT TTC 2884 946 Leu Gly Leu Ile Ile Cys Ala Ile Ser Tyr Tyr Leu Glu Phe Phe 960 147 O. DE SMIDT et al. FIG. 5 (Continued) 2885 GGT AAA ATT TGG GGA ACA TTA TCT TTT GTG ATG TTT CCT ATT TCC 2929 961 Gly Lys Ile Trp Gly Thr Leu Ser Phe Val Met Phe Pro Ile Ser 975 2930 GGT GCA TTC TTT TTA GTG AAT AGT TTG CCA AAC AAT CTG CAA TCT 2974 976 Gly Ala Phe Phe Leu Val Asn Ser Leu Pro Asn Asn Leu Gln Ser 990 2975 ATT TTG CTT TGG TTT CCA ATG GTT CAC GGT ACG GAA ATG TTT CGT 3019 991 Ile Leu Leu Trp Phe Pro Met Val His Gly Thr Glu Met Phe Arg 1005 Region B (990–1009) 3020 CAC GGT TAT TTT GGT TCT TCA GTT ATT ACA ATG GAA TCA CCG AGT 3064 1006 His Gly Tyr Phe Gly Ser Ser Val Ile Thr Met Glu Ser Pro Ser 1020 3065 TAT TTA TTT ATT TGT GAT TTG GTG ATG TTA TTA ATC GGT CTA CTG 3109 1021 Tyr Leu Phe Ile Cys Asp Leu Val Met Leu Leu Ile Gly Leu Leu 1035 hctA 3110 ATG GTG GGT AGT TTT AGT AAT AGG ATT AAT GCA AGA TG ATT AGT 3153 1036 Met Val Gly Ser Phe Ser Asn Arg Ile Asn Ala Arg *** Met Ile Ser 1050 3154 GTA GAC CAC GTT TAT AAA AAA TAT CAA ACA CGG ACA GGT TCG GTA 3198 1051 Val Asp His Val Tyr Lys Lys Tyr Gln Thr Arg Thr Gly Ser Val 1065 3299 CCC GTA TTA AAT GAT ATT AAT TTT AGC CTT ACC AAA GAA GAA AAA 3243 1066 Pro Val Leu Asn Asp Ile Asn Phe Ser Leu Thr Lys Glu Glu Lys 1080 3244 ATT GGT ATT TTA GGT CGC AAC GGA GCA GGA AAA TCA CCA TTA ATT 3288 1081 Ile Gly Ile Leu Gly Arg Asn Gly Ala Gly Lys Ser Pro Leu Ile 1095 Region C1 (1080–1092) 3289 CGT TTA ATG AGT GGT GTT GAA GCT CCA ACT TCA GGA ATA ATT CGA 3333 1096 Arg Leu Met Ser Gly Val Glu Ala Pro Thr Ser Gly Ile Ile Arg 1110 3334 CGA GAA ATG AGC ATT TCT TGG CCA TTA GCC TTT AGC GGT GCA TTC 3378 1111 Arg Glu Met Ser Ile Ser Trp Pro Leu Ala Phe Ser Gly Ala Phe 1125 3379 CAA GGT AGC TTA ACG GGA ATG GAT AAT TTA CGC TTC ATT TGT CGT 3423 1126 Gln Gly Ser Leu Thr Gly Met Asp Asn Leu Arg Phe Ile Cys Arg 1140 Region C2 (1131–1138) 3424 ATT TAT AAT GCT GAT ATT AAT TAT GTT ACT GAA TTT ACG GAA TCC 3468 1141 Ile Tyr Asn Ala Asp Ile Asn Tyr Val Thr Glu Phe Thr Glu Ser 1155 3469 TTT TCC GAA TTG GGC AAT TAT TTA TAT GAG CCT GTA AAA AAT TAT 3513 1156 Phe Ser Glu Leu Gly Asn Tyr Leu Tyr Glu Pro Val Lys Asn Tyr 1170 3514 TCT TCA GGA ATG AAA GCA CGC TTA GCT TTT GCA TTG TCG TTA TCC 3558 1171 Ser Ser Gly Met Lys Ala Arg Leu Ala Phe Ala Leu Ser Leu Ser 1185 3559 GTT GAG TTT GAT TGC TAT CTC ATT GAT GAA GTG ATT GCC GTT GGA 3603 1186 Val Glu Phe Asp Cys Tyr Leu Ile Asp Glu Val Ile Ala Val Gly 1200 3604 GAT TCT CGT TTT AGT GAT AAA TGT CGC TAT GAA CTT TTT GAA AAA 3648 1201 Asp Ser Arg Phe Ser Asp Lys Cys Arg Tyr Glu Leu Phe Glu Lys 1215 3649 CGC AAA GAT CGT TCC ATT ATT TTA GTT TCT CAT AGT CCA ACC GCT 3693 1216 Arg Lys Asp Arg Ser Ile Ile Leu Val Ser His Ser Pro Thr Ala 1230 3694 ATT AGA CAA TAT TGT GAT AAT GCA AAA GTA TTA GAT AAA GGA AAA 3738 1229 Ile Arg Gln Tyr Cys Asp Asn Ala Lys Val Leu Asp Lys Gly Lys 1245 3739 TTG TTA GAT TTC TCT TCT ATT GAT GAG GCT TAT CAA TAT TAT AAT 3783 1246 Leu Leu Asp Phe Ser Ser Ile Asp Glu Ala Tyr Gln Tyr Tyr Asn 1260 3784 CAG ACA TAG AGG TTA GAT TTT AAA ATA AAA TAA CGT TAC TTT CTT 3883 1261 Gln Thr *** 1260 3829 GCT TTA TCA TAA ATT TCA ATG GCT ATA GTT AAG TTC GAA ATA AAT 3873 3874 CAA GGT AAC AAG CTG AAT ACA GTG AAA AAT AGC ACT TTT TAT GCC 3918 3919 AAG GT… 148 Genetic organisation of capsule transport gene region from Haemophilus paragallinarum acids. hctB terminates with a TGA stop codon where it overlaps with the hctA start codon. hctA contains 648 nucleotides, encodes a putative protein of 215 amino acids and terminates at a TAG stop codon. Downstream of hctA all reading frames in both direc- tions are closed with multiple stop codons. Part of an open reading frame is present upstream of hctD, which showed considerable homology with the P. multocida hyaA biosynthesis gene. The overlapping stop and start codons in the hct genes (Fig. 4 and 5) indicate that these four genes are probably tran- scriptionally coupled. DISCUSSION Analysis of the H. paragallinarum hctDCBA gene cluster revealed a clear bias toward codons rich in nucleotides A and T (37 % GC content) consistent with the 39 % GC content of the H. influenzae cap- sule gene cluster (Kroll, Loynds, Brophy & Moxon 1990) and 37 % GC content of the H. influenzae genome overall (Roy & Smith 1973). It also corre- lates with the calculated GC contents of A. pleuro- pneumoniae (40 %) and P. multocida (37 %). The gene lengths and region size correlate well with those of related organisms, all belonging to the fam- ily Pasteurellaceae (Table 2). Blast searches of the combined, non-redundant nucleotide and protein databases at the National Centre for Biotechnology Information (NCBI) indicated that H. paragallinarum hctDCBA were highly homologous at both the nu- cleotide and amino acid levels to H. influenzae bexDCBA (Kroll et al. 1990), A. pleuropneumoniae cpxDCBA (Ward & Inzana 1997), P. multocida hexDCBA (Chung, Zhang & Adler 1998) and Neis- 149 O. DE SMIDT et al. HctB (207) S I L L W F P M V H G T E M F R H G Y F BexB (208) S I A L W F P M I H G T E M F R H G Y F OppB (209) R T A R A K G L P M R R I I F R H A L K FIG. 6 Alignment of the relatively hydrophilic portions of HctB, BexB and OppB. The number in brackets is the position of the first amino acid in each sequence. Identical amino acids in all three genes are boxed, and the matches of the OppB sequence to the Dassa/Hofnung consensus are underlined TABLE 2 Comparison of capsular transport gene and protein sizes and % identity and similarity between proteins from H. para- gallinarum 1742 with those of related bacterial species H. paragallinarum Related bacterial species Comparison on protein level ORFa Proteinb ORFa Accession no.c % Identity % Similarity hctA (648) HctA (215) bexA (654) (H. influenzae) P10640 77.2 85.1 cpxA (615) (A. pleuropneumoniae) U36397 78.6 85.6 ctrD (651) (N. meningitidis) M57677 78.6 87.4 hexA (660) (P. multocida) AF067175 75.3 85.6 hctB (798) HctB (265) bexB (798) (H. influenzae) P19391 57.7 86.0 cpxB (798) (A. pleuropneumoniae U36397 57.7 84.9 ctrC (798) (N. meningitidis) M57677 58.1 86.7 hexB (798) (P. multocida) AF067175 58.5 82.3 hctC (1164) HctC (387) bexC (1134) (H. influenzae) P22930 43.7 67.2 cpxC (1167) (A. pleuropneumoniae) U36397 43.9 65.6 ctrB (1164) (N. meningitidis) M57677 38.7 58.1 hexC (1137) (P. multocida) AF067175 49.9 71.8 hctD (1188) HctD (395) bexD (1182) (H. influenzae) P22236 42.5 63.0 cpxD (1212) (A. pleuropneumoniae) U36397 42.5 65.0 ctrA (1176) (N. meningitidis) M57677 41.5 62.5 hexD (1182) (P. multocida) AF067175 43.0 68.1 a Open reading frame of each capsule transport gene and corresponding nucleotide size in base pairs b Predicted proteins for each capsule transport gene and protein size in amino acids c GenBank accession numbers of capsular transport sequences from related bacterial species seria meningitidis ctrABCD (Frosch, Muller, Bousset & Muller 1992) (Table 2). The predicted amino acid sequences of the hct genes showed significant identity with the capsule transport genes of related organisms. The predicted HctA protein showed on average 77 % identity and 85.4 % similarity with the A proteins of H. influenzae, A. pleuropneumoniae and P. multocida. HctA con- tains the ATP-binding domains A (GXLGRXGXGKS) and B (XXDNLRFI) (Walker, Sarste, Runswick & Gay 1982) at amino acids 1080–1092 and 1131– 1138 respectively (Fig. 5, regions C1 and C2), which are conserved in the BexA and CpxA homologues (Kroll et al. 1990; Fath & Kolter 1993; Ward & Inzana 1997). The nucleotide homology as well as the high degree of similarity between homologous proteins, supports the speculation that hctA might encode an ATP-binding protein component of a polysaccharide export apparatus. HctB protein showed an average of 58 % identity and 84.4 % similarity with its corresponding homo- logues and is predicted to be a hydrophobic protein over most of its length, containing at least six poten- tial membrane-spanning a-helical domains (Kyte & Doolittle 1982; Kroll et al. 1990 ). A short relatively hydrophilic region starting at amino acid 990 (Fig. 5, region B) aligned with a similar region in OppB of Salmonella typhimurium (Hiles, Gallagher, Jamie- son & Higgins 1987) and BexB from H. influenzae (Kroll et al. 1990). Furthermore, each showed a mar- ginal sequence similarity to a consensus thought to be involved in intermolecular interactions in the oligopeptide transporter (Dassa & Hofnung 1985). Fig. 5 (region B) shows the position of this sequence on HctB and Fig. 6 shows an alignment of the rela- tively hydrophilic portions of HctB, BexB and OppB. HctB is therefore a candidate for an integral inner- membrane component of the putative polysaccha- ride exporter. The multiple protein sequence alignment of HctC with the respective C proteins of H. influenzae, A. pleuropneumoniae and P. multocida showed a lower homology (average of 45.8 % identity and 68.2 % similarity) in comparison to HctA and B with their corresponding homologues. Transposon mutagen- esis of bexC (Kroll et al. 1990) suggested that this gene might be a periplasmic protein. Prediction of protein subcellular localisation of the HexC protein performed with PSORT (Nakai & Kahehisa 1991), suggested an inner membrane protein, possibly with a periplasmic domain, concurring with the transpo- son mutagenesis data on BexC (Chung et al. 1998). The N-terminus of BexC containing phosphatase activity suggests that the protein is either excreted into the periplasm with cleavage of an N-terminal leader peptide or anchored in the bacterial inner membrane by an uncleaved N-terminal domain to protrude into the periplasm. It is therefore a candi- date for a periplasmically orientated component of a capsular polysaccharide exporter. Ward & Inzana (1997) predicted the CpxC protein of A. pleurop- neumoniae to be relatively hydrophilic with hydro- phobic domains near the N and C-termini that may serve as membrane anchors. Three long hydropho- bic stretches of amino acid sequence with mem- brane-spanning potential allowing the possibility of anchoring at more than one site have been identi- fied in BexC (Kroll et al. 1990). Similar stretches of sequence are present in HctC at amino acids 421– 442, 458–475 (Fig. 5, regions A1 and A2) at the proposed N-terminal and 753–777 at the C-terminal (Fig. 5, region A3). Considering this information and the facts known about the HctC homologues, it is proposed that this protein serves as the second component of a protein complex involved in poly- saccharide export across the cytoplasmic mem- brane (Reizer, Reizer & Saver 1992). HctD showed an average of 42.6 % identity and 65.3 % similarity with the predicted D proteins of H. influenzae, A. pleuropneumoniae and P. multocida. HctD showed similarity of 63 % with BexD and 62.5 % with CrtA from H. influenzae and N. menin- gitidis respectively. CtrA from Neisseria meningitidis is believed to be an outer membrane protein with porin properties (Frosch et al. 1992). In addition, BexD and its homologues is believed to be outer membrane associated (Kroll et al. 1990; Rosenow, Esumah, Roberts & Jann 1995), mutations in the bexD gene coding for this corresponding protein accumulated polysaccharides in the periplasmic space (Bronner, Clarke & Whitfield 1994). Based on these similarities with CtrA and BexD, HctD it is probably an outer membrane protein involved in capsular polysaccharide transport across the outer membrane, possibly with porin properties. These data are therefore consistent with the hy- pothesis that the hctABCD gene cluster encodes proteins that form an export complex for capsule polysaccharides. The findings will greatly facilitate the investigation at molecular level of the role of the H. paragallinarum capsule in pathogenesis. How- ever, confirmation of the importance of each gene product and elucidation of the function of each pro- tein will require characterization of the phenotypic impact of in-frame deletions or other mutations in the respective genes. In-frame deletions might lead 150 Genetic organisation of capsule transport gene region from Haemophilus paragallinarum 151 to reduced virulence with the possible use as a live vaccine. ACKNOWLEDGEMENTS This study was supported financially by the National Research Foundation (NRF) as a student scholar- ship to O. de Smidt and core funding to J. Albertyn. 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