Department of Microbiology and Parasitology, The University of Queensland, Brisbane, Queensland 4072, Australia1
Author for correspondence: Michael P. Jennings. Tel: +61 7 3365 4879. Fax: +61 7 3365 4620. e-mail: jennings{at}biosci.uq.edu.au
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ABSTRACT |
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Keywords: pilin, glycosylation, glycosyltransferase, lipopolysaccharide, Neisseria meningitidis
Abbreviations: DATDH, 2,4-diacetimido-2,4,6-trideoxyhexose; DIG, digoxigenin
The GenBank accession number for the sequence determined in this work is AF014804.
a Present address: Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A 0R6.
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INTRODUCTION |
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METHODS |
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Recombinant DNA techniques and nucleotide sequence analysis.
Most recombinant DNA techniques were used as described in Sambrook et al. (1989) . Nucleotide sequence analysis was performed using the PRISM Dye Terminator Sequencing Kit with AmpliTaq DNA polymerase FS (Perkin Elmer) in conjunction with a model 373a automated sequencer (Applied Biosystems). Oligonucleotide primers were synthesized on a model ABI392 synthesizer (Applied Biosystems). PCR was essentially done as described by Saiki et al. (1988)
. Nucleotide sequence analysis was done using MacVector (Oxford Molecular).
Southern blotting and hybridization.
Restriction endonuclease (ClaI) digested genomic DNA was separated on 0·7% agarose gels and transferred to Hybond-N+ Nylon membrane (Amersham), essentially as described in Sambrook et al. (1989) . Primers shown in Table 1
were used in the PCR reactions to amplify probes for pglB, pglC, pglD, and avtA (described in the legend to Fig. 1
). The products were purified from agarose using a Qiaex gel extraction kit (Qiagen). The fragments were then DIG-labelled and hybridization was done using the DIG DNA Labelling and Detection Kit (Boehringer Mannheim) as recommended by the manufacturer. All restriction endonucleases and ligases were obtained from New England Biolabs.
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Production of antisera specific for the glycosylated pilin of C311#3 and C311#3pglA.
Antisera directed against the pili expressed by C311#3 and C311#3pglA were raised in Lop/New Zealand White rabbits as follows. Cells collected from plate cultures of each strain were resuspended in PBS at a density of 1011 c.f.u. ml-1, vortexed for 1 min to shear the pili, and centrifuged at 14000 g for 15 min. The resulting supernatants contained similar amounts of semi-purified native pilin as confirmed by SDS-PAGE and Western analysis using the pilin-specific monoclonal antibody SM1. Each preparation of pili was combined with an equal volume of adjuvant (MPL + TDM + CWS; Sigma M6661) to a final concentration of approximately 2·5 mg ml-1 for inoculation into rabbits. Each rabbit received five 0·4 ml inoculations at intervals of 2 weeks, and blood was harvested by cardiac puncture 6 weeks after the final inoculation.
Using a modification of the method of Gruber & Zingales (1995) , these sera were absorbed against meningococcal cells to enhance their specificity for glycosylated pili. Briefly, C311#3 and C311#3 pglA cells were harvested from five plates into 10 ml PBS and heat-killed at 56 °C for 1 h before being washed twice in PBS and resuspended in 1·5 ml PBS. The rabbit sera were diluted 1:50 with 1% BSA in PBS, after which anti-C311#3 serum was absorbed against C311#3pglA cells and anti-C311#3pglA serum was absorbed against C311#3 cells. In each case 20 ml of the diluted serum preparation was inoculated with 0·3 ml of the cell suspension, agitated at 4 °C for 4 h, centrifuged at 3500 r.p.m. for 15 min, and the pellet discarded. Each serum underwent five cycles of absorption, followed by filtration through a 22 µm Millipore filter and storage at -20 °C until used. Specificity of the absorbed anti-C311#3 serum for the pilin-linked trisaccharide (anti-trisaccharide serum) was confirmed in three independent Western blot experiments against whole-cell lysates of C311#3, C311#3pglA and C311#3galE mutants. Similarly, specificity of the absorbed anti-C311#3pglA serum to the truncated glycan modification present on the pilin of C311#3plgA (anti-pglA serum) was also confirmed.
SDS-PAGE, Western blotting and LPS analysis.
Analysis of LPS and pilin migration using SDS-PAGE and detection of pilin-specific bands in Western blots using SM1 have been described previously (Virji et al., 1993 ). The anti-trisaccharide and anti-pglA sera were diluted twofold (to a total dilution of 1:100) in 5% skimmed milk powder in TBS before use as primary antibody in Western analysis. Western analyses using the anti-trisaccharide and anti-pglA sera were carried out as for SM1 above but using the anti-rabbit IgG secondary antibody (Sigma). All Western analysis results were confirmed in three independent experiments.
Selective detection of glycoproteins containing terminal galactose moieties.
Galactose oxidase was used to create specific aldehyde groups at the C6 position of terminal galactose residues based on the method of Haselbeck & Hösel (1993) . Briefly, Western membranes were blocked overnight at 4 °C with 1% BSA in PBS (pH 7·5) and then washed for 5 min with 50 ml buffer 1 (0·1 M potassium phosphate, pH 6·0). The membranes were then incubated for 15 h at 37 °C with 7·5 U galactose oxidase (Worthington) and 2 µl 5 mM biotinamidocaproyl hydrazide (Sigma) in 10 ml buffer 1, followed by three 50 ml washes with PBS. The membranes were then blocked a second time for 1 h in 0·5% casein in PBS followed by three 50 ml washes with TBS (Tris-buffered saline, pH 7·5), before immersion in conjugate solution (1 µg ml-1 streptavidinalkaline phosphatase in TBS) for 1 h at room temperature. Three 10 ml washes with TBS were then carried out prior to immersion in colour-development solution (NBT/BCIP). Development was stopped by rinsing the membranes several times with TBS and air drying. The controls used in these experiments were pilin isolated from the C311#3galE mutant (which is unable to synthesize UDP-galactose) and duplicate blots either without galactose oxidase or without biotinamidocaproyl hydrazide. The control blots without biotinamidocaproyl hydrazide detected a non-specific protein band slightly smaller than pilin already bound to biotin (possibly the biotin carboxyl carrier protein).
Electron microscopy.
Cells were grown overnight on BHI agar as described above. Two colonies per plate were resuspended in a 10 µl drop of sterile PBS (pH 7·2). Pioloform-coated copper grids were gently placed on the drop for 1 min, then for 3 min on a 50 µl drop of 3% glutaraldehyde (in PBS), followed by washing twice in 20 µl PBS and three times in 50 µl water. Staining was carried out for 20 s in a 20 µl drop of ammonium molybdate (1% in PBS) and the grids dried by gently blotting on filter paper. Micrographs were taken on a JEOL1010 operating at 6080 kV.
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RESULTS |
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The homologies found with these genes are displayed in Table 2 and described below.
pglB.
The pglB ORF is 1242 nt in length, starting at nucleotide 556 of the determined sequence (GenBank entry AF014804) and terminating at nucleotide 1797. The putative PglB protein is 414 aa in length and has a calculated molecular mass of 44·5 kDa. A BLASTX homology search (Altschul et al., 1997 ) revealed two regions of distinct homology within the pglB ORF.
The first region, consisting of the N-terminal 201 aa of PglB, had highest similarity with a family of presumed glycosyltransferases. The highest scores were to a group of putative transferases from Campylobacter that were first identified as necessary for LPS biosynthesis (50/63% identity/similarity: Wood et al., 1999 ; Fry et al., 1998
). These genes have recently (Szymanski et al., 1999
) been shown to be involved in flagellin glycosylation (see Discussion and Table 3
). There is also similarity to genes involved in O-antigen synthesis in Anabaena sp. (Korolik et al., 1997
) and capsule biosynthesis in Staphylococcus aureus (Sau & Lee, 1996
; Sau et al., 1997a
, b
). The activity of this family of genes is based upon similarity to RfbP of Salmonella typhimurium (Wang & Reeves, 1994
, 1996
), which acts to transfer galactose to an undecaprenol phosphate in the first steps of O-polysaccharide biosynthesis. The second region of homology consists of the C-terminal 166 amino acids (from amino acid 248). This region is homologous to a family of acetyltransferases. The highest similarity with this region is LpsB of Caulobacter crescentus (41/60%), which is believed to be an UDP-N-acetylglucosamine acyltransferase involved in LPS biosynthesis (Awram & Smit, 1998
). In common with this family of genes, this area of similarity contains an isoleucine patch which is speculated to play an important structural role in the function of these enzymes (Dicker & Seetharam, 1992
). The two regions of the putative PglB also have distinct G+C contents, the N-terminal region having a G+C content of 52 mol% and the C-terminal region a G+C content of 62·2 mol%.
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pglD.
The third ORF, pglD, begins at nucleotide 3153 and terminates at nucleotide 5063. This constitutes an ORF of 1911 bp and translation yields a peptide of 637 aa with a calculated molecular mass of 71·0 kDa. The putative PglD protein is highly similar to a family of proteins proposed to be dehydratases involved in LPS and O-antigen biosynthesis. It is most similar (55/72%) to TrsG of Yersinia enterocolitica (Skurnik et al., 1995 ).
avtA.
The last ORF starts at nucleotide 5119 of the determined sequence and is 1293 bp in length, terminating at nucleotide 6411. The putative AvtA protein is 431 aa long, has a calculated molecular mass of 47·1 kDa and is homologous (46/65%) with AvtA from E. coli (Sofia et al., 1994 ). AvtA is an alanine-to-valine aminotransferase and has not been implicated in any saccharide biosynthesis.
Construction and analysis of pgl::kan mutant strains
In order to determine whether pglB, pglC or pglD played a role in either LPS biosynthesis or pilin glycosylation, mutants were constructed by the insertion of a kanamycin-resistance (kanR) cassette into restriction endonuclease sites present in each of the coding regions (see Fig. 1). These insertional mutant constructs were then used to transform strain C311#3, a meningococcal strain with a well-characterized pilin structure, so that the inactive alleles were transferred to the chromosome by homologous recombination. The presence of the inactive allele was confirmed, both by Southern hybridization and by PCR of the mutated alleles (see Methods; results not shown).
LPS phenotype of pglB, pglC and pglD::kan mutant strains.
Many of the genes involved in meningococcal LPS biosynthesis have been described (Jennings et al., 1999 ). However, there is still the potential for novel structures to be identified. To determine whether pglB, pglC or pglD affected LPS biosynthesis, the LPS of the mutant derivatives was analysed by SDS-PAGE. This revealed that there were no alterations in migration of the LPS molecule isolated from any of the mutant strains when compared to the parental strain and the control strain C311#3galE::kan (Fig. 2b
). These results indicate that neither pglB, pglC or pglD is involved in LPS biosynthesis.
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Sequencing of the pilE gene of the pglB, pglC and pglD::kan mutants of strain C311#3.
The gene encoding the pilin subunit, pilE, displays a high degree of sequence variation, particularly towards the carboxy-terminal end of the protein (Seifert, 1996 ). Alterations in the sequence of pilE result in alterations in pilin migration in SDS-PAGE. Therefore, the pilE genes of the pglB, pglC and pglD::kan mutants of C311#3 were sequenced and the sequences compared with the pilE sequence of the parental strain C311#3 (GenBank accession number L22639). The sequences (results not shown) confirmed that there was no difference in the pilE genes of the mutants compared to the parental gene, indicating that the differences observed in pilin migration from these mutants must be due to alterations in post-translational modifications.
Pilin of the pglB, pglC and pglD mutants is not detected by a terminal galactose-specific method.
In order to determine whether the change in pilin migration was due to an alteration in the trisaccharide modification, as opposed to one of the other known pilin modifications, we examined the pglB, pglC, and pglD mutant derivatives using a terminal-galactose-specific detection system (see Methods). Semi-purified pilin (see Methods) extracted from C311#3, and its galE, pglA, pglB, pglC and pglD mutants, was separated by SDS-PAGE and transferred to PVDF membrane. Pilins containing terminal galactose moieties were then selectively detected by the use of biotinamidocaproyl hydrazide following galactose oxidase treatment, which creates aldehyde groups specifically on terminal galactose residues, resulting in the incorporation of biotin specifically into structures containing a terminal galactose residue (Haselbeck & Hösel, 1993 ). On establishing this method it became clear that the semi-purified pilin preparations contained a non-specific protein band (possibly a biotin carboxyl carrier protein) that was detected in the absence of biotin hydrazide (results not shown). The predicted molecular mass of the biotin carboxyl carrier protein in N. meningitidis strain Z2491, based on the preliminary Sanger genome sequence data, is 15·9 kDa, which is consistent with its migration in SDS-PAGE. The presence of this non-specific band is evidently due to the co-isolation of this protein in the semi-purified pilin preparations. To demonstrate that this non-specific band did not co-migrate with pilin, and therefore that pilin could be detected by the galactose-specific method, a split-band experiment was carried out (Fig. 3
). In this experiment, following separation of pilin by SDS-PAGE, each lane was cut vertically. Pilin was then detected using SM1 and the non-specific band was detected with biotin/streptavidin. In all cases the non-specific band did not co-migrate with pilin. The increased concentration of the non-specific band in the pglB and pglC mutant pilin preparations resulted from a higher proportion being co-purified due to a smaller difference between the molecular mass of pilin from these mutant strains and the non-specific band.
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Electron micrographs of the pili produced by the pglB, pglC and pglD::kan mutants show no effect on pilus morphology.
To determine whether the pglB, pglC and pglD mutant derivatives of strain C311#3 still expressed pili located on the cell surface, and whether these pili had altered morphology, the pili of the pglB, pglC and pglD::kan mutants were observed by electron microscopy. All mutant derivatives were found to surface-express pili. A large degree of variation (within samples) in both piliation level and presence or absence of bundles was encountered. In general, there were no consistent differences in pilus morphology or piliation level of the pglB, pglC and pglD::kan mutants in comparison to wild-type. A representative electron micrograph of the pili of the pglB mutant compared to C311#3 is shown in Fig. 6.
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DISCUSSION |
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In this study we have characterized a genetic locus, pglBCD, from N. meningitidis that appears to encode additional enzymes involved in pilin glycosylation. Analysis of the incomplete N. meningitidis strain Z2491 genome (Sanger Centre) revealed that the pglA and the pglBCD loci are unlinked (600 kb apart). Three of the four ORFs identified in the new locus show significant homology to proteins implicated in LPS and capsule biosynthesis. Analysis of the migration of the pilin subunit of each of the pglB, pglC and pglD mutants indicated an alteration in molecular mass. Nucleotide sequence analysis confirmed that this was not due to amino acid sequence variation of PilE, indicating post-translational modification as the most likely cause of the altered migration This was confirmed by two independent methods. Firstly, use of a terminal galactose-specific stain confirmed that the post-translational modification that was affected was the pilin-linked trisaccharide, which was either absent or altered in all three of the mutant strains. The second method confirmed the absence of the complete pilin-linked trisaccharide from the pilin of each of the pglB, pglC and pglD mutants using antisera specific for either the complete trisaccharide or a truncated form.
The structure of the pilin-linked trisaccharide of C311#3 is shown in Fig. 8. To make this structure, two classes of enzyme are required: glycosyltransferases and enzymes involved in the biosynthesis of the unusual 2,4-diacetimido-2,4,6-trideoxyhexose residue. Glycosyltransferases are required for the Gal(ß1-4)Gal linkage, Gal(
1-3)DATDH linkages and the O-linkage of DATDH to serine 63. In our previous work we have proposed that pglA catalyses the Gal(
1-3)DATDH linkage; therefore two further transferases remain to be identified. Galactose is not specific to the pilin-linked trisaccharide structure, also being present in LPS, but the DATDH residue is pilin-specific.
Deoxy and dideoxy sugars, in particular 2,6- and 4,6-dideoxyhexoses, are found as components of glycoproteins, bacterial cell walls, and numerous secondary metabolites (Thorson et al., 1993 ). However, acetamido sugars are unusual. The biosynthesis of a 4-acetamido-4,6-dideoxyhexose in E. coli has been described by Dietzler & Strominger (1973)
and Matsuhashi & Strominger (1964)
. The biosynthesis of this sugar first requires a dehydratase step at the carbon at position C4 of the hexose residue, followed by transamination and acetylation. The stereospecificity of the N. meningitidis DATDH residue, and therefore its likely precursor molecule, is unknown. However, we predict that the biosynthesis of this sugar will follow the pathway described (above) for acetamido sugars. Therefore, the biosynthesis of this residue would require several specific enzyme activities, including dehydratases, transaminases and acetyltransferases. Homologues found to the pglB, pglC and pglD genes suggest they have roles in the biosynthesis of this amino sugar residue.
The first ORF identified in the locus, pglB, possesses two regions of distinct homology which also have distinct G+C contents. This suggests a recent in-frame fusion of two ORFs resulting in a bifunctional protein. For this reason, the two distinct regions will be discussed separately. The amino-terminal region of the putative PglB protein is highly homologous with a family of putative glycosyltransferases. These glycosyltransferases are proposed to transfer a sugar residue (usually galactose) to the lipid precursor undecaprenol phosphate (Wood et al., 1999 ; Fry et al., 1998
; Drummelsmith & Whitfield, 1999
). This region of PglB also contains the hydrophobic domain that is required by these proteins for interaction with the undecaprenol phosphate lipid carrier (Wood et al., 1999
). The transfer of a nucleotide sugar to a lipid carrier is often the first step in oligosaccharide biosynthesis. It is therefore suggested that PglB may be required in the transfer of the first sugar residue either to an undecaprenol phosphate lipid carrier or directly to the serine 63 of pilin. This transfer may represent the initial step in the glycosylation of pilin.
The carboxy-terminal 168 aa stretch of the putative PglB protein shows strong homology with a family of acetyltransferases (e.g. UDP-N-acetylglucosamine and sialic acid transfer) required for LPS and capsular polysaccharide biosynthesis (Awram & Smit, 1998 ; Fry et al., 1998
; Annunziato et al., 1995
). Typical of this family of acetyltransferases, PglB contains an isoleucine patch which is a common signature associated with acetyltransferase function (Dicker & Seetharam, 1992
). The biosynthesis of acetamido sugars such as the 2,4-diacetamido sugar residue found in the pilin-linked trisaccharide requires acetylation of the amino groups. Therefore, this region of PglB is suggested to be involved in the acetylation at C2 or C4 of the diacetamido sugar constituent of the pilin-linked trisaccharide.
Thus it appears that PglB may be a bifunctional protein involved in the early steps of trisaccharide biosynthesis, namely in the biosynthesis of the first sugar residue and its transfer either to an undecaprenol phosphate lipid carrier or to serine 63 of the pilin. The pilin produced by the pglB::kan mutant would be expected to be devoid of the complete trisaccharide structure. The altered migration, the absence of a terminal galactose residue and the failure of pilin isolated from the pglB::kan mutant to react with either anti-trisaccharide or anti-pglA sera support this hypothesis.
The second ORF identified in the locus, pglC, shows significant homology with a family of aminotransferases involved in the synthesis of amino sugars (Stroeher et al., 1995 ). The putative PglC protein is strongly similar to RfbE of E. coli, which is a probable perosamine synthetase. RfbE is believed to catalyse the transamination of GDP-4-keto-6-deoxymannose to GDP-4-amino-4,6-dideoxymannose (perosamine; Stroeher et al., 1995
). PglC is also highly similar to NikC from Streptomyces Tü901, which has been shown to encode an L-lysine 2-aminotransferase required for the biosynthesis of nikkomycin (Bruntner & Bormann, 1998
). All of these proteins belong to a novel class of pyridoxamine- or pyridoxal-phosphate-dependent dehydrases and aminotransferases, based on the presence of a pyridoxal-phosphate-binding motif (PS00600; Appel et al., 1994
). This is consistent with the findings of Matsuhashi & Strominger (1966)
, where a pyridoxal-phosphate-dependent transaminase is required for the biosynthesis of an acetamido sugar. This leads us to propose that in N. meningitidis PglC is required for the transamination of either C2 or C4. Therefore, we would expect the pglC::kan mutant to be unable to synthesize a complete DATDH residue, resulting in pilin with either a single sugar residue or no glycosylation depending on the substrate specificity of the glycosyltransferases. The possibility of complete absence of glycosylation on the serine 63 of pilin isolated from the pglC::kan mutant is supported by its increased migration, its failure to be detected by a terminal galactose-specific stain and its failure to be detected by either anti-trisaccharide or anti-pglA sera.
The pglD gene product is highly similar to proteins essential for either LPS or O-antigen amino sugar biosynthesis (Skurnik et al., 1995 ; Comstock et al., 1996
). These proteins all have maximum homology in their carboxy-termini and are divergent in their amino-termini. Fallarino et al. (1997)
suggested that this was due to the amino-terminus being involved in specificity and the carboxy-terminus incorporating the functional domain of these proteins. The putative PglD protein shares these characteristics and also contains the two distinct motifs, both for binding NAD, that have been suggested to be a requirement for enzyme function (Skurnik et al., 1995
). PglD demonstrated high similarity to the TrsG from Y. enterocolitica, which is proposed to be an epimerase or dehydratase involved in N-acetylgalactosamine biosynthesis (Skurnik et al., 1995
). The BplL protein of Bordetella pertussis is also highly similar to PglD and itself has some strong similarities to a number of dTDP-glucose 4,6-dehydrases (Bechthold et al., 1995
; Linton et al., 1995
). Based on this observation, Allen & Maskell (1996)
suggest that BplL is responsible for the biosynthesis of FucNAcMe (a 6-deoxy derivative of galactose). They further suggest that this group of enzymes are generally required for the synthesis of 6-deoxy and dideoxy sugars.
It seems likely, therefore, that PglD is involved in synthesis of the DATDH moiety of the pilin-linked trisaccharide as a dehydratase acting on C2, C4 or C6. Therefore, we would expect a similar modification (i.e. one or no sugar residues) to be present on the pglD::kan mutant pilin, as is expected for the pglC::kan mutant. This hypothesis is again supported both by the decreased migration of pilin isolated from the mutant compared to the wild-type, and also by the failure of this pilin to be detected by a terminal galactose-specific method, anti-trisaccharide or anti-pglA sera.
The final ORF in the identified locus, AvtA, has a high degree of similarity to putative alanine-to-valine aminotransferases (e.g. AvtA of E. coli; Sofia et al., 1994 ). These genes are involved in amino acid biosynthesis and have not been implicated in sugar biosynthesis. Several attempts were made to knock out the avtA gene of N. meningitidis, but they were unsuccessful, indicating a possible essential housekeeping function for this protein. Therefore, the role (if any) of this gene in pilin glycosylation could not be determined.
The functions proposed for the pglB, pglC and pglD genes, and their close proximity to each other, suggest that they act together in the biosynthesis of the acetamido sugar residue of the pilin-linked trisaccharide. The biosynthesis of an acetamido sugar, as described by Matsuhashi & Strominger (1966) , is initiated by dehydration (PglD dependent), followed by transamination (PglC dependent) and transacetylation (PglB dependent) of a single carbon. This acetamido sugar could then be transferred to either a lipid carrier or the serine 63 of pilin by PglB. As the DATDH sugar residue is a diacetamido sugar, we suggest that pglB, pglC and pglD may act on a precursor that is either a C2 or a C4 acetamido sugar (for example glucosamine or fucosamine).
A survey to detect the presence of pglB, pglC, pglD and avtA, using Southern analysis, revealed that these genes were present in all of the N. meningitidis strains tested, in N. gonorrhoeae and also in other species of Neisseria. This was also found with the previously identified pglA gene, suggesting that pilin glycosylation may be common amongst neisserial species (Jennings et al., 1998 ). During the preparation of this paper, Szymanski et al. (1999)
reported the identification of a Campylobacter jejuni locus involved in the biosynthesis of an undefined glycosylation on the flagella of this organism. Although the sequence identities were not amongst the highest matches found, the genes were present in the same order as the pglBCD locus described here (see Table 3
). Szymanski et al. (1999)
not only implicated the locus in glycosylation of flagella, but also of other proteins, confirming that their locus represents a general glycosylation pathway.
In conclusion, this study has identified three genes, pglB, pglC and pglD, involved in pilin glycosylation, in addition to the previously identified pglA gene (Jennings et al., 1998 ). To date, the precise functions that we have predicted for pglB, pglC and pglD are based on amino acid sequence homologies with genes in the databases that are involved in the biosynthesis of similar chemical structures to the pilin-linked trisaccharide. Currently we are obtaining structural data and biochemical evidence to support the gene functions that we have proposed.
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ACKNOWLEDGEMENTS |
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Received 22 July 1999;
revised 1 December 1999;
accepted 16 December 1999.