Isolation of the gene encoding pilin of Bacteroides nodosus (strain 198), the causal organism of ovine footrot

The gene for pilin, the monomeric protein subunit from which the pilus of Bacteroides nodosus is constructed, has been isolated. Isolation was achieved by cloning the fragmented genome of B. nodosus in Escherichia coli RR1 using the plasmid vector pBR322. Pilin-producing colonies were identified by screening with a colony immunoassay using antiserum from a sheep immunized against purified pili from B. nodosus strain 198, and were further characterized by immunoblot analysis. Final confirmation of the presence of the pilin gene was by nucleotide sequence data which translated to the known pilin amino acid sequence.     

Nucleotide sequence of the gene encoding the two-subunit pilin of Bacteroides nodosus 265.

The nucleotide sequence of the gene encoding pilin from Bacteroides nodosus 265 has been determined. The pilin is encoded by a single-copy gene, from which can be predicted a prepilin comprising a single protein chain of Mr 16,637. The prepilin sequence differs in several respects from the mature protein sequence. Seven additional N-terminal amino acid residues are present in prepilin, whereas residue 8, phenylalanine, undergoes posttranslational modification to become the N-methylated amino-terminal residue of mature pilin. In addition, further processing occurs through internal cleavage to produce two noncovalently linked subunits characteristic of pilins from serogroup H of B. nodosus, of which strain 265 is a member. The position of cleavage has been identified between alanine residues at positions 72 and 73 of the mature 149-residue pilin protein. The predicted pilin sequence of B. nodosus 265 shows extensive N-terminal amino acid sequence homology with other pilins of the N-methylphenylalanine type. In addition this sequence also shows homology with these N-methylphenylalanine-type pilins in the C-terminal region of the molecule, especially with pilin from Pseudomonas aeruginosa PAK.     

Sequence of pilin from Bacteroides nodosus 351 (Serogroup H) and implications for serogroup classification

The nucleotide sequence of the pilin gene from Bacteroides nodosus strain 351, currently classified as serogroup H, subgroup 2 (H2) has been determined. The gene encodes a single polypeptide (prepilin) of 160 amino acids and Mr 17,150. However, pilin isolated from B. nodosus 351 migrates as two distinct bands in sodium dodecyl sulphate-polyacrylamide gel electrophoresis, due to an internal peptide bond cleavage. Amino acid sequence studies of pilin from B. nodosus 351 have established that the cleavage occurs between 72Ala and 73Ser of the mature protein sequence. Comparisons of gene and amino acid sequences of pilin from B. nodosus 351 with the corresponding sequences from strains of serogroups D and H1 indicate that these sequences share a close relationship. However, the level of sequence identity between B. nodosus 351 pilin and pilin from strain 265 of serogroup H1 is lower than anticipated for strains within a serogroup and suggests that B. nodosus 265 and B. nodosus 351 should not be classified within the same serogroup.     

Molecular analysis of one of multiple protease-encoding genes from the prototype virulent strain of Bacteroides nodosus

The aim of these studies was to examine the organization of the Bacteroides nodosus protease-encoding gene(s). The extracellular serine proteases (38 kDa) from the prototype virulent strain of B. nodosus were purified and used to raise a specific antiserum in rabbits. This antiserum was used in a colony immunoassay to screen a genomic DNA library constructed in Escherichia coli using BamHI-digested B. nodosus DNA and the plasmid pBR322. An E. coli clone expressing a 50-kDa immunoreactive polypeptide was identified. No protease activity was detected in the culture media, or in crude soluble and membrane fractions prepared from this clone. Restriction mapping and deletion analysis of the recombinant plasmid, pEKM2, was used to locate the coding region to a 1.4-kb EcoRI-BamHI fragment which was subsequently sequenced. A large open reading frame was found to extend through the BamHI site from a putative start codon just downstream from the EcoRI site, which indicated that the complete gene was not isolated. Southern blotting demonstrated that there were at least three B. nodosus BamHI fragments which were homologous to the 0.4-kb PstI-BamHI fragment of pEKM2. Based on these results the existence of multiple protease genes in B. nodosus was postulated.     

Nucleotide sequence of the pilin gene of Bacteroides nodosus 340 (serogroup D) and implications for the relatedness of serogroups

The gene encoding pilin of Bacteroides nodosus 340 has been isolated and the nucleotide sequence determined. The gene is present as a single copy within the B. nodosus genome and a protein of Mr 16683 can be predicted from the proposed coding region. A comparison of the predicted amino acid sequence with pilin from other strains of B. nodosus indicated that the protein of strain 340 (serogroup D) has a high degree of similarity with pilin of strain 265 (serogroup H). The degree of similarity between pilins from these strains and from other B. nodosus serogroups is no greater than that between B. nodosus pilins and the homologous proteins of several different bacterial species. These findings suggest that serogroups D and H may form a subset of B. nodosus serogroups.     

Isolation and characterization of Bacteroides nodosus fimbriae: structural subunit and basal protein antigens

We examined the isolation of fimbriae from Bacteroides nodosus. It was found that the best preparations were obtained from the supernatant of washed cells cultured on solid medium, from which fimbriae could be recovered in high yield and purity by a simple one-step procedure. Analysis of such preparations by sodium dodecyl sulfate gel electrophoresis showed that greater than 98% of the protein consisted of fimbrial structural subunits whose molecular weight was ca. 17,000. These preparations also usually exhibited minor contamination with a polypeptide of ca. 80,000 molecular weight, as well as trace amounts of lipopolysaccharide. Attempts to release additional fimbriae by the traditional means of subjecting the bacterial cells to physical stress, such as shearing or heating, resulted primarily in an increase in the level of contamination, without significant gain in the yield of fimbriae. Removal of the 80,000-dalton component could not be achieved by any of a variety of techniques normally used in fimbriae purification, including isoelectric precipitation, MgCl2 precipitation, and CsCl gradient ultracentrifugation, implying a direct physical association with the fimbrial strand. Electron micrographs of fractions containing this protein show cap-shaped structures attached to the ends of what appeared to be fimbrial stubs. These observations suggest that the 80,000-dalton polypeptide may actually constitute the basal attachment site which anchors the fimbria to the outer membrane, analogous to a similar protein recently described in enterotoxigenic strains of Escherichia coli. In B. nodosus, this 80,000-dalton protein is a major surface antigen, and like the fimbrial subunit, exhibited variation in electrophoretic mobility between serotypically different isolates.     

Fimbriae of Bacteroides nodosus: protein engineering of the structural subunit for the production of an exogenous peptide

The pattern of sequence variation between Bacteroides nodosus fimbrial subunits of different serotypes suggests a degree of flexibility, which might be exploited for protein engineering approaches for the expression of other peptides. We have tested this using the well-characterized peptide epitope from VP1 of foot-and-mouth disease virus (FMDV), residues 144-159: LRGDLQVLAQKVARTL (strain 01-BFS). Using bacterial codon usage, several oligonucleotides were designed for the substitution of this sequence internally at hypervariable regions of the fimbrial subunit (aligned for maximum homology), and for its addition at the carboxyterminus with a diglycine spacer as a flexible hinge. Following site-directed mutagenesis in phage M13, the modified genes were placed under PL promoter control and placed in a broad host range vector. Analysis of the variant proteins expressed in E. coli showed that these substitutions affected, to varying extents, recognition by both anti-fimbrial and anti-FMDV antibodies, indicating that hypervariable region 2 is a major antigenic determinant of the fimbrial subunit and that local stereochemical effects can influence antibody binding to the FMDV peptide antigenic determinant. In Pseudomonas aeruginosa, viable transformants could only be obtained with the mutant gene encoding the carboxy-terminal graft. These cells provided fimbrial preparations comprised of the modified subunit. This then constitutes the prototype for the development of a general expression system for the production of vaccine epitopes and other bioactive peptides. Furthermore, there is considerable scope for further modification of the system, for example by engineering specific chemical or protease cleavage sites for release of the grafted peptide. Alternatively, the fimbriae themselves may serve as a useful supramolecular carrier or adjuvant for immune provocation.     

Ultrastructure of the Bacteroides nodosus cell envelope layers and surface.

The surface structure and cell envelope layers of various virulent Bacteroides nodosus strains were examined by light microscopy and by electron microscopy by using negative staining, thin-section, and freeze-fracture-etch techniques. Three surface structures were described: pili and a diffuse material, both of which emerged from one or both poles of the bacteria (depending on the stage of growth and division), and large rodlike structures (usually 30 to 40 nm in diameter) associated with a small proportion of the bacterial population. No capsule was detected. The cell envelope consisted of four layers: a plasma membrane, a peptidoglycan layer, an outer membrane, and an outermost additional layer. The additional layer was composed of subunits, generally hexagonally packed with center-to-center spacing of 6 to 7 nm. The outer membrane and plasma membrane freeze-fractured through their hydrophobic regions revealing four fracture faces with features similar to those of other gram-negative bacteria. However, some unusual features were seen on the fracture faces of the outer membrane: large raised ring structure (11 to 12 nm in diameter) on cw 3 at the poles of the bacteria; complementary pits or ring-shaped depressions on cw 2; and small raised ring structures (7 to 8 nm in diameter) all over cw 2.     

Primary structure of the F-gene from Rinderpest virus strain K

Synthesis, cDNA cloning, and nucleotide sequencing of F gene of rinderpest virus strain K was carried out. Analysis of nucleotide sequence showed the only open reading frame coding for protein from 546 a.o. with mol. weight 58.6 kDa. The mean percentage of identical nucleotide residues between F genes of strains K, Kabete O, and L is 76.4% for 5'-untranslated region and 90.5% for translated region, the share of similar amino acid residues in the respective proteins is 92.9%. The structure of restriction site of F0 precursor protein in rinderpest strains with different virulence is similar. Protein F of rinderpest virus strain K has 3 potential glycosylation sites and 13 cystein residues in positions identical to those of F protein of rinderpest strains Kabete O and L.     

The complete primary structure of pilin fromHaemophilus influenzae type b strain Eagan

Adherence ofHaemophilus influenzae type b (Hib) to human oropharyngeal cells is mediated by pili which are proteinaceous filaments that extend outward from the bacterial cell surface. Pili from Hib strain Eagan were purified, and the primary structure of the major subunit, pilin, was determined. Sequencing of overlapping peptides showed the mature protein to be comprised of 196 amino acids and to have an M_r of 21,152. The amino terminal sequence was found to be homologous with the sequence previously reported for Hib strain M43 and also to have significant homology to pilins of other gram-negative pathogenic bacteria. Furthermore, Hib pilin had two cysteinyl residues in the amino terminal portion of the protein which were separated by 40 residues (positions 21 and 61); a motif found in other bacterial pilins. The data show that Hib pilin has structural features common to other bacterial pilins.     

Analyses of the vrl gene cluster in Desulfococcus multivorans: homologous to the virulence-associated locus of the ovine footrot pathogen Dichelobacter nodosus strain A198

Major parts of the virulence-associated vrl locus known from the gammaproteobacterium Dichelobacter nodosus, the causative agent of ovine footrot, were analyzed in the genome of the sulfate-reducing deltaproteobacterium Desulfococcus multivorans. In the genome of D. multivorans 13 of the 19 vrl genes described for D. nodosus are present and highly conserved with respect to gene sequence and order. The vrl locus and its flanking regions suggest a bacteriophage-mediated transfer into the genome of D. multivorans. Comparative analysis of the deduced Vrl proteins reveals a wide distribution of parts of the virulence-associated vrl locus in distantly related bacteria. Horizontal transfer is suggested as driving mechanism for the circulation of the vrl genes in bacteria. Except for the vrlBMN genes D. multivorans and Desulfovibrio desulfuricans G20 together contain all vrl genes displaying a high degree of similarity. For D. multivorans it could be shown that guanine plus cytosine (GC) content, GC skew, di-, tri- or tetranucleotide distribution did not differ between the vrl locus and its flanking sequences. This could be a hint that the vrl locus originated from a related organism or at least a genome with similar characteristics. The conspicuous high degree of conservation of the analyzed vrl genes may result from a recent transfer event or reflect a function of the vrl genes, which is still unknown and not necessarily disease associated. The latter is supported by the evidence for expression of the vrl genes in D. multivorans, which has not been described as pathogen or to be associated to any disease pattern before.     

Molecular cloning of the fimbrial subunit gene from a benign type B isolate of Bacteroides nodosus

Abstract The fimbrial subunit gene from the benign type B Bacteroides nodosus isolate AC/6 was cloned into the Sph I site of the multicopy vector plasmid pUC19. Five Escherichia coli recombinants that were positive in a colony immunoassay were shown, by Western transfer analysis, to produce an immunologically cross-reacting protein of identical molecular size to fimbrial subunits prepared from B. nodosus AC/6. Restriction endonuclease analysis showed that 4 of the recombinant plasmids carried a 6.7 kb Sph I fragment. Recloning experiments showed that the fimbrial subunit gene was located within a 2.5 kb Eco RI- Sph I fragment and that there was a Pst I site located within the structural gene or its regulatory region. These recombinant clones will prove useful for the construction of a multivalent recombinant vaccine for the control of ovine footrot.     

Evidence that Bacteroides nodosus belongs in subgroup gamma of the class Proteobacteria, not in the genus Bacteroides: partial sequence analysis of a B. nodosus 16S rRNA gene

The taxonomic status of the anaerobe Bacteroides nodosus has for some time been uncertain. To resolve this uncertainty, the distal portion of a 16S rRNA gene from this important ovine pathogen was cloned, mapped, and sequenced. A comparison of the sequence with the sequences of 16S rRNA molecules from other bacteria indicated that B. nodosus is more closely related to Escherichia coli and other members of the class Proteobacteria than to Bacteroides fragilis or the bacteroides-flavobacterium-cytophaga phylum. The evidence from the comparison of sequence signatures suggests that B. nodosus is not a member of the genus Bacteroides but that it belongs in subgroup gamma of the class Proteobacteria.     

Activities and partial purification of extracellular proteases of Bacteroides nodosus from virulent and benign footrot

In an attempt to differentiate virulent and benign strains of B. nodosus, the extracellular proteolytic activity of these cultures was assayed with elastin, casein and hide powder azure, and the stability to heating at 55 degrees C was determined. Broth cultures of both strains hydrolysed 125I-labelled elastin, indicating that this activity is not a unique marker of virulence. When cultures were grown in Trypticase-arginine-serine broth medium modified by omitting Na2CO3 and thioglycollic acid, the total proteolytic activity and its stability at 55 degrees C could be used to differentiate isolates causing virulent or benign footrot lesions. However, when other broth cultures were used, these parameters could no longer be used to make such a distinction. The proteases of a virulent and benign strain of B. nodosus were partially purified and characterized. Four to five closely related proteases were detected by polyacrylamide gel electrophoresis at pH 8.8 in both types of isolates. The proteases are serine-type enzymes requiring a divalent metal ion such as calcium for activity. The proteases of the benign strain were somewhat less stable to heat than the enzymes of the virulent strain. Differences in the relative mobilities of the proteases of virulent and benign strains of B. nodosus, on electrophoresis at pH 8.8, suggest that this property may be used to distinguish virulent and benign strains.     

Western blot (immunoblot) analysis of the fimbrial antigens of Bacteroides nodosus

The roles of the fimbrial subunit and the putative basal protein antigens in the serological classification of Bacteroides nodosus have been examined by Western blot (immunoblot)-antibody binding studies of fimbriae isolated from a wide range of strains representative of different serogroups and serotypes. Fimbrial subunits were recognized by antiserum against the homologous serogroup but not generally by heterologous antisera, whereas recognition of the basal antigen was independent of serological classification. Secondary cross-reaction patterns among fimbrial subunits indicated that some serogroups may be more closely related than others. Examples include serogroups C and G and serogroups D and H. Similar analyses of isolates classified within serotypes A1 and A2, with serotype-specific antisera, showed that this subdivision is also determined by the fimbrial subunit and that significant variation does occur even at this level. These studies suggest that the various serogroups and serotypes of B. nodosus comprise a series of overlapping sets of antigenically related strains.     

The complete primary structure of pilin fromHaemophilus influenzae type b strain Eagan

Adherence ofHaemophilus influenzae type b (Hib) to human oropharyngeal cells is mediated by pili which are proteinaceous filaments that extend outward from the bacterial cell surface. Pili from Hib strain Eagan were purified, and the primary structure of the major subunit, pilin, was determined. Sequencing of overlapping peptides showed the mature protein to be comprised of 196 amino acids and to have an M_r of 21,152. The amino terminal sequence was found to be homologous with the sequence previously reported for Hib strain M43 and also to have significant homology to pilins of other gram-negative pathogenic bacteria. Furthermore, Hib pilin had two cysteinyl residues in the amino terminal portion of the protein which were separated by 40 residues (positions 21 and 61); a motif found in other bacterial pilins. The data show that Hib pilin has structural features common to other bacterial pilins.