Cloning of a novel pilin-like gene from Bordetella pertussis: homology to the fim2 gene

A search for pilin genes in a Bordetella pertussis (Bp) genomic library has led to the identification of several clones which hybridize to synthetic oligonucleotides with sequences derived from amino acid sequences of Bp fimbrial subunits. One of these clones (corresponding to a gene we have named fimX) contains an open reading frame encoding a protein with a molecular weight of about 20 kD and a sequence similar but not identical to the fimbrial subunit fim2 and to other fimbrial protein sequences. In this communication we present the cloning and nucleotide sequence of the fimX gene and its homology to the fim2 gene. A genomic analysis on the positional relationship between the two genes is also presented.     

Characterization of a Bordetella pertussis fimbrial gene cluster which is located directly downstream of the filamentous haemagglutinin gene

The biosynthesis of fimbriae is a complex process requiring multiple genes which are generally found clustered on the chromosome. In Bordetella pertussis, only major fimbrial subunit genes have been identified, and no evidence has yet been found that they are located in a fimbrial gene cluster. To locate additional genes involved in the biosynthesis of B. pertussis fimbriae, we used TnphoA mutagenesis. A PhoA+ mutant (designated B176) was isolated which was affected in the production of both serotype 2 and 3 fimbriae. Cloning and sequencing of the DNA region harbouring the transposon insertion revealed the presence of at least three additional fimbrial genes, designated fimB, fimC and fimD. The transposon was found to be located in fimD. Analysis of PhoA activity indicated that the fimbrial gene cluster was positively regulated by the bvg locus. A potential binding site for BvgA was observed upstream of fimB. FimB showed homology with the so-called chaperone-like fimbrial proteins, while FimC was homologous with a class of fimbrial proteins located in the outer membrane and presumed to be involved in transport and anchorage of fimbrial subunits. An insertion mutation in fimB abolished the expression of fimbrial subunits, implicating this gene in the biosynthesis of both serotype 2 and 3 fimbriae. Upstream of fimB a pseudogene (fimA) was observed which showed homology with the three major fimbrial subunit genes, fim2, fim3 and fimX. The construction of a phylogenetic tree suggested that fimA may be the primordial major fimbrial subunit gene from which the other three were derived by gene duplication. Interestingly, the fimbrial gene cluster was found to be located directly downstream from the gene coding for the filamentous haemagglutinin, an important B. pertussis adhesin, possibly suggesting co-operation between the two loci in the pathogenesis of pertussis.     

Characterization of fimN, a new Bordetella bronchiseptica major fimbrial subunit gene

Fimbrial proteins play an important role in the binding of Bordetella bronchiseptica to mammalian cells, an event that is key to the pathogenesis of this organism. The fimbrial phenotype of B. bronchiseptica isolates is usually defined serologically by Fim2 and Fim3 antigens. In this study, a previously unidentified fimbrial gene, fimN, was cloned and sequenced. The identity of fimN is based on several observations. The predicted FimN protein has 59.4 and 52. 2% homology with B. bronchiseptica Fim2 and Fim3, respectively, and is similar in size to these fimbriae. fimN, expressed as a recombinant protein, is recognized by mAb prepared against Fim2 from Bordetella pertussis. The fimN promoter region contains a stretch of cytosine residues similar in length to those of other fimbrial genes expressed by Bordetella species. It also has an activator binding region, upstream from the C-stretch, that closely resembles a corresponding bvg regulated region in fim2, fim3, and fimX. The fimN gene was isolated from a cosmid prepared with B. bronchiseptica genomic DNA that restored normal properties of cellular adhesion to an adhesion deficient strain of B. bronchiseptica. As such, FimN may be a previously overlooked fimbrial antigen and may play an important role in the pathogenicity of B. bronchiseptica.     

Serotype variation in Bordetella pertussis is governed by cis-acting elements

Bordetella pertussis contains two genes encoding the serospecific fimbrial subunit proteins 2 and 3 which are assembled into completed fimbriae, which elicit the formation of agglutinating antibodies. Expression of these agglutinogens can vary independently of each other. A gene library from a B. pertussis strain (fimbrial serotype 0.3) was probed with an oligonucleotide probe specific for fimbrial subunit genes. Three homologous genetic loci were identified; an active fim 3 gene, an inactive fim 2 gene and an unknown fim-homologous region. The fim 3 gene carried on a cosmid produced agglutinating fimbrial structures in B. parapertussis and in variants of B. pertussis which had lost the capacity to produce the agglutinogen. This indicated that cis-acting factors are associated with serotype variation in B. pertussis rather than the production of trans-acting repressor molecules.     

Nucleotide sequences of the genes encoding type 1 fimbrial subunits of Klebsiella pneumoniae and Salmonella typhimurium.

The nucleotide sequences of the genes encoding the subunits of Klebsiella pneumoniae and Salmonella typhimurium type 1 fimbriae were determined. Comparison of the predicted amino acid sequences of the two subunits revealed domains in which the sequences were highly conserved. Both gene products possessed signal peptides, a fact consistent with the transport of the fimbrial subunit across the membrane, but these regions showed no amino acid homology between the two proteins. The predicted N-terminal amino acid sequences of the processed fimbrial subunits were in good agreement with those obtained by purification of the fimbrial subunits.     

Porphyromonas gingivalis FimA Fimbriae: Fimbrial Assembly by fimA Alone in the fim Gene Cluster and Differential Antigenicity among fimA Genotypes

The periodontal pathogen Porphyromonas gingivalis colonizes largely through FimA fimbriae, composed of polymerized FimA encoded by fimA. fimA exists as a single copy within the fim gene cluster (fim cluster), which consists of seven genes: fimX, pgmA and fimA-E. Using an expression vector, fimA alone was inserted into a mutant from which the whole fim cluster was deleted, and the resultant complement exhibited a fimbrial structure. Thus, the genes of the fim cluster other than fimA were not essential for the assembly of FimA fimbriae, although they were reported to influence FimA protein expression. It is known that there are various genotypes for fimA, and it was indicated that the genotype was related to the morphological features of FimA fimbriae, especially the length, and to the pathogenicity of the bacterium. We next complemented the fim cluster-deletion mutant with fimA genes cloned from P. gingivalis strains including genotypes I to V. All genotypes showed a long fimbrial structure, indicating that FimA itself had nothing to do with regulation of the fimbrial length. In FimA fimbriae purified from the complemented strains, types I, II, and III showed slightly higher thermostability than types IV and V. Antisera of mice immunized with each purified fimbria principally recognized the polymeric, structural conformation of the fimbriae, and showed low cross-reactivity among genotypes, indicating that FimA fimbriae of each genotype were antigenically different. Additionally, the activity of a macrophage cell line stimulated with the purified fimbriae was much lower than that induced by Escherichia coli lipopolysaccharide.     

Bordetella species are distinguished by patterns of substantial gene loss and host adaptation

Pathogens of the bacterial genus Bordetella cause respiratory disease in humans and animals. Although virulence and host specificity vary across the genus, the genetic determinants of this diversity remain unidentified. To identify genes that may underlie key phenotypic differences between these species and clarify their evolutionary relationships, we performed a comparative analysis of genome content in 42 Bordetella strains by hybridization of genomic DNA to a microarray representing the genomes of three Bordetella species and by subtractive hybridization. Here we show that B. pertussis and B. parapertussis are predominantly differentiated from B. bronchiseptica by large, species-specific regions of difference, many of which encode or direct synthesis of surface structures, including lipopolysaccharide O antigen, which may be important determinants of host specificity. The species also exhibit sequence diversity at a number of surface protein-encoding loci, including the fimbrial major subunit gene, fim2. Gene loss, rather than gene acquisition, accompanied by the proliferation of transposons, has played a fundamental role in the evolution of the pathogenic bordetellae and may represent a conserved evolutionary mechanism among other groups of microbial pathogens.     

Molecular epidemiology of Bordetella pertussis in Taiwan, 1993-2004: suggests one possible explanation for the outbreak of pertussis in 1997

Pertussis reemerges periodically despite high pertussis vaccination coverage in many countries. We used prn and fim3 gene sequences and pulsed-field gel electrophoresis (PFGE) to analyze the molecular epidemiology of 168 clinical isolates of Bordetella pertussis during 1993-2004, and deduced possible reasons for an outbreak in 1997 in Taiwan. In Taiwan, during 1996-1997, a shift of prn1 to prn2 was reflected in a transition of PFGE group I to group IIIa; during 2000-2001, the change from fim3A to fim3B was displayed in transition of PFGE group IIIa to group IIIb. These changes were also consistent with the two peaks of pertussis incidence in 1997 and 2000. In 1997, a larger than expected increase in the incidence of pertussis occurred and isolates were characterized by complicated pulsotypes, appearance of many new profiles and an unusual presence of prn3. Based on a high resemblance of PFGE profiles and the same virulence genes, a similar shift of circulating strains was observed in European countries as well as Taiwan; thus, the high incidence of pertussis in 1997 may be due to an international expansion of B. pertussis strains from a similar source. This study provides further elucidation of the global molecular epidemiology of B. pertussis.     

Application of the hypervariable region of the 16S rDNA sequence as an index for the rapid identification of species in the genus Alicyclobacillus

A comparison of the 16S rRNA gene (rDNA) sequences of seven type strains belonging to different Alicyclobacillus species (i.e., five validated species, one proposed species and one genomic species) suggested that the 5' end hypervariable region (259-273 bases in length) of 16S rDNA was specific for the respective type strains. Further phylogenetic analysis based on DNA sequences of the hypervariable region using 24 Alicyclobacillus strains revealed that the strains could be categorized into five species and the A. acidocaldarius-Alicyclobacillus genomic species 1 group. The hypervariable region was highly conserved among the five species: A. acidiphilus, A. acidoterrestris, A. cycloheptanicus, A. herbarius, and A. hesperidum. The strains in the A. acidocaldarius-Alicyclobacillus genomic species 1 group were subdivided into two clusters (Clusters I and II) based on DNA sequences in the hypervariable region. On the basis of phenotypic characteristics, chemotaxonomic and phylogenetic analyses, and DNA-DNA hybridization data, strains in Cluster I were grouped as Alicyclobacillus genomic species 1 and strains in Cluster II were re-identified as A. acidocaldarius, thereby demonstrating that the hypervariable regions were also highly conserved within these two species. These results suggest that as is the case with Bacillus, the hypervariable region is significantly species-specific in the genus Alicyclobacillus to distinguish Alicyclobacillus species by DNA sequence comparison of the hypervariable region.     

Common accessory genes for the Bordetella pertussis filamentous hemagglutinin and fimbriae share sequence similarities with the papC and papD gene families.

The Bordetella pertussis filamentous hemagglutinin (FHA) is a major virulence factor responsible for attachment, one of the early events in bacterial pathogenesis. Deletion of its structural gene, fhaB, or a Tn5 insertion in fhaA, downstream of fhaB, resulted in a FHA- and fimbriae- phenotype, although fhaB and the fim genes are not linked. The fhaB downstream region therefore most likely encodes accessory proteins required for the biosynthesis of FHA and fimbriae, despite the lack of sequence similarities between these two proteins. The nucleotide sequence of this area contains the open reading frames fhaD and fhaA, whose products share sequence similarities with the papD and papC gene products, respectively. PapD is a periplasmic chaperone protein able to bind to the Escherichia coli P pilin subunits and to transport them towards the outer membrane protein PapC which is responsible for pilus membrane translocation. An additional open reading frame, fhaE, is located downstream of fhaA. Its amino acid sequence shares similarities with those of the fimbrial subunits. Deletion analyses suggest that fhaB and the downstream genes can be transcribed as a polycistronic operon, and primer extension analysis revealed the presence of a second promoter between fhaB and fhaD.     

Contribution of horizontal gene transfer and deletion events to development of distinctive patterns of fimbrial operons during evolution of Salmonella serotypes.

Only certain serotypes of Salmonella represent 99% of all human clinical isolates. We determined whether the phylogenetic distribution of fimbrial operons would account for the host adaptations observed for Salmonella serotypes. We found that three fimbrial operons, fim, lpf, and agf, were present in a lineage ancestral to Salmonella. While the fim and agf fimbrial operons were highly conserved among all Salmonella serotypes, sequence analysis suggested that the lpf operon was lost from many distantly related lineages. As a consequence, the distribution of the lpf operon cannot be explained easily and may be a consequence of positive and negative selection in different hosts for the presence of these genes. Two other fimbrial operons, sef and pef, each entered two distantly related Salmonella lineages and each is present only in a small number of serotypes. These results show that horizontal gene transfer and deletion events have created unique combinations of fimbrial operons among Salmonella serotypes. The presence of sef and pef correlated with serotypes frequently isolated from common domesticated animals.     

TGA cysteine codons and intron sequences in conserved and nonconserved positions are found in macronuclear RNA polymerase genes of Euplotes octocarinatus.

The gene sequences of the second largest subunits of RNA polymerases I and II of Euplotes octocarinatus, RPA2 and RPB2, were determined and compared to the respective known sequences of Saccharomyces cerevisiae. The similarity of the derived polypeptide sequences permitted their assignment to the respective polymerases and allowed the comparison of the zinc binding regions. In frame TGA codons were detected, which are likely to encode conserved cysteinyl residues in the putative zinc-finger region of the RPA2 gene. They were also found in other positions in both the RPA2 and RPB2 genes. The RPB2 gene contains a 30 bp intron close to the 5'-end of its coding region. The 5'-ends of the coding regions of all three genes encoding the largest subunits of the three different polymerases were also analyzed. The zinc finger structures again show the use of TGA codons for conserved cysteinyl residues in two of the genes. An N-terminal intron is located in the RPB1 gene at a conserved position as compared to the respective genes of several other eucarya.     

Use of an immunoblotting method for identifying the individual proteins in the antigenic complexes of Bordetella pertussis

The composition of antigenic complexes isolated from the supernatant fluid of B. pertussis culture has been studied by means of immunoblotting techniques. In preparations obtained from B. pertussis strains 305 and 475 fragments of the molecule of fimbrial hemagglutinin, three subunits of B. pertussis toxin and agglutinogens 2 and 3 have been detected with the use of antisera to B. pertussis protective substances.     

Genetic manipulation of major P-fimbrial subunits and consequences for formation of fimbriae.

The influence of genetic manipulation of the structural genes coding for major P-fimbrial subunits on the formation of fimbriae in Escherichia coli was studied. Deletion of two regions that code for hypervariable parts of the P fimbrillin resulted in strong reduction or total absence of fimbria production. Replacement of deleted amino acids by other amino acid residues restored the formation of fimbriae. The hypervariable regions may be important for biogenesis of fimbriae by imposing correct spacing between conserved regions of the protein. The potential for substituting amino acids in the P-fimbrial subunit opens interesting possibilities for use of fimbriae as carriers of foreign antigenic determinants. An antigenic determinant of foot-and-mouth disease virus (FMDV) was incorporated in the F11 fimbrial subunit. Hybrid fimbriae, recognized by an FMDV-specific neutralizing monoclonal antibody directed against FMDV, were formed.     

fimA-folD genes linkage in Salmonella identifies a putative junctional site of chromosomal rearrangement in the enterobacterial genome

Abstract Analysis of the Salmonella chromosomal region located upstream of the fimA gene (coding for the major type 1 fimbrial subunit) showed a close linkage of this gene to the folD gene (coding for the enzyme 5,10-methylenetetrahydrofolate dehydrogenase/5, 10-methenyltetrahydrofolate cyclohydrolase), indicating that the fim gene cluster of Salmonella , unlike that of Escherichia coli , has no regulatory genes located upstream of fimA and apparently terminates with this gene. The respective locations of the fim and folD genes in the E. coli and Salmonella genetic maps suggests that the fimA-folD intergenic region of Salmonella encompasses a junctional site of a genetic rearrangement that probably originated from the different chromosomal location of the fim genes in these species.     

Variable copy number, intra-genomic heterogeneities and lateral transfers of the 16S rRNA gene in Pseudomonas

Even though the 16S rRNA gene is the most commonly used taxonomic marker in microbial ecology, its poor resolution is still not fully understood at the intra-genus level. In this work, the number of rRNA gene operons, intra-genomic heterogeneities and lateral transfers were investigated at a fine-scale resolution, throughout the Pseudomonas genus. In addition to nineteen sequenced Pseudomonas strains, we determined the 16S rRNA copy number in four other Pseudomonas strains by Southern hybridization and Pulsed-Field Gel Electrophoresis, and studied the intra-genomic heterogeneities by Denaturing Gradient Gel Electrophoresis and sequencing. Although the variable copy number (from four to seven) seems to be correlated with the evolutionary distance, some close strains in the P. fluorescens lineage showed a different number of 16S rRNA genes, whereas all the strains in the P. aeruginosa lineage displayed the same number of genes (four copies). Further study of the intra-genomic heterogeneities revealed that most of the Pseudomonas strains (15 out of 19 strains) had at least two different 16S rRNA alleles. A great difference (5 or 19 nucleotides, essentially grouped near the V1 hypervariable region) was observed only in two sequenced strains. In one of our strains studied (MFY30 strain), we found a difference of 12 nucleotides (grouped in the V3 hypervariable region) between copies of the 16S rRNA gene. Finally, occurrence of partial lateral transfers of the 16S rRNA gene was further investigated in 1803 full-length sequences of Pseudomonas available in the databases. Remarkably, we found that the two most variable regions (the V1 and V3 hypervariable regions) had probably been laterally transferred from another evolutionary distant Pseudomonas strain for at least 48.3 and 41.6% of the 16S rRNA sequences, respectively. In conclusion, we strongly recommend removing these regions of the 16S rRNA gene during the intra-genus diversity studies.     

Identification and sequence analysis of lpfABCDE, a putative fimbrial operon of Salmonella typhimurium.

A chromosomal region present in Salmonella typhimurium but absent from related species was identified by hybridization. A DNA probe originating from 78 min on the S. typhimurium chromosome hybridized with DNA from Salmonella enteritidis, Salmonella heidelberg, and Salmonella dublin but not with DNA from Salmonella typhi, Salmonella arizonae, Escherichia coli, and Shigella serotypes. Cloning and sequence analysis revealed that the corresponding region of the S. typhimurium chromosome encodes a fimbrial operon. Long fimbriae inserted at the poles of the bacterium were observed by electron microscopy when this fimbrial operon was introduced into a nonpiliated E. coli strain. The genes encoding these fimbriae were therefore termed lpfABCDE, for long polar fimbriae. Genetically, the lpf operon was found to be most closely related to the fim operon of S. typhimurium, both in gene order and in conservation of the deduced amino acid sequences.     

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.