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.     

Identification of ancillary fim genes affecting fimA expression in Salmonella typhimurium.

Regulation of the gene, fimA, encoding the major fimbrial subunit of S. typhimurium S6704 was examined by using a lambda fimA-lacZ lysogen. Transformation of the lambda fimA-lacZ lysogen with various derivatives of the recombinant plasmid that encodes type 1 fimbrial expression, pISF101, indicated that two regions of this plasmid alter beta-galactosidase production. One plasmid is a deletion resulting in the loss of a 28-kDa polypeptide downstream of fimA, while the other plasmid encodes a 24- and a 27-kDa polypeptide. Northern (RNA) blot analyses indicated that the steady-state fimA mRNA levels of these transformants were high. In addition, phenotypic expression of type 1 fimbriae by agar-grown cultures is observed only in those transformants bearing plasmids which show increased beta-galactosidase and fimA mRNA levels.     

The Type IV Fimbrial Subunit Gene (fimA) of Dichelobacter nodosus Is Essential for Virulence, Protease Secretion, and Natural Competence

Dichelobacter nodosus is the essential causative agent of footrot in sheep. The major D. nodosus-encoded virulence factors that have been implicated in the disease are type IV fimbriae and extracellular proteases. To examine the role of the fimbriae in virulence, allelic exchange was used to insertionally inactivate the fimA gene, which encodes the fimbrial subunit protein, from the virulent type G D. nodosus strain VCS1703A. Detailed analysis of two independently derived fimA mutants revealed that they no longer produced the fimbrial subunit protein or intact fimbriae and did not exhibit twitching motility. In addition, these mutants were no longer capable of undergoing natural transformation and did not secrete wild-type levels of extracellular proteases. These effects were not due to polar effects on the downstream fimB gene because insertionally inactivated fimB mutants were not defective in any of these phenotypic tests. Virulence testing of the mutants in a sheep pen trial conducted under controlled environmental conditions showed that the fimA mutants were avirulent, providing evidence that the fimA gene is an essential D. nodosus virulence gene. These studies represent the first time that molecular genetics has been used to determine the role of virulence genes in this slow growing anaerobic bacterium.     

Proteus mirabilis MR/P fimbriae: molecular cloning, expression, and nucleotide sequence of the major fimbrial subunit gene.

Proteus mirabilis, a cause of serious urinary tract infection and acute pyelonephritis, produces several putative virulence determinants, among them, fimbriae. Principally, two fimbrial types are produced by this species: mannose-resistant/Proteus-like (MR/P) fimbriae and mannose-resistant/Klebsiella-like (MR/K) fimbriae. To isolate MR/P fimbrial gene sequences, a P. mirabilis cosmid library was screened by immunoblotting and by hybridization with an oligonucleotide probe based on the N-terminal amino acid sequence of the isolated fimbrial polypeptide, ADQGHGTVKFVGSIIDAPCS. One clone, pMRP101, reacted strongly with a monoclonal antibody specific for MR/P fimbriae and with the DNA probe. This clone hemagglutinated both tannic acid-treated and untreated chicken erythrocytes with or without 50 mM D-mannose and was shown to be fimbriated by transmission electron microscopy. A 525-bp open reading frame, designated mrpA, predicted a 175-amino-acid polypeptide including a 23-amino-acid hydrophobic leader peptide. The unprocessed and processed polypeptides are predicted to be 17,909 and 15,689 Da, respectively. The N-terminal amino acid sequence of the processed fimbrial subunit exactly matched amino acid residues 24 to 43 predicted by the mrpA nucleotide sequence. The MrpA polypeptide shares 57% amino acid sequence identity with SmfA, the major fimbrial subunit of Serratia marcescens mannose-resistant fimbriae.     

Cloning and sequence of the gene encoding the major structural component of mannose-resistant fimbriae of Serratia marcescens.

Serratia marcescens US46, a human urinary tract isolate, exhibits mannose-resistant hemagglutination and agglutinates yeast cells, thereby indicating that it has two types of adhesins. We constructed a cosmid library for the DNA of this organism and isolated DNA clones carrying genes for mannose-sensitive (MS) and mannose-resistant (MR) fimbriae. On introduction of the cloned genes into Escherichia coli K-12, MS and MR fimbriae were formed. These fimbriae were functionally and morphologically indistinguishable from those of S. marcescens. Subcloning of these gene clusters revealed that the genes encoding MS fimbriae reside on a 9-kilobase (kb) DNA fragment, while those encoding MR fimbriae are present on a 12-kb fragment. Transposon insertion and maxicell analyses revealed that formation of MR fimbriae is controlled by several genes which reside on the 9-kb fragment. The nucleotide sequence of smfA, the gene encoding the major structural component of MR fimbriae, revealed that this gene encodes a 174-amino-acid polypeptide with a typical procaryotic signal peptide. The primary structure of the smfA product showed significant homology with the primary structure of the E. coli fimbrial subunit.     

A previously uncharacterized gene stm0551 plays a repressive role in the regulation of type 1 fimbriae in Salmonella enterica serotype Typhimurium

ABSTRACT: BACKGROUND: Salmonella enterica serotype Typhimurium produces surface-associated fimbriae that facilitate adherence of the bacteria to a variety of cells and tissues. Type 1 fimbriae with binding specificity to mannose residues are the most commonly found fimbrial type. In vitro, static-broth culture favors the growth of S. Typhimurium with type 1 fimbriae, whereas non-type 1 fimbriate bacteria are obtained by culture on solid-agar media. Previous studies demonstrated that the phenotypic expression of type 1 fimbriae is the result of the interaction and cooperation of the regulatory genes fimZ, fimY, fimW, and fimU within the fim gene cluster. Genome sequencing revealed a novel gene, stm0551, located between fimY and fimW that encodes an 11.4-kDa putative phosphodiesterase specific for the bacterial second messenger cyclic-diguanylate monophosphate (c-di-GMP). The role of stm0551 in the regulation of type 1 fimbriae in S. Typhimurium remains unclear. RESULTS: A stm0551-deleted stain constructed by allelic exchange constitutively produced type 1 fimbriae in both static-broth and solid-agar medium conditions. Quantative RT-PCR revealed that expression of the fimbrial major subunit gene, fimA, and one of the regulatory genes, fimZ, were comparably increased in the stm0551-deleted strain compared with those of the parental strain when grown on the solid-agar medium, a condition that normally inhibits expression of type 1 fimbriae. Following transformation with a plasmid possessing the coding sequence of stm0551, expression of fimA and fimZ decreased in the stm0551 mutant strain in both culture conditions, whereas transformation with the control vector pACYC184 relieved this repression. A purified STM0551 protein exhibited a phosphodiesterase activity in vitro while a point mutation in the putative EAL domain, substituting glutamic acid (E) with alanine (A), of STM0551 or a FimY protein abolished this activity. CONCLUSIONS: The finding that the stm0551 gene plays a negative regulatory role in the regulation of type 1 fimbriae in S. Typhimurium has not been reported previously. The possibility that degradation of c-di-GMP is a key step in the regulation of type 1 fimbriae warrants further investigation.     

The fimA gene encoding the type-1 fimbrial subunit of Escherichia coli. Nucleotide sequence and primary structure of the protein

The nucleotide sequence was determined of a region of 1450 base pairs encompassing the fimA gene for the subunit of type 1 fimbriae of Escherichia coli as well as flanking regions containing potential regulator sequences. The 'translated' protein contains a 23-residue signal peptide; the processed fimbrial subunit consists of 158 amino acid residues yielding a relative molecular mass of 15706. The elucidated sequence shows significant homology with those of other E. coli fimbrial proteins.     

Type 1C fimbriae of a uropathogenic Escherichia coli strain: cloning and characterization of the genes involved in the expression of the 1C antigen and nucleotide sequence of the subunit gene

The genes responsible for expression of type 1C fimbriae have been cloned from the uropathogenic Escherichia coli strain AD110 in the plasmid vector pACYC184. Analysis of deletion mutants from these plasmids showed that a 7-kb DNA fragment was required for biosynthesis of 1C fimbriae. Further analysis of this DNA fragment showed that four genes are present encoding proteins of 16, 18.5, 21 and 89 kDal. A DNA fragment encoding the 16-kDal fimbrial subunit has been cloned. The nucleotide sequence of the structural gene and of the C- and N-terminal flanking regions was determined. The structural gene codes for a polypeptide of 181 amino acids, including a 24-residue N-terminal signal sequence. The nucleotide sequence and the deduced amino acid sequence of the 1C subunit gene were compared with the sequences of the fimA gene, encoding the type 1 fimbrial subunit of E. coli K-12. The data show absolute homology at the N- and C-termini; there is less, but significant homology in the region between the N- and C-termini. Comparison of the amino acid compositions of the 1C and FimA subunit proteins with those of the F72 and PapA proteins (subunits for P-fimbriae) revealed that homology between these two sets of fimbrial subunits is also maximal at the N- and C-termini.     

PCR amplification of the fimA gene sequence of Salmonella typhimurium, a specific method for detection of Salmonella spp.

The goal of this study was to evaluate the suitability of the fimA gene amplification by PCR as a specific method for detection of Salmonella strains. Salmonella typhimurium and other pathogenic members of the family Enterobacteriaceae produce morphologically and antigenically related, thin, aggregative, type 1 fimbriae. A single gene, fimA, encodes the major fimbrial unit. In order to obtain higher specificity, we have selected a series of primers internal to the fimA gene sequence and have developed a PCR method for detecting Salmonella strains. A collection of 376 strains of Salmonella comprising over 80 serovars, isolated from animals and humans in Canada, have been used to evaluate this PCR method. Forty non-Salmonella strains were also tested by the same procedure. Cultures were screened by inoculating a single colony of bacteria directly into a PCR mixture containing a pair of primers specific for the fimA gene. The specific PCR product is an 85-bp fragment which was visualized by polyacrylamide gel electrophoresis and ethidium bromide staining. All Salmonella strains gave positive results by the PCR. Feed and milk samples contaminated by Salmonella strains were also detected by this procedure. The detection of all Salmonella strains tested and the failure to amplify the fragment from non-Salmonella strains confirm that the fimA gene contains sequences unique to Salmonella strains and demonstrate that this gene is a suitable PCR target for detection of Salmonella strains in food samples.     

Analysis of the Salmonella fim gene cluster: Identification of a new gene (fimI) encoding a fimbrin-like protein and located downstream from the fimA gene

Abstract The fimA gene coding for the major component (fimbrin) of type 1 fimbriae was mapped within the Salmonella typhi fim gene cluster, and its nucleotide sequence determined. The deduced amino acid sequence of S. typhi fimbrin is highly homologous to that of S. typhimurium type 1 fimbrin and showed similarity to that of other enterobacterial type 1 fimbrins. Downstream of fimA , an open reading frame was found, named fimI , able to encode a fimbrin-like protein. The fimI product could represent the counterpart, in type 1 fimbriae, of the PapH protein involved in cell anchoring and length modulation of Escherichia coli Pap pili. This genetic organization was found to be common to other Salmonella serovars, including S. typhimurium and S. choleraesuis .     

Two regulatory fim genes, fimB and fimE, control the phase variation of type 1 fimbriae in Escherichia coli.

The expression of type 1 fimbriae in Escherichia coli is phase dependent, i.e. a cell is either completely fimbriated or bald. This phenomenon is due to the periodic inversion of a specific 300-bp DNA segment containing the promoter for the fimbrial subunit gene, fimA. The phase switch is controlled by the products of two regulatory genes, fimB and fimE, located upstream of fimA. The fimB and fimE proteins direct the phase switch into the 'on' and 'off' position, respectively. The DNA sequence of a 3000-bp region containing the two genes has been determined. The fimB and fimE proteins exhibit strong homology and have most likely originated by duplication of an ancestral gene. They are highly basic implying that they control the phase switch through interaction at the DNA level.     

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.     

Identification and characterization of the genes encoding the type 3 and type 1 fimbrial adhesins of Klebsiella pneumoniae.

Strains of Klebsiella pneumoniae are known to express two morphologically and functionally distinct filaments, the type 3 and the type 1 fimbriae. The gene (mrkD) encoding the adhesion of K. pneumoniae type 3 fimbriae was identified by transcomplementation analysis with the pap fimbrial gene cluster of Escherichia coli. The nucleotide sequence of the mrkD gene was determined. In addition, the determinant coding for the K. pneumoniae type 1 fimbrial adhesion was identified, and its nucleotide sequence was deduced. The predicted amino acid sequences of the K. pneumoniae adhesion proteins are compared, and similarities with the major fimbrial structural proteins (MrkA and FimA) are discussed.     

Characterization of FimA in Porphyromonas gingivalis genotype IV

It has been reported that a large majority of periodontitis patients carry organisms with either type II or IV-fimA, while type I is the most prevalent fimA genotype among Porphyromonas gingivalis-positive healthy adults. Here we report characterization of recombinant fimbrial protein (rFimA) produced in Escherichia coli from genotype IV-fimA. In SDS-PAGE and immunoblot analysis after partial dissociation, type IV-rFimA showed a ladder-like pattern representing oligomeric/polymeric forms of native fimbrial structure. Unlike anti-type I-native fimbriae which can only recognize conformational epitopes of the respective proteins, both anti-type IV-native fimbriae and anti-type IV-rFimA antibodies recognized conformational as well as linear epitopes in type IV-fimbriae. These results suggest that the type IV-rFimA proteins retain the native fimbrial antigenicity and the antigenicity of type IV-fimbriae is different from that of type I-fimbriae.     

Inactivation of the Porphyromonas gingivalis fimA gene blocks periodontal damage in gnotobiotic rats.

Fimbrial production by Porphyromonas gingivalis was inactivated by insertion-duplication mutagenesis, using the cloned gene for the P. gingivalis major fimbrial subunit protein, fimA. by several criteria, this insertion mutation rendered P. gingivalis unable to produce fimbrilin or an intact fimbrial structure. A nonfimbriated mutant, DPG3, hemagglutinated sheep erythrocytes normally and was unimpaired in the ability to coaggregate with Streptococcus gordonii G9B. The cell surface hydrophobicity of DPG3 was also unaffected by the loss of fimbriae. However, DPG3 was significantly less able to bind to saliva-coated hydroxyapatite than wild-type P. gingivalis 381. This suggested that P. gingivalis fimbriae are important for adherence of the organism to saliva-coated oral surfaces. Further, DPG3 was significantly less able to cause periodontal bone loss in a gnotobiotic rat model of periodontal disease. These observations are consistent with other data suggesting that P. gingivalis fimbriae play an important role in the pathogenesis of human periodontal disease.     

Fimbrial phase variation in Escherichia coli: dependence on integration host factor and homologies with other site-specific recombinases

Expression of fimA, the structural gene for type 1 fimbriae of Escherichia coli, is phase variable. Significant homologies were identified between the recombinases which control fimbrial phase variation, FimB and FimE, and the integrase class of site-specific recombinases. Normal expression of fimA was shown to require the integration host factor (IHF). Mutations in either the himA-or the himD (hip) gene, which encode the alpha and beta subunits of IHF, respectively, prevented phase variation and locked expression of fimA in either the "on" or "off" phase. In addition, both himA and himD lesions caused a sevenfold reduction in expression of a phi(fimA-lacZ) operon fusion in strains in which fimA was locked in the on phase. Thus, IHF plays a dual role in controlling fimA expression: it is required both for inversion of the fimA control region and for efficient expression from the fimA promoter. A mechanism by which IHF may exert control over fimA expression is discussed.