Roles of fimB and fimE in site-specific DNA inversion associated with phase variation of type 1 fimbriae in Escherichia coli.

Evidence obtained with an improved in vivo assay of fimbrial phase variation in Escherichia coli supported a revised understanding of the roles of fimB and fimE in the site-specific DNA rearrangement with which they are associated. A previously proposed model argued that fimB and fimE play antagonistic, unidirectional roles in regulating the orientation of the invertible DNA element located immediately upstream of fimA, the gene encoding the major subunit of type 1 fimbriae. This conclusion, though, is based on an in vivo DNA inversion assay using recombinant plasmid substrates under conditions that, among other things, were incapable of detecting recombination of the fim invertible element from the on to the off orientation. Using a modified system that overcome this and several additional technical problems, we confirmed that fimB acts independently of fimE on the invertible element and that the additional presence of fimE results in the preferential rearrangement of the element to the off orientation. It is now demonstrated that fimE can act in the absence of fimB in this recombination to promote inversion primarily from on to off. In contrast to the previous studies, the effect of fimB on a substrate carrying the invertible element in the on orientation could be examined. It was found that fimB mediates DNA inversion from on to off, as well as from off to on, and that, contrary to prior interpretations, the fimB-associated inversion occurs with only minimal orientational preference to the on phase.     

The molecular basis for the specificity of fimE in the phase variation of type 1 fimbriae of Escherichia coli K-12

The expression of type 1 fimbriae in Escherichia coli is phase variable, with cells switching between fimbriate (ON) and afimbriate (OFF) phases. The phase variation is dependent on the orientation of a 314 bp DNA element (the switch) that undergoes DNA inversion. DNA inversion requires either fimB or fimE, site-specific recombinases that differ in both specificity and activity. Whereas fimB promotes recombination with little orientational bias, fimE promotes recombination in the ON-to-OFF direction exclusively. In wild-type cells, fimE activity predominates and, hence, most bacteria are afimbriate. Here, it is shown that fimE specificity is caused by two different, but complementary, mechanisms. First, FimE shows a strong preference for the switch in the ON orientation as a substrate for recombination. Differences in the nucleotide sequence of the recombinase binding sites is a key factor in determining FimE specificity, although one or more additional cis-active sites that flank the fim switch also appear to be involved. Secondly, the orientation of the switch controls fimE in cis, most probably to control recombinase expression.     

FimE-catalyzed off-to-on inversion of the type 1 fimbrial phase switch and insertion sequence recruitment in an Escherichia coli K-12 fimB strain

We have investigated the capacity of a well-defined Escherichia coli fimB strain, AAEC350 (a derivative of MG1655), to express type 1 fimbriae under various growth conditions. The expression of type 1 fimbriae is phase-variable due to the inversion of a 314-bp DNA segment. Two tyrosine recombinases, FimB and FimE, mediate the inversion of the phase switch. FimB can carry out recombination in both directions, whereas the current evidence suggests that FimE-catalyzed switching is on-to-off only. We show here that AAEC350 is in fact capable of off-to-on phase switching and type 1 fimbrial expression under aerobic static growth conditions. The phase switching is mediated by FimE, and allows emerging fimbriate AAEC350 to outgrow their non-fimbriate counterparts by pellicle formation. Following inversion of the phase switch, this element can remain phase-locked in the on orientation due to integration of insertion sequence elements, viz. IS1 or IS5, at various positions in either the fimE gene or the phase switch.     

Characterization of the fimA gene encoding bundle-forming fimbriae of the plant pathogen Xanthomonas campestris pv. vesicatoria.

The fimA gene of Xanthomonas campestris pv. vesicatoria was identified and characterized. A 20-mer degenerate oligonucleotide complementary to the N-terminal amino acid sequence of the purified 15.5-kDa fimbrillin was used to locate fimA on a 2.6-kb SalI fragment of the X. campestris pv. vesicatoria 3240 genome. The nucleotide sequence of a 1.4-kb fragment containing the fimA region revealed two open reading frames predicting highly homologous proteins FimA and FimB. FimA, which was composed of 136 amino acids and had a calculated molecular weight of 14,302, showed high sequence identity to the type IV fimbrillin precursors. fimB predicted a protein product of 135 amino acids and a molecular weight of 13,854. The open reading frame for fimB contained near the 5' end a palindromic sequence with a terminator loop potential, and the expression level of fimB in vitro and in Xanthomonas was considerably lower than that of fimA. We detected an efficiently transcribed fimA-specific mRNA of 600 bases as well as two weakly expressed, longer mRNA species that reacted with both fimA and fimB. A homolog of fimA but not of fimB was detected by Southern hybridization in strains of X. campestris pv. vesicatoria, campestris, begoniae, translucens, and graminis. A fimA::omega mutant of strain 3240 was not significantly reduced in virulence or adhesiveness to tomato leaves. However, the fimA mutant was dramatically reduced in cell aggregation in laboratory cultures and on infected tomato leaves. The fimA mutant strain also exhibited decreased tolerance to UV light.     

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.     

Environmental regulation of the fim switch controlling type 1 fimbrial phase variation in Escherichia coli K-12: effects of temperature and media.

Expression of type 1 fimbriae in Escherichia coli K-12 is phase variable and associated with the inversion of a short DNA element (switch). The fim switch requires either fimB (on-to-off or off-to-on switching) or fimE (on-to-off switching only) and is affected by the global regulators leucine-responsive regulatory protein (Lrp), integration host factor (IHF), and H-NS. Here it is shown that switching frequencies are regulated by both temperature and media and that these effects appear to be independent. fimE-promoted on-to-off switching occurs far more rapidly than previously estimated (0.3 per cell per generation in defined rich medium at 37 degrees C) and faster at lower than at higher temperatures. In direct contrast, fimB-promoted switching increases with temperature, with optima between 37 and 41 degrees C. Switching promoted by both fimB and fimE is stimulated by aliphatic amino acids (alanine, isoleucine, leucine, and valine), and this stimulation requires lrp. Furthermore, lrp appears to differentially regulate fimB- and fimE-promoted switching in different media.     

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.     

Type 1 fimbriation and phase switching in a natural Escherichia coli fimB null strain, Nissle 1917

Escherichia coli Nissle 1917 has been used as a probiotic against intestinal disorders for many decades. It is a good colonizer of the human gut and has been reported to be able to express type 1 fimbriae. Type 1 fimbriae are surface organelles which mediate alpha-D-mannose-sensitive binding to various host cell surfaces. The expression is phase variable, and two tyrosine recombinases, FimB and FimE, mediate the inversion of the fimbrial phase switch. Current evidence suggests that FimB can carry out recombination in both directions, whereas FimE-catalyzed switching is on to off only. We show here that under liquid shaking growth conditions, Nissle 1917 did not express type 1 fimbriae, due to a truncation of the fimB gene by an 1,885-bp insertion element. Despite its fimB null status, Nissle 1917 was still capable of off-to-on switching of the phase switch and expressing type 1 fimbriae when grown under static conditions. This phase switching was not catalyzed by FimE, by truncated FimB, or by information residing within the insertion element. No further copies of fimB seemed to be present on the chromosome of Nissle 1917, suggesting that another tyrosine recombinase in Nissle 1917 is responsible for the low-frequency off-to-on inversion of the phase switch that is strongly favored under static growth conditions. This is the first report documenting the non-FimB- or non-FimE-catalyzed inversion of the fim switch.     

Interaction of FimB and FimE with the fim switch that controls the phase variation of type 1 fimbriae in Escherichia coli K-12

The phase variation of type 1 fimbriae in Escherichia coli is associated with the site-specific inversion of a short DNA element. Recombination at fim requires fimB and fimE , and their products are considered to be the fim recombinases. In this study, FimB and FimE were overproduced and extracts containing the proteins were shown to (i) bind to and (ii) invert the fim switch in vitro . Phenanthroline-copper protection of DNA–protein complexes showed that both FimB and FimE bind to half-sites that flank, and overlap with, the left and right inverted repeats (IRL and IRR, respectively) of the fim switch. Alignment of the four half-sites identified a conserved 5'-CA doublet; mutation of these two bases lowers the affinity of binding of both FimB and FimE to the inverted repeats, and greatly diminishes inversion of the fim switch in vivo . The specificity of the fim recombinases observed in vivo (FimB switching in both directions; FimE switching from on-to-off only) was maintained in vitro Furthermore, the different binding affinities of FimB and FimE for the various half-site combinations suggests that the specificity of FimE could arise, in part, from the low affinity of FimE for IRL (off).     

Genetic analysis and morphological identification of pilus-like structures in members of the genus <Emphasis Type="Italic">Bifidobacterium</Emphasis>

BACKGROUND: Cell surface pili in Gram positive bacteria have been reported to orchestrate the colonization of host tissues, evasion of immunity and the development of biofilms. So far, little if any information is available on the presence of pilus-like structures in human gut commensals like bifidobacteria. RESULTS AND DISCUSSION: In this report, Atomic Force Microscopy (AFM) of various bifidobacterial strains belonging to Bifidobacterium bifidum, Bifidobacterium longum subsp. longum, Bifidobacterium dentium, Bifidobacterium adolescentis and Bifidobacterium animalis subsp. lactis revealed the existence of appendages resembling pilus-like structures. Interestingly, these microorganisms harbour two to six predicted pilus gene clusters in their genome, with each organized in an operon encompassing the major pilin subunit-encoding gene (designated fimA or fimP) together with one or two minor pilin subunit-encoding genes (designated as fimB and/or fimQ), and a gene encoding a sortase enzyme (strA). Quantitative Real Time (qRT)-PCR analysis and RT-PCR experiments revealed a polycistronic mRNA, encompassing the fimA/P and fimB/Q genes, which are differentially expressed upon cultivation of bifidobacteria on various glycans.