PapB paralogues and their effect on the phase variation of type 1 fimbriae in Escherichia coli

Recent work has demonstrated that expression of type 1 fimbriae is repressed by PapB, a regulator of pyelonephritis-associated pili (P-pili). PapB belongs to family of related adhesin regulators, for which consensus residues required for DNA binding and oligomerization have been identified. Of the regulators tested in this study, PapB, SfaB (S-fimbriae) and PefB (Salmonella enterica serovar Typhimurium--plasmid-encoded fimbriae) repressed FimB-promoted off-to-on inversion of the fim switch, although complete repression was only demonstrated by PapB. DaaA, FaeB, FanA, FanB and ClpB had no effect on fim switching. In addition, only PapB stimulated FimE-promoted on-to-off inversion. Deletion analysis demonstrated that this specificity resides in the carboxy terminal of the protein, and not the amino terminal, with the central region being homologous among the family members. Exchange of Leu(82) and Ile(83) of PapB for the equivalent residues from the DaaA protein (Phe and Gln) within the carboxy terminal virtually abolished cross-talk activity. Whereas PapB can bind to a region around the left inverted repeat of the fim switch, DaaA and the PapB double mutant were effectively unable to bind this region. A previously characterized PapB DNA binding mutant also failed to bind to this region and failed to inhibit FimB activity at the fim switch. Thus, repression of fim expression appears unique to PapB and SfaB within E. coli and requires DNA binding involving amino acid residues located both within the homologous core and in the heterogeneous carboxy terminus. The variation in the carboxy terminus between the PapB family members explains their differential effects on fim. This mechanism of cross-talk seems restricted to the P and S family adhesins with type 1 fimbriae and may ensure variable and sequential expression of adhesins during urinary tract infections.     

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).     

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.     

Regulation of type 1 fimbrial expression in uropathogenic Escherichia coli : heterogeneity of expression through sequence changes in the fim switch region

Over 80% of uropathogenic Escherichia coli express type 1 fimbriae. Expression is phase variable, and regulation of phase switching can differ between isolates. Previously, this was explained by differences in the expression of the fim recombinases, FimB and FimE. Our study of 50 uropathogenic E . coli isolates confirms variation in the regulation of type 1 fimbriae but, in many cases, the variation could be accounted for by sequence changes within and adjacent to the fim switch, rather than by differences in recombinase expression. This was demonstrated by moving the switch from the isolates into an isogenic background and comparing the switching behaviour with that of the original isolate. Isolates could be arranged into groups based on fim switch regulation and sequence similarity. In certain cases, the altered regulation was located to specific basepair changes within the fim switch. Sequence changes were found that had a marked effect on the activity of either FimB or FimE switching, while others affected FimB switching in only one direction. These results emphasize the value of using naturally selected sequence variation to further the understanding of gene regulation.     

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.     

Integration host factor stimulates both FimB- and FimE-mediated site-specific DNA inversion that controls phase variation of type 1 fimbriae expression in Escherichia coli

The site-specific DNA inversion that controls phase variation of type 1 fimbriation in E. coli is catalysed by two recombinases, FimB and FimE. Efficient inversion by either recombinase also requires the leucine-responsive regulatory protein (Lrp). In addition, FimB recombination is stimulated by the integration host factor (IHF). The effect of IHF on FimE inversion has not previously been reported. Here it is shown that IHF stimulates FimE recombination; in strain MG1655, mutants containing lesions in either the α ( ihfA ) or β ( ihfB ) subunits of IHF show a marked decrease in both FimB- (100-fold) and FimE (15 000-fold)-promoted switching. IHF is shown to bind with high affinity to sites both adjacent to (site I) and within (site II) the fim invertible element. Furthermore, mutations in site I or site II that lower the affinity of IHF binding in vitro were found to lower the frequency of FimE and/or FimB recombination in vivo . Although site I and site II mutations in combination have an effect on FimB-promoted switching comparable to that of IHF knockout mutations (100-fold), the cis site mutations have a much less marked effect (100-fold) on FimE-promoted switching.     

DNA sequence heterogeneity in Fim tyrosine-integrase recombinase-binding elements and functional motif asymmetries determine the directionality of the fim genetic switch in Escherichia coli K-12

Phase-variable expression of type 1 fimbriae in Escherichia coli K-12 involves inversion by site-specific recombination of a 314 bp sequence containing the promoter for fim structural gene expression. The invertible sequence is flanked by 9 bp inverted repeats, and each repeat is in turn flanked by non-identical recombinase-binding elements (RBEs) to which the FimB or FimE site-specific recombinases bind. These proteins have distinct DNA inversion preferences: FimB inverts the switch in the ON-to-OFF and OFF-to-ON directions with similar efficiencies, whereas FimE inverts it predominantly in the ON-to-OFF direction. We have found that FimB and FimE invert the switch through a common mechanism. A genetic investigation involving base-by-base substitution combined with a biochemical study shows that the same DNA cleavage and religation sites are used within the 9 bp inverted repeats, and that each recombination involves a common 3 bp spacer region. A comprehensive programme of RBE exchanges and replacements reveals that FimB is much more tolerant of RBE sequence variation than FimE. The asymmetric location of conserved 5'-CA motifs at either side of each spacer region allows the inside and outside of the switch to be differentiated while the RBE sequence heterogeneity permits its ON and OFF forms to be distinguished by the recombinases.     

Identification of two ancillary subunits of Escherichia coli type 1 fimbriae by using antibodies against synthetic oligopeptides of fim gene products.

We have chemically synthesized oligopeptides corresponding to the NH2-terminal stretch of two gene products, designated FimG and FimH, of the fim gene cluster of Escherichia coli. These synthetic peptides, designated S-T1FimG(1-16) and S-T1FimH(1-25)C, evoked antibodies in rabbits that reacted with 14- and 29-kilodalton subunits, respectively, of dissociated fimbriae encoded by the recombinant plasmid pSH2 carrying the genetic information for the synthesis and expression of functional type 1 fimbriae. Neither of these fimbrial proteins was detected in dissociated fimbrial preparations from nonadhesive E. coli cells carrying the mutant plasmid pUT2002, containing a restriction site-specific deletion of fimG and fimH. Anti-S-T1FimH(1-25)C inhibited the adherence of type 1 fimbriated E. coli to epithelial cells. Immunoelectron microscopy revealed that anti-S-T1FimH(1-25)C, but not anti-S-T1FimG(1-16), bound to intact type 1 fimbriae of E. coli at the fimbrial tips and at long intervals along the fimbrial filaments. Anti-S-T1FimG(1-16) appeared to be directed at epitopes not accessible on the intact fimbriae and consequently failed to bind to intact fimbriae or to block fimbrial attachment. Our results suggest that the fimG and fimH gene products are components of type 1 fimbriae and that FimH may be the tip adhesin mediating the binding of type 1 fimbriated E. coli to D-mannose residues on mucosal surfaces.     

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.     

DNA supercoiling and the Lrp protein determine the directionality of fim switch DNA inversion in Escherichia coli K-12

Site-specific recombinases of the integrase family usually require cofactors to impart directionality in the recombination reactions that they catalyze. The FimB integrase inverts the Escherichia coli fim switch (fimS) in the on-to-off and off-to-on directions with approximately equal efficiency. Inhibiting DNA gyrase with novobiocin caused inversion to become biased in the off-to-on direction. This directionality was not due to differential DNA topological distortion of fimS in the on and off phases by the activity of its resident P(fimA) promoter. Instead, the leucine-responsive regulatory (Lrp) protein was found to determine switching outcomes. Knocking out the lrp gene or abolishing Lrp binding sites 1 and 2 within fimS completely reversed the response of the switch to DNA relaxation. Inactivation of either Lrp site alone resulted in mild on-to-off bias, showing that they act together to influence the response of the switch to changes in DNA supercoiling. Thus, Lrp is not merely an architectural element organizing the fim invertasome, it collaborates with DNA supercoiling to determine the directionality of the DNA inversion event.     

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.