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.     

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.     

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.     

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 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.     

A newly identified, essential catalytic residue in a critical secondary structure element in the integrase family of site-specific recombinases is conserved in a similar element in eucaryotic type IB topoisomerases.

The integrase family of site-specific recombinases catalyzes conservative rearrangements between defined segments of DNA. A highly conserved tetrad (RHRY) of catalytic residues is essential for this process. This tetrad is dispersed in two motifs in the linear sequence, but is configured appropriately in the catalytic pocket to execute the strand cleavage and rejoining reactions. A third conserved motif has been identified in the Xer subgroup of the integrase family. Mutational analysis of 12 conserved residues in this motif in the XerD protein from Salmonella typhimurium led to the identification of an essential fifth catalytic residue (lysine 172) which is implicated in strand cleavage or exchange. This lysine residue occupies part of the turn of an antiparallel beta-hairpin which forms one side of the catalytic cleft in XerD, and is found at similar positions among evolutionarily diverse integrase family members. Related antiparallel beta-hairpins are present in eucaryotic type IB topoisomerase enzymes which also contain a critical lysine residue in the turn of the hairpin. In both the integrase family and eucaryotic type IB topoisomerases, the catalytic lysine residues are in close contact with the substrates and may play similar roles in influencing the reactivity of the phosphotyrosine intermediates formed during reactions catalyzed by both enzymes.     

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.     

Fifteen minutes of fim: control of type 1 pili expression in E. coli

Pili are used by Escherichia coli to attach to and invade mammalian tissues during host infection and colonization. Expression of type 1 pili, believed to act as virulence factors in urinary tract infections, is under control of the 'firm' genetic network. This network is able to sense the environment and actuate phase variation control. It is a prime exemplar of an integrative regulatory system because of its role in mediating a complex infection process, and because it instantiates a number of regulatory motifs, including DNA inversion and stochastic variation. With the help of a mathematical model, we explore the mechanisms and architecture of the fim network. We explain (1) basic network operation, including the roles of the recombinase and global regulatory protein concentrations, their DNA binding affinities, and their switching rates in observed phase variation behavior; (2) why there are two recombinases when one would seem to suffice; (3) the source of on-to-off switching specificity of FimE; (4) the role of fimE orientational control in switch dynamics; and (5) how temperature tuning of piliation is achieved. In the process, we identify a general regulatory motif that tunes phenotype to an environmental variable, and explain a number of apparent experimental inconsistencies.     

Piv site-specific invertase requires a DEDD motif analogous to the catalytic center of the RuvC Holliday junction resolvases

Piv, a unique prokaryotic site-specific DNA invertase, is related to transposases of the insertion elements from the IS110/IS492 family and shows no similarity to the site-specific recombinases of the tyrosine- or serine-recombinase families. Piv tertiary structure is predicted to include the RNase H-like fold that typically encompasses the catalytic site of the recombinases or nucleases of the retroviral integrase superfamily, including transposases and RuvC-like Holliday junction resolvases. Analogous to the DDE and DEDD catalytic motifs of transposases and RuvC, respectively, four Piv acidic residues D9, E59, D101, and D104 appear to be positioned appropriately within the RNase H fold to coordinate two divalent metal cations. This suggests mechanistic similarity between site-specific inversion mediated by Piv and transposition or endonucleolytic reactions catalyzed by enzymes of the retroviral integrase superfamily. The role of the DEDD motif in Piv catalytic activity was addressed using Piv variants that are substituted individually or multiply at these acidic residues and assaying for in vivo inversion, intermolecular recombination, and DNA binding activities. The results indicate that all four residues of the DEDD motif are required for Piv catalytic activity. The DEDD residues are not essential for inv recombination site recognition and binding, but this acidic tetrad does appear to contribute to the stability of Piv-inv interactions. On the basis of these results, a working model for Piv-mediated inversion that includes resolution of a Holliday junction is presented.     

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 the FimB integrase with the fimS invertible DNA element in Escherichia coli in vivo and in vitro

The FimB protein is a site-specific recombinase that inverts the fimS genetic switch in Escherichia coli. Based on amino acid sequence analysis alone, FimB has been assigned to the integrase family of tyrosine recombinases. We show that amino acid substitutions at positions R47, H141, R144, and Y176, corresponding to highly conserved members of the catalytic motif of integrase proteins, render FimB incapable of inverting the fimS element in vivo. The arginine substitutions reduced the ability of FimB to bind to fimS in vivo or in vitro, while the substitution R144Q resulted in a protein unable to bind independently to the half sites located at the left end of fimS in phase-on bacteria. These data confirm that FimB is an integrase and suggest that residue R144 has a role in binding to a specific component of the fim switch.     

Similarities and differences among 105 members of the Int family of site-specific recombinases.

Alignments of 105 site-specific recombinases belonging to the Int family of proteins identified extended areas of similarity and three types of structural differences. In addition to the previously recognized conservation of the tetrad R-H-R-Y, located in boxes I and II, several newly identified sequence patches include charged amino acids that are highly conserved and a specific pattern of buried residues contributing to the overall protein fold. With some notable exceptions, unconserved regions correspond to loops in the crystal structures of the catalytic domains of lambda Int (Int c170) and HP1 Int (HPC) and of the recombinases XerD and Cre. Two structured regions also harbor some pronounced differences. The first comprises beta-sheets 4 and 5, alpha-helix D and the adjacent loop connecting it to alpha-helix E: two Ints of phages infecting thermophilic bacteria are missing this region altogether; the crystal structures of HPC, XerD and Cre reveal a lack of beta-sheets 4 and 5; Cre displays two additional beta-sheets following alpha-helix D; five recombinases carry large insertions. The second involves the catalytic tyrosine and is seen in a comparison of the four crystal structures. The yeast recombinases can theoretically be fitted to the Int fold, but the overall differences, involving changes in spacing as well as in motif structure, are more substantial than seen in most other proteins. The phenotypes of mutations compiled from several proteins are correlated with the available structural information and structure-function relationships are discussed. In addition, a few prokaryotic and eukaryotic enzymes with partial homology with the Int family of recombinases may be distantly related, either through divergent or convergent evolution. These include a restriction enzyme and a subgroup of eukaryotic RNA helicases (D-E-A-D proteins).     

Mutational analysis of a site-specific recombinase: characterization of the catalytic and dimerization domains of the β recombinase of pSM19035

The β recombinase encoded by the streptococcal plasmid pSM19035, which shows 28 to 34% identity with DNA resolvases and DNA invertases, can catalyze formation of deletions or inversions between properly oriented target sites. We have constructed a number of site-directed mutations at residues that are conserved between the β protein and other DNA recombinases of the resolvase/invertase family. The analysis of the recombination and DNA-binding ability of each mutant protein shows that the mutations affect the catalytic activity and, in two cases, the dimerization of the protein. The results suggest that the β protein probably mediates recombination by a catalytic mechanism similar to that proposed for the resolvase/invertase family. Since the β recombinase differs from DNA resolvases and DNA invertases in its lack of bias towards either of these reactions, the results presented support the hypothesis that its unique properties might depend on details of the architecture or assembly of the recombination complex. In addition, two β protein mutants that can no longer form dimers in solution have provided new insights into the way the protein binds to DNA     

The integrase family of recombinase: organization and function of the active site

The integrase family of site-specific recombinases (also called the tyrosine recombinases) mediate a wide range of biological outcomes by the sequential exchange of two pairs of DNA strands at defined phosphodiester positions.The reaction produces a recombinant arrangement of the DNA sequences flanking the cross-over region. The crystal structures of four integrase family members have revealed very similar three-dimensional protein folds that belie the large diversity in amino acid sequences among them. The active sites are similar in organization to those seen in structures of eukaryotic type IB topoisomerases, and conservation of catalytic mechanism is expected. The crystal structures, combined with previous biochemical knowledge, allow the refinement of models for recombination and the assignment of catalytic function to the active site residues. However, each system has its own peculiarities, and the exact sequence of events that allows the reaction to proceed from the first exchange reaction to the second is still unclear for at least some family members.     

Integron integrases possess a unique additional domain necessary for activity.

Integrons are genetic elements capable of integrating genes by a site-specific recombination system catalyzed by an integrase. Integron integrases are members of the tyrosine recombinase family and possess the four invariant residues (RHRY) and conserved motifs (boxes I and II and patches I, II, and III). An alignment of integron integrases compared to other tyrosine recombinases shows an additional group of residues around the patch III motif. We have analyzed the DNA binding and recombination properties of class I integron integrase (IntI1) variants carrying mutations at residues that are well conserved among all tyrosine recombinases and at some residues from the additional motif that are conserved among the integron integrases. The well-conserved residues studied were H277 from the conserved tetrad RHRY (about 90% conserved), E121 found in the patch I motif (about 80% conserved in prokaryotic recombinases), K171 from the patch II motif (near 100% conserved), W229 and F233 from the patch III motif, and G302 of box II (about 80% conserved in prokaryotic recombinases). Additional IntI1 mutated residues were K219 and a deletion of the sequence ALER215. We observed that E121, K171, and G302 play a role in the recombination activity but can be mutated without disturbing binding to DNA. W229, F233, and the conserved histidine (H277) may be implicated in protein folding or DNA binding. Some of the extra residues of IntI1 seem to play a role in DNA binding (K219) while others are implicated in the recombination activity (ALER215 deletion).