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

Abstract

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.

FIG. 1.

DNA relaxation and fim switch inversion preferences. (A) Phase-on and -off fim switches, showing inverted repeats IRL and IRR, the P_fimA promoter (−10, −35), the fimA transcription start site, and Lrp binding sites 1 and 2. (B) Topoisomers of plasmid pUC18 isolated from E. coli K-12 strain VL386 treated with novobiocin at the concentrations shown. (C) Switch inversion preferences with and without a functional P_fimA promoter (structures summarized above each panel) at increasing concentrations of novobiocin. Densitometric data from PCR switch assay gels (insets) were used to plot the graphs. Numbers above each gel lane are μg/ml of novobiocin. Bands corresponding to phase-on or -off are labeled.

FIG. 2.

Inactivation of the lrp gene reverses the response of the fim switch to DNA relaxation. (A) Treatment with increasing concentrations of novobiocin results in a strong on-to-off bias in an lrp::Tn10 knockout mutant, the opposite of the pattern seen in the wild type. (B) Complementation of the lrp knockout mutation with a functional copy of the lrp gene restores the wild-type pattern of response to novobiocin treatment. Densitometric data from PCR switch assay gels (insets) were used to plot the graphs. Numbers above each gel lane are μg/ml of novobiocin. Bands corresponding to phase-on or -off are labeled.

FIG. 3.

Inactivation of the Lrp binding sites 1 and 2 within the fim switch. Data are shown from an electrophoretic mobility shift assay monitoring interaction of purified Lrp protein with the wild-type fim switch and mutants deficient in site 1, site 2, or sites 1 and 2. The protein concentrations used are given at the top of each lane. Lrp-fimS complexes are labeled at the right: complexes C1 and C2 and higher-order complexes (H.C.).

FIG. 4.

Removal of the Lrp binding sites alters the response of the fim switch to DNA relaxation. With sites 1 and 2 intact, fimS becomes biased in favor of phase-on in response to increasing concentrations of novobiocin (A). Elimination of site 1 (B) or 2 (C) prevents on-biasing in response to novobiocin. When Lrp sites 1 and 2 are both disrupted, the response of the switch to novobiocin treatment is reversed (D) and resembles that seen in the lrp::Tn10 mutant (Fig. 2). The y axes of the graphs report the percentage of on and off switches in the population. The dosage of novobiocin is reported on the x axes. Densitometric data from PCR switch assay gels (insets) were used to plot the graphs. Numbers above each gel lane are μg/ml of novobiocin. Bands corresponding to phase-on or -off are labeled.

FIG. 5.

A model to account for trapping of the switch in the on orientation following DNA relaxation. The relevant features of the fim switch are shown: inverted repeats IRL and IRR, Lrp sites 1 and 2 (numbered hexagons), the P_fimA promoter, and a putative reference point (Ref) located in the noninvertible region of the chromosome adjacent to IRL. Communication between Ref and the occupied Lrp binding sites facilitates the formation of an inhibitory complex (gray box). Negative supercoiling of the DNA assists extrusion of IRL from the inhibitory complex (top). Removal of negative superhelicity following gyrase inhibition abrogates extrusion of IRL, and the switch is trapped in the on orientation (middle). Elimination of Lrp binding disrupts the inhibitory complex, and rapid on-to-off switching occurs (bottom).