RecX facilitates homologous recombination by modulating RecA activities

Abstract

The Bacillus subtilis recH342 strain, which decreases interspecies recombination without significantly affecting the frequency of transformation with homogamic DNA, carried a point mutation in the putative recX (yfhG) gene, and the mutation was renamed as recX342. We show that RecX (264 residues long), which shares partial identity with the Proteobacterial RecX (<180 residues), is a genuine recombination protein, and its primary function is to modulate the SOS response and to facilitate RecA-mediated recombinational repair and genetic recombination. RecX-YFP formed discrete foci on the nucleoid, which were coincident in time with RecF, in response to DNA damage, and on the poles and/or the nucleoid upon stochastic induction of programmed natural competence. When DNA was damaged, the RecX foci co-localized with RecA threads that persisted for a longer time in the recX context. The absence of RecX severely impaired natural transformation both with plasmid and chromosomal DNA. We show that RecX suppresses the negative effect exerted by RecA during plasmid transformation, prevents RecA mis-sensing of single-stranded DNA tracts, and modulates DNA strand exchange. RecX, by modulating the "length or packing" of a RecA filament, facilitates the initiation of recombination and increases recombination across species.

Figure 1. Survival curves of B. subtilis cells after exposure to an acute dose of MMS or H₂O₂.

Cells were grown to OD₅₆₀ = 0.4 in LB medium and exposed to increasing concentrations of MMS (A and C) or H₂O₂ (B and D) for 15 min. The strains used are indicated by the relevant mutant phenotype. The recX342 (pRecX) strain bears a plasmid-borne recX gene. The results are the average of at least five independent experiments and the standard errors are indicated.

Figure 2. B. subtilis RecA protein accumulation upon SOS induction in different genetic backgrounds.

Cells were grown to OD₅₆₀ = 0.4 in LB medium and exposed to increasing concentrations of MMC for 30 min. The cells were lysed and equivalent protein amounts subjected to 10% SDS-PAGE, followed by immunoblot transfer (see Materials and Methods). (A) rec ⁺, ΔrecX, ΔlexA and recX342 cells; (B) rec ⁺, recF15, ΔrecX recF15, ΔrecO and ΔrecX ΔrecO cells. The results are the average of at least four independent experiments and the standard errors are indicated.

Figure 3. Fluorescence microscopy of growing B. subtilis cells expressing RecX-YFP.

(A) RecX-YFP in exponentially growing cells. (B and C) Cells at 60 (B) and 180 min (C) after addition of 0.15 µM MMC. Shown are membrane stain, DNA and the corresponding RecX-YFP fluorescence. White arrowheads on the overlay denote the few RecX foci visible 180 min after MMC addition. White bars 2 µm.

Figure 4. B. subtilis RecX-YFP has a similar subcellular position to CFP-RecA threads and does not co-localize with sites of DSBs.

(A) RecX-YFP, CFP-RecA and overlay in exponentially growing cells. (B–D) Cells at 60 (B), 120 min (C) and 180 min (D) after addition of 0.15 µM MMC. Shown is the corresponding RecX-YFP or CFP-RecA fluorescence, and an overlay of both signals (RecX in red, RecA in green). White triangles indicate examples of colocalization at 60 min. (E) Fluorescent microscopy of cells during mid-exponential growth (upper panels) or after a defined break (lower panels). After induction of HO endonuclease cutting close to origin regions decorated with LacI-CFP (60 min induced), RecX foci (white arrows on the overlay) generally do not coincide with the cut sites. White bars 2 µm.

Figure 5. Fluorescence microscopy of B. subtilis rec⁺ and ΔrecX cells expressing CFP-RecA.

(A) CFP-RecA in exponentially growing cells. (B–E) Cells at 90 (B), 120 (C), 180 (D) and 210 min (E) after induction of DSBs by 0.15 µM MMC. CFP-RecA threads are visible on the nucleoid (some cells whose outline is unclear are denoted by a dotted line) in ΔrecX cells 210 min after MMC addition. Note that this panel is a composite image. White bars 2 µm.

Figure 6. Dynamic B. subtilis RecA assembly into SsbA-coated ssDNA.

It is proposed that RecO (perhaps in concert with RecR) mediates RecA nucleation, and RecF and RecX modulate RecA filament extension. SsbA binds to ssDNA and limits RecA nucleation (step a). SsbA recruits RecO (RecOR_Eco) onto SsbA-coated ssDNA. RecO might recruit RecR. RecO interacting with ssDNA promotes limited dislodging of SsbA, and facilitates RecA nucleation (step c). RecF and RecX lead to an effectual RecA filament (step d). In the absence of RecX, the contribution of RecF on RecA nucleation and/or filament extension is poorly understood (step e). In the absence of RecF, RecX facilitates the dynamic disassembly of the RecA filaments (step f).