Bacillus subtilis RecA with DprA–SsbA antagonizes RecX function during natural transformation

Abstract

Bacillus subtilis DprA and RecX proteins, which interact with RecA, are crucial for efficient chromosomal and plasmid transformation. We showed that RecA, in the rATP·Mg2+ bound form (RecA·ATP), could not compete with RecX, SsbA or SsbB for assembly onto single-stranded (ss)DNA, but RecA·dATP partially displaced these proteins from ssDNA. RecX promoted reversible depolymerization of preformed RecA·ATP filaments. The two-component DprA–SsbA mediator reversed the RecX negative effect on RecA filament extension, but not DprA or DprA and SsbB. In the presence of DprA–SsbA, RecX added prior to RecA·ATP inhibited DNA strand exchange, but this inhibition was reversed when RecX was added after RecA. We propose that RecA nucleation is more sensitive to RecX action than is RecA filament growth. DprA–SsbA facilitates formation of an active RecA filament that directly antagonizes the inhibitory effects of RecX. RecX and DprA enable chromosomal transformation by altering RecA filament dynamics. DprA–SsbA and RecX proteins constitute a new regulatory network of RecA function. DprA–SsbA contributes to the formation of an active RecA filament and directly antagonizes the inhibitory effects of RecX during natural transformation.

Bacillus subtilis RarA Acts as a Positive RecA Accessory Protein

“…The negative modulator RecX has been shown to disassemble RecA nucleoprotein filaments (Cárdenas et al, 2012;Le et al, 2017), but little is known about RecD2, whose function in HR is poorly understood (Walsh et al, 2014;Torres et al, 2017). Investigating the genetic connection between RarA and RecX or RecD2, we found recX and recD2 mutants to be sensitive to acute H 2 O 2 or MMS exposure (Figures 3D, 4D), as described earlier (Cárdenas et al, 2012;Torres et al, 2017).…”

“…The two-component mediator SsbA and RecO (in conjunction with RecR), together with positive (RecF) and negative modulators (RecX, RecU), load RecA on a ssDNA gap or a 3tailed duplex ssDNA, regulate RecA filament growth, and activate RecA to catalyze DNA strand exchange (Figure 1b) (Kidane et al, 2004;Cárdenas et al, 2012;Lenhart et al, 2014;Le et al, 2017).…”

“…Thus, a lower dose of DNA damage should be sufficient for RecA to stimulate LexA autocleavage, so maximal RecA levels are obtained at lower MMC doses in the absence of negative regulators. For example, in the absence of the positive modulator RecF, an MMC dose higher than the one needed in the rec + control was required to have maximal RecA expression levels, but in the absence of negative modulator RecX, a lower MMC dose was sufficient ( Figure 5A) (Cárdenas et al, 2012;Le et al, 2017).…”

Viral SPP1 DNA is infectious in naturally competent Bacillus subtilis cells: inter‐ and intramolecular recombination pathways

“…These and previous data suggest that during CT, RecX (or RecU [in the Δ recX context]) promotes RecA disassembly from the incoming ssDNA that has homology with the chromosome (Le et al ., ; Serrano et al ., ). In addition, RecX and RecU have to overcome the negative effect of the unproductive RecA filament formed on linear SPP1 or pUB110 ssDNA that do not have homology with recipient genome.…”

A new sRNA‐mediated posttranscriptional regulation system for Bacillus subtilis

“…Because of the repeated sequences in sgRNAs, the construction process should be quite laborious and challenging. Moreover, due to the high recombination capacity of B. subtilis (Le et al, ; Tsuge, Matsui, & Itaya, ), unexpected recombination between the tandem CRISPR DNA may lead to the instability of recombinant plasmids and the loss of some sgRNA encoding genes (Supporting Information Table S3). On the contrary, the constructed MOS‐DOS toolbox here achieved simultaneous precise repression of multiple genes only with the introduction of a single antisense RNA SR4, which is quite different from other reported interfering systems (Si et al, ).…”