RecA Regulation by RecU and DprA During Bacillus subtilis Natural Plasmid Transformation

Abstract

Natural plasmid transformation plays an important role in the dissemination of antibiotic resistance genes in bacteria. During this process, Bacillus subtilis RecA physically interacts with RecU, RecX, and DprA. These three proteins are required for plasmid transformation, but RecA is not. In vitro, DprA recruits RecA onto SsbA-coated single-stranded (ss) DNA, whereas RecX inhibits RecA filament formation, leading to net filament disassembly. We show that a null recA (ΔrecA) mutation suppresses the plasmid transformation defect of competent ΔrecU cells, and that RecU is essential for both chromosomal and plasmid transformation in the ΔrecX context. RecU inhibits RecA filament growth and facilitates RecA disassembly from preformed filaments. Increasing SsbA concentrations additively contributes to RecU-mediated inhibition of RecA filament extension. DprA is necessary and sufficient to counteract the negative effect of both RecU and SsbA on RecA filament growth onto ssDNA. DprA-SsbA activates RecA to catalyze DNA strand exchange in the presence of RecU, but this effect was not observed if RecU was added prior to RecA. We propose that DprA contributes to RecA filament growth onto any internalized SsbA-coated ssDNA. When the ssDNA is homologous to the recipient, DprA antagonizes the inhibitory effect of RecU on RecA filament growth and helps RecA to catalyze chromosomal transformation. On the contrary, RecU promotes RecA filament disassembly from a heterologous (plasmid) ssDNA, overcoming an unsuccessful homology search and favoring plasmid transformation. The DprA–DprA interaction may promote strand annealing upon binding to the complementary plasmid strands and facilitating thereby plasmid transformation rather than through a mediation of RecA filament growth.

Bacillus subtilis PcrA Couples DNA Replication, Transcription, Recombination and Segregation

“…RecX is a negative modulator of RecA filament growth (Cárdenas et al, 2012 ; Le et al, 2017 ), whereas RecU has two activities: to negatively modulate RecA filament growth and to cleave Holliday junctions (HJs) at a cognate site in concert with the RuvAB branch migration translocase (Ayora et al, 2004 ; Carrasco et al, 2005 ; Cárdenas et al, 2012 ; Cañas et al, 2014 ; Serrano et al, 2018 ). The absence of RecX or RecU did not restore viability of PcrA depleted cells grown in rich medium agar plates containing IPTG.…”

“…First, B. subtilis cells have two RecQ-like helicases, RecQ and RecS, with the latter potentially masking the outcome, whereas E. coli has only RecQ. Second, E. coli cells have two proteins (UvrD and RecX) to actively dismantle a RecA nucleoprotein filament (Petrova et al, 2015 ; Le et al, 2017 ), whereas B. subtilis cells have four different proteins (PcrA, RecX, RecU, and RecD2) to do this job (Anand et al, 2007 ; Le et al, 2017 ; Torres et al, 2017 ; Serrano et al, 2018 ). PcrA was also found to be necessary to survive DNA damage.…”

Bacillus subtilis RarA Acts as a Positive RecA Accessory Protein

“…It is unknown whether RecU can cleave the reversed forks generated by RecG in B. subtilis. In any event, RecU has two activities: to mediate HJ cleavage in concert with a branch migration translocase (Cañas et al, 2014), and to modulate RecA nucleoprotein filament formation by its interaction with the RecA protein (Carrasco et al, 2005;Serrano et al, 2018).…”

“…We can envision that RarA, RecO and RecF contribute to RecA * accumulation, but not RecX (Figure 5A), which in vitro acts as a negative modulator of RecA nucleoprotein filament formation as RecU (Le et al, 2017;Serrano et al, 2018). The role of RecU in RecA * accumulation in vivo is poorly understood, thus it was addressed.…”

“FAGOMA: Spanish Network of Bacteriophages and Transducer Elements”—V Meeting Report

“…She addressed the molecular mechanisms by which the RecA mediator (DprA) and modulators (RecX and RecU) regulate RecA filament growth on the internalized single-stranded DNA. These accessory proteins control overlapping mechanisms to process the horizontal transfer of exogenous DNA of different nature, including viral DNA [8,9].…”