Chromosomal transformation inBacillus subtilisis a non-polar recombination reaction

Abstract

Natural chromosomal transformation is one of the primary driving forces of bacterial evolution. This reaction involves the recombination of the internalized linear single-stranded (ss) DNA with the homologous resident duplex via RecA-mediated integration in concert with SsbA and DprA or RecO. We show that sequence divergence prevents Bacillus subtilis chromosomal transformation in a log-linear fashion, but it exerts a minor effect when the divergence is localized at a discrete end. In the nucleotide bound form, RecA shows no apparent preference to initiate recombination at the 3′- or 5′-complementary end of the linear duplex with circular ssDNA, but nucleotide hydrolysis is required when heterology is present at both ends. RecA·dATP initiates pairing of the linear 5′ and 3′ complementary ends, but only initiation at the 5′-end remains stably paired in the absence of SsbA. Our results suggest that during gene transfer RecA·ATP, in concert with SsbA and DprA or RecO, shows a moderate preference for the 3′-end of the duplex. We show that RecA-mediated recombination initiated at the 3′- or 5′-complementary end might have significant implication on the ecological diversification of bacterial species with natural transformation.

Adaptive evolution of hybrid bacteria by horizontal gene transfer

“…S3 for details. This result is in agreement with previous work (14, 15). In our system, the dependence on sequence similarity already generates a significant variation of transfer rates independently of selection on specific genes.…”

“…The efficiency of the DNA uptake machinery is a major determinant of the probability of transformation (11). At the level of recombination, this probability decreases exponentially as a function of the local sequence divergence, likely because nucleotide mismatches suppress sequence pairing at the initiation of the recombination step (14–16). As shown in a recent study, laboratory experiments can induce genome-wide transformation between close lineages (17).…”

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

“…Some of these factors are specific for genetic recombination, for recombinational repair, or for both ( 3 ). During genetic recombination, the accessory proteins that act before homology search can again be divided into those that promote (DprA, RecO[R]), limit RecA (SsbA, SsbB) or activate RecA nucleation to catalyze DSE in the presence of adenosine triphosphate (ATP) (SsbA and DprA or RecO[R], two-component mediators ( 9 , 10 , 15 – 17 ). The proteins that act during homology search are RecX and RecU ( 3 ).…”

“…In their ATP-bound form, these recombinases can nucleate on protein-free ssDNA, but they cannot catalyze DSE in the absence of accessory factors (inactive RecA) ( 10 , 32 – 34 ). Unlike RecA Eco ·ATP ( 11 – 13 ) or RecA·dATP ( 32 , 35 ), RecA·ATP cannot nucleate or polymerize in the SsbA– or SsbB–ssDNA complexes ( 10 , 16 , 17 , 36 ). The presence of DprA reverses the negative effect of SsbA or SsbB on RecA filament growth, and DprA–SsbA are necessary and sufficient to activate RecA·ATP to catalyze bidirectional DSE, with greater efficiency in the 5′→3′ direction ( 10 , 17 ).…”

Bacillus subtilis single-stranded DNA-binding protein SsbA is phosphorylated at threonine 38 by the serine/threonine kinase YabT

“…Physiological roles of both these proteins have been under extensive investigation for over a decade, and they are currently known to participate in DNA replication (2), recombination (3), damage repair (4) and the process of transformation of the B. subtilis chromosome in the state of natural competence (5). We have previously reported that B. subtilis SsbA and SsbB can undergo phosphorylation at tyrosine residues, and this phosphorylation is catalyzed by the B. subtilis BY-kinase PtkA (6).…”