Abstract
The efficiency of horizontal gene transfer, which contributes to acquisition and spread of antibiotic resistance and pathogenicity traits, depends on nucleotide sequence and different mismatch-repair (MMR) proteins participate in this process. To study how MutL and MutS MMR proteins regulate recombination across species boundaries, we have studied natural chromosomal transformation with DNA up to ∼23% sequence divergence. We show that Bacillus subtilis natural chromosomal transformation decreased logarithmically with increased sequence divergence up to 15% in wild type (wt) cells or in cells lacking MutS2 or mismatch repair proteins (MutL, MutS or both). Beyond 15% sequence divergence, the chromosomal transformation efficiency is ∼100-fold higher in ΔmutS and ΔmutSL than in ΔmutS2 or wt cells. In the first phase of the biphasic curve (up to 15% sequence divergence), RecA-catalyzed DNA strand exchange contributes to the delineation of species, and in the second phase, homology-facilitated illegitimate recombination might aid in the restoration of inactivated genes. To understand how MutS modulates the integration process, we monitored DNA strand exchange reactions using a circular single-stranded DNA and a linear double-stranded DNA substrate with an internal 77-bp region with ∼16% or ∼54% sequence divergence in an otherwise homologous substrate. The former substrate delayed, whereas the latter halted RecA-mediated strand exchange. Interestingly, MutS addition overcame the heterologous barrier. We propose that MutS assists DNA strand exchange by facilitating RecA disassembly, and indirectly re-engagement with the homologous 5′-end of the linear duplex. Our data supports the idea that MutS modulates bidirectional RecA-mediated integration of divergent sequences and this is important for speciation.
Highlights
Genetic surveys of bacterial populations show that horizontal gene transfer (HGT) is important in acquiring genetic diversity, which provides a central role in the evolution and spread of antibiotic resistance and pathogenicity traits (Doolittle, 1999; Gogarten et al, 2002)
After Worth et al (1994) and Tham et al (2013), we propose that a heterologous region forms a blockage to RecA filament growth and RecA-mediated DNA strand exchange, and MutS bound to the barrier indirectly facilitates the spontaneous RecA nucleoprotein filament disassembly
These data, which are similar to previous reports (Rossolillo and Albertini, 2001; Pillon et al, 2015; Lenhart et al, 2016; Burby and Simmons, 2017), show that RifR mutations accumulated in the absence of mismatch repair (MMR)
Summary
Genetic surveys of bacterial populations show that horizontal gene transfer (HGT) is important in acquiring genetic diversity, which provides a central role in the evolution and spread of antibiotic resistance and pathogenicity traits (Doolittle, 1999; Gogarten et al, 2002). Generalized transduction involves virus-mediated injection of linear double-stranded (ds) DNA; chromosomal conjugation is a cell contact-dependent transfer mechanism of linear single-stranded (ss) DNA between cells and subsequent conversion to linear dsDNA. In both cases, DNA transfer is mediated by episomal proteins, and the host recombination machinery catalyzes DNA strand exchange between a linear, end-resected 3 -tailed donor duplex and the recipient duplex genome (Kowalczykowski et al, 1994; Matic et al, 1996; Fraser et al, 2007). If sufficient homology is provided, RecA-mediated bidirectional DNA strand exchange integrates the incoming linear ssDNA into the recipient circular duplex genome (Kidane et al, 2012; Carrasco et al, 2016)
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