MutS2 Promotes Homologous Recombination in Bacillus subtilis

Abstract

Bacterial MutS proteins are subdivided into two families, MutS1 and MutS2. MutS1 family members recognize DNA replication errors during their participation in the well-characterized mismatch repair (MMR) pathway. In contrast to the well-described function of MutS1, the function of MutS2 in bacteria has remained less clear. In Helicobacter pylori and Thermus thermophilus, MutS2 has been shown to suppress homologous recombination. The role of MutS2 is unknown in the Grampositive bacterium Bacillus subtilis. In this work, we investigated the contribution of MutS2 to maintaining genome integrity in B. subtilis. We found that deletion of mutS2 renders B. subtilis sensitive to the natural antibiotic mitomycin C (MMC), which requires homologous recombination for repair. We demonstrate that the C-terminal small MutS-related (Smr) domain is necessary but not sufficient for tolerance to MMC. Further, we developed a CRISPR/Cas9 genome editing system to test if the inducible prophage PBSX was the underlying cause of the observed MMC sensitivity. Genetic analysis revealed that MMC sensitivity was dependent on recombination and not on nucleotide excision repair or a symptom of prophage PBSX replication and cell lysis. We found that deletion of mutS2 resulted in decreased transformation efficiency using both plasmid and chromosomal DNA. Further, deletion of mutS2 in a strain lacking the Holliday junction endonuclease gene recU resulted in increased MMC sensitivity and decreased transformation efficiency, suggesting that MutS2 could function redundantly with RecU. Together, our results support a model where B. subtilis MutS2 helps to promote homologous recombination, demonstrating a new function for bacterial MutS2.IMPORTANCE Cells contain pathways that promote or inhibit recombination. MutS2 homologs are Smr-endonuclease domain-containing proteins that have been shown to function in antirecombination in some bacteria. We present evidence that B. subtilis MutS2 promotes recombination, providing a new function for MutS2. We found that cells lacking mutS2 are sensitive to DNA damage that requires homologous recombination for repair and have reduced transformation efficiency. Further analysis indicates that the C-terminal Smr domain requires the N-terminal portion of MutS2 for function in vivo. Moreover, we show that a mutS2 deletion is additive with a recU deletion, suggesting that these proteins have a redundant function in homologous recombination. Together, our study shows that MutS2 proteins have adapted different functions that impact recombination.

Transcriptional responses of Streptococcus gordonii and Fusobacterium nucleatum to coaggregation

“…The genes involved in DNA metabolism are predicted to encode a regulatory protein (RecX), an endonuclease (MutS2), a cell division protein (DivIB) and a DNA repair protein (RecN). In Bacillus subtilis MutS2 promotes homologous recombination and protects cells from DNA damage . It is possible that the changes in DNA metabolism prime the S. gordonii cells for uptake and incorporation of foreign DNA following sensing of a different species.…”

CRISPR/Cas9 Editing of the Bacillus subtilis Genome

“…A proto-spacer (highlighted blue and capital letters) and proto-spacer adjacent motif (PAM site; highlighted red); B. Oligonucleotides with proper overhangs (lower case letters) necessary to ligate into pPB41 (Jiang et al ., 2013; Burby and Simmons, 2017); C. Annealing and phosphorylation reaction to prepare a dsDNA proto-spacer for ligation into pPB41.…”

“…Therefore, although the methods described above work, we were in search of a genome editing method with higher efficiency that also required less time at the bench. These criteria prompted us to adapt a CRISPR/Cas9 genome editing system (Jiang et al ., 2013) to B. subtilis (Burby and Simmons, 2017). CRISPR/Cas9 can be used to introduce a variety of mutations including gene deletions, fusions, and even point mutations (Sternberg and Doudna, 2015).…”

“…Prepare naturally competent B. subtilis cultures (Burby and Simmons, 2017) (Figure 5A). Streak out strain from frozen 20% glycerol stock on LB agar plate for single colonies using a wooden stick and incubate plate at 30 or 37 °C overnight in an incubator.…”

Molecular basis for the functions of a bacterial MutS2 in DNA repair and recombination

“…MutS2 in H. pylori as well as in some other bacteria was shown to have anti-recombination activity, playing a role in maintaining genome integrity by suppressing homologous and homeologous DNA recombination [13, 16–18]. However, B. subtilis MutS2 was recently shown to function in promoting homologous recombination [19]. The other major function of MutS2 is repair of oxidative DNA damage, which was first demonstrated in H. pylori [12], and subsequently revealed in T. thermophilus and D. radiodurans [20, 21].…”