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 Gram-positive 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.

Keywords: CRISPR; DNA damage; DNA recombination; MutS.

FIG 1

MutS2 is a MutS paralog expressed throughout exponential growth and has no role in mutagenesis. (A) A predicted domain alignment of MutS and MutS2 proteins. (B) Western blot of MutS2 protein levels throughout exponential growth. Cultures were grown at 37°C, and samples of equivalent OD₆₀₀ values were taken at increasing optical densities (OD₆₀₀ from 0.8 to 5.9). Lane 1, molecular mass marker (M); 2, WT (OD₆₀₀ of 5.9); 3, ΔmutS2 control (OD₆₀₀ of 5.6); 4 to 9, samples taken at increasing OD₆₀₀ (from 0.8 to 3.8). An antibody for DnaN was used as a loading control. The lower panel is a growth curve of B. subtilis at 37°C. Data are the means from six independent replicates from two experiments, and the error bars are the standard errors of the means. (C) A trimethoprim resistance assay to measure mutagenesis. Data were plotted as averages from three independent experiments, and error bars represent the standard errors of the means.

FIG 2

ΔmutS2 cells are sensitive to mitomycin C chronic exposure. (A) Spot-titer assay of WT and ΔmutS2 cells. Cultures were normalized via OD₆₀₀ and serially diluted, and the indicated dilutions were spotted on an untreated control plate and the indicated concentrations of mitomycin C (MMC). (B) Plating efficiency assay. Cultures were normalized via OD₆₀₀, serially diluted, and plated on either LB or LB with 30 ng/ml MMC to determine CFU. Plating efficiency is the percent CFU on MMC relative to the LB control. A Mann-Whitney U test was used to determine statistical significance. An asterisk indicates a one-sided P value of <0.05. (C) Survival assay. WT and ΔmutS2 cultures were normalized via OD₆₀₀ and then treated with the indicated concentration of MMC for 30 min in saline, followed by plating for viable cells on LB. Percent survival is the percentage of CFU from the treatment relative to the untreated control.

FIG 3

C-terminal Smr domain is necessary but not sufficient for MMC tolerance. (A) MutS2 constructs used for complementation experiments in panels B, C, and D. (B) A spot-titer assay was done as described for Fig. 2. Complementation constructs were under the control of the IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible P_spac promoter. (C) Plating efficiency assay performed as described for Fig. 2 with 1 mM IPTG or without IPTG. The Mann-Whitney U test was used to determine statistical significance. ns, not significant; *, one-sided P value of <0.05. (D) Western blot using a MutS2 antibody and a DnaN antibody as a loading control. Cultures of the indicated genotypes were grown in the absence (lanes 1 to 5) or presence (lanes 6 to 8) of IPTG. The cultures were normalized via OD₆₀₀ and analyzed via Western blotting.

FIG 4

ΔmutS2 sensitivity is independent of uvrA and recU and shows no additive effects with recA. (A to C) Plating efficiency assays performed as described for Fig. 2 with the indicated genotypes and the indicated concentrations of MMC. The Mann-Whitney U test was used to determine statistical significance. ns, not significant; *, one-sided P value of <0.05. Note that lower concentrations of MMC were used for this experiment and ΔmutS2 cells do not show a phenotype.

FIG 5

MutS2 is important for plasmid transformation, and mutS2 deletion is additive with a recU deletion in chromosomal DNA (chrDNA) transformation. (A) Transformation efficiency assay using plasmid DNA. (B) Transformation efficiency using chromosomal DNA. (C) Transformation efficiency assay using chromosomal DNA in the presence of 1 mM IPTG. Transformation efficiency is the ratio of transformants (CFU) to viable cells (CFU). The Mann-Whitney U test was used to determine statistical significance. ns, not significant; *, two-sided P value of <0.05.

FIG 6

Functional GFP-MutS2 fusion has a diffuse cytosolic distribution. (A) A spot-titer assay showing that GFP fused to the N terminus of MutS2 expressed from the native locus is functional. (B) Western blot using a MutS2 antibody shows that GFP-MutS2 is expressed and we do not detect degradative products, indicating MutS2 is intact. (C) Fluorescence microscopy to visualize GFP-MutS2 under vehicle control and 100-ng/ml MMC treatment conditions (red, membranes stained with FM4-64; green, GFP-MutS2; scale bar is 2 μm).