Dataset Information

Protoplast fusion in Bacillus species produces frequent, unbiased, genome-wide homologous recombination.

ABSTRACT: In eukaryotes, fine-scale maps of meiotic recombination events have greatly advanced our understanding of the factors that affect genomic variation patterns and evolution of traits. However, in bacteria that lack natural systems for sexual reproduction, unbiased characterization of recombination landscapes has remained challenging due to variable rates of genetic exchange and influence of natural selection. Here, to overcome these limitations and to gain a genome-wide view on recombination, we crossed Bacillus strains with different genetic distances using protoplast fusion. The offspring displayed complex inheritance patterns with one of the parents consistently contributing the major part of the chromosome backbone and multiple unselected fragments originating from the second parent. Our results demonstrate that this bias was in part due to the action of restriction-modification systems, whereas genome features like GC content and local nucleotide identity did not affect distribution of recombination events around the chromosome. Furthermore, we found that recombination occurred uniformly across the genome without concentration into hotspots. Notably, our results show that species-level genetic distance did not affect genome-wide recombination. This study provides a new insight into the dynamics of recombination in bacteria and a platform for studying recombination patterns in diverse bacterial species.

SUBMITTER: Vasileva DP

PROVIDER: S-EPMC9226520 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Protoplast fusion in Bacillus species produces frequent, unbiased, genome-wide homologous recombination.

Vasileva Delyana P DP Streich Jared C JC Burdick Leah H LH Klingeman Dawn M DM Chhetri Hari B HB Brelsford Christa M CM Ellis J Christopher JC Close Dan M DM Jacobson Daniel A DA Michener Joshua K JK

Nucleic acids research 20220601 11

In eukaryotes, fine-scale maps of meiotic recombination events have greatly advanced our understanding of the factors that affect genomic variation patterns and evolution of traits. However, in bacteria that lack natural systems for sexual reproduction, unbiased characterization of recombination landscapes has remained challenging due to variable rates of genetic exchange and influence of natural selection. Here, to overcome these limitations and to gain a genome-wide view on recombination, we c ...[more]

PMID: 35061904

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Project description: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.ImportanceCells 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.

Project description:BACKGROUND:A frequent event in the evolution of prokaryotic genomes is homologous recombination, where a foreign DNA stretch replaces a genomic region similar in sequence. Recombination can affect the relative position of two genomes in a phylogenetic reconstruction in two different ways: (i) one genome can recombine with a DNA stretch that is similar to the other genome, thereby reducing their pairwise sequence divergence; (ii) one genome can recombine with a DNA stretch from an outgroup genome, increasing the pairwise divergence. While several recombination-aware phylogenetic algorithms exist, many of these cannot account for both types of recombination; some algorithms can, but do so inefficiently. Moreover, many of them reconstruct the ancestral recombination graph (ARG) to help infer the genome tree, and require that a substantial portion of each genome has not been affected by recombination, a sometimes unrealistic assumption. METHODS:Here, we propose a Coarse-Graining approach for Phylogenetic reconstruction (CGP), which is recombination-aware but forgoes ARG reconstruction. It accounts for the tendency of a higher effective recombination rate between genomes with a lower phylogenetic distance. It is applicable even if all genomic regions have experienced substantial amounts of recombination, and can be used on both nucleotide and amino acid sequences. CGP considers the local density of substitutions along pairwise genome alignments, fitting a model to the empirical distribution of substitution density to infer the pairwise coalescent time. Given all pairwise coalescent times, CGP reconstructs an ultrametric tree representing vertical inheritance. RESULTS:Based on simulations, we show that the proposed approach can reconstruct ultrametric trees with accurate topology, branch lengths, and root positioning. Applied to a set of E. coli strains, the reconstructed trees are most consistent with gene distributions when inferred from amino acid sequences, a data type that cannot be utilized by many alternative approaches. CONCLUSIONS:The CGP algorithm is more accurate than alternative recombination-aware methods for ultrametric phylogenetic reconstructions.

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Protoplast fusion in Bacillus species produces frequent, unbiased, genome-wide homologous recombination.

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Protoplast fusion in Bacillus species produces frequent, unbiased, genome-wide homologous recombination.

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