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Pervasive prophage recombination occurs during evolution of spore-forming Bacilli


Phages are the main source of within-species bacterial diversity and drivers of horizontal gene transfer, but we know little about the mechanisms that drive genetic diversity of these mobile genetic elements (MGEs). Recently, we showed that a sporulation selection regime promotes evolutionary changes within SPβ prophage of Bacillus subtilis, leading to direct antagonistic interactions within the population. Herein, we reveal that under a sporulation selection regime, SPβ recombines with low copy number phi3Ts phage DNA present within the B. subtilis population. Recombination results in a new prophage occupying a different integration site, as well as the spontaneous release of virulent phage hybrids. Analysis of Bacillus sp. strains suggests that SPβ and phi3T belong to a distinct cluster of unusually large phages inserted into sporulation-related genes that are equipped with a spore-related genetic arsenal. Comparison of Bacillus sp. genomes indicates that similar diversification of SPβ-like phages takes place in nature. Our work is a stepping stone toward empirical studies on phage evolution, and understanding the eco-evolutionary relationships between bacteria and their phages. By capturing the first steps of new phage evolution, we reveal striking relationship between survival strategy of bacteria and evolution of their phages.

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Fig. 1: Changes within B. subtilis prophage sequence and integration site observed after prolonged sporulation selection regime.
Fig. 2: Hybrid phages and extrachromosomal fragments of phage DNA, detected in the evolved strains.
Fig. 3: Detection of phi3Ts DNA in the ancestor strain B. subtilis 168 through mapping of raw sequencing reads.
Fig. 4: Effect of phi3T lysogenization on B. subtilis sporulation and germination dynamics.
Fig. 5: Overview of prophage elements of natural Bacillus sp. isolates.
Fig. 6: Natural diversity of SPβ-like phages.


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The authors thank M. Kilstrup, P. Sazinas, K. Middleboe, D. Castillio and P. Stefanic for their valuable comments. We are profoundly grateful to O. Kuipers, A. de Jong and W. Overkamp from University of Groningen, for sharing their raw sequencing data and all relevant information, which allowed us to finalize the paper. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 713683 (H.C. Ørsted COFUND to AD), Individual grant from Friedrich Schiller University Jena to support postdoc researchers to AD, and supported by the Danish National Research Foundation (DNRF137) for the Center for Microbial Secondary Metabolites. Funded in part by NIH R01GM121865 to BMB.

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AD and ATK designed the study. AD, PB, ZH, CK performed experiments. AD and MLS performed bioinformatics analysis. PK performed electron microscopy, GM performed genome sequencing and analyzed the data, BB and BMB shared sequencing data. AD wrote the paper. All authors contributed to final version of the paper.

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Correspondence to Anna Dragoš or Ákos T. Kovács.

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Dragoš, A., Priyadarshini, B., Hasan, Z. et al. Pervasive prophage recombination occurs during evolution of spore-forming Bacilli. ISME J 15, 1344–1358 (2021).

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