Disruption of bacteriophage integration site promotes rapid diversification of multicellular traits in Bacillus subtilis

Certain bacteria are known for their remarkable genetic and phenotypic diversity, as well as rapid morphological diversification during evolution experiments. An example is Bacillus subtilis , which can switch motility, biofilm or antagonistic interaction patterns either as a result of spontaneous mutations or due to changes in prophage elements. Prophages can integrate into conserved and functional loci, disrupting host genes and modulating their phenotypic traits. In B. subtilis , the SPβ prophage integrates into the sporulation-associated gene spsM , whose reversible inactivation during lysogeny has also been implicated in modulating biofilm formation and host development. Here, we investigated the evolutionary and phenotypic consequences of spsM disruption through both SPβ integration and artificial mutagenesis ( spsM::kan ) in B. subtilis natural isolates. We observed that spsM::kan mutants frequently developed spontaneous mutations, particularly in swrA and comP , key regulators of swarming motility and biofilm development. These mutations reproducibly gave rise to altered colony morphotypes and impaired surface motility, suggesting strong selection for loss-of-function mutations in regulatory genes under laboratory conditions. In contrast, SPβ lysogens exhibited minimal mutational diversification, indicating that dynamic prophage excision may preserve genomic stability. In this work spsM inactivation alone did not significantly impair biofilm formation or motility. However, we reveal a novel role of prophage integration sites as possible evolutionary hotspots that influence genome integrity and adaptive potential. This work highlights the interplay between prophage integration, host genome architecture, and the selective pressures shaping bacterial multicellular communities.