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

Abstract

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 SP{beta} 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 SP{beta} 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, SP{beta} 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. IMPORTANCEProphages, defined as viruses integrated into bacterial genomes, can reshape bacterial physiology and evolution. Previous studies suggested that disruption of an integration site (spsM) by the SP{beta} prophage impairs biofilm formation in Bacillus subtilis, yet the functional basis for this remained unclear. Here, we show that spsM disruption across diverse natural isolates promotes the rapid emergence of spontaneous mutations in key regulatory genes like swrA and comP, which do influence biofilm morphology and motility. Strikingly, when spsM is disrupted by a prophage capable of precise excision, such diversification is minimized, indicating a protective role for dynamic prophage integration. These findings reconcile data from earlier work and identify prophage integration sites as evolutionary hotspots, affecting host genome stability. This has broader implications for how we understand the genetic basis of microbial adaptation and the evolutionary roles of prophages.