Fuente: PubMed "swarm" Microbiol Spectr. 2026 Apr 20:e0250425. doi: 10.1128/spectrum.02504-25. Online ahead of print.ABSTRACTCertain 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. Here, we investigated how different forms of disruption at the spsM locus, including SPβ integration, insertional mutagenesis (spsM::kan), and markerless spsM deletion, influence colony morphology, motility, and the emergence of spontaneous variants in B. subtilis natural isolates. We reassessed a previously reported biofilm defect of an spsM::kan mutant and found that the phenotype stemmed from an undetected secondary mutation rather than from loss of spsM. We observed that spsM::kan mutants frequently developed spontaneous mutations in key regulators of swarming motility and biofilm development. Consistently, we show that spsM::kan significantly elevates mutation rates, explaining why unnoticed mutations can arise rapidly during strain construction and phenotyping. In contrast, a markerless ΔspsM strain did not show a detectable increase in mutation rate relative to wild type, indicating that the elevated mutation rate is not attributable to loss of SpsM function. The SPβ lysogen produced far fewer visible variant morphotypes, indicating that reversible prophage integration does not lead to the same degree of diversification observed in the spsM::kan background. Our findings show that different modes of disrupting the spsM locus can alter the likelihood of selecting recurrent regulatory mutations, highlighting how local genomic context shapes phenotypic diversification. This work highlights the interplay between prophage integration, local genome architecture, and the selective pressures that influence diversification of bacterial multicellular behaviors.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 SPβ prophage impairs biofilm formation in Bacillus subtilis. Here, we show that insertion of a kanamycin resistance cassette at the native spsM locus (spsM::kan) promotes the rapid emergence of spontaneous mutations in key regulatory genes. In contrast, a markerless ΔspsM strain does not show a detectable increase in mutation rate, indicating that elevated mutation supply is not a general consequence of spsM loss. Our results indicate that different modes of spsM disruption have distinct consequences for phenotypic diversification. These findings help clarify earlier observations and show that phenotypic diversification depends strongly on the mode of spsM disruption and the genetic background. This has broader implications for how we understand the genetic basis of microbial adaptation, the genetic manipulation, and the evolutionary roles of prophages.PMID:42007703 | DOI:10.1128/spectrum.02504-25