Prokaryotic viruses of Spbetavirus genus are temperate bacteriophages targeting Bacillus subtilis. As these phages carry genes encoding for bacteriocins, communication systems and regulators of sporulation/germination processes, they substantially alter host phenotype during lysogeny.  The impact they exert on the bacterial host is strengthened by their ability of active lysogeny (or regulatory switch mechanism, RS), an alternative lysogenic life cycle characterized by integration into specific bacterial functional genes, resulting in inactivation/reactivation of the target genes by phage integration/excision, respectively. Temperate phages characterized by following this specific life cycle are also named RS-phages. While investigating the impact of RS-phages on B. subtilis physiology, we observed a cell morphology change caused by a specific Spbetavirus, resulting in an aberrant spherical shape as opposed to a conventional rod shape showed by the wild type strain. This phenotype manifests in late exponential phase and during sporulation, being stable and heritable. Furthermore, we also discovered that superinfection with a second homologous Spbetavirus restores the conventional rod-shaped morphology, suggesting a phage-phage interplay which results in protecting the host from the cell morphology shifting. We investigated potential differences between the wild type and its spherical-shaped lysogenic version through Transmission Electron Microscopy analysis and Fluorescence Microscopy’s time-lapse experiments using various reporter fusion constructs, questioning mainly processes like cell elongation, cell division and sporulation. Moreover, we obtained relevant results investigating the single and double lysogeny influence on phage/s and host fitness through plaque and growth assays, observing variety of plaque morphologies correlated to peculiar growth dynamics. Additionally, the sequencing analysis helped to explore the impact of the double lysogeny on the host genome, in comparison with the single lysogens. Interestingly, the effect on host morphology does not seem unique of a specific prophage, but it is exerted by other closely related phages belonging to the Spbetavirus genus, as we observed while screening our lysogens collection.

Exploring the mechanism behind the host’s morphology change by specific Spbetaviruses will increase the current limited knowledge on lysogenic conversion, likewise interference of prophages with fundamental cellular processes. The outcome of this research will bring us closer to understanding the role of prophages in controlling bacterial hosts, highlighting mechanisms still unknown, crucial knowledge to control bacteria for biotechnological and medical applications.