To date, only limited knowledge is available on how complex cellular behaviors are regulated in P. polymyxa . In this study, we investigate several regulatory proteins which play a role in governing different physiological traits.

Despite being unicellular organisms, bacteria undergo complex regulation mechanisms which coordinate different physiological traits. Among others, DegU, DegS, and Spo0A are the pleiotropic proteins which govern various cellular responses and behaviors. However, the functions and regulatory networks between these three proteins are rarely described in the highly interesting bacterium Paenibacillus polymyxa. In this study, we investigate the roles of DegU, DegS, and Spo0A by introduction of targeted point mutations facilitated by a CRISPR-Cas9-based system. In total, five different mutant strains were generated: the single mutants DegU Q218*, DegS L99F, Spo0A A257V, the double mutant DegU Q218* DegS L99F, and the triple mutant DegU Q218* DegS L99F Spo0A A257V. Characterization of the wild type and the engineered strains revealed differences in swarming behavior, genetic competence, sporulation, and viscosity formation of the culture broth. In particular, the double mutant DegU Q218* DegS L99F showed significant increase in regard to the genetic competence as well as a stable exopolysaccharides formation. Furthermore, we highlight similarities and differences of the roles of DegU, DegS, and Spo0A between P. polymyxa and related species. Finally, this study provides novel insights in the complex regulatory system of P. polymyxa DSM 365.