Insights in the complex DegU, DegS, Spo0A regulation system of Paenibacillus polymyxa by CRISPR-Cas9-based targeted point mutations

Abstract 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. Importance To date, only limited knowledge is available on how complex cellular behaviors are regulated in P. polymyxa . In this study, we investigate three regulatory proteins which play a role in governing different physiological traits. Precise targeted point mutations are introduced to their respective genes by employing a highly efficient CRISPR-Cas9-based system. Characterization of the strains revealed some similarities, but also differences, with the model bacterium Bacillus subtilis in regard to the regulation of cellular behaviors. Furthermore, we identified several strains which have superior performance in comparison to the wild type strain. Overall, our study provides novel insights which will be of importance in understanding how multiple cellular processes are regulated in Paenibacillus species.