Institute of Molecular Microbiology and Biotechnology, University of Münstergrid.5949.1, Münster, Germany.
Department of Biotechnology and Food Science, Norwegian University of Science and Technology, Trondheim, Norway.
Appl Environ Microbiol. 2022 Jun 14;88(11):e0016422. doi: 10.1128/aem.00164-22. Epub 2022 May 19.
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, and 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, conjugation efficiency, sporulation, and viscosity formation of the culture broth. In particular, the double mutant DegU Q218* DegS L99F showed a significant increase in conjugation efficiency as well as a stable exopolysaccharides formation. Furthermore, we highlight similarities and differences in the roles of DegU, DegS, and Spo0A between P. polymyxa and related species. Finally, this study provides novel insights into the complex regulatory system of P. polymyxa DSM 365. 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. Precise targeted point mutations were introduced to their respective genes by employing a highly efficient CRISPR-Cas9-based system. Characterization of the strains revealed some similarities, but also differences, to the model bacterium Bacillus subtilis with regard to the regulation of cellular behaviors. Furthermore, we identified several strains which have superior performance over the wild-type. The applicability of the CRISPR-Cas9 system as a robust genome editing tool, in combination with the engineered strain with increased genetic accessibility, would boost further research in P. polymyxa and support its utilization for biotechnological applications. Overall, our study provides novel insights, which will be of importance in understanding how multiple cellular processes are regulated in species.
尽管是单细胞生物，但细菌经历复杂的调控机制，协调不同的生理特征。其中，DegU、DegS 和 Spo0A 是调节各种细胞反应和行为的多效蛋白。然而，这三种蛋白质之间的功能和调控网络在高度有趣的多粘类芽孢杆菌中很少被描述。在这项研究中，我们通过基于 CRISPR-Cas9 的系统引入靶向点突变来研究 DegU、DegS 和 Spo0A 的作用。总共生成了五个不同的突变菌株，分别是单个突变菌株 DegU Q218*、DegS L99F 和 Spo0A A257V，双突变菌株 DegU Q218* DegS L99F，以及三突变菌株 DegU Q218* DegS L99F Spo0A A257V。野生型和工程菌株的表征揭示了在群集行为、 conjugation 效率、 sporulation 和培养液粘度形成方面的差异。特别是，双突变菌株 DegU Q218* DegS L99F 显示出 conjugation 效率的显著提高以及稳定的胞外多糖形成。此外，我们还强调了多粘类芽孢杆菌和相关物种中 DegU、DegS 和 Spo0A 作用的相似性和差异性。最后，这项研究为多粘类芽孢杆菌复杂调控系统提供了新的见解。迄今为止，关于多粘类芽孢杆菌如何调控复杂细胞行为的知识有限。在这项研究中，我们研究了几种在调节不同生理特征中起作用的调控蛋白。通过使用高效的基于 CRISPR-Cas9 的系统，在它们各自的基因中引入了精确的靶向点突变。菌株的表征揭示了与模式细菌枯草芽孢杆菌在细胞行为调控方面的一些相似性，但也存在差异。此外，我们还鉴定了一些在某些方面表现优于野生型的菌株。CRISPR-Cas9 系统作为一种强大的基因组编辑工具的适用性，结合具有增强遗传可及性的工程菌株，将促进多粘类芽孢杆菌的进一步研究，并支持其在生物技术应用中的利用。总的来说，我们的研究提供了新的见解，这对于理解多种细胞过程如何在物种中被调控具有重要意义。
