CRISPR interference-based gene repression in the plant growth promoter Paenibacillus sonchi genomovar Riograndensis SBR5

Abstract

Gene repression using the endonucleolytically deactivated dCas9 protein and sgRNAs (CRISPR interference or CRISPRi) is a useful approach to study gene functions. Here, we established CRISPRi in Paenibacillus sonchi genomovar Riograndensis SBR5, a plant growth promoting bacterium. CRISPRi system with sgRNAs targeting SBR5 endogenous genes spo0A, yaaT and ydjJ and plasmid-borne gfpUV was constructed and analyzed. Flow cytometry analysis revealed a significant decrease of reporter protein GFPUV signal in P. sonchi strains expressing gfpUV sgRNA in comparison with non-targeting controls. CRISPRi-based repression of chromosomal genes for regulation of sporulation spo0A and yaaT decreased sporulation and increased biofilm formation in SBR5. Repression of the sorbitol catabolic gene ydjJ revealed decreased specific activity of YdjJ in crude cell extracts and reduced biomass formation from sorbitol in growth experiments. Our work on CRISPRi-based gene repression serves as basis for gene function studies of the plant growth promoter P. sonchi SBR5. To our knowledge, the present study presents the first tool for gene repression established in Paenibacillus species. Key points • CRISPRi toward gene repression was applied for the first time in Paenibacillus. • CRISPRi of spo0A and yaaT depleted spores and increased biofilms in SBR5. • CRISPRi-based ydjJ repression decreased specific activity of sorbitol dehydrogenase.

Inorganic Phosphate Solubilization by Rhizosphere Bacterium Paenibacillus sonchi: Gene Expression and Physiological Functions

“…RNAseq has been used to chart its global transcriptomic landscape (Brito et al, 2017a). Moreover, molecular tools for gene expression (Brito et al, 2017b) and CRISPR interference-based gene repression (Brito et al, 2020) were developed for this organism. Hence, a methodology is in place to study the poorly characterized PS by SBR5.…”

CRISPRi in Xanthomonas demonstrates functional convergence of transcription activator‐like effectors in two divergent pathogens

“…CRISPR interference platforms have been established in many human and animal pathogens including Vibrio cholerae , Yersinia pestis , Streptococcus pneumoniae , and Staphylococcus aureus (Zhang et al ., 2021). To the best of our knowledge, such system has never been applied to plant‐pathogenic bacteria, while a few examples were reported for plant‐associated microorganisms such as Pseudomonas fluorescens (Tan et al ., 2018) and Paenibacillus sonchi (Brito et al ., 2020). Here, we demonstrated the potential of CRISPRi for rapid and efficient knockdown of an entire gene family of major virulence factors from the plant‐pathogenic bacterium Xanthomonas , applying only one sgRNA which targets a highly conserved sequence in their promoters or 5′‐UTR regions.…”

Development of a Biosensor for Crotonobetaine-CoA Ligase Screening Based on the Elucidation of Escherichia coli Carnitine Metabolism

“…The cell pellets were immediately frozen and stored at −80 °C. RNA isolation and qRT-PCR were performed as previously described 57 with slight modifications.…”