Optimized CRISPR Interference System for Investigating <i>Pseudomonas alloputida</i> Genes Involved in Rhizosphere Microbiome Assembly

<i>Pseudomonas alloputida</i> KT2440 (formerly <i>P. putida</i>) has become both a well-known chassis organism for synthetic biology and a model organism for rhizosphere colonization. Here, we describe a CRISPR interference (CRISPRi) system in KT2440 for exploring microbe-microbe interactions in the rhizosphere and for use in industrial systems. Our CRISPRi system features three different promoter systems (XylS/<i>P</i>_m, LacI/<i>P</i>_lac, and AraC/<i>P</i>_BAD) and a dCas9 codon-optimized for Pseudomonads, all located on a mini-Tn7-based transposon that inserts into a neutral site in the genome. It also includes a suite of pSEVA-derived sgRNA expression vectors, where the expression is driven by synthetic promoters varying in strength. We compare the three promoter systems in terms of how well they can precisely modulate gene expression, and we discuss the impact of environmental factors, such as media choice, on the success of CRISPRi. We demonstrate that CRISPRi is functional in bacteria colonizing the rhizosphere, with repression of essential genes leading to a 10-100-fold reduction in <i>P. alloputida</i> cells per root. Finally, we show that CRISPRi can be used to modulate microbe-microbe interactions. When the gene <i>pvdH</i> is repressed and <i>P. alloputida</i> is unable to produce pyoverdine, it loses its ability to inhibit other microbes <i>in vitro.</i> Moreover, our design is amendable for future CRISPRi-seq studies and in multispecies microbial communities, with the different promoter systems providing a means to control the level of gene expression in many different environments.