Optimized CRISPR Interference System for Investigating Pseudomonas alloputida Genes Involved in Rhizosphere Microbiome Assembly
Year: 2024
Type: article
Abstract: Pseudomonas alloputida KT2440 (formerly P. putida ) 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/ P m, LacI/ P lac, and AraC/ P 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 P. alloputida cells per root. Finally, we show that CRISPRi can be used to modulate microbe–microbe interactions. When the gene pvdH is repressed and P. alloputida is unable to produce pyoverdine, it loses its ability to inhibit other microbes in vitro. 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.
Source: ACS Synthetic Biology
Institutions: Iowa State University, Ames National Laboratory
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $4,500
Funders: National Science Foundation, U.S. Department of Energy, U.S. Department of Agriculture, Iowa State University, Joint Genome Institute, National Institute of Food and Agriculture, Office of Science, Division of Research on Learning in Formal and Informal Settings, Biological and Environmental Research
Awards: G1117004060, G1286236842, G1337544421, G1519358529, G155191036, G1863003419, MILK INCOME LOSS CONTRACTS; TO MAINTAIN AND EXPAND EXISTING MARKETS FOR DAIRY WHICH ARE VITAL TO THE WELFARE OF MILK PRODUCERS IN THE UNITED STATES, G2132461622, G223002938, G3083819904, G3961521387, G419881185, G42916606, G4501827968, G498139845, Interdisciplinary Approaches to Teaching Computational Environmental Science, G573842556, G6652958823, G6734632886, G7205239661, G7787287669, G7947762707, G834277795, G876836414, G969889393
OpenAlex ID: W4401729275