ACS synthetic biologyAug 20, 2024

Improved CRISPR Tool to Study Pseudomonas alloputida Genes That Help Build Root Microbe Communities

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Updated Jun 27, 2026

Abstract

Essence

An optimized toolkit enabled tunable gene repression in Pseudomonas alloputida to probe fitness and microbe-microbe interactions.

Evidence

A bacterial platform experiment in Pseudomonas alloputida KT2440 compared three promoter-controlled CRISPRi systems and showed rhizosphere essential-gene knockdowns caused 10-100-fold fewer cells per root and pvdH repression changed microbial inhibition in vitro.

Caveat

The work validates selected genes and conditions; the abstract says media and environment affect CRISPRi success and leaves CRISPRi-seq/multispecies use as future work.

Simplified

KT2440 (formerly) has become both a well-known chassis organism for synthetic biology and a model organism for colonization. Here, we describe a (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/, LacI/, and AraC/) and a 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 incells per root. Finally, we show that CRISPRi can be used to modulate microbe-microbe interactions. When the geneis repressed andis unable to produce pyoverdine, it loses its ability to inhibit other microbesMoreover, 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. Pseudomonas alloputida P. putida P P P P. alloputida pvdH P. alloputida in vitro. m lac BAD

Key numbers

10-100×

Reduction in Bacterial Cells

Repression of essential genes led to this reduction in cells per root.

73-fold

Concentration Comparison

Fewer cells colonized the roots at 1 day post-planting with arabinose compared to the control.

6-fold

CFUs Reduction

Fewer CFUs per root were observed at 1 day after planting with .

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Improved CRISPR Tool to Study Pseudomonas alloputida Genes That Help Build Root Microbe Communities

Abstract

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