Optimized CRISPR Interference System for Investigating Genes Involved in Rhizosphere Microbiome Assembly

Overview

Journal ACS Synth Biol

Publisher American Chemical Society

Specialties Biotechnology
Molecular Biology

Date 2024 Aug 20

PMID 39163848

Authors

Marissa N Roghair Stroud

Dua X Vang

Larry J Halverson

Affiliations

Soon will be listed here.

Abstract

KT2440 (formerly ) 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/, LacI/, and AraC/) 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 cells per root. Finally, we show that CRISPRi can be used to modulate microbe-microbe interactions. When the gene is repressed and is unable to produce pyoverdine, it loses its ability to inhibit other microbes 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.

References

Matilla M, Espinosa-Urgel M, Rodriguez-Herva J, Ramos J, Ramos-Gonzalez M . Genomic analysis reveals the major driving forces of bacterial life in the rhizosphere. Genome Biol. 2007; 8(9):R179. PMC: 2375017. DOI: 10.1186/gb-2007-8-9-r179. View

Banerjee D, Eng T, Lau A, Sasaki Y, Wang B, Chen Y . Genome-scale metabolic rewiring improves titers rates and yields of the non-native product indigoidine at scale. Nat Commun. 2020; 11(1):5385. PMC: 7584609. DOI: 10.1038/s41467-020-19171-4. View

Fernandez M, Conde S, Duque E, Ramos J . In vivo gene expression of Pseudomonas putida KT2440 in the rhizosphere of different plants. Microb Biotechnol. 2013; 6(3):307-13. PMC: 3815925. DOI: 10.1111/1751-7915.12037. View

Gauttam R, Mukhopadhyay A, Simmons B, Singer S . Development of dual-inducible duet-expression vectors for tunable gene expression control and CRISPR interference-based gene repression in Pseudomonas putida KT2440. Microb Biotechnol. 2021; 14(6):2659-2678. PMC: 8601191. DOI: 10.1111/1751-7915.13832. View

Todor H, Silvis M, Osadnik H, Gross C . Bacterial CRISPR screens for gene function. Curr Opin Microbiol. 2020; 59:102-109. PMC: 8331264. DOI: 10.1016/j.mib.2020.11.005. View

Lopez-Guerrero M, Wang P, Phares F, Schachtman D, Alvarez S, van Dijk K . A glass bead semi-hydroponic system for intact maize root exudate analysis and phenotyping. Plant Methods. 2022; 18(1):25. PMC: 8897885. DOI: 10.1186/s13007-022-00856-4. View

Cabrefiga J, Bonaterra A, Montesinos E . Mechanisms of antagonism of Pseudomonas fluorescens EPS62e against Erwinia amylovora, the causal agent of fire blight. Int Microbiol. 2007; 10(2):123-32. View

Coppens L, Lavigne R . SAPPHIRE: a neural network based classifier for σ70 promoter prediction in Pseudomonas. BMC Bioinformatics. 2020; 21(1):415. PMC: 7510298. DOI: 10.1186/s12859-020-03730-z. View

Stolle A, Meader B, Toska J, Mekalanos J . Endogenous membrane stress induces T6SS activity in . Proc Natl Acad Sci U S A. 2021; 118(1). PMC: 7817224. DOI: 10.1073/pnas.2018365118. View

10.

Ma L, Shi Y, Siemianowski O, Yuan B, Egner T, Mirnezami S . Hydrogel-based transparent soils for root phenotyping in vivo. Proc Natl Acad Sci U S A. 2019; 116(22):11063-11068. PMC: 6561166. DOI: 10.1073/pnas.1820334116. View

11.

Calles B, Goni-Moreno A, de Lorenzo V . Digitalizing heterologous gene expression in Gram-negative bacteria with a portable ON/OFF module. Mol Syst Biol. 2019; 15(12):e8777. PMC: 6920698. DOI: 10.15252/msb.20188777. View

12.

Blanco-Romero E, Duran D, Garrido-Sanz D, Rivilla R, Martin M, Redondo-Nieto M . Transcriptomic analysis of F113 reveals the antagonistic roles of AmrZ and FleQ during rhizosphere adaption. Microb Genom. 2022; 8(1). PMC: 8914362. DOI: 10.1099/mgen.0.000750. View

13.

Wang T, Guan C, Guo J, Liu B, Wu Y, Xie Z . Pooled CRISPR interference screening enables genome-scale functional genomics study in bacteria with superior performance. Nat Commun. 2018; 9(1):2475. PMC: 6018678. DOI: 10.1038/s41467-018-04899-x. View

14.

Sivakumar R, Ranjani J, Vishnu U, Jayashree S, Lozano G, Miles J . Evaluation of INSeq To Identify Genes Essential for PGPR2 Corn Root Colonization. G3 (Bethesda). 2019; 9(3):651-661. PMC: 6404608. DOI: 10.1534/g3.118.200928. View

15.

Liu Z, Beskrovnaya P, Melnyk R, Hossain S, Khorasani S, OSullivan L . A Genome-Wide Screen Identifies Genes in Rhizosphere-Associated Required to Evade Plant Defenses. mBio. 2018; 9(6). PMC: 6222131. DOI: 10.1128/mBio.00433-18. View

16.

McMackin E, Corley J, Karash S, Marden J, Wolfgang M, Yahr T . Cautionary Notes on the Use of Arabinose- and Rhamnose-Inducible Expression Vectors in Pseudomonas aeruginosa. J Bacteriol. 2021; 203(16):e0022421. PMC: 8297530. DOI: 10.1128/JB.00224-21. View

17.

Cole B, Feltcher M, Waters R, Wetmore K, Mucyn T, Ryan E . Genome-wide identification of bacterial plant colonization genes. PLoS Biol. 2017; 15(9):e2002860. PMC: 5627942. DOI: 10.1371/journal.pbio.2002860. View

18.

Biessy A, Novinscak A, St-Onge R, Leger G, Zboralski A, Filion M . Inhibition of Three Potato Pathogens by Phenazine-Producing spp. Is Associated with Multiple Biocontrol-Related Traits. mSphere. 2021; 6(3):e0042721. PMC: 8265658. DOI: 10.1128/mSphere.00427-21. View

19.

Haas A, Zemke A, Melvin J, Armbruster C, Hendricks M, Moore J . Iron bioavailability regulates Pseudomonas aeruginosa interspecies interactions through type VI secretion expression. Cell Rep. 2023; 42(3):112270. PMC: 10586262. DOI: 10.1016/j.celrep.2023.112270. View

20.

Casavant N, Beattie G, Phillips G, Halverson L . Site-specific recombination-based genetic system for reporting transient or low-level gene expression. Appl Environ Microbiol. 2002; 68(7):3588-96. PMC: 126813. DOI: 10.1128/AEM.68.7.3588-3596.2002. View