ENGINEERING A DIGUANYLATE CYCLASE-FREE Pseudomonas aeruginosa STRAIN BY MULTIPLEXED GENOME EDITING

Congreso; Congreso Conjunto SAIB-SAMIGE 2021; 2021

SAIB-SAMIGE

The second messenger bis-(3′→5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) governs a wide range of physiological functions in bacteria including biofilm formation, motility, and virulence. In response to internal or external cues, c-di-GMP levels are modulated by diguanylate cyclases (DGCs, with the canonical GGDEF motif) and phosphodiesterases (PDEs, with either EAL or HD-GYP domains). DGCs and PDEs catalyze c-di-GMP synthesis and breakdown, respectively. Exploring the role of these enzymes has traditionally involved the sequential deletion of genes encoding DGCs or PDEs. In few cases, these efforts led to mutant strains devoid of a small number of DGC/PDE (e.g. in Salmonella). However, the construction of multiple mutants by targeted mutagenesis is both laborious and expensive, mainly in species containing a high amount of c-di-GMP?synthesizing proteins. The opportunistic human pathogen Pseudomonas aeruginosa falls in this category, with 40 genes encoding DCGs, PDEs, or dual domains predicted to be likewise involved in c-di-GMP metabolism. In this work, we developed a CRISPR/Cas9-based, multiplex genome edition tool for Pseudomonas species to target all 32 genes encoding proteins displaying a GGDEF domain in P. aeruginosa UCBPP-PA14. In silico prediction of spacer sequences enabled targeting up to 6 DGC simultaneously. The gene knockout efficiency was confirmed by whole-genome sequencing of the resulting clones, and the DGC-free mutant had no detectable levels of c-di-GMP. Phenotypic characterization of this strain indicated that growth rates were not affected by the absence of the messenger nucleotide across experimental conditions. However, bacterial growth into structured biofilm communities was severely impaired in the mutant. Motility assays showed that swimming and twitching were likewise unaffected, whereas the DGC-free strain failed to swarm. Furthermore, we determined that in the absence of c-di-GMP, the production of pyoverdin, the main siderophore mediating P. aeruginosa´s iron-gathering capacity, was drastically reduced. In all, this study provides insights into the multilayered signaling architecture of the highly complex c-di-GMP networks of a priority bacterial pathogen?offering, at the same time, tools specifically tailored for the genetic exploration of bacterial regulatory networks.