FimX regulates type 4 pilus assembly and twitching motility in the absence of cyclic-di-GMP in Pseudomonas aeruginosa

Los Cocos

Congreso; XVII Congreso Argentino de Microbiología General; 2022

SAMIGE

The second messenger bis-(3´→5´)-cyclic dimeric guanosine monophosphate (c-di-GMP) governs a wide range of cellular responses including biofilm formation, motility, siderophore production 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 orHD-GYP domains). DGCs and PDEs catalyze c-di-GMP synthesis and breakdown, respectively.Pseudomonas aeruginosa is endowed with 40 enzymes including DGC, PDE, and others carrying both domains. Understanding how this complex network is regulated and works orchestrating bacterial life is challenging. While the intracellular level of c-di-GMP is known to be tightly regulated, the molecular mechanisms of control are still unclear. It has been proposed that five DGCs (bifA, dgcH, dipA, PA14_03720 y PA14_03790), highly conserved along the genus Pseudomonas and with invariable expression profiles, form a core of enzymes required for the maintenance of the basal level of c-diGMP in P. aeruginosa. To inquire into the mechanism controlling c-di-GMP levels, we have previously engineered a P. aeruginosa UCBPP-PA14 diguanylate cyclase-free strain (DGC-free) by a CRISPR/Cas9-based multiplex genome edition tool. This strain allows us to study the role of single DGCs in the absence of all the other ones. Here, we analyzed the role of a DGC encoded in ORF PA14_23130, one of the few cytoplasmatic enzymes holding the classical domain GGEEF and has been barely studied inP. aeruginosa. Overexpression of PA14_23130 from the pJN23130 plasmid in a PA14 wild-type (WT) background did not affect biofilms formation nor modify swarming behavior. However, when expressed in the DGC-free strain, PA14_23130 intriguingly induced biofilms biomass production up to two times greater than in the WT strain. Facing this observation, we are currently exploring thehypothesis that in the WT strain, c-di-GMP particularly produced by PA14_23130 is regulated by one or more of the enzymes that belong to the core antagonizing PA14_23130. This regulation would control the global cellular response and buffer DGC PA14_23130 output. This study provides insights into the role of the DGC PA14_23130 and exemplified, at the same time, the use of the DGC-freeinnovative tool for the genetic exploration of the multilayered and highly complex c-di-GMP network in the opportunistic pathogen Pseudomonas aeruginosa.