Phenotypic and integrated analysis of a comprehensive Pseudomonas aeruginosa PAO1 library of mutants lacking cyclic-di-GMP-related genes

Abstract

Pseudomonas aeruginosa is a Gram-negative bacterium that is able to survive and adapt in a multitude of niches as well as thrive within many different hosts. This versatility lies within its large genome of ca. 6 Mbp and a tight control in the expression of thousands of genes. Among the regulatory mechanisms widespread in bacteria, cyclic-di-GMP signaling is one which influences all levels of control. c-di-GMP is made by diguanylate cyclases and degraded by phosphodiesterases, while the intracellular level of this molecule drives phenotypic responses. Signaling involves the modification of enzymes’ or proteins’ function upon c-di-GMP binding, including modifying the activity of regulators which in turn will impact the transcriptome. In P. aeruginosa, there are ca. 40 genes encoding putative DGCs or PDEs. The combined activity of those enzymes should reflect the overall c-di-GMP concentration, while specific phenotypic outputs could be correlated to a given set of dgc/pde. This notion of specificity has been addressed in several studies and different strains of P. aeruginosa. Here, we engineered a mutant library for the 41 individual dgc/pde genes in P. aeruginosa PAO1. In most cases, we observed a significant to slight variation in the global c-di-GMP pool of cells grown planktonically, while several mutants display a phenotypic impact on biofilm including initial attachment and maturation. If this observation of minor changes in c-di-GMP level correlating with significant phenotypic impact appears to be true, it further supports the idea of a local vs global c-di-GMP pool. In contrast, there was little to no effect on motility, which differs from previous studies. Our RNA-seq analysis indicated that all PAO1 dgc/pde genes were expressed in both planktonic and biofilm growth conditions and our work suggests that c-di-GMP networks need to be reconstructed for each strain separately and cannot be extrapolated from one to another.

Identification of echinacoside as a tobramycin potentiator againstPseudomonas aeruginosaaggregates

“…However, despite its thoroughly proven DGC activity 30,54 , lacking WspR per se did not influence the overall biomass but more on biofilm 3-D structure 23,24 . Therefore, the inactivation of WspR in strains without mutations in wspF may not be the optimal strategy solely from the perspective of its role in biofilm formation.…”

“…To generate a more in vivo- like environment, P. aeruginosa aggregates were grown in synthetic cystic fibrosis medium 2 (SCFM2), which is regarded as the most accurate pre-clinical model developed to mimic CF mucus so far 22 . In P. aeruginosa , 42 proteins were reported to contain GG(D/E)EF, EAL/HD-GYP, or dual domains 23,24 , of which WspR has been used as the target for all previously identified DGC inhibitors in P. aeruginosa . However, we found that deleting wspR in PAO1 did not affect the overall auto-aggregation level in SCFM2.…”

Control of Biofilm Formation by anAgrobacterium tumefaciensPterin-Binding Periplasmic Protein Conserved Among Pathogenic Bacteria

“…In combination they may provide similar, but independently regulated DGC/PDE activities, as does the dual functionality of DcpA in A. tumefaciens. In the two major model strains of P. aeruginosa , PA01 and PA14, mutants for their dcpA homologs (PA2780/PA1426970), both impact cdGMP-dependent phenotypes, although to different extents (42, 43). In all of these cases, the positioning of a pruR -type gene immediately upstream is consistent with a regulatory relationship with the downstream gene.…”

Divergent molecular strategies drive convergent evolutionary adaptation to kin competition in biofilms

“…3a). In P. aeruginosa, BifA is a highly conserved PDE responsible for c-di-GMP hydrolysis, so its dysfunction would elevate c-di-GMP and increase bio lm formation [36][37][38] . Although this mutation was not predicted to modify BifA structure (Fig.…”