Abstract
The second messenger c-di-GMP regulates the switch between motile and sessile bacterial lifestyles. A general feature of c-di-GMP metabolism is the presence of a surprisingly large number of genes coding for diguanylate cyclases and phosphodiesterases, the enzymes responsible for its synthesis and degradation respectively. However, the physiological relevance of this apparent redundancy is not clear, emphasizing the need for investigating the functions of each of these enzymes. Here we focused on the phosphodiesterase PA2133 from Pseudomonas aeruginosa, an important opportunistic pathogen. We phenotypically characterized P. aeruginosa strain K overexpressing PA2133 or its inactive mutant. We showed that biofilm formation and motility are severely impaired by overexpression of PA2133. Our quantitative proteomic approach applied to the membrane and exoprotein fractions revealed that proteins involved in three processes were mostly affected: flagellar motility, type III secretion system and chemotaxis. While inhibition of biofilm formation can be ascribed to the phosphodiesterase activity of PA2133, down-regulation of flagellar, chemotaxis, and type III secretion system proteins is independent of this enzymatic activity. Based on these unexpected effects of PA2133, we propose to rename this gene product FcsR, for Flagellar, chemotaxis and type III secretion system Regulator.
Highlights
The cyclic nucleotide bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) is a global second messenger that has a central role in the regulation of bacterial adhesiveness, controlling both cell–cell and cell–surface interactions
P. aeruginosa strain K (PAK)/pNJ-FcsR showed the highest phosphodiesterase activity. c-di-GMP levels drop approximately five-fold after 3 h of incubation in protein extracts from this strain, while under the same conditions the levels of this cyclic nucleotide remained practically unchanged in protein extracts from PAK and PAK/pJN-FcsRE60A (Supplementary Figure 2b)
Our results clearly showed that while biofilm defective phenotype is mediated by FcsR enzymatic activity, the regulation of flagellar motility, chemotaxis, and type III secretion system (TTSS) does not rely on c-di-GMP hydrolysis
Summary
The cyclic nucleotide bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) is a global second messenger that has a central role in the regulation of bacterial adhesiveness, controlling both cell–cell and cell–surface interactions. Analysis of the phenotypes related to the overexpression or deletion of each GGDEF and EAL containing gene in P. aeruginosa suggested that not all the diguanylate cyclases or phosphodiesterases have the same role on biofilm formation and cytotoxicity[13]. These observations points to a c-di-GMP mediated signalling network far more complex than previously thought; deserving further investigation of the molecular function(s) of each GGDEF and EAL containing protein. We suggest renaming PA2133 FcsR, for Flagellar, chemotaxis and type III secretion Regulator
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