Post-translational regulation of the exopolysaccharide alginate in Pseudomonas aeruginosa
Parasitology and Biology of Tropical Infectious Disease Pathogens
Final Report Abstract
The majority of Pseudomonas aeruginosa strains isolated from chronically infected cystic fibrosis (CF) patients display a mucoid phenotype. Mucoidy is caused by the overproduction of alginate, an exopolysaccharidethat provides protection against antibacterial compounds amongst others. Oxygen deprived niches are common to the chronically infected CF lung and it has been shown that anoxia stimulates the production of alginate in the non mucoid isolate PAO1. The regulatory pathway(s) that is triggered by oxygen limitation and stimulates the synthesis of alginate remained elusive. We showed that increasing alginate levels in response to anoxia are not based on de novo protein synthesis. Consistent with this observation we found that the diguanylate cyclase SadC (PA4332), which synthesizes the second messenger c-di-GMP, is required for the stimulation of alginate production under anaerobic conditions. The purification of the cytosolic fragment of SadC containing the DGC domain revealed that the protein is highly active specifically in the absence of oxygen. SadC does not appear to contain oxygen sensing activity.However, the sadC gene is located in a conserved operon annotated as an enoyl-CoA hydratase domain (PA4330) and a Fe-S binding ferredoxin oxidoreductase (PA4331), respectively, which could be involved in oxygen sensing and signaling. We demonstrate that a PAO1 deletion mutant of PA4330 was defective in alginate production, whereas the deletion of PA4331 resulted in alginate production also under aerobic conditions, which normally inhibit alginate synthesis. Using HPLC-MS/MS to measure cyclic-di-GMP produced by the purified DGC fragment of SadC, we found that the addition of purified PA4331 but not PA4330 significantly decreased the amount of cyclic di-GMP. The incubation of a synthetic peptide comprising the DGC domain and downstream amino acids of SadC with PA4331 resulted in hydroxylation of the proline residue 410. We propose a model in which SadC, PA4330 and PA4331 form a signaling module that directly senses oxygen and modulates cyclic di-GMP levels produced by SadC to control alginate synthesis.
