Post-translational regulation of the exopolysaccharide alginate in Pseudomonas aeruginosa

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The opportunistic pathogen Pseudomonas aeruginosa causes life threatening lung infections in patients with the hereditary disease cystic fibrosis (CF). In the viscous mucus secretions of the patients’ lungs, P. aeruginosa forms alginate-rich biofilms, which are held responsible for the chronicity of the infections. P. aeruginosa does not express the exopolysaccharide alginate under normoxic conditions, however its expression is highly increased under low oxygen conditions which are present in the CF mucus. In the centre of this project is the elucidation of a novel covalent enzyme modification mechanism, which we have discovered and which differentially regulates alginate production in P. aeruginosa. Two complementary work strategies are followed. Firstly, the diguanylate cyclase (DGC) PA4332 which activates alginate expression via the formation of the second messenger cyclic bis-(3-5)-diguanylate monophosphate (c-di-GMP) is purified as a mannose binding protein adduct (PA4332-MBP) and incubated with the purified hydroxylase/reductase PA4331 and the dehydratase PA4330, respectively. Evidence for covalent modifications of Pro-410 in P4332-MBP has already been obtained (hydroxyprolin, dehydroproline). These modifications will be analyzed in vitro using HPLC und mass spectroscopy; the kinetic and essential co-factors of the reactions will be investigated and the physical interaction of the reaction partners is assessed. Secondly, in the P. aeruginosa strain PAO1 and in its isogenic PA4331 and PA4330 mutants the PA4332- MBP will be over-expressed, isolated and investigated for covalent protein modifications using the same methods. Finally, we investigate the physical interaction of the DGC PA4332 with the target protein Alg44 and ask whether DGCs of other bacterial species are similarly regulated in a post-translational manner. The novel covalent enzyme modification mechanism provides an important example for the known phenomenon of the rapid adaptation of P. aeruginosa to changing environmental condi.