Abstract
In Pseudomonas aeruginosa, the transition between planktonic and biofilm lifestyles is modulated by the intracellular secondary messenger cyclic dimeric-GMP (c-di-GMP) in response to environmental conditions. Here, we used gene deletions to investigate how the environmental stimulus nitric oxide (NO) is linked to biofilm dispersal, focusing on biofilm dispersal phenotype from proteins containing putative c-di-GMP turnover and Per-Arnt-Sim (PAS) sensory domains. We document opposed physiological roles for the genes ΔrbdA and Δpa2072 that encode proteins with identical domain structure: while ΔrbdA showed elevated c-di-GMP levels, restricted motility and promoted biofilm formation, c-di-GMP levels were decreased in Δpa2072, and biofilm formation was inhibited, compared to wild type. A second pair of genes, ΔfimX and ΔdipA, were selected on the basis of predicted impaired c-di-GMP turnover function: ΔfimX showed increased, ΔdipA decreased NO induced biofilm dispersal, and the genes effected different types of motility, with reduced twitching for ΔfimX and reduced swimming for ΔdipA. For all four deletion mutants we find that NO-induced biomass reduction correlates with increased NO-driven swarming, underlining a significant role for this motility in biofilm dispersal. Hence P. aeruginosa is able to differentiate c-di-GMP output using structurally highly related proteins that can contain degenerate c-di-GMP turnover domains.
Highlights
Pseudomonas aeruginosa is a gram-negative bacterium known for its environmental versatility
The recurrence of proteins with similar architecture which may play a role in biofilm regulation poses a key question: If Diguanylate cyclases (DGCs) and PDEs regulate the transition from sessility to the planktonic state, do these proteins provide an element of redundancy within the cell or do they coordinate individual behaviours that contribute to this phenotypic change? To address this question, data on how different DGC/PDEs diversify in regulating biofilm dispersal are urgently needed
Antibiotics were used at the following concentrations as previously described[30]: for P. aeruginosa PAO1, gentamicin was used at 60 μg/ml, carbenicillin at 400 μg/ml, kanamycin at 300 μg/ml and tetracycline at 60 μg/ml; for E. coli S17-1, gentamicin was used at 15 μg/ml, ampicillin at 100 μg/ml, tetracycline at 30 μg/ml, kanamycin at 50 μg/ml and streptomycin at 50 μg/ml
Summary
Pseudomonas aeruginosa is a gram-negative bacterium known for its environmental versatility. The DGC SadC and the PDE BifA are involved in the regulation of swarming motility[14,15], required for formation and dispersal of biofilms[16,17,18,19]. The two DGCs, SadC and RoeA both promote biofilm formation, but individually control flagellar motility or the production of EPS, respectively[21]. These examples illustrate how multiple proteins have discrete phenotypic outputs to adjust the intracellular c-di-GMP and regulate biofilm formation and dispersal. Using deletion mutants we present data on c-di-GMP levels, motility, EPS production, biofilm structure and nitric oxide induced dispersal for four PAS domain containing DGC-PDE proteins. This study highlights how P. aeruginosa is able to differentiate signals from multiple c-di-GMP outputs in order to regulate complex biological processes associated with biofilm development
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