Abstract
Protein phosphorylation has a major role in controlling the life-cycle and infection stages of bacteria. Proteome-wide occurrence of S/T/Y phosphorylation has been reported for many prokaryotic systems. Previously, we reported the phosphoproteome of Pseudomonas aeruginosa and Pseudomonas putida. In this study, we show the role of S/T phosphorylation of one motility protein, FliC, in regulating multiple surface-associated phenomena of P. aeruginosa PAO1. This is the first report of occurrence of phosphorylation in the flagellar protein, flagellin FliC in its highly conserved N-terminal NDO domain across several Gram negative bacteria. This phosphorylation is likely a well-regulated phenomenon as it is growth phase dependent in planktonic cells. The absence of phosphorylation in the conserved T27 and S28 residues of FliC, interestingly, did not affect swimming motility, but affected the secretome of type 2 secretion system (T2SS) and biofilm formation of PAO1. FliC phosphomutants had increased levels and activities of type 2 secretome proteins. The secretion efficiency of T2SS machinery is associated with flagellin phosphorylation. FliC phosphomutants also formed reduced biofilms at 24 h under static conditions and had delayed biofilm dispersal under dynamic flow conditions, respectively. The levels of type 2 secretome and biofilm formation under static conditions had an inverse correlation. Hence, increase in type 2 secretome levels was accompanied by reduced biofilm formation in the FliC phosphomutants. As T2SS is involved in nutrient acquisition and biofilm dispersal during survival and spread of P. aeruginosa, we propose that FliC phosphorylation has a role in ecological adaptation of this opportunistic environmental pathogen. Altogether, we found a system of phosphorylation that affects key surface related processes such as proteases secretion by T2SS, biofilm formation and dispersal.
Highlights
Successful survival of environmental microbes in diverse niches requires highly co-ordinated switching of life strategies
In order to confirm the presence of T27 and S28 phosphorylation in FliC, we carried out its phosphopeptide enrichment and nano LC-MS/MS from early and late log phase grown PAO1 WT
S/T/Y phosphorylation has been described at proteome-wide level for a number of species across both Gram positive and negative bacteria [22, 24,25,26,27, 29, 48, 49]
Summary
Successful survival of environmental microbes in diverse niches requires highly co-ordinated switching of life strategies. The two pre-dominant lifestyles of bacteria are the free-swimming planktonic state and the surface-associated biofilm state [1, 2]. The development of biofilm involves five stages, namely, initial reversible attachment, irreversible attachment, microcolony formation, maturation of microcolonies to mushroomstalk architecture, and dispersal of cells from the biofilm and return to the planktonic state [7,8,9]. Surface appendages such as flagella and pili can facilitate these lifestyle switches [10,11,12]. Flagella, which consists of multimers of flagellin, are important in the initial attachment after which flagellin is shed and in the final detachment stage, where cells again start synthesizing flagellin and return to planktonic state [14,15,16]
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