The role of the c-di-GMP specific phosphodiesterase NbdA in NO-induced biofilm dispersal in Pseudomonas aeruginosa

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Dispersion is a process by which bacteria transit from a biofilm to a motile growth state to spawn novel communities in new locales. Alterations in c-di-GMP levels have been shown to be associated with biofilm dispersal in a number of different bacteria. The signaling molecule nitric oxide (NO) is one factor known to induce biofilm dispersion through stimulation of c-di-GMP degrading phosphodiesterase (PDE) activity. We have recently identified a membrane-anchored protein specifically involved in NO-induced biofilm dispersal in the opportunistic pathogen Pseudomonas aeruginosa. NO-induced biofilm dispersion locus A is a multidomain protein consisting of a MHYT-GGDEF-EAL fusion. In addition to NbdA, several other proteins were shown to be involved in P. aeruginosa biofilm dispersal, including the chemotaxis transducer BdlA and the PDE DipA. In order to understand the mechanism of NO-induced biofilm dispersal we intend to verify the sensory function of NbdA using purified recombinant protein. Based on bioinformatic predictions the MHYT-domain is proposed to be a gas sensor domain functioning through bound copper ions. UV-Vis and inductively coupled plasma optical emission spectroscopy in combination with site-directed mutagenesis will be used to identify the nature of cofactor and the coordinating amino acid residues. In addition, PDE assays will be performed to establish whether NO has a stimulatory effect on PDE activity. As an nbdA knock-out mutant is unable to disperse upon NO treatment we will perform cross-complementation experiments in biofilm tube reactors using known dispersion loci in order to identify the order of signal transduction. In addition, an in vivo membrane-Strep-tagged protein interaction experiment (membrane-SPINE) will be used to find direct interaction partner(s) to NbdA. Previous data also suggested a transcriptional regulation as nbdA mRNA levels in NO-dispersed cells were elevated. Transcriptional regulation will be investigated employing LOV-based fluorescent reporter fusions and monitored using fluorescence microscopy in biofilms and NO-dispersed cells. Several transcriptional regulators known to be involved in NO-sensing will be tested for their contribution in transcriptional regulation. In this regard we will also test whether the source of NO (exogenous vs. endogenous) has an influence on dispersal. Here we will make use of a P. aeruginosa mutant strain that contains a structurally intact, but catalytically inactive nitrite reductase. As a tool for the SPP community and to study biofilm dispersal independent of chemical, potentially harmful signals (like NO) we will construct a light-controllable PDE based on the bacterial phytochrome of P. aeruginosa. As a long term goal we will investigate how the perceived signal is transduced into a cellular response using a combination of fluorescent reporter gene fusion and nucleotide capture compounds to identify missing links in the signal transduction pathway.