NO-induced biofilm dispersion in Pseudomonas aeruginosa is mediated by an MHYT domain-coupled phosphodiesterase

Abstract

Dispersion is a process used by bacteria to successfully transit from a biofilm to a planktonic growth state and to spawn novel communities in new locales. Alterations in bis-(3'-5')-cyclic dimeric GMP (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 known to induce biofilm dispersion through stimulation of c-di-GMP-degrading phosphodiesterase (PDE) activity. However, no c-di-GMP modulating enzyme directly involved in NO-induced dispersion has yet been described in the opportunistic pathogen Pseudomonas aeruginosa. Here, we characterized MucR (PA1727) and NbdA (PA3311, NO-induced biofilm dispersion locus A), two membranebound proteins with identical domain organization consisting of MHYT-GGDEF-EAL, with respect to their role in NO-induced dispersion. Inactivation of mucR impaired biofilm dispersion in response to NO and glutamate, whereas inactivation of nbdA only impaired biofilm dispersion upon exposure to NO. A specific role of NbdA in NO-induced dispersion was supported by increased PDE activity, resulting in decreased c-di-GMP levels in biofilms expressing nbdA upon exposure to NO, a response that was absent in the δnbdA strain. Moreover, increased PDE activity was mainly due to a transcriptional activation of nbdA upon addition of NO. Biochemical analyses of recombinant protein variants lacking the membrane-anchored MHYT domain support NbdA being an active PDE. In contrast, MucR displayed both diguanylate cyclase and PDE activity in vitro, which seemed regulated in a growth-dependent manner in vivo. This is the first description of a PDE specifically involved in NO-induced biofilm dispersion in P. aeruginosa. © 2013, American Society for Microbiology.