NO-Induced Biofilm Dispersion in Pseudomonas aeruginosa Is Mediated by an MHYT Domain-Coupled Phosphodiesterase

Abstract

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, N O-induced b iofilm d ispersion locus A ), two membrane-bound 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 Δ nbdA 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 .