Nitric oxide regulation of cyclic di-GMP synthesis and hydrolysis in Shewanella woodyi.

ABSTRACT

Although several reports have documented nitric oxide (NO) regulation of biofilm formation, the molecular basis of this phenomenon is unknown. In many bacteria, an H-NOX (heme-nitric oxide/oxygen-binding) gene is found near a diguanylate cyclase (DGC) gene. H-NOX domains are conserved hemoproteins that are known NO sensors. It is widely recognized that cyclic di-GMP (c-di-GMP) is a ubiquitous bacterial signaling molecule that regulates the transition between motility and biofilm. Therefore, NO may influence biofilm formation through H-NOX regulation of DGC, thus providing a molecular-level explanation for NO regulation of biofilm formation. This work demonstrates that, indeed, NO-bound H-NOX negatively affects biofilm formation by directly regulating c-di-GMP turnover in Shewanella woodyi strain MS32. Exposure of wild-type S. woodyi to a nanomolar level of NO resulted in the formation of thinner biofilms, and less intracellular c-di-GMP, than in the absence of NO. Also, a mutant strain in the gene encoding SwH-NOX showed a decreased level of biofilm formation (and a decreased amount of intracellular c-di-GMP) with no change observed upon NO addition. Furthermore, using purified proteins, it was demonstrated that SwH-NOX and SwDGC are binding partners. SwDGC is a dual-functioning DGC; it has diguanylate cyclase and phosphodiesterase activities. These data indicate that NO-bound SwH-NOX enhances c-di-GMP degradation, but not synthesis, by SwDGC. These results support the biofilm growth data and indicate that S. woodyi senses nanomolar NO with an H-NOX domain and that SwH-NOX regulates SwDGC activity, resulting in a reduction in c-di-GMP concentration and a decreased level of biofilm growth in the presence of NO. These data provide a detailed molecular mechanism for NO regulation of c-di-GMP signaling and biofilm formation.