H-NOX Regulates Biofilm Formation in Agrobacterium Vitis in Response to NO.

Transitions between motile and biofilm lifestyles are highly regulated and fundamental to microbial pathogenesis. H-NOX (heme-nitric oxide/oxygen-binding domain) is a key regulator of bacterial communal behaviors, such as biofilm formation. A predicted bifunctional cyclic di-GMP metabolizing enzyme, composed of diguanylate cyclase and phosphodiesterase (PDE) domains ( avi _3097), is annotated downstream of an hnoX gene in Agrobacterium vitis S4. Here, we demonstrate that av H-NOX is a nitric oxide (NO)-binding hemoprotein that binds to and regulates the activity of avi _3097 ( av HaCE; H-NOX-associated cyclic di-GMP processing enzyme). Kinetic analysis of av HaCE indicates a ∼four-fold increase in PDE activity in the presence of NO-bound av H-NOX. Biofilm analysis with crystal violet staining reveals that low concentrations of NO reduce biofilm growth in the wild-type A. vitis S4 strain, but the mutant Δ hnoX strain has no NO phenotype, suggesting that H-NOX is responsible for the NO biofilm phenotype in A. vitis . Together, these data indicate that av H-NOX enhances cyclic di-GMP degradation to reduce biofilm formation in response to NO in A. vitis .