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Abstract

Biofilms form when bacteria aggregate in a self-secreted
exopolysaccharide matrix; they are resistant to antibiotics and
implicated in disease. Nitric oxide (NO) is known to mediate biofilm
formation in many bacteria via ligation to H-NOX (heme-NO/oxygen
binding) domains. Most NO-responsive bacteria, however, lack H-NOX
domain-containing proteins. We have identified another NO-sensing
protein (NosP), which is predicted to be involved in two-component
signaling and biofilm regulation in many species. Here, we demonstrate
that NosP participates in the previously described H-NOX/NO-responsive
multicomponent c-di-GMP signaling network in Shewanella oneidensis.
Strains lacking either nosP or its co-cistronic kinase nahK (previously
hnoS) produce immature biofilms, while hnoX and hnoK (kinase responsive
to NO/H-NOX) mutants result in wild-type biofilm architecture. We
demonstrate that NosP regulates the autophosphorylation activity of NahK
as well as HnoK. HnoK and NahK have been shown to regulate three
response regulators (HnoB, HnoC, and HnoD) that together comprise a
NO-responsive multicomponent c-di-GMP signaling network. Here, we
propose that NosP/NahK adds regulation on top of H-NOX/HnoK to modulate
this c-di-GMP signaling network, and ultimately biofilm formation, by
governing the flux of phosphate through both HnoK and NahK. In addition,
it appears that NosP and H-NOX act to counter each other in a push-pull
mechanism; NosP/NahK promotes biofilm formation through inhibition of
H-NOX/HnoK signaling, which itself reduces the extent of biofilm
formation. Addition of NO results in a reduction of c-di-GMP and biofilm
formation, primarily through disinhibition of HnoK activity.