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.
Bibliographical noteFunding Information:
L.-M.N., L.B., B.B., S.H., N.J.K., E.D.B., R.H., K.D., D.P.A., S.M., K.M.T., J.A.G., and E.M.B. have made major contributions to the acquisition, analysis, or interpretation of the data; L.-M.N., K.M.T., J.A.G., and E.M.B. to the conception or design of the study; and L.-M.N. and E.M.B. to the writing of the manuscript. This work was supported by the Stony Wold-Herbert Fund, the National Science Foundation (Grant CHE-1607532 to E.M.B.), and the National Institutes of Health (Grant GM118894-01A1 to E.M.B. and Grant T32GM092714 to L.-M.N.). The authors declare no competing financial interest.