H-NOX regulation of c-di-GMP metabolism and biofilm formation in Legionella pneumophila

P>Haem Nitric oxide/OXygen (H-NOX) binding domains are a family of haemoprotein sensors that are widespread in bacterial genomes, but limited information is available on their function. Legionella pneumophila is the only prokaryote found, thus far, to encode two H-NOX proteins. This paper presents data supporting a role for one of the L. pneumophila H-NOXs in the regulation of biofilm formation. In summary: (i) unmarked deletions in the hnox1 gene do not affect growth rate in liquid culture or replication in permissive macrophages; (ii) the Delta hnox1 strain displays a hyper-biofilm phenotype; (iii) the gene adjacent to hnox1 is a GGDEF-EAL protein, lpg1057, and overexpression in L. pneumophila of this protein, or the well-studied diguanylate cyclase, vca0956, results in a hyper-biofilm phenotype; (iv) the Lpg1057 protein displays diguanylate cyclase activity in vitro and this activity is inhibited by the Hnox1 protein in the Fe(II)-NO ligation state, but not the Fe(II) unligated state; and (v) consistent with the Hnox1 regulation of Lpg1057, unmarked deletions of lpg1057 in the Delta hnox1 background results in reversion of the hyper-biofilm phenotype back to wild-type biofilm levels. Taken together, these results suggest a role for hnox1 in regulating c-di-GMP production by lpg1057 and biofilm formation in response to NO.