UC Berkeley

Heme-Nitric oxide/OXygen binding (H-NOX) domains are distributed widely in bacterial genomes, but limited information is available on their function. In facultative aerobic prokaryotes, H-NOX domains are stand-alone proteins, and H-NOX genes are typically found in the same predicted operon as genes coding for proteins involved in signal transduction. Some H-NOX genes are adjacent to genes coding for c-di-GMP metabolism genes and others are adjacent to histidine kinases. Histidine-aspartic acid phosphotransfer pathways are central components of prokaryotic signal transduction pathways, and are also found in many eukaryotes. Tools to study histidine kinases, however, are currently quite limited. We developed a new method to study histidine-aspartic acid phosphotransfer pathways. We show that many histidine kinases will accept ATPγS as a substrate to form a stable thiophosphohistidine, even when they do not form stable phosphohistidines using the natural substrate ATP. An antibody that has previously been used to detect thiophosphorylated serine, threonine and tyrosine residues is shown to recognize thiophosphohistidine and thiophophoaspartic acid residues. Histidine kinase autothiophosphorylation is regulated by other protein sensor domains in the same way as autophosphorylation, and thiophosphate is transferred to downstream aspartic acid containing response regulators. We used this method to demonstrate H-NOX regulation of a histidine kinase that would not accept ATP to form a stable phosphohistidine.

Legionella pneumophila is unique among prokaryotes because its genome encodes two H-NOX proteins. We show that Hnox2, but not Hnox1, regulates the autophosphorylation of the histidine kinase, Lpg2458, and the phosphorylation of the CheY-like response regulator, Lpg2457. We provide UV-Vis spectral evidence that Lpg2458 alters the conformation of the Hnox2 Fe(II)-NO complex. We also performed phosphorylation timecourses which reveal that the Hnox2 does not directly compete with Lpg2457 for binding to Lpg2458. These results have implications for biophysical and functional studies focusing on H-NOX domain signaling.

Finally, we present data to support the role for a Legionella pneumophila H-NOX protein in the regulation of biofilm formation. We show the following: (i) Clean deletions in the hnox1 gene do not affect growth rate in liquid culture or replication in permissive macrophages. (ii) The Δhnox1 strain displays a hyper-biofilm phenotype. (iii) The gene adjacent to hnox1 codes for a GGDEF-EAL protein, lpg1057, and overexpression in Legionella 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. (v) Consistent with the Hnox1 regulation of Lpg1057, clean deletions of lpg1057 in the Δhnox1 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 nitric oxide.