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Deciphering the Functional Role Bacterial H-NOX Proteins


Acute and specific sensing of diatomic gas molecules is an essential facet of biological signaling. Heme-Nitric oxide/Oxygen sensing (H-NOX) proteins are a family of gas sensors found in diverse classes of bacteria and eukaryotes. In mammals, the H-NOX is the sensor domain of soluble guanylate cyclase (sGC). The H-NOX of sGC binds the diatomic radical gas NO with picomolar affinity and activates production of the second messenger cyclic GMP from GTP. Bacterial H-NOX domains are divided into two subfamilies. In facultative anaerobes, H-NOX domains are encoded as single domain proteins that regulate an effector protein expressed on the same operon. These H-NOX proteins specifically sense NO, inhibit the effector, either a histidine kinase or cyclic-di-GMP phosphodiesterase, and regulate a biofilm formation phenotype. In obligate anaerobes, H-NOX domains are fused to methyl-accepting chemotaxis proteins. These H-NOX domains have a conserved H-bonding network in the gas ligand-binding pocket that allows formation of a stable bond with O2. The function of these O2-binding H-NOX domains is unknown. However, they are likely involved in regulating a repellent chemotaxis response. Because both NO and O2 are toxic to obligate anaerobes, the physiological ligand for these H-NOX domains is not yet known. These H-NOX domains stably bind NO, but the conservation of the H-bonding network suggests a role in O2 signaling.

In this dissertation, biological function of both subfamilies of H-NOX domains is explored. The chemotaxis signaling circuit of Caldanaerobacter subterraneus was reconstituted in vitro, but no gas ligand or H-NOX dependence was identified. Crystal structures of C. subterraneus H-NOX and orthogonal functional assays with the histidine kinase from Vibrio cholerae indicate specific O2 signaling function. Additionally, the role of two conserved glycine residues termed the “glycine hinge” was examined. In structures of both NO and O2 sensing H-NOX domains, a conformational change occurs upon ligand binding along this glycine hinge. However, some H-NOX domains from Flavobacteriaceae have a native alanine substitution in the position of one glycine residue. It was predicted that the steric bulk from the alanine methyl group would prevent the conformational shift. However, H-NOX dependent histidine kinase assays display NO dependent regulation. Mutation of the alanine to a glycine however, destabilizes the H-NOX conformation and allows kinase inhibition independent of NO. Finally, function of the NO sensing H-NOX from Vibrio cholerae was explored. Deletions of V. cholerae H-NOX and its histidine kinase show that the H-NOX controls biofilm formation. Proteomics experiments show that the cholera toxin master regulator TcpN is upregulated in an NO and H-NOX dependent manner. Finally deletion mutants of both the H-NOX and its kinase have a modest but significant competitive advantage compared to wild type V. cholerae during infant mouse infections.

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