UC Berkeley

The gaseous free radical nitric oxide (NO) is firmly established as a unique signaling agent in nature. Eukaryotes employ this freely diffusible molecule in low transient concentrations as a cardiovascular signaling agent and neurotransmitter. Macrophages produce higher, cytotoxic concentrations of NO to serve as an integral piece of the host-defense against pathogens. NO signaling in vertebrates is well characterized and involves the Heme-Nitric oxide/Oxygen binding (H-NOX) domain of soluble guanylate cyclase (sGC) as a selective NO sensor. H-NOX domains are also present in many bacteria including a number of pathogens. Bacterial H-NOX proteins are often found in the same operon as signaling proteins such as histidine kinases, suggesting a role for H-NOX proteins as sensors in prokaryotic NO signaling pathways.

H-NOX-dependent control of histidine kinase autophosphorylation has been demonstrated, but little was known about the biological role or output of H-NOX two-component signaling pathways in bacteria. Here, molecular details of a bacterial H-NOX signaling network in Shewanella oneidensis are presented. NO regulates biofilm formation by controlling the levels of the bacterial secondary messenger cyclic diguanosine monophosphate (cyclic-di-GMP) through an unusually complex multi-component signaling network. Homologous pathways exist in the pathogen Vibrio cholerae and in additional gammaproteobacteria. This work and other recent studies highlight a more general role for NO in restructuring bacterial communal behavior, influencing motility and biofilm formation, host-symbiont interactions, and quorum sensing.

The connectivity of the signaling network in S. oneidensis was mapped by phosphotransfer profiling, which demonstrated signal integration from two histidine kinases and branching to three response regulators: HnoB, HnoC, and HnoD. Phosphodiesterase assays showed that a feed-forward loop between HnoB and HnoD response regulators with phosphodiesterase domains and phosphorylation-mediated activation intricately regulated c-di-GMP levels. In vivo phenotypic characterization established a direct link between NO signaling and increased biofilm formation. Cellular adhesion in biofilms may provide a general protection mechanism for bacteria against high concentrations of reactive and damaging NO. HnoC functions as transcription factor controlling expression of the signaling components in the network. Mechanistic studies of HnoC revealed an unprecedented regulation mechanism, involving phosphorylation-induced dissociation of the response regulator tetramer. The transcriptional feedback loop created by HnoC further regulates the dynamics of the H-NOX signaling network in response to NO stimuli.

One of the phosphotransfer targets of the H-NOX-associated histidine kinase in the signaling network in S. oneidensis and V. cholerae contains a HD-GYP domain: a predicted but poorly characterized phosphodiesterase domain for cyclic-di-GMP hydrolysis. The HnoD HD-GYP domains contain degenerate residues that cause them to be catalytically inactive. To understand the functional consequence of the degeneracy and to gain general insights on the catalytic mechanism of HD-GYP domains, two catalytically active HD-GYP enzymes were characterized. The enzymes contained a binuclear iron center, and reconstitution experiments demonstrated that a heterovalent Fe(III)-Fe(II) cluster is likely required for catalysis. The absence of the metal-coordination site in HnoD eliminates any phosphodiesterase activity.

The prevalence of orphan H-NOX/histidine kinase pairs highlights the necessity for improved methods to map connectivities in two-component signaling. The identity of cognate response regulator(s) is needed to define the biological function of the signaling system. Analogous to approaches in eukaryotic kinases, protein engineering was applied to histidine kinases to permit the use of unnatural ATP analogs as substrates, which are unreactive with other enzymes. Analog-sensitive alleles of two model histidine kinases were developed, and the phosphotransfer transfer reactions to their respective response regulators were optimized. Additionally, a panel of kinase inhibitors was screened for specificity against the analog-sensitive alleles. The analog-sensitive histidine kinases could become useful for in situ identification of phosphotransfer partners of orphan histidine kinases.