H‐NOX Signaling and Biofilm Formation in Caulobacter crescentus

Abstract

Bacterial Heme‐Nitric Oxide/Oxygen binding (H‐NOX) proteins utilize heme to act as diatomic gas sensors. These proteins often interact with a signaling partner to govern communal behaviors such as quorum sensing and biofilm formation in response to nitric oxide in facultative anaerobes. H‐NOX from Vibrio cholerae (Vc H‐NOX) in the Fe(III) and Fe(II)‐NO states was demonstrated to inhibit the activity of a cognate histidine kinase (HK). Heme‐free Vc H‐NOX could also act as an inhibitor of HK signaling through reversible oxidation of cysteine residues at a zinc binding site, suggesting that H‐NOX proteins may act as redox sensors in some organisms. The zinc‐binding Cys residues are conserved across numerous species, including the aquatic organism and obligate aerobe, Caulobacter crescentus (Cc H‐NOX). Using UV‐Vis spectroscopy, our lab has characterized heme complexes from Cc H‐NOX as purified, reduced, CO‐bound, and NO‐bound. ICP‐OES has provided insight to the degree of heme binding in Cc H‐NOX samples as well as zinc content. Circular dichroism has provided an estimate of secondary structure and a melt curve using single wavelength readings with variable temperatures. Lastly, we have generated a knockout mutant to investigate the importance of H‐NOX in biofilm regulation and the impact this knockout has on the Caulobacter crescentus proteome. Our results have shown complete zinc binding for Cc H‐NOX, the formation of a stable NO‐complex, and a mild hyper‐biofilm phenotype among Caulobacter crescentus mutants. The mutant also exhibits differences in protein expression levels and phosphorylation reflecting signaling pathways altered in the absence of H‐NOX. This has led to an improved understanding of the roles of H‐NOX signaling across bacterial species.