A Bacterial Hemerythrin Domain Regulates the Activity of a Vibrio cholerae Diguanylate Cyclase

Abstract

The first demonstrated example of a regulatory function for a bacterial hemerythrin (Bhr) domain is reported. Bhrs have a characteristic sequence motif providing ligand residues for a type of non-heme diiron site that is known to bind O2 and undergo autoxidation. The amino acid sequence encoded by the gene, VC1216, from Vibrio cholerae O1 biovar El Tor str. N16961 contains an N-terminal Bhr domain connected to a C-terminal domain characteristic of bacterial di-guanylate cyclases (DGCs) that catalyze formation of cyclic di-(3′,5′)-guanosine monophosphate (c-di-GMP) from GTP. This protein, Vc Bhr-DGC, was found to contain two tightly bound non-heme iron atoms per protein monomer. The as-isolated protein showed the spectroscopic signatures of oxo/dicarboxylato-bridged non-heme diferric sites of previously characterized Bhr domains. The diiron site was capable of cycling between diferric and diferrous forms, the latter of which was stable only under anaerobic conditions, undergoing rapid autoxidation upon exposure to air. Vc Bhr-DGC showed approximately 10-times higher DGC activity in the diferrous relative to the diferric form. The level of intracellular c-di-GMP is known to regulate biofilm formation in Vibrio cholerae. The higher DGC activity of the diferrous Vc Bhr-DGC is consistent with induction of biofilm formation in low dioxygen environments. The non-heme diiron cofactor in the Bhr domain thus represents an alternative to heme or flavin for redox and/or diatomic gas sensing and regulation of DGC activity.

Dioxygen and nitric oxide scavenging by Treponema denticola flavodiiron protein: a mechanistic paradigm for catalysis

“…This codon-optimized gene was synthesized and subcloned into pAG8H [34] by GenScript (Piscataway, NJ, USA). The plasmid encodes an 8×His-tag at the N-terminal end of the protein.…”

Microbial Surface Colonization and Biofilm Development in Marine Environments

“…Surface-associated bacteria usually harbor more c-di-GMP regulators than free-living bacteria, presumably as an adaptive strategy (120). O 2 , H 2 O 2 , NO, redox potential, light, sucrose, amino acids, polyamines (such as norspermidine and spermidine), Zn 2ϩ , bile acids, bicarbonate, indole, QS autoinducers, cis-2-dodecenoic acid and cis-11-methyl-dodecenoic acid (unsaturated fatty acids that serve as bacterial diffusible signal factors), and nutritional conditions that cause starvation (or depletion of a specific carbon source such as glucose or glycerol) have been identified as environmental cues that induce the bacterial response via altering the intracellular c-di-GMP concentration (480,(492)(493)(494)(495)(496)(497)(498)(499)(500)(501)(502)(503)(504)(505)(506)(507)(508). However, the vast majority of the environmental signals that modulate the activity of the DGCs and PDEs remain unidentified.…”

Oxygen sensing strategies in mammals and bacteria

“…Recently, O 2 -sensing was demonstrated for the protein known as Bhr-DGC, a bacterial hemerythrin from Vibrio cholerae [143]. The rate for producing cyclic di-GMP, a secondary messenger in bacteria, by Bhr-DGC decreased by 10-fold when the protein was oxidized to the [Fe(III) Fe(III)] oxidation state, indicating that oxidation by O 2 to form met Bhr-DGC led to a detectable change in an important signaling molecule [143]. …”

“…Bacterial Hr-like proteins bind O 2 and undergo rapid auto-oxidation [141, 143, 147], forming a stable [Fe(III) Fe(III)] cofactor. In contrast, FBXL5 does not form an observable O 2 -adduct, suggesting that cofactor oxidation may involve an alternate chemical mechanism.…”