Bacterial Heme-Based Sensors of Nitric Oxide

Abstract

Significance: The molecule nitric oxide (NO) has been shown to regulate behaviors in bacteria, including biofilm formation. NO detection and signaling in bacteria is typically mediated by hemoproteins such as the bis-(3',5')-cyclic dimeric adenosine monophosphate-specific phosphodiesterase YybT, the transcriptional regulator dissimilative nitrate respiration regulator, or heme-NO/oxygen binding (H-NOX) domains. H-NOX domains are well-characterized primary NO sensors that are capable of detecting nanomolar NO and influencing downstream signal transduction in many bacterial species. However, many bacteria, including the human pathogen Pseudomonas aeruginosa, respond to nanomolar concentrations of NO but do not contain an annotated H-NOX domain, indicating the existence of an additional nanomolar NO-sensing protein (NosP). Recent Advances: A newly discovered bacterial hemoprotein called NosP may also act as a primary NO sensor in bacteria, in addition to, or in place of, H-NOX. NosP was first described as a regulator of a histidine kinase signal transduction pathway that is involved in biofilm formation in P. aeruginosa.

Critical issues: The molecular details of NO signaling in bacteria are still poorly understood. There are still many bacteria that are NO responsive but do encode either H-NOX or NosP domains in their genomes. Even among bacteria that encode H-NOX or NosP, many questions remain.

Future directions: The molecular mechanisms of NO regulation in many bacteria remain to be established. Future studies are required to gain knowledge about the mechanism of NosP signaling. Advancements on structural and molecular understanding of heme-based sensors in bacteria could lead to strategies to alleviate or control bacterial biofilm formation or persistent biofilm-related infections.

Keywords: H-NOX; NO sensor; NosP; hemoprotein; nitric oxide; signal transduction.

FIG. 1.

Ferrous-unligated-and-ligated forms of heme for H-NOX domains. (A) H-NOX domains from facultative aerobes form 5-coordinate ferrous-unligated complexes, 5-coordinate ferrous-NO complexes with NO present in either the distal or proximal heme pocket, and 6-coordinate ferrous-CO complexes with CO present in the distal pocket. (B) H-NOX domains from obligate anaerobes form 5-coordinate ferrous-unligated complexes and 6-coordinate ferrous-ligated complexes with NO, CO, and O₂. CO, carbon monoxide; H-NOX, heme-NO/oxygen binding; NO, nitric oxide.

FIG. 2.

Crystal structure of the Fe(II)-O₂ complex of Cs-H-NOX (PDB ID: 1U55). (A) Full protein structure showing six out of the seven α-helices (αA–D, αF), a four-stranded antiparallel β-sheet, and heme. (B) Zoomed-in view of the proximal heme pocket featuring the conserved residues Y131, S133, and R135 of the YxS/TxR motif, and (C) the heme-distorting residue P115 and the heme proximal ligand H102. (D) Zoomed-in view of the distal heme pocket featuring the W9/N74/Y140 hydrogen-bonding network with O₂. Cs, Caldanaerobacter subterraneus.

FIG. 3.

General NO activation mechanism of H-NOX proteins. Before NO binding, the H-NOX heme cofactor is distorted (1). NO binds, leading to histidine dissociation and the histidine and proline shift away from the Fe(II) heme complex, resulting in a flattened H-NOX heme cofactor (2). This conformational rearrangement results in a signal output to the functional domain (3).

FIG. 4.

Effect of H-NOX domains on diguanylate cyclase activity. (A) Ferrous-unligated H-NOX does not change the diguanylate cyclase activity of the L. pneumophilia HaCE protein. Ferrous-unligated H-NOX in S. woodyi upregulates diguanylate cyclase activity, whereas the phosphodiesterase activity remains unchanged. (B) Ferrous-NO bound H-NOX downregulates diguanylate cyclase activity in L. pneumophilia, S. woodyi, and A. vitis, and it upregulates phosphodiesterase activity in S. woodyi and A. vitis. Bold arrows signify upregulation of enzymatic activity. c-di-GMP, cyclic diguanylate monophosphate; EAL or HD-GYP, conserved amino acids in the catalytic site of some phosphodiesterases; GGDEF, conserved amino acids in the catalytic sites of diguanylate cyclases; GTP, guanosine triphosphate; HaCE, H-NOX-associated c-di-GMP processing enzymes; pGpG, 5′-phosphoguanylyl-(3′-5′)-guanosine.

FIG. 5.

H-NOX domains and two-component signaling. (A) Ferrous-NO bound H-NOX inhibits autokinase activity of HahK which, in turn, decreases phosphate flux to the EAL, HtH, and HD-GYP response regulator proteins in S. oneidensis and V. cholerae (V. cholerae only encodes the EAL and HD-GYP response regulator homologs). (B) Ferrous-NO-bound H-NOX inhibits HahK and, therefore, phosphate flux to the HD-GYP response regulator. Both the EAL and the HtH response regulator homologs are present in P. atlantica, but they have not yet been characterized. ADP, adenosine diphosphate; ATP, adenosine triphosphate; HahK, H-NOX-associated histidine kinase.

FIG. 6.

H-NOX domains involved in quorum sensing and symbiosis. (A) In V. harveyi and V. parahaemolyticus, ferrous-NO-bound H-NOX inhibits phosphate flux through a quorum-sensing circuit. (B) In Silicibacter sp. TrichCH4B and V. fischeri, ferrous-NO-bound H-NOX inhibits phosphate flux through a HqsK-signaling pathway, which ultimately regulates the symbiosis of Silicibacter sp. TrichCH4B and V. fischeri with T. erythraeum and E. scolopes, respectively. HqsK, H-NOX-associated quorum-sensing histidine kinase; Hpt, histidine-containing phosphotransfer protein.

FIG. 7.

NosP-associated signal transduction pathways in bacteria. (A) In P. aeruginosa, NO/NosP inhibits NahK autokinase activity and therefore phosphate flux through the associated signaling pathway. This leads to a change in biofilm formation in P. aeruginosa. (B) In V. cholerae, NO/NosP inhibits Vc-NqsK autokinase activity and, therefore, quorum sensing. (C) In L. pneumophilia, NO/NosP inhibits NahK activity, which, in turn, decreases phosphate flux through the associated signaling pathway and may result in the regulation of biofilm formation in this bacterium. (D) In S. oneidensis, NO/NosP inhibits NahK autokinase activity, which decreases phosphate flux to three response regulator proteins: EAL, HtH, and HD-GYP. ?, uncharacterized pathway; NahK, NosP-associated histidine kinase; NosP, NO-sensing protein; Nqsk, NosP-associated quorum-sensing histidine kinase; Vc, Vibrio cholerae.

FIG. 8.

The domain architecture of YybT proteins. YybT is composed of two N-terminal TM helices, a heme-binding PAS domain, a degenerate diguanylate cyclase (GGDEF) domain, and a C-terminal DHH/DHHA1 phosphodiesterase domain. In the presence of NO, phosphodiesterase activity increases threefold compared with unligated-heme-bound YybT. c-di-AMP, bis-(3′,5′)-cyclic dimeric adenosine monophosphate; pApA, 5′-phosphoadenylyl-(3′→5′)-adenosine; TM, transmembrane.

FIG. 9.

A general scheme of anaerobic denitrification. The transcriptional regulator ANR activates DNR under hypoxic conditions. NO, a product of nitrite (NO₂⁻) reduction by NIR, activates DNR. Activated DNR induces the expression of the enzymes nitrate (NO₃⁻) NAR, NIR, NOR, and nitrous oxide (N₂O) reductase (N₂OR). ANR, anaerobic regulation of arginine deiminase and nitrate reduction; DNR, dissimilative nitrate respiration regulator; NAR, nitrate reductase; NIR, nitrite reductase; NOR, nitric oxide reductase.