Gas Sensing and Signaling in the PAS-Heme Domain of the Pseudomonas aeruginosa Aer2 Receptor

Abstract

The Aer2 chemoreceptor from Pseudomonas aeruginosa contains a PAS sensing domain that coordinates b-type heme and signals in response to the binding of O 2 , CO, or NO. PAS-heme structures suggest that Aer2 uniquely coordinates heme via a His residue on a 3 10 helix (H234 on E), stabilizes O 2 binding via a Trp residue (W283), and signals via both W283 and an adjacent Leu residue (L264). Ligand binding may displace L264 and reorient W283 for hydrogen bonding to the ligand. Here, we clarified the mechanisms by which Aer2-PAS binds heme, regulates ligand binding, and initiates conformational signaling. H234 coordinated heme, but additional hydrophobic residues in the heme cleft were also critical for stable heme binding. O 2 appeared to be the native Aer2 ligand (dissociation constant [K d ] of 16 M). With one exception, mutants that bound O 2 could signal, whereas many mutants that bound CO could not. W283 stabilized O 2 binding but not CO binding, and it was required for signal initiation; W283 mutants that could not stabilize O 2 were rapidly oxidized to Fe(III). W283F was the only Trp mutant that bound O 2 with wildtype affinity. The size and nature of residue 264 was important for gas binding and signaling: L264W blocked O 2 binding, L264A and L264G caused O 2 -mediated oxidation, and L264K formed a hexacoordinate heme. Our data suggest that when O 2 binds to Aer2, L264 moves concomitantly with W283 to initiate the conformational signal. The signal then propagates from the PAS domain to regulate the C-terminal HAMP and kinase control domains, ultimately modulating a cellular response.IMPORTANCE Pseudomonas aeruginosa is a ubiquitous environmental bacterium and opportunistic pathogen that infects multiple body sites, including the lungs of cystic fibrosis patients. P. aeruginosa senses and responds to its environment via four chemosensory systems. Three of these systems regulate biofilm formation, twitching motility, and chemotaxis. The role of the fourth system, Che2, is unclear but has been implicated in virulence. The Che2 system contains a chemoreceptor called Aer2, which contains a PAS sensing domain that binds heme and senses oxygen. Here, we show that Aer2 uses unprecedented mechanisms to bind O 2 and initiate signaling. These studies provide both the first functional corroboration of the Aer2-PAS signaling mechanism previously proposed from structure as well as a signaling model for Aer2-PAS receptors.KEYWORDS chemoreceptor, PAS domain, signal transduction, Pseudomonas aeruginosa, heme, oxygen P seudomonas aeruginosa is a common environmental bacterium and a significant cause of opportunistic human disease. It survives in complex environments with the aid of 26 chemoreceptors and four chemosensory systems that collectively sense environmental conditions and modify bacterial behavior. The roles of three of these chemosensory systems are known: one modulates type IV pili production and twitching motility (Pil-Chp system), another controls biofilm formation (Wsp system), and a third

Sensory Repertoire of Bacterial Chemoreceptors

“…A recent study revealed that oxygen binds to the heme-containing PAS domain with a K D (equilibrium dissociation constant) of 16 M, which is comparable to the affinities of other PAS-heme O 2 sensors (152). This and the fact that amino acid substitutions in the gas binding site affected primarily the binding of O 2 and not of other gases led to the proposition that O 2 is the natural ligand for this chemoreceptor (152). Comparison of the structures containing ferric, ligand-free heme (153) and cyanide-bound ferric heme (154) also led to a model of receptor function (153).…”

“…The Aer-2/McpB PAS domain contains heme, which enables this chemoreceptor to recognize O 2 , NO, CO, and cyanide. A recent study revealed that oxygen binds to the heme-containing PAS domain with a K D (equilibrium dissociation constant) of 16 M, which is comparable to the affinities of other PAS-heme O 2 sensors (152). This and the fact that amino acid substitutions in the gas binding site affected primarily the binding of O 2 and not of other gases led to the proposition that O 2 is the natural ligand for this chemoreceptor (152).…”

Repurposing a macromolecular machine: Architecture and evolution of the F7 chemosensory system

“…While we did not test different growth conditions for M. alcaliphilum, we did observe that formation of F7 arrays in S. oneidensis was dependent on culture conditions. Another clue is that both P. aeruginosa and V. cholerae are capable of sensing oxygen, which binds to the PAS-heme domains of Aer2 receptors to activate signaling (24,45). We therefore favor the working model that the older F7 systems are part of an emergency response system activated by stress conditions, perhaps related to the availability of oxygen.…”

Blue‐light perception by epiphytic Pseudomonas syringae drives chemoreceptor expression, enabling efficient plant infection

“…Our model is supported by the demonstration that the inactivation of two of these chemoreceptors reduces virulence. One of these chemoreceptors, PSPTO_1008, has a cytosolic location, contains two PAS sensor domains, and is similar to the chemoreceptors that bind oxygen (Garcia et al, 2017) and mediate aerotaxis (Garcia F I G U R E 6 Methyl-accepting chemotaxis proteins (MCPs) PSPTO-1008 and PSPTO-2526 are required for full virulence of Pseudomonas syringae pv. tomato (PsPto) in tomato plants.…”

“…Our model is supported by the demonstration that the inactivation of two of these chemoreceptors reduces virulence. One of these chemoreceptors, PSPTO_1008, has a cytosolic location, contains two PAS sensor domains, and is similar to the chemoreceptors that bind oxygen (Garcia et al ., 2017) and mediate aerotaxis (Garcia et al ., 2016), a process that permits efficient pathogenesis in a number of species (Yao and Allen, 2007). The PSPTO_2526 chemoreceptor contains a periplasmic sensor domain that shares 52% sequence similarity with the ligand‐binding domain of the inorganic phosphate (Pi) chemoreceptor, CtpH, chemoreceptor of P. aeruginosa (Rico‐Jimenez et al ., 2016).…”