Delineating PAS-HAMP interaction surfaces and signalling-associated changes in the aerotaxis receptor Aer.

The Escherichia coli aerotaxis receptor, Aer, monitors cellular oxygen and redox potential via FAD bound to a cytosolic PAS domain. Here, we show that Aer-PAS controls aerotaxis through direct, lateral interactions with a HAMP domain. This contrasts with most chemoreceptors where signals propagate along the protein backbone from an N-terminal sensor to HAMP.

Different conformations of the kinase-on and kinase-off signaling states in the Aer HAMP domain.

HAMP domains are sensory transduction modules that connect input and output domains in diverse signaling proteins from archaea, bacteria, and lower eukaryotes. Here, we employed in vivo disulfide cross-linking to explore the structure of the HAMP domain in the Escherichia coli aerotaxis receptor Aer. Using an Aer HAMP model based on the structure of Archaeoglobus fulgidus Af1503-HAMP, the closest residue pairs at the interface of the HAMP AS-1 and AS-2' helices were determined and then replaced with cysteines and cross-linked in vivo.

PAS/poly-HAMP signalling in Aer-2, a soluble haem-based sensor.

Poly-HAMP domains are widespread in bacterial chemoreceptors, but previous studies have focused on receptors with single HAMP domains. The Pseudomonas aeruginosa chemoreceptor, Aer-2, has an unusual domain architecture consisting of a PAS-sensing domain sandwiched between three N-terminal and two C-terminal HAMP domains, followed by a conserved kinase control module. The structure of the N-terminal HAMP domains was recently solved, making Aer-2 the first protein with resolved poly-HAMP structure.

Role of the F1 region in the Escherichia coli aerotaxis receptor Aer.

In Escherichia coli, the aerotaxis receptor Aer is an atypical receptor because it senses intracellular redox potential. The Aer sensor is a cytoplasmic, N-terminal PAS domain that is tethered to the membrane by a 47-residue F1 linker. Here we investigated the function, topology, and orientation of F1 by employing random mutagenesis, cysteine scanning, and disulfide cross-linking.

Gain-of-function mutations cluster in distinct regions associated with the signalling pathway in the PAS domain of the aerotaxis receptor, Aer.

The Aer receptor monitors internal energy (redox) levels in Escherichia coli with an FAD-containing PAS domain. Here, we randomly mutagenized the region encoding residues 14-119 of the PAS domain and found 72 aerotaxis-defective mutants, 24 of which were gain-of-function, signal-on mutants. The mutations were mapped onto an Aer homology model based on the structure of the PAS-FAD domain in NifL from Azotobacter vinlandii.

Structure-function relationships in the HAMP and proximal signaling domains of the aerotaxis receptor Aer.

Aer, the Escherichia coli aerotaxis receptor, faces the cytoplasm, where the PAS (Per-ARNT-Sim)-flavin adenine dinucleotide (FAD) domain senses redox changes in the electron transport system or cytoplasm. PAS-FAD interacts with a HAMP (histidine kinase, adenylyl cyclase, methyl-accepting protein, and phosphatase) domain to form an input-output module for Aer signaling. In this study, the structure of the Aer HAMP and proximal signaling domains was probed to elucidate structure-function relationships important for signaling.

Aer on the inside looking out: paradigm for a PAS-HAMP role in sensing oxygen, redox and energy.

Aer, the Escherichia coli aerotaxis (oxygen-sensing) receptor, is representative of a small class of receptors that face the cytoplasm in bacteria. Instead of sensing oxygen directly, Aer detects redox changes in the electron transport system or cytoplasm when the bacteria enter or leave a hypoxic microniche. As a result, Aer sensing also enables bacteria to avoid environments where carbon deficiency, unfavourable reduction potential or other insults would limit energy production.

Organization of the aerotaxis receptor aer in the membrane of Escherichia coli.

The Aer receptor guides Escherichia coli to specific oxygen and energy-generating niches. The input sensor in Aer is a flavin adenine dinucleotide-binding PAS domain, which is separated from a HAMP/signaling output domain by two membrane-spanning segments that flank a short (four-amino-acid) periplasmic loop. In this study, we determined the overall membrane organization of Aer by introducing combinations of residues that allowed us to differentiate intradimeric collisions from interdimeric collisions.

Oxygen and redox sensing by two-component systems that regulate behavioral responses: behavioral assays and structural studies of aer using in vivo disulfide cross-linking.

A remarkable increase in the number of annotated aerotaxis (oxygen-seeking) and redox taxis sensors can be attributed to recent advances in bacterial genomics. However, in silico predictions should be supported by behavioral assays and genetic analyses that confirm an aerotaxis or redox taxis function. This chapter presents a collection of procedures that have been highly successful in characterizing aerotaxis and redox taxis in Escherichia coli.

Differentiation between electron transport sensing and proton motive force sensing by the Aer and Tsr receptors for aerotaxis.

Aerotaxis (oxygen-seeking) behaviour in Escherichia coli is a response to changes in the electron transport system and not oxygen per se. Because changes in proton motive force (PMF) are coupled to respiratory electron transport, it is difficult to differentiate between PMF, electron transport or redox, all primary candidates for the signal sensed by the aerotaxis receptors, Aer and Tsr. We constructed electron transport mutants that produced different respiratory H+/e- stoichiometries.

Function of the N-terminal cap of the PAS domain in signaling by the aerotaxis receptor Aer.

Aer, the Escherichia coli receptor for behavioral responses to oxygen (aerotaxis), energy, and redox potential, contains a PAS sensory-input domain. Within the PAS superfamily, the N-terminal segment (N-cap) is poorly conserved and its role is not well understood. We investigated the role of the N-cap (residues 1 to 19) in the Aer PAS domain by missense and truncation mutagenesis.

Minimal requirements for oxygen sensing by the aerotaxis receptor Aer.

The PAS and HAMP domain superfamilies are signal transduction modules found in all kingdoms of life. The Aer receptor, which contains both domains, initiates rapid behavioural responses to oxygen (aerotaxis) and other electron acceptors, guiding Escherichia coli to niches where it can generate optimal cellular energy. We used intragenic complementation to investigate the signal transduction pathway from the Aer PAS domain to the signalling domain.

Topology and boundaries of the aerotaxis receptor Aer in the membrane of Escherichia coli.

Escherichia coli chemoreceptors are type I membrane receptors that have a periplasmic sensing domain, a cytosolic signaling domain, and two transmembrane segments. The aerotaxis receptor, Aer, is different in that both its sensing and signaling regions are proposed to be cytosolic. This receptor has a 38-residue hydrophobic segment that is thought to form a membrane anchor.

Genetic analysis of the HAMP domain of the Aer aerotaxis sensor localizes flavin adenine dinucleotide-binding determinants to the AS-2 helix.

HAMP domains are signal transduction domains typically located between the membrane anchor and cytoplasmic signaling domain of the proteins in which they occur. The prototypical structure consists of two helical amphipathic sequences (AS-1 and AS-2) connected by a region of undetermined structure. The Escherichia coli aerotaxis receptor, Aer, has a HAMP domain and a PAS domain with a flavin adenine dinucleotide (FAD) cofactor that senses the intracellular energy level.

The Aer protein of Escherichia coli forms a homodimer independent of the signaling domain and flavin adenine dinucleotide binding.

In vivo cross-linking between native cysteines in the Aer receptor of Escherichia coli showed dimer formation at the membrane anchor and in the putative HAMP domain. Dimers also formed in mutants that did not bind flavin adenine dinucleotide and in truncated peptides without a signaling domain and part of the HAMP domain.

Interactions between the PAS and HAMP domains of the Escherichia coli aerotaxis receptor Aer.

The Escherichia coli energy-sensing Aer protein initiates aerotaxis towards environments supporting optimal cellular energy. The Aer sensor is an N-terminal, FAD-binding, PAS domain. The PAS domain is linked by an F1 region to a membrane anchor, and in the C-terminal half of Aer, a HAMP domain links the membrane anchor to the signaling domain.

PAS domain of the Aer redox sensor requires C-terminal residues for native-fold formation and flavin adenine dinucleotide binding.

The Aer protein in Escherichia coli is a membrane-bound, FAD-containing aerotaxis and energy sensor that putatively monitors the redox state of the electron transport system. Binding of FAD to Aer requires the N-terminal PAS domain and residues in the F1 region and C-terminal HAMP domain. The PAS domains of other PAS proteins are soluble in water.

Aer and Tsr guide Escherichia coli in spatial gradients of oxidizable substrates.

The Aer and Tsr chemoreceptors in Escherichia coli govern tactic responses to oxygen and redox potential that are parts of an overall behaviour known as energy taxis. They are also proposed to mediate responses to rapidly utilized carbon sources, glycerol and succinate, via the energy taxis mechanism. In this study, the Aer and Tsr proteins were individually expressed in an 'all-transducer-knockout' strain of E.

Aerotactic responses in bacteria to photoreleased oxygen.

Bacterial aerotaxis is a rapid response towards or away from oxygen. Here we report on the use of computer-assisted motion analysis coupled to flash photolysis of caged oxygen to quantify aerotactic responses in bacteria. The caged compound (mu-peroxo)(mu-hydroxo)bis[bis(bipyridyl) cobalt(III)] perchlorate liberates molecular oxygen upon irradiation with near-UV light.