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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 residues 210 to 290 were individually replaced with cysteine and then cross-linked in vivo. The results confirmed that the Aer HAMP domain is composed of two [alpha]-helices separated by a structured loop. The proximal signaling domain consisted of two [alpha]-helices separated by a short undetermined structure. The Af1503 HAMP domain from Archaeoglobus fulgidus was recently shown to be a four-helix bundle. To test whether the Af1503 HAMP domain is a prototype for the Aer HAMP domain, the latter was modeled using coordinates from Af1503. Several findings supported the hypothesis that Aer has a four-helix HAMP structure: (i) cross-linking independently identified the same residues at the dimer interface that were predicted by the model, (ii) the rate of cross-linking for residue pairs was inversely proportional to the [beta]-carbon distances measured on the model, and (iii) clockwise lesions that were not contiguous in the linear Aer sequence were clustered in one region in the folded HAMP model, defining a potential site of PAS-HAMP interaction during signaling. In silico modeling of mutant Aer proteins indicated that the four-helix HAMP structure was important for Aer stability or maturation. The significance of the HAMP and proximal signaling domain structure for signal transduction is discussed.

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. Collisions between proximal residues in the membrane anchor were exclusively intra- or interdimeric but, with one exception, not both. Cross-linking profiles were consistent, with a rigid rather than flexible periplasmic loop and a tilted TM2 helix that crossed TM2' at residue V197C, near the center of the lipid bilayer. The periplasmic loop formed a stable neighborhood that (i) included a maximum of three Aer dimers, (ii) did not swap neighbors over time, and (iii) appeared to be constrained by interactions in the cytosolic signaling domain.

Aer, the Escherichia coil 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. Aer-PAS N-cap truncations and an Aer-M21P substitution resulted in low cellular levels of the mutant proteins, suggesting that the N-terminal region was important for stabilizing the structure of the PAS domain. The junction of the N-cap and PAS core was critical for signaling in Aer. Mutations and truncations in the sequence encoding residues 15 to 21 introduced a range of phenotypes, including defects in FAD binding, constant tumbling motility, and an inverse response in which E. coli cells migrated away from oxygen concentrations to which they are normally attracted. The proximity of two N-cap regions in an Aer dimer was assessed in vivo by oxidatively cross-linking serial cysteine substitutions. Cross-linking of several cysteine replacements at 23[degrees]C was attenuated at 10[degrees]C, indicating contact was not at a stable dimer interface but required lateral mobility. We observed large muitimers of Aer when we combined cross-linking of N-cap residues with a cysteine replacement that cross-links exclusively at the Aer dimer interface. This suggests that the PAS N-cap faces outwards in a dimer and that PAS-PAS contacts can occur between adjacent dimers.

Escherichia coil 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. Most transmembrane prediction programs predict a single transmembrane-spanning segment, but such a topology is inconsistent with recent studies indicating that there is direct communication between the membrane flanking PAS and HAMP domains. We studied the overall topology and membrane boundaries of the Aer membrane anchor by a cysteine-scanning approach. The proximity of 48 cognate cysteine replacements in Aer dimers was determined in vivo by measuring the rate and extent of disulfide cross-linking after adding the oxidant copper phenanthroline, both at room temperature and to decrease lateral diffusion in the membrane, at 4[degrees]C. Membrane boundaries were identified in membrane vesicles using 5-iodoacetamidofluorescein and methoxy polyethylene glycol 5000 (mPEG). To map periplasmic residues, accessible cysteines were blocked in whole cells by pretreatment with 4-acetamido-4'-maleimidylstilbene-2, 2' disulfonic acid before the cells were lysed in the presence of mPEG. The data were consistent with two membrane-spanning segments, separated by a short periplasmic loop. Although the membrane anchor contains a central proline residue that reaches the periplasm, its position was permissive to several amino acid and peptide replacements.

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. Previous studies reported mutations in the HAMP domain that abolished FAD binding to the PAS domain. In this study, using random and site-directed mutagenesis, we identified the distal helix, AS-2, as the component of the HAMP domain that stabilizes FAD binding. AS-2 in Aer is not amphipathie and is predicted to be buried. Mutations in the sequence coding for the contiguous proximal signaling domain altered signaling by Aer but did not affect FAD binding. The V264M residue replacement in this region resulted in an inverted response in which E. coli cells expressing the mutant Aer protein were repelled by oxygen. Bioinformaties analysis of aligned HAMP domains indicated that the proximal signaling domain is conserved in other HAMP domains that are not involved in chemotaxis or aerotaxis. Only one null mutation was found in the coding sequence for the HAMP AS-1 and connector regions, suggesting that these are not active signal transduction sites. We consider a model in which the signal from FAD is transmitted across a PAS-HAMP interface to AS-2 or the proximal signaling domain.

In vivo cross-linking between native cysteines in the Aer receptor of Eseherichia 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.

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. The F1 region, membrane anchor, and HAMP domain are required for FAD binding. Presumably, alterations in the redox potential of FAD induce conformational changes in the PAS domain that are transmitted to the HAMP and C-terminal signaling domains. In this study we used random mutagenesis and intragenic pseudoreversion analysis to examine functional interactions between the HAMP domain and the N-terminal half of Aer. Missense mutations in the HAMP domain clustered in the AS-2 [alpha]-helix and abolished FAD binding to Aer, as previously reported. Three amino acid replacements in the Aer-PAS domain, S28G, A65V, and A99V, restored FAD binding and aerotaxis to the HAMP mutants. These suppressors are predicted to surround a cleft in the PAS domain that may bind FAD. On the other hand, suppression of an Aer-C253R HAMP mutant was specific to an N34D substitution with a predicted location on the PAS surface, suggesting that residues C253 and N34 interact or are in close proximity. No suppressor mutations were identified in the F1 region or membrane anchor. We propose that functional interactions between the PAS domain and the HAMP AS-2 helix are required for FAD binding and aerotactic signaling by Aer.

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. To investigate properties of the PAS domain, we subcloned segments of the aer gene from E. coli that encode the PAS domain with and without [His.sub.6] tags and expressed the PAS peptides in E. coli. The 20-kDa [His.sub.6]-[Aer.sub.2-166] PAS-F1 fragment was purified as an 800-kDa complex by gel filtration chromatography, and the associating protein was identified by N-terminal sequencing as the chaperone protein GroEL. None of the N-terminal fragments of Aer found in the soluble fraction was released from GroEL, suggesting that these peptides do not fold correctly in an aqueous environment and require a motif external to the PAS domain for proper folding. Consistent with this model, peptide fragments that included the membrane binding region and part ([Aer.sub.2-231) or all ([Aer.sub-2.285]) of the HAMP domain inserted into the membrane, indicating that they were released by GroEL [Aer.sub-2.285], but not [Aer.sub.2-231], bound FAD, confirming the requirement for the HAMP domain in stabilizing FAD binding. The results raise an interesting possibility that residues outside the PAS domain that are required for FAD binding are essential for formation of the PAS native fold.

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. coli and taxis was analysed in gradients of various oxidizable carbon sources. In addition to the known response to glycerol and succinate, it was found that Aer directed taxis towards ribose, galactose, maltose, malate, proline and alanine as well as the phosphotransferase system (PTS) carbohydrates glucose, mannitol, mannose, sorbitol and fructose, but not to aspartate, glutamate, glycine and arabinose. Tsr directed taxis towards sugars (including those transported by the PTS), but not to organic acids or amino acids. When a mutated Aer protein unable to bind the FAD cofactor was expressed in the receptor-less strain, chemotaxis was not restored to any substrate. Aer appears to mediate responses to rapidly oxidizable substrates, whether or not they are effective growth substrates, whereas Tsr appears to mediate taxis to substrates that support maximal growth, whether or not they are rapidly oxidizable. This correlates with the hypothesis that Aer and Tsr sense redox and proton motive force, respectively. Taken together, the results demonstrate that Aer and Tsr mediate responses to a broad range of chemicals and their attractant repertoires overlap with those of specialized chemoreceptors, namely Trg (ribose, galactose) and Tar (maltose).

Research was conducted to examine the respiratory pathway of laccase-positive strain of a proteobacterium. The loss of respiratory cytochrome c oxidase in this strain is attributed to a defect in the heme component biosynthesis and an adaptive strategy to tolerate the laccase generated oxidized quinone inside the cells.

The oxygen-dependent growth of the obligate anaerobe Desulfovibrio vulgaris Hildenborough was analyzed. Hildenborough, a sulfate-reducing bacterium, was classified as an obligate anaerobe and subjected to a preferred oxygen concentration of 0.02 to 0.04%. Results led to the proposition that zones of transition from an oxic to anoxic environment, D. vulgaris protects anoxic microenvironments from intrusion of oxygen.

An analysis of glycerol was conducted to determine its role in the eliciting of energy taxis of Escherichia coli and Salmonella typhimurium. The study utilized both spatial and temporal behavioral assays to demonstrate that glycerol is an attractant for both E. coli and S. typhimurium. Results suggests that methylation of a chemotaxis receptor is needed for positive glycerol chemotaxis in E. coli or S. typhimurium.

The aerophilic and aerophobic responses of Azospirillum brasilense to oxygen depends on variations in proton motive force. The nitrogen-fixing bacteria maintains an aerotactic band that ranges from 3 to 5 micromolars and avoids higher or lower oxygen concentrations. The bacteria also exhibited a decrease in proton motive force when moving from higher to lower oxygen concentrations. Furthermore, the probability of reversal in the direction of swimming increased when the bacteria sensed a change in the proton motive force.

The movement of the archaeon bacteria Halobacterium salinarium away from low O2 concentration areas is controlled by the methylation of methyl accepting proteins. Aerotactic responses are absent in the absence of methyl donors and in methyltransferase-deficient mutants. The aerotactic response consists of smooth swimming and a brief tumbling stage. The response is greatest at the time of high respiration such as the exponential growth phase. Methanol is released in response to both increases or decreases in O2 concentration.

The methylation-independent and methylation- dependent pathways for chemotaxis to oxyen and sugars of Eschirichia coli appear to converge at the histidine kinase CheA. Researchers tested both pathways for chemotaxis by expressing cheA, cheW, and cheY genes in an E. coli strain that lacks chemoreceptors and chemotaxis proteins but but has a flagellar motor. The products of all three genes were required for both methylation-independent and methylation-dependent chemotaxis.

Bacillus subtilis has unusual amino acid efflux of methanol when responding to chemotactic and osmotic signals. The observed tumbling response to osmotic upshift supports an optimal osmolarity attraction and repulsion by either high or low osmolarity despite smaller tumbling responses than Escherichia coli. The chemotaxic release of methanol is most likely related to a methyl-accepting chemotaxis protein's methylation while the osmotaxic response may be methylation dependent.

Aerotaxis is associated with methylation in Bacillus subtilis, but not in Escherichia coli or Salmonella typhimurium. A decrease in oxygen concentrations for B. subtilis, E. coli and S. typhimurium results in tumbling, while smooth swimming occurs in response to increased oxygen concentrations, accompanied by methylation in B. subtilis. In B. subtilis, depletion of methionine removes intracellular S-adenosylmethionine, which is required as a substrate by cheR methyltransferase, a chemotaxis-specific protein. Aerotaxis is a migratory response of motile bacteria to move to the oxygen concentration optimal for survival.

Histidine starvation technique, arsenate treatment and alpha-methylglucoside-plus-azide treatment are the three methods which induce depletion of ATP in Salmonella typhimurium. Arsenate and alpha-methylglucoside-plus-azide treatment restrict the cellular processes influenced by triphosphonucleotide, and the levels of nucleotide triphosphate are reduced. Depletion of ATP is characterized by histidine starvation and this method is capable of distinguishing processes influenced by ATP from those influenced by other nucleotides.

S1 and S2 boxes which are highly conserved regions in PAS domains and occurring in numerous sensor proteins including Archaea have been identified. The S1 box comprises the middle one-third of the known sequences for PAS-A and PAS-B direct repeats. The S2 box extends by 50 residues the carboxyl boundary of the PAS-A and B repeats. PAS domains are involved in light reception and regulation and protein-to-protein interactions.

Barry L. Taylor [1] Igor B. Zhulin [2] Mark S. Johnson [3] Department of Microbiology and Molecular Genetics, School of Medicine, Loma Linda University, Loma Linda, California 92350; e-mail: [1] [...]

PAS domains are signaling domains distributed in proteins from members of the Archeae and Bacteria and from fungi, insects, plants, and vertebrates. The domains function as input modules in proteins that sense redox potential, oxygen, light and other types of stimuli. PAS proteins are located intracellular and observes external and internal environments. The transduction of redox signal may be a common mechanistic theme in several PAS domains.