The newly discovered aer locus of Escherichia coli encodes a 506-residue protein with an N terminus that resembles the NifL aerosensor and a C terminus that resembles the flagellar signaling domain of methylaccepting chemoreceptors. Deletion mutants lacking a functional Aer protein failed to congregate around air bubbles or follow oxygen gradients in soft agar plates. Membranes with overexpressed Aer protein also contained high levels of noncovalently associated flavin adenine dinucleotide (FAD). We propose that Aer is a flavoprotein that mediates positive aerotactic responses in E. coli. Aer may use its FAD prosthetic group as a cellular redox sensor to monitor environmental oxygen levels.Aerotaxis, the movement of a cell or organism toward or away from oxygen, was first described in bacteria more than a century ago by Engelmann (8), Pfeffer (24), and Beijerinck (3), who observed accumulation of cells near air bubbles or other sources of oxygen. Despite considerable study, particularly in Escherichia coli (22,28,30), the molecular mechanism underlying this behavior has remained elusive. Does the organism detect oxygen directly, or does it instead sense some metabolic consequence of different oxygen environments, such as changes in electron transport activity (19), cellular redox potential (4), or proton motive force (20, 32)? We describe here a gene, dubbed aer for aerotaxis, that encodes a likely flavoprotein signal transducer for aerotaxis in E. coli. Studies of the Aer protein promise to provide a definitive answer to the longstanding puzzle of how cells detect oxygen gradients during aerotaxis.Sequence features of the aer locus. We initially identified the aer gene as an open reading frame (ORF506) discovered in the E. coli genome sequencing project (7). Its conceptual translation product, a 506-amino-acid Aer protein, exhibits several hallmarks of an aerosensing function (Fig. 1). Aer residues 10 to 110 are similar to parts of NifL, FixL, and related bacterial proteins that trigger regulatory responses to changes in environmental oxygen levels (5, 11). Residues 168 to 209 are predominantly hydrophobic and could serve to anchor Aer in the cytoplasmic membrane. Aer residues 259 to 506 are about 50% identical to the cytoplasmic signaling domains of methyl-accepting chemotaxis proteins (MCPs), the principal chemoreceptors of E. coli (14). These features suggested that Aer might generate chemotactic signals in response to oxygen gradients.Construction of an aer mutant. We constructed a large inframe deletion lacking codons 5 to 505 of the aer coding region by PCR amplification of chromosomal sequences flanking the aer locus in strain RP437 (23) by using primer pairs NSB19-NSB20 and NSB25-NSB22 (Fig. 1). The two PCR fragments were ligated at their common XbaI site, joining aer codon 4 to codon 506, and inserted into the pMAK705 vector, whose replication is temperature-sensitive (13), producing plasmid pSB25. RP437 carrying pSB25 was grown at 44°C to select recombinational insertions and then at 30°C for recombin...

Aerotactic responses inredox sensing ͉ prosthetic group ͉ PAS domain ͉ membrane topology M otile bacteria exhibit many adaptive locomotor behaviors, the best studied of which is chemotaxis in Escherichia coli (see refs. 1-3 for recent reviews). These organisms use transmembrane chemoreceptors, known as methyl-accepting chemotaxis proteins (MCPs), to monitor and respond to changes in their chemical environment as they swim about. MCPs have a periplasmic ligand-binding domain that communicates via membrane-spanning segments with a cytoplasmic signaling domain, which forms stable complexes with the CheA and CheW proteins to transmit sensory information to the flagellar motors. Changes in receptor occupancy modulate conformation of the MCP signaling domain, thereby controlling the CheA histidine kinase, whose protein phosphorylation activity regulates the direction of motor rotation. MCPs are excellent models for exploring the molecular mechanisms of transmembrane signaling and sensory adaptation, but, despite extensive study, there are still significant gaps in our understanding of these important processes.The recently discovered Aer protein is an MCP-like transducer that mediates aerotactic (oxygen-seeking) behavior in E. coli (4, 5). The sequence features of Aer suggest that it has an unorthodox domain organization and membrane topology whose study may shed light on the signaling mechanisms in more conventional MCPs (Fig. 1). The N terminus of Aer resembles a segment of NifL, an O 2 -responsive regulatory protein that employs a bound flavin adenine dinucleotide (FAD) molecule as a redox sensor (6, 7).Initial work on Aer suggested that it also bound FAD (4), presumably for sensing aerotactic stimuli in the form of cellular redox changes (8,9). The N termini of Aer and NifL were subsequently shown to contain a PAS motif (10, 11), which in some proteins is known to comprise a binding pocket for a prosthetic group (reviewed in ref. 12). The putative NifL͞PAS aerosensing domain of Aer is followed by a block of predominantly hydrophobic amino acids that may serve to anchor the protein to the cytoplasmic side of the inner membrane (4). The C terminus of Aer has high similarity to the signaling domains of MCPs (4, 5) and may form ternary complexes with CheA and CheW to control the cell's flagellar motors in response to aerotactic stimuli.Here we report initial biochemical and genetic studies of Aer that examine key predictions of this working model. We show that Aer binds FAD noncovalently and that the N-terminal 290 residues of the protein are sufficient for this activity. Fusion of the FAD-binding portion of Aer to the flagellar signaling domain of Tsr, the serine chemoreceptor, yielded a functional aerotaxis transducer, demonstrating that the FAD-binding portion of Aer is sufficient for detecting aerotactic stimuli. We also describe a colony morphology assay for aerotaxis and its use in isolating non-aerotactic point mutants. We found aerotaxisAbbreviations: FAD, flavin adenine dinucleotide; MCP, methyl-accepting che...

Aer is a membrane-associated protein that mediates aerotactic responses in Escherichia coli. Its C-terminal half closely resembles the signaling domains of methyl-accepting chemotaxis proteins (MCPs), which undergo reversible methylation at specific glutamic acid residues to adapt their signaling outputs to homogeneous chemical environments. MCP-mediated behaviors are dependent on two specific enzymes, CheR (methyltransferase) and CheB (methylesterase). The Aer signaling domain contains unorthodox methylation sites that do not conform to the consensus motif for CheR or CheB substrates, suggesting that Aer, unlike conventional MCPs, might be a methylation-independent transducer. Several lines of evidence supported this possibility. (i) The Aer protein was not detectably modified by either CheR or CheB. (ii) Amino acid replacements at the putative Aer methylation sites generally had no deleterious effect on Aer function. (iii) Aer promoted aerotactic migrations on semisolid media in strains that lacked all four of the E. coli MCPs. CheR and CheB function had no influence on the rate of aerotactic movements in those strains. Thus, Aer senses and signals efficiently in the absence of deamidation or methylation, methylation changes, methylation enzymes, and methyl-accepting chemotaxis proteins. We also found that chimeric transducers containing the PAS-HAMP sensing domain of Aer joined to the signaling domain and methylation sites of Tar, an orthodox MCP, exhibited both methylationdependent and methylation-independent aerotactic behavior. The hybrid Aear transducers demonstrate that methylation independence does not emanate from the Aer signaling domain but rather may be due to transience of the cellular redox changes that are thought to trigger Aer-mediated behavioral responses.Methyl-accepting chemotaxis proteins (MCPs) mediate many of the chemotactic behaviors of bacteria and archaea (see references 6 and 20 for recent reviews). MCPs typically possess an extracellular ligand-binding domain for monitoring environmental chemoeffector levels and an intracellular signaling domain that controls motor responses and undergoes reversible methylation at multiple sites. Methylation enables MCPs to detect chemical changes over time by comparing current chemoeffector levels, reflected in the fraction of ligand-occupied molecules, with chemoeffector levels during the past few seconds, represented by the average methylation state of the molecules. Whenever current conditions differ from those of the recent past, MCPs produce a feedforward excitation signal that modulates the organism's motility and a feedback adaptation signal that updates MCP methylation state to correspond to the new chemical environment (see references 12 and 33 for recent reviews). The ability of MCPs to adapt to homogeneous chemical environments via methylation changes endows these chemoreceptors with a wide dynamic range for stimulus detection and signaling (45).Escherichia coli has four transmembrane MCPs that share a highly conserved signaling and methy...

After the light-induced charge separation in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides, the electron reaches, via the tightly bound ubiquinone QA, the loosely bound ubiquinone Q(B) After two subsequent flashes of light, Q(B) is reduced to ubiquinol Q(B)H2, with a semiquinone anion Q-(B) formed as an intermediate after the first flash. We studied Q(B)H2 formation in chromatophores from Rb. sphaeroides mutants that carried Arg-->Ile substitution at sites 207 and 217 in the L-subunit. While Arg-L207 is 17 A away from Q(B), Arg-L217 is closer (9 A) and contacts the Q(B)-binding pocket. From the pH dependence of the charge recombination in the RC after the first flash, we estimated deltaG(AB), the free energy difference between the Q-(A)Q(B) and Q(A)Q-(B) states, and pK212, the apparent pK of Glu-L212, a residue that is only 4 A away from Q(B). As expected, the replacement of positively charged arginines by neutral isoleucines destabilized the Q-(B) state in the L217RI mutant to a larger extent than in the L207RI one. Also as expected, pK212 increased by approximately 0.4 pH units in the L207RI mutant. The value of pK212 in the L217RI mutant decreased by 0.3 pH units, contrary to expectations. The rate of the Q-(A)Q-(B)-->Q(A)Q(B)H2 transition upon the second flash, as monitored by electrometry via the accompanying changes in the membrane potential, was two times faster in the L207RI mutant than in the wild-type, but remained essentially unchanged in the L217RI mutant. To rationalize these findings, we developed and analyzed a kinetic model of the Q-(A)Q-(B)-->Q(A)Q(B)H2 transition. The model properly described the available experimental data and provided a set of quantitative kinetic and thermodynamic parameters of the Q(B) turnover. The non-electrostatic, 'chemical' affinity of the QB site to protons proved to be as important for the attracting protons from the bulk, as the appropriate electrostatic potential. The mutation-caused changes in the chemical proton affinity could be estimated from the difference between the experimentally established pK2J2 shifts and the expected changes in the electrostatic potential at Glu-L212, calculable from the X-ray structure of the RC. Based on functional studies, structural data and kinetic modeling, we suggest a mechanistic scheme of the QB turnover. The detachment of the formed ubiquinol from its proximal position next to Glu-L212 is considered as the rate-limiting step of the reaction cycle.

To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used.