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In the bacterial chemotaxis network, receptor clusters process input (1-3), and flagellar motors generate output (4). Receptor and motor complexes are coupled by the diffusible protein CheY-P. Receptor output (the steady-state concentration of CheY-P) varies from cell to cell (5). However, the motor is ultrasensitive, with a narrow operating range of CheY-P concentrations (6). How the match between receptor output and motor input might be optimized is unclear. Here we show that the motor can shift its operating range by changing its composition. The number of FliM subunits in the C-ring increases in response to a decrement in the concentration of CheY-P, increasing motor sensitivity. This shift in sensitivity explains the slow partial adaptation observed in mutants that lack the receptor methyltransferase and methylesterase (7,8) and why motors show signal-dependent FliM turnover (9). Adaptive remodelling is likely to be a common feature in the operation of many molecular machines., The chemotaxis signalling pathway allows bacterial cells to sense and respond to changes in concentrations of chemical attractants or repellents (1,2). Binding of chemicals by receptors modulates the activity of [...]

Remote sensing of thermal radiation (infrared wave-lengths) has been reported only in some terrestrial animals and is known to have significant physiological and ecological meaning. In aquatic animals, however, it [...]

Hereditary deficiency of the protein α-1 antitrypsin (AAT) causes a chronic lung disease in humans that is characterized by excessive mobilization of neutrophils into the lung. However, the reason for the increased neutrophil burden has not been fully elucidated. In this study we have demonstrated using human neutrophils that serum AAT coordinates both CXCR1- and soluble immune complex (sIC) receptor-mediated chemotaxis by divergent pathways. We demonstrated that glycosylated AAT can bind to IL-8 (a ligand for CXCR1) and that AAT-IL-8 complex formation prevented IL-8 interaction with CXCR1. Second, AAT modulated neutrophil chemotaxis in response to sIC by controlling membrane expression of the glycosylphosphatidylinositolanchored (GPI-anchored) Fc receptor FcγRIIIb. This process was mediated through inhibition of ADAM-17 enzymatic activity. Neutrophils isolated from clinically stable AAT-deficient patients were characterized by low membrane expression of FcγRIIIb and increased chemotaxis in response to IL-8 and sIC. Treatment of AATdeficient individuals with AAT augmentation therapy resulted in increased AAT binding to IL-8, increased AAT binding to the neutrophil membrane, decreased FcγRIIIb release from the neutrophil membrane, and normalization of chemotaxis. These results provide new insight into the mechanism underlying the effect of AAT augmentation therapy in the pulmonary disease associated with AAT deficiency., Introduction α-1 Antitrypsin (AAT), a 52-kDa glycosylated protein primarily synthesized in the liver, is the major physiological inhibitor of a range of serine proteases, and within the lung maintains a [...]

Motile bacteria bias the random walk of their motion in response to chemical gradients by the process termed chemotaxis, which allows cells to accumulate in favorable environments and disperse from less favorable ones. In this work, we describe a simple microchannel-nanopore device that establishes a stable chemical gradient for chemotaxis assays in [less than or equal to] 1 min. Chemoattractant is dispensed by diffusion through 10 nm diameter pores at the intersection of two microchannels. This design requires no external pump and minimizes the effect of transmembrane pressure, resulting in a stable, reproducible gradient. The microfluidic platform facilitates microscopic observation of individual cell trajectories, and chemotaxis is quantified by monitoring changes in cell swimming behavior in the vicinity of the intersection. We validate this system by measuring the chemotactic response of an aquatic bacterium, Caulobacter crescentus, to xylose concentrations from 1.3 [micro]M to 1.3 M. Additionally, we make an unanticipated observation of increased turn frequency in a chemotaxis-impaired mutant which provides new insight into the chemotaxis pathway in C. crescentus. 10.1021/ac101977f

We present a diffusion-driven and long-range concentration gradient generator that uses hydrogel as a porous membrane to prevent convection flows but allow the diffusion of cell signaling molecules for the study of bacterial chemotaxis in a microfluidic device. Using this device, we characterized the critical concentrations associated with the chemotactic responses of cells that initially created a population band and then migrated in bands in the presence of multiconcentration gradients. In addition, this device can be used to study the preferential chemotaxis of bacterial cells toward different carbon sources: glucose, galactose, and mannose were preferred over arabinose and xylose, in this order. This was possible since the device is able to simultaneously produce long-range concentration gradients of different chemicals as well. The method presented in this study is easy to perform and the device is cheap to fabricate, so that we believe that these characteristics not only make this device a very useful tool to study the chemotaxis of various, motile microorganisms but also permit parallel experimentation and reduce the time and effort needed in characterizing bacterial responses to various chemicals. 10.1021/ac102022q

The chemotaxis system of Escherichia coil is sensitive to small relative changes in ambient chemoattractant concentrations over a broad range. Interactions among receptors are crucial to this sensitivity, as is precise adaptation, the return of chemoreceptor activity to prestimulus levels in a constant chemoeffector environment through methylation and demethylation of receptors. Signal integration and cooperativity have been attributed to strongly coupled, mixed teams of receptors, but receptors become individually methylated according to their ligand occupancy states. Here, we present a model of dynamic signaling teams that reconciles strong coupling among receptors with receptor-specific methylation. Receptor trimers of dimers couple to form a honeycomb lattice, consistent with cryo-electron microscopy (cryoEM) tomography, within which the boundaries of signaling teams change rapidly. Our model helps explain the inferred increase in signaling team size with receptor modification, and indicates that active trimers couple more strongly than inactive trimers. adaptation | modeling | receptor clustering doi/ 10.1073/pnas.1005017107

Neutrophils are potent contributors to the lung pathophysiological changes occurring in allergic airway inflammation, which typically involve T helper type 2 (Th2) cytokine overexpression. We have previously reported that equine pulmonary endothelial cells are activated by the Th2 cytokine IL-4 and express chemotactic factors for neutrophils after stimulation. We have further explored the possible mechanisms linking Th2-driven inflammation and neutrophilia by studying the effects of recombinant equine IL-4, a prototypical Th2 cytokine, on peripheral blood neutrophils (PBN) isolated from normal animals and from horses with asthmatic airway inflammation (equine heaves). We found that IL-4 induced morphological changes in PBN, dose- and time-dependent expression of IL-8 mRNA, as well as the release of chemotactic factors for neutrophils in culture supernatants. Also, IL-4 induced a mixed inflammatory response in PBN from control and asthmatic-animals with increased expression of proinflammatory IL-8 and TNF-[alpha] but a marked inhibition of IL-1[beta] IL-4 type I receptor (IL-4R[alpha]) and CD23 (Fc[member of]RII) expression were also upregulated by IL-4. Importantly, disease as well as chronic antigenic exposure modified gene expression by PBN. Finally, we found that activation of equine neutrophils with IL-4 involved STAT6 phosphorylation and p38 MAPK and phosphatidylinositol 3-kinase (PI3K); the pharmacological inhibitors, SB-203580 and LY-294002, respectively, significantly reversed IL-4-induced gene modulation in PBN. Overall, results from this study add to the link between Th2-driven inflammation and neutrophilia in the equine model and further extend the characterization of IL-4 effects on neutrophils. cytokines; allergy; equine asthma doi: 10.1152/ajplung.00135.2009.

Bacteria have evolved the ability to monitor changes in various physico-chemical parameters and to adapt their physiology and metabolism by implementing appropriate cellular responses to these changes. Energy taxis is a metabolism-dependent form of taxis and is the directed movement of motile bacteria in gradients of physico-chemical parameters that affect metabolism. Energy taxis has been described in diverse bacterial species and several dedicated energy sensors have been identified. The molecular mechanism of energy taxis has not been studied in as much detail as chemotaxis, but experimental evidence indicates that this behaviour differs from metabolismindependent taxis only by the presence of dedicated energy taxis receptors. Energy taxis receptors perceive changes in energy-related parameters, including signals related to the redox and/or intracellular energy status of the cell. The best-characterized energy taxis receptors are those that sense the redox state of the electron transport chain via non-covalently bound FAD cofactors. Other receptors shown to mediate energy taxis lack any recognizable redox cofactor or conserved energy-sensing motif, and some have been suggested to monitor changes in the proton motive force. The exact energy-sensing mechanism(s) involved are yet to be elucidated for most of these energy sensors. By monitoring changes in energy-related parameters, energy taxis receptors allow cells to couple motility behaviour with metabolism under diverse environmental conditions. Energy taxis receptors thus provide fruitful models to decipher how cells integrate sensory behaviours with metabolic activities. DOI 10.1099/mic.0.039214-0

In chemotactic cells, G protein--coupled receptors activate Ras proteins, but it is unclear how Ras-associated pathways link extracellular signaling to cell migration. We show that, in Dictyostelium discoideum, activated forms of RasC prolong the time course of TORC2 (target of rapamycin [Tor] complex 2)-mediated activation of a myristoylated protein kinase B (PKB; PKBR1) and the phosphorylation of PKB substrates, independently of phosphatidylinositol-(3,4,5)-trisphosphate. Paralleling these changes, the kinetics of chemoattractant-induced adenylyl cyclase activation and actin polymerization are extended, pseudopodial activity is increased and mislocalized, and chemotaxis is impaired. The effects of activated RasC are suppressed by deletion of the TORC2 subunit PiaA. In vitro [RasC.sup.Q62L]-dependent PKB phosphorylation can be rapidly initiated by the addition of a PiaA-associated immunocomplex to membranes of TORC2-deficient cells and blocked by TOR-specific inhibitor PP242. Furthermore, TORC2 binds specifically to the activated form of RasC. These results demonstrate that RasC is an upstream regulator of TORC2 and that the TORC2-PKB signaling mediates effects of activated Ras proteins on the cytoskeleton and cell migration. doi/ 10.1083/jcb.201001129

In this paper, we describe the regulatory roles of VisN, VisR and Rem in the expression of flagellar, motility and chemotaxis genes in Rhizobium leguminosarum biovar viciae strains VF39SM and 3841. Individual mutations in the genes encoding these proteins resulted in a loss of motility and an absence of flagella, indicating that these regulatory genes are essential for flagellar synthesis and function. Transcriptional experiments involving gusA--gene fusions in wild-type and mutant backgrounds were performed to identify the genes under VisN/R and Rem regulation. Results showed that the chemotaxis and motility genes of R. leguminosarum could be separated into two groups: one group under VisN/R-Rem regulation and another group that is independent of this regulation. VisN and VisR regulate the expression of rem, while Rem positively regulates the expression of flaA, flaB, flaC, flaD, motA, motB, che1 and mcpD. All of these genes except mcpD are located within the main motility and chemotaxis gene cluster of R. leguminosarum. Other chemotaxis and motility genes, which are found outside of the main motility gene cluster (che2 operon, flaH for VF39SM, and flaG) or are plasmid-borne (flaE and mcpC), are not part of the VisN/R-Rem regulatory cascade. In addition, all genes exhibited the same regulation pattern in 3841 and in VF39SM, except flaE and flail, flaE is not regulated by VisN/R-Rem in 3841 but it is repressed by Rem in VF39SM. flail is under VisN/R-Rem regulation in 3841, but not in VF39SM. A kinetics experiment demonstrated that a subset of the flagellar genes is continuously expressed in all growth phases, indicating the importance of continuous motility for R. leguminosarum under free-living conditions. On the other hand, motility is repressed under symbiotic conditions. Nodulation experiments showed that the transcriptional activators VisN and Rem are dramatically downregulated in the nodules, suggesting that the symbiotic downregulation of motility-related genes could be mediated by repressing the expression of VisN/R and Rem. DOI 10.1099/mic.0.035386-0

Chemotaxis, the chemically guided movement of cells, plays an important role in several biological processes including cancer, wound healing, and embryogenesis. Chemotacting cells are able to sense shallow chemical gradients where the concentration of chemoattractant differs by only a few percent from one side of the cell to the other, over a wide range of local concentrations. Exactly what limits the chemotactic ability of these cells is presently unclear. Here we determine the chemotactic response of Dictyostelium cells to exponential gradients of varying steepness and local concentration of the chemoattractant cAMP. We find that the ceils are sensitive to the steepness of the gradient as well as to the local concentration. Using information theory techniques, we derive a formula for the mutual information between the input gradient and the spatial distribution of bound receptors and also compute the mutual information between the input gradient and the motility direction in the experiments. A comparison between these quantities reveals that for shallow gradients, in which the concentration difference between the back and the front of a 10-[micro]m-diameter cell is mutual information | noise limits | Dictyostelium | microfluidics www.pnas.org/cgi/doi/10.1073/pnas.0911178107

The direction of rotation of the Escherichia coli flagellum is controlled by an assembly called the switch complex formed from multiple subunits of the proteins FliG, FliM, and FliN. Structurally, the switch complex corresponds to a drum-shaped feature at the bottom of the basal body, termed the C-ring. Stimulus-regulated reversals in flagellar motor rotation are the basis for directed movement such as chemotaxis. In E. coli, the motors turn counterclockwise (CCW) in their default state, allowing the several filaments on a cell to join together in a bundle and propel the cell smoothly forward. In response to the chemotaxis signaling molecule phospho-CheY ([CheY.sup.P]), the motors can switch to clockwise (CW) rotation, causing dissociation of the filament bundle and reorientation of the cell. [CheY.sup.P] has previously been shown to bind to a conserved segment near the N terminus of FliM. Here, we show that this interaction serves to capture [CheY.sup.P] and that the switch to CW rotation involves the subsequent interaction of [CheY.sup.P] with FliN. FliN is located at the bottom of the C-ring, in close association with the C-terminal domain of FliM ([FliM.sub.C]), and the switch to CW rotation has been shown to involve relative movement of FliN and [FliM.sub.C]. Using a recently developed structural model for the FliN/[FliM.sub.C] array, and the [CheY.sup.P]-binding site here identified on FliN, we propose a mechanism by which [CheY.sup.P] binding could induce the conformational switch to CW rotation. switching | cell motility | signal transduction | molecular motors

Chemotaxis is an important virulence factor for Helicobacter pylori colonization and infection. The chemotactic system of H. pylori is marked by the presence of multiple response regulators: CheY1, one CheY-like-containing CheA protein (CheAY2), and three CheV proteins. Recent studies have demonstrated that these molecules play unique roles in the chemotactic signal transduction mechanisms of H. pylori. Here we report the crystal structures of Be[F.sub.3]--activated CheY1 from H. pylori resolved to 2.4 [Angstrom]. Structural comparison of CheY1 with active-site residues of Be[F.sub.3]--bound CheY from Escherichia coli and fluorescence quenching experiments revealed the importance of Thr84 in the phosphotransfer reaction. Complementation assays using various nonchemotactic E. coli mutants and pull-down experiments demonstrated that CheY1 displays differential association with the flagellar motor in E. coli. The structural rearrangement of helix 5 and the C-terminal loop in CheY1 provide a different interaction surface for FliM. On the other hand, interaction of the CheA-P2 domain with CheYl, but not with CheY2/CheV proteins, underlines the preferential recognition of CheY1 by CheA in the phosphotransfer reaction. Our results provide the first structural insight into the features of the H. pylori chemotactic system as a model for Epsilonproteobacteria. doi: 10.1128/JB.00603-09

MyTH/FERM (myosin tail homology 4/band 4.1, ezrin, radixin, and moesin) myosins have roles in cellular adhesion, extension of actin-filled projections such as filopodia and stereocilia, and directional migration. The amoeba Dictyostelium discoideum expresses a simple complement of MyTH/FERM myosins, a class VII (M7) myosin required for cell-substrate adhesion and a unique myosin named MyoG. Mutants lacking MyoG exhibit a wide range of normal actin-based behaviors, including chemotaxis to folic acid, but have a striking defect in polarization and chemotaxis to cAMP. Although the myoG mutants respond to cAMP stimulation by increasing persistence and weakly increasing levels of cortical F-actin, they do not polarize; instead, they maintain a round shape and move slowly and randomly when exposed to a chemotactic gradient. The mutants also fail to activate and localize PI3K to the membrane closest to the source of chemoattractant. These data reveal a role for a MyTH/FERM myosin in mediating early chemotactic signaling and suggest that MyTH/FERM proteins have conserved roles in signaling and the generation of cell polarity. actin cytoskeleton | cell signaling | cell motility | cytoskeletal dynamics doi/ 10.1073/pnas.0909796107

Guidance of axons by molecular gradients is crucial for wiring up the developing nervous system. It often is assumed that the unique signature of such guidance is immediate and biased turning of the axon tip toward or away from the gradient. However, here we show that such turning is not required for guidance. Rather, by a combination of experimental and computational analyses, we demonstrate that growth-rate modulation is an alternative mechanism for guidance. Furthermore we show that, although both mechanisms may operate simultaneously, biased turning dominates in steep gradients, whereas growth-rate modulation may dominate in shallow gradients. These results suggest that biased axon turning is not the only method by which guidance can occur. chemotaxis | growth cone | nerve growth factor | neural development | computational neuroscience doi: 10.1073/pnas.0909254107

Transmembrane chemoreceptors are central components in bacterial chemotaxis. Receptors couple ligand binding and adaptational modification to receptor conformation in processes that create transmembrane signaling. Homodimers, the fundamental receptor structural units, associate in trimers and localize in patches of thousands. To what degree do conformational coupling and transmembrane signaling require higher-order interactions among dimers? To what degree are they altered by such interactions? To what degree are they inherent features of homodimers? We addressed these questions using nanodiscs to create membrane environments in which receptor dimers had few or no potential interaction partners. Receptors with many, few, or no interaction partners were tested for conformational changes and transmembrane signaling in response to ligand occupancy and adaptational modification. Conformation was assayed by measuring initial rates of receptor methylation, a parameter independent of receptor-receptor interactions. Coupling of ligand occupancy and adaptational modification to receptor conformation and thus to transmembrane signaling occurred with essentially the same sensitivity and magnitude in isolated dimers as for dimers with many neighbors. Thus, we conclude that the chemoreceptor dimer is the fundamental unit of conformational coupling and transmembrane signaling. This implies that in signaling complexes, coupling and transmembrane signaling occur through individual dimers and that changes between dimers in a receptor trimer or among trimer-based signaling complexes are subsequent steps in signaling. doi: 10.1128/JB.01391-09

Extracellular adenosine is a potent immunosuppressor that accumulates during tumor growth. We performed proof-of-concept studies investigating the therapeutic potential and mechanism of action of monoclonal antibody (mAb)-based therapy against CD73, an ecto-enzyme overexpressed on breast-cancer cells that catalyzes the dephosphorylation of adenosine monophosphates into adenosine. We showed that anti-CD73 mAb therapy significantly delayed primary 4T1.2 and E0771 tumor growth in immune-competent mice and significantly inhibited the development of spontaneous 4T1.2 lung metastases. Notably, anti-CD73 mAb therapy was essentially dependent on the induction of adaptive anti-tumor immune responses. Knockdown of CD73 in 4T1.2 tumor cells confirmed the tumor-promoting effects of CD73. In addition to its immunosuppressive effect, CD73 enhanced tumor-cell chemotaxis, suggesting a role for CD73-derived adenosine in tumor metastasis. Accordingly, administration of adenosine-5'-N-ethylcarboxamide to tumorbearing mice significantly enhanced spontaneous 4T1.2 lung metastasis. Using selective adenosine-receptor antagonists, we showed that activation of A2B adenosine receptors promoted 4T1.2 tumorcell chemotaxis in vitro and metastasis in vivo. In conclusion, our study identified tumor-derived CD73 as a mechanism of tumor immune escape and tumor metastasis, and it also established the proof of concept that targeted therapy against CD73 can trigger adaptive anti-tumor immunity and inhibit metastasis of breast cancer. adenosine | cancer | chemotaxis | immunosuppression | regulatory doi/ 10.1073/pnas.0908801107

Regular environmental conditions allow for the evolution of specifically adapted responses, whereas complex environments usually lead to conflicting requirements upon the organism's response. A relevant instance of these issues is bacterial chemotaxis, where the evolutionary and functional reasons for the experimentally observed response to chemoattractants remain a riddle. Sensing and motility requirements are in fact optimized by different responses, which strongly depend on the chemoattractant environmental profiles. It is not clear then how those conflicting requirements quantitatively combine and compromise in shaping the chemotaxis response. Here we show that the experimental bacterial response corresponds to the maximin strategy that ensures the highest minimum uptake of chemoattractants for any profile of concentration. We show that the maximin response is the unique one that always outcompetes motile but nonchemotactic bacteria. The maximin strategy is adapted to the variable environments experienced by bacteria, and we explicitly show its emergence in simulations of bacterial populations in a chemostat. Finally, we recast the contrast of evolution in regular vs. complex environments in terms of minimax vs. maximin game-theoretical strategies. Our results are generally relevant to biological optimization principles and provide a systematic possibility to get around the need to know precisely the statistics of environmental fluctuations. biological optimization | E. coli | evolution | game theory | strategy doi/ 10.1073/pnas.0909673107

Phosphate is essential for life on earth, since it is an integral part of important biomolecules. The mechanisms applied by bacteria and eukarya to combat phosphate limitation are fairly well understood. However, it is not known how archaea sense phosphate limitation or which genes are regulated upon limitation. We conducted a microarray analysis to explore the phosphate-dependent gene expression of Halobacterium salinarum strain R1. We identified a set of 17 genes whose transcript levels increased up to several hundredfold upon phosphate limitation. Analysis of deletion mutants showed that this set of genes, the PHO stimulon, is very likely independent of signaling via two-component systems. Our experiments further indicate that PHO stimulon induction might be dependent on the intracellular phosphate concentration, which turned out to be subject to substantial changes. Finally, the study revealed that H. salinarum exhibits a phosphate-directed chemotaxis, which is induced by phosphate starvation.

We investigated the regulatory role of 14-kDa secretory group V phospholipase [A.sub.2] ([gVPLA.sub.2]) in the development of acute lung injury (ALI) and neutrophilic inflammation (NI) caused by intratracheal administration of LPS. Experiments were conducted in [gVPLA.sub.2] knockout ([pla2g5.sup.-/-] ) mice, which lack the gene, and [gVPLA.sub.2] wild-type littermate control ([pla2g5.sup.+/+]) mice. Indices of pulmonary injury were evaluated 24 h after intratracheal administration of LPS. Expression of [gVPLA.sub.2] in microsections of airways and mRNA content in lung homogenates were increased substantially in [pla2g5.sup.+/+] mice after LPS-administered compared with saline-treated [pla2g5.sup.+/+] mice. By contrast, expression of [gVPLA.sub.2] was neither localized in LPS- nor saline-treated [pla2g5.sup.-/-] mice. LPS also caused 1) reduced transthoracic static compliance, 2) lung edema, 3) neutrophilic infiltration, and 4) increased neutrophil myeloperoxidase activity in [pla2g5.sup.+/+] mice. These events were attenuated in [pla2g5.sup.-/-] mice exposed to LPS or in [pla2g5.sup.+/+] mice receiving MCL-3G1, a neutralizing MAb directed against [gVPLA.sub.2], before LPS administration. Our data demonstrate that [gVPLA.sub.2] is an inducible protein in [pla2g5.sup.+/+] mice but not in [pla2g5.sup.-/-] mice within 24 h after LPS treatment. Specific inhibition of [gVPLA.sub.2] with MCL-3GI or gene-targeted mice lacking [gVPLA.sub.2] blocks ALI and attenuates NI caused by LPS. airway inflammation; chemotaxis; pulmonary compliance

We developed a high-throughput quantitative capillary assay and demonstrated that Pseudomonas putida strains F1 and PRS2000 were attracted to cytosine, but not thymine or uracil. In contrast, Pseudomonas aeruginosa PAO1 was not chemotactic to any pyrimidines. Chemotaxis assays with a mutant strain of F1 in which the putative methyl-accepting chemotaxis protein-encoding gene Pput_0623 was deleted revealed that this gene (designated mcpC) encodes a chemoreceptor for positive chemotaxis to cytosine. P. putida F1 also responded weakly to cytidine, uridine, and thymidine, but these responses were not mediated by mcpC. Complementation of the F1 [DELTA]mcpC mutant XLF004 with the wild-type gene restored chemotaxis to cytosine. In addition, introduction of this gene into P. aeruginosa PAO1 conferred the ability to respond to cytosine. To our knowledge, this is the first report of a chemoreceptor for cytosine.

Helicobacter pylori is a chemotactic bacterium that has three CheV proteins in its predicted chemotaxis signal transduction system. CheV proteins contain both CheW- and responseregulator-like domains. To determine the function of these proteins, we developed a fixed-time diffusion method that would quantify bacterial direction change without needing to define particular behaviours, to deal with the many behaviours that swimming H. pylori exhibit. We then analysed mutants that had each cheV gene deleted individually and found that the behaviour of each mutant differed substantially from wild-type and the other mutants, cheV1 and cheV2 mutants displayed smooth swimming behaviour, consistent with decreased cellular CheY-P, similar to a cheW mutant. In contrast, the cheV3 mutation had the opposite effect and the mutant cells appeared to change direction frequently. Additional analysis showed that the cheV mutants displayed aberrant behaviour as compared to the wild-type in the soft-agar chemotaxis assay. The soft-agar assay phenotype was less extreme compared to that seen in the fixed-time diffusion model, suggesting that the cheV mutants are able to partially compensate for their defects under some conditions. Each cheV mutant furthermore had defects in mouse colonization that ranged from severe to modest, consistent with a role in chemotaxis. These studies thus show that the H. pylori CheV proteins each differently affect swimming behaviour.

Directed cell migration, which is critical for embryonic development, leukocyte trafficking, and cell metastasis, depends on chemoattraction. 3-hydroxy-3-methylglutaryl coenzyme A reductase regulates the production of an attractant for Drosophila germ cells that may itself be geranylated. Chemoattractants are commonly secreted through a classical, signal peptide--dependent pathway, but a geranyl-modified attractant would require an alternative pathway. In budding yeast, pheromones produced by a-cells are farnesylated and secreted in a signal peptide--independent manner, requiring the adenosine triphosphate--binding cassette (ABC) transporter Ste6p. Here we show that Drosophila germ cell migration uses a similar pathway, demonstrating that invertebrate germ cells, like yeast cells, are attracted to lipid-modified peptides. Components of this unconventional export pathway are highly conserved, suggesting that this pathway may control the production of similarly modified chemoattractants in organisms ranging from yeast to humans.

Dickeya dadantii 3937 (ex Erwinia chrysanthemi), a member of the Enterobacteriaceae, causes soft rot in many economically important crops. A successful pathogen has to reach the interior of the plant in order to cause disease. To study the role of motility and chemotaxis in the pathogenicity of D. dadantii 3937, genes involved in the chemotactic signal transduction system (cheW, cheB, cheY and cheZ) and in the structure of the flagellar motor (motA) were mutagenized. All the mutant strains grew like the wild-type in culture media, and the production and secretion of pectolytic enzymes was not affected. As expected, the swimming ability of the mutant strains was reduced with respect to the wild-type: motA (94%), cheY (80%), cheW (74%), cheB (54%) and cheZ (48%). The virulence of the mutant strains was analysed in chicory, Saintpaulia and potato. The mutant strains were also tested for their capability to enter into Arabidopsis leaves. All the mutants showed a significant decrease of virulence in certain hosts; however, the degree of virulence reduction varied depending on the virulence assay. The ability to penetrate Arabidopsis leaves was impaired in all the mutants, whereas the capacity to colonize potato tubers after artificial inoculation was affected in only two mutant strains. In general, the virulence of the mutants could be ranked as motA

PDGF B chain or PDGF receptor (PDGFR)-[beta]-deficient (-/-) mice lack mesangial cells. To study responses of [alpha]- and [beta]-receptor activation to PDGF ligands, metanephric mesenchymal cells (MMCs) were established from embryonic day E11.5 wild-type (+/+) and -/- mouse embryos. PDGF BB stimulated cell migration in +/+ cells, whereas PDGF AA did not. Conversely, PDGF AA was chemotactic for -/- MMCs. The mechanism by which PDGFR-[beta] inhibited AA-induced migration was investigated. PDGF BB, but not PDGF AA, increased intracellular [Ca.sup.2+] and the production of reactive oxygen species (ROS) in +/+ cells. Transfection of -/- MMCs with the wild-type [beta]-receptor restored cell migration and ROS generation in response to PDGF BB and inhibited AA-induced migration. Inhibition of [Ca.sup.2+] signaling facilitated PDGF AA-induced chemotaxis in the wild-type cells. The antioxidant N-acetyl-L-cysteine (NAC) or the NADPH oxidase inhibitor diphenyleneiodonium (DPI) abolished the BB-induced increase in intracellular [Ca.sup.2+] concentration, suggesting that ROS act as upstream mediators of [Ca.sup.2+] in suppressing PDGF AA-induced migration. These data indicate that ROS and [Ca.sup.2+] generated by active PDGFR-[beta] play an essential role in suppressing PDGF AA-induced migration in +/+ MMCs During kidney development, PDGFR [beta]-mediated ROS generation and [Ca.sup.2+] influx suppress PDGF AA-induced chemotaxis in metanephric mesenchyme. reactive oxygen species; calcium

[Ca.sup.2+] is known to have important roles in sperm chemotaxis, although the relationship between intracellular [Ca.sup.2+] concentration ([Ca.sup.2+].sub.i]) and modulation of the swimming and chemotactic behavior of spermatozoa has not been elucidated. Using a high-speed [Ca.sup.2+] imaging system, we examined the chemotactic behavior and [[Ca.sup.2+].sub.i] in individual ascidian sperm cells exhibiting chemotactic responses toward sperm activating and attracting factor (SAAF), a chemoattractant released by eggs. In this study, we found that transient [[Ca.sup.2+].sub.i] increased in the flagellum ([Ca.sup.2+] bursts) concomitantly with a change in the swimming direction in an SAAF gradient field. During the initial phase of the [Ca.sup.2+] bursts, the flagellum of the spermatozoon exhibited highly asymmetric waveforms enabling the quick turning of the swimming path. However, the flagellum subsequently changed to symmetric beating, causing the spermatozoon to swim straight. Interestingly, during such responses, [[Ca.sup.2+].sub.i] remained higher than the basal level, indicating that the series of responses was not simply determined by [Ca.sup.2+] concentrations. Also, we found that [Ca.sup.2+] bursts were consistently evoked at points at which the spermatozoon attained around a temporally minimal value for a given SAAF concentration. We concluded that [Ca.sup.2+] bursts induced around a local minimal SAAF concentration trigger a sequence of flagellar responses comprising quick turning followed by straight swimming to direct spermatozoa efficiently toward eggs. ascidian | calcium | fertilization | flagella | chemotaxis

Chemotactic eukaryotic cells are able to detect chemoattractant gradients that are both shallow and have a low background concentration. Under these conditions, the noise in the number of bound receptors can be significant and needs to be taken into account in determining the directional sensing process. Here, we quantify numerically the number of bound receptors on the membrane of a disk-shaped cell by using a numerical Monte Carlo tool. The obtained time traces of the receptor occupancy can be used as inputs for any directional sensing model. We investigate the response of the local excitation global inhibition model and a recently developed balanced inactivation model. We determine a measure for the motility of the cell for each model, based on the relevant output variable, as a function of experimental parameters, resulting in several experimentally testable predictions. Furthermore, we show that these two models behave in a qualitatively different fashion when the background concentration is varied. Thus, to properly characterize the sensitivity of cells to receptor occupancy, it is not sufficient to examine the input signal. Rather, one needs to take into account the response of the second messenger pathway. chemotaxis | motility

Phosphorylation-based signaling pathways employ dephosphorylation mechanisms for signal termination. Histidine to aspartate phosphosignaling in the two-component system that controls bacterial chemotaxis has been studied extensively. Rhodobacter sphaeroides has a complex chemosensory pathway with multiple homologues of the Escherichia coil chemosensory proteins, although it lacks homologues of known signal-terminating CheY-P phosphatases, such as CheZ, CheC, FliY or CheX. Here, we demonstrate that an unusual CheA homologue, Che[A.sub.3], is not only a phosphodonor for the principal CheY protein, Che[Y.sub.6], but is also is a specific phosphatase for Che[Y.sub.6]-P. This phosphatase activity accelerates Che[Y.sub.6]-P dephosphorylation to a rate that is comparable with the measured stimulus response time of approximately 1 s. Che[A.sub.3] possesses only two of the five domains found in classical CheAs, the Hpt (P1) and regulatory (PS) domains, which are joined by a 794-amino acid sequence that is required for phosphatase activity. The P1 domain of Che[A.sub.3] is phosphorylated by Che[A.sub.4], and it subsequently acts as a phosphodonor for the response regulators. A Che[A.sub.3] mutant protein without the 794-amino acid region lacked phosphatase activity, retained phosphotransfer function, but did not support chemotaxis, suggesting that the phosphatase activity may be required for chemotaxis. Using a nested deletion approach, we showed that a 200-amino acid segment of Che[A.sub.3] is required for phosphatase activity. The phosphatase activity of previously identified nonhybrid histidine protein kinases depends on the dimerization and histidine phosphorylation (DHp) domains. However, Che[A.sub.3] lacks a DHp domain, suggesting that its phosphatase mechanism is different from that of other histidine protein kinases. response regulator | signal termination | two-component | Rhodobacter sphaeroides | histidine protein kinase

We found exaggerated chemotaxis in plasma treated with EDTA and thought that the EDTA might itself be inhibiting a tonic inhibitor(s) of chemotaxis. Our plasma fractionations suggested that evidence should be sought for a lipid moiety carrying this activity, and on spectrometry (LC-MS-MS together with GC-MS analyses), the biologically active but not the inactive fraction contained oleic and arachidonic acids. Because fatty acids are largely protein bound, we flooded plasma preparations with delipidated albumin, reasoning that it would bind enough fatty acids, including inhibitory ones, to counter their tonic inhibition. Indeed, we observed dramatic increases in chemotaxis. Hence, adding delipidated albumin to plasma has a similar effect to that of adding EDTA--amplification of the chemotactic response. Oleic acid in physiologic concentrations diminishes the magnifying effects of both EDTA and of delipidated albumin, and in fact diminishes the chemotactic response even without the presence of the amplifiers of chemotaxis. In contrast, arachidonic acid amplifies further the effect of EDTA but not of delipidated albumin, and this augmentation appears to be caused by an EDTA-dependent enrichment of the chemotactic gradient with leukotriene B4 (LTB4). We conclude that oleic acid, the blood levels of which vary among individuals, is at least one tonic inhibitor of chemotaxis in plasma. fatty acids | oleic acid | neutrophils

Spatial organization of cells is important for both multicellular development and tactic responses to a changing environment. We find that the social bacterium, Myxococcus xanthus utilizes a chemotaxis (Che)-like pathway to regulate multicellular rippling during predation of other microbial species. Tracking of GFP-labeled cells indicates directed movement of M. xanthus cells during the formation of rippling wave structures. Quantitative analysis of rippling indicates that ripple wavelength is adaptable and dependent on prey cell availability. Methylation of the receptor, FrzCD is required for this adaptation: a frzF methyltransferase mutant is unable to construct ripples, whereas a frzG methylesterase mutant forms numerous, tightly packed ripples. Both the frzF and frzG mutant strains are defective in directing cell movement through prey colonies. These data indicate that the transition to an organized multicellular state during predation in M. xanthus relies on the tactic behavior of individual cells, mediated by a Che-like signal transduction pathway. chemotaxis | morphogenesis | predation | rippling | chemosensory

Bacterial chemoreceptors undergo conformational changes in response to variations in the concentration of extracellular ligands. These changes in chemoreceptor structure initiate a series of signaling events that ultimately result in regulation of rotation of the flagellar motor. Here we have used cryo-electron tomography combined with 3D averaging to determine the in situ structure of chemoreceptor assemblies in Escherichia coli cells that have been engineered to overproduce the serine chemoreceptor Tsr. We demonstrate that chemoreceptors are organized as trimers of receptor dimers and display two distinct conformations that differ principally in arrangement of the HAMP domains within each trimer. Ligand binding and methylation alter the distribution of chemoreceptors between the two conformations, with serine binding favoring the 'expanded' conformation and chemoreceptor methylation favoring the 'compact' conformation. The distinct positions of chemoreceptor HAMP domains within the context of a trimeric unit are thus likely to represent important aspects of chemoreceptor structural changes relevant to chemotaxis signaling. Based on these results, we propose that the compact and expanded conformations represent the 'kinase-on' and 'kinase-off' states of chemoreceptor trimers, respectively. cryo-electron tomography | molecular architecture | signal transduction

Many types of ceils are able to accurately sense shallow gradients of chemicals across their diameters, allowing the ceils to move toward or away from chemical sources. This chemotactic ability relies on the remarkable capacity of cells to infer gradients from particles randomly arriving at cell-surface receptors by diffusion. Whereas the physical limits of concentration sensing by cells have been explored, there is no theory for the physical limits of gradient sensing. Here, we derive such a theory, using as models a perfectly absorbing sphere and a perfectly monitoring sphere, which, respectively, infer gradients from the absorbed surface particle density or the positions of freely diffusing particles inside a spherical volume. We find that the perfectly absorbing sphere is superior to the perfectly monitoring sphere, both for concentration and gradient sensing, because previously observed particles are never remeasured. The superiority of the absorbing sphere helps explain the presence at the surfaces of cells of signal-degrading enzymes, such as PDE for cAMP in Dictyostelium discoideum (Dicty) and BAR1 for mating factor [alpha] in Saccharomyces cerevisiae (budding yeast). Quantitatively, our theory compares favorably with recent measurements of Dicty moving up a cAMP gradient, suggesting these cells operate near the physical limits of gradient detection. chemotaxis | receptors | noise

We have characterized and quantified a form of bacterial chemotaxis that manifests only as an emergent property by measuring symmetry breaking in a swarm of Myxococcus xanthus exposed to a two-dimensional nutrient gradient from within an agar substrate. M. xanthus chemotaxis requires cell-cell contact and coordinated motility, as individual motile cells exhibit only nonvectorial movement in the presence of a nutrient gradient. Genes that specifically affect M. xanthus chemotaxis include at least 10 of the 53 that express enhancer binding proteins of the NtrC-like class, an indication that this behavior is controlled through transcription, most likely by a complex signal transduction network.

Chemoreceptor arrays are macromolecular complexes that form extended assemblies primarily at the poles of bacterial cells and mediate chemotaxis signal transduction, ultimately controlling cellular motility. We have used cryo-electron tomography to determine the spatial distribution and molecular architecture of signaling molecules that comprise chemoreceptor arrays in wild-type Caulobacter crescentus cells. We demonstrate that chemoreceptors are organized as trimers of receptor dimers, forming partially ordered hexagonally packed arrays of signaling complexes in the cytoplasmic membrane. This novel organization at the threshold between order and disorder suggests how chemoreceptors and associated molecules are arranged in signaling assemblies to respond dynamically in the activation and adaptation steps of bacterial chemotaxis.

A chemotaxis signal transduction pathway (hereafter called Che1) has been previously identified in the alphaproteobacterium Azospirillum brasilense. Previous experiments have demonstrated that although mutants lacking CheB and/or CheR homologs from this pathway are defective in chemotaxis, a mutant in which the entire chemotaxis pathway has been mutated displayed a chemotaxis phenotype mostly similar to that of the parent strain, suggesting that the primary function of this Che1 pathway is not the control of motility behavior. Here, we report that mutants carrying defined mutations in the cheA1 (strain AB101) and the cheY1 (strain AB102) genes and a newly constructed mutant lacking the entire operon [[DELTA](cheA1-cheR1)::Cm] (strain AB103) were defective, but not null, for chemotaxis and aerotaxis and had a minor defect in swimming pattern. We found that mutations in genes of the Che1 pathway affected the cell length of actively growing cells but not their growth rate. Cells of a mutant lacking functional cheB1 and cheR1 genes (strain BS104) were significantly longer than wild-type cells, whereas cells of mutants impaired in the cheA1 or cheY1 genes, as well as a mutant lacking a functional Che1 pathway, were significantly shorter than wild-type cells. Both the modest chemotaxis defects and the observed differences in cell length could be complemented by expressing the wild-type genes from a plasmid. In addition, under conditions of high aeration, cells of mutants lacking functional cheA1 or cheY1 genes or the Che1 operon formed clumps due to cell-to-cell aggregation, whereas the mutant lacking functional CheB1 and CheR1 (BS104) clumped poorly, if at all. Further analysis suggested that the nature of the exopolysaccharide produced, rather than the amount, may be involved in this behavior. Interestingly, mutants that displayed clumping behavior (lacking cheA1 or cheY1 genes or the Che1 operon) also flocculated earlier and quantitatively more than the wild-type cells, whereas the mutant lacking both CheB1 and CheR1 was delayed in flocculation. We propose that the Che1 chemotaxis-like pathway modulates the cell length as well as clumping behavior, suggesting a link between these two processes. Our data are consistent with a model in which the function of the Che1 pathway in regulating these cellular functions directly affects flocculation, a cellular differentiation process initiated under conditions of nutritional imbalance.

All cells possess transmembrane signaling systems that function in the environment of the lipid bilayer. In the Escherichia coli chemotaxis pathway, the binding of attractants to a two-dimensional array of receptors and signaling proteins simultaneously inhibits an associated kinase and stimulates receptor methylation--a slower process that restores kinase activity. These two opposing effects lead to robust adaptation toward stimuli through a physical mechanism that is not understood. Here, we provide evidence of a counterbalancing influence exerted by receptor density on kinase stimulation and receptor methylation. Receptor signaling complexes were reconstituted over a range of defined surface concentrations by using a template-directed assembly method, and the kinase and receptor methylation activities were measured. Kinase activity and methylation rates were both found to vary significantly with surface concentration--yet in opposite ways: samples prepared at high surface densities stimulated kinase activity more effectively than low-density samples, whereas lower surface densities produced greater methylation rates than higher densities. FRET experiments demonstrated that the cooperative change in kinase activity coincided with a change in the arrangement of the membrane-associated receptor domains. The counterbalancing influence of density on receptor methylation and kinase stimulation leads naturally to a model for signal regulation that is compatible with the known logic of the E. coli pathway. Density-dependent mechanisms are likely to be general and may operate when two or more membrane-related processes are influenced differently by the two-dimensional concentration of pathway elements. biological cooperativity | methyl-accepting chemotaxis protein | signal transduction | liposome | phosphorylation

Methyl-accepting chemotaxis proteins (MCPs) are receptors that play an important role in bacterial chemotaxis. Methylation of Tsr, the MCP that mediates chemotaxis towards serine in Escherichia coli, is thought to be facilitated by binding of the methyltransferase to a flexible tether region at the C-terminal end of Tsr. This study analysed natural length variants of the tether that occur in E. coli due to genetic instability in tandem repeat DNA sequences that code for glutaminyl (Q) residues, creating polyQ sequences of variable lengths in the tether region. The tsr gene of E. coil K-12 (strain MG1655) codes for 4Q at the beginning of its 35 aa tether region. The tether varies in length from 35 to 47 residues among pathogenic and non-pathogenic strains of Escherichia, Shigella spp., Salmonella, Yersinia and Photorhabdus. Among previous sequences, Escherichia and Shigella mostly have 4Q and 7Q variants, and one strain (E. coil HS) has 10Q. In E. coil isolated from 50 humans and 75 animals (dogs, cats, horses, birds, etc.), polyQ up to 13Q (44 aa tether) were identified (6 strains); relative frequencies were 7Q (~77% of the total) >4Q (14%) > 13Q (5%) > 10Q (4%). Phylogenetic analysis revealed that E. coil strains with 10Q or 13Q largely fell within two clusters. Serine chemotaxis was not significantly different among 7Q, 10Q and 13Q strains, and was comparable to chemotaxis in the frequently studied K-12 strain. These results are consistent with models indicating that polyQ sequences from 7Q to 13Q are flexible, and that longer tethers, within this range, would not change the precision of adaptation mediated by methylation. Studies of this naturally variable polyQ region in E. coil may also have relevance to mechanisms that mediate polyQ instability in human genetic diseases.

Pseudomonas aeruginosa demonstrates type IV pilus-mediated directional twitching motility up a gradient of phosphatidylethanolamine (PE). Only one of four extracellular phospholipases C of P. aeruginosa (i.e., PlcB), while not required for twitching motility per se, is required for twitching-mediated migration up a gradient of PE or phosphatidylcholine. Whether other lipid metabolism genes are associated with this behavior was assessed by analysis of transcription during twitching up a PE gradient in comparison to transcription during twitching in the absence of any externally applied phospholipid. Data support the hypothesis that PE is further degraded and that the long-chain fatty acid (LCFA) moieties of PE are completely metabolized via [beta]-oxidation and the glyoxylate shunt. It was discovered that P. aeruginosa exhibits twitching-mediated chemotaxis toward unsaturated LCFAs (e.g., oleic acid), but not saturated LCFAs (e.g., stearic acid) of corresponding lengths. Analysis of mutants that are deficient in glyoxylate shunt enzymes, specifically isocitrate lyase ([DELTA]aceA) and malate synthase ([DELTA]aceB), suggested that the complete metabolism of LCFAs through this pathway was required for the migration of P. aeruginosa up a gradient of PE or unsaturated LCFAs. At this point, our data suggested that this process should be classified as energy taxis. However, further evaluation of the ability of the [DELTA]aceA and [DELTA]aceB mutants to migrate up a gradient of PE or unsaturated LCFAs in the presence of an alternative energy source clearly indicated that metabolism of LCFAs for energy is not required for chemotaxis toward these compounds.

Responses to extracellular stress directly confer survival fitness by means of complex regulatory networks. Despite their complexity, the networks must be evolvable because of changing ecological and environmental pressures. Although the regulatory networks underlying stress responses are characterized extensively, their mechanism of evolution remains poorly understood. Here, we examine the evolution of three candidate stress response networks (chemotaxis, competence for DNA uptake, and endospore formation) by analyzing their phylogenetic distribution across several hundred diverse bacterial and archaeal lineages. We report that genes in the chemotaxis and sporulation networks group into well defined evolutionary modules with distinct functions, phenotypes, and substitution rates as compared with control sets of randomly chosen genes. The evolutionary modules vary in both number and cohesiveness among the three pathways. Chemotaxis has five coherent modules whose distribution among species shows a clear pattern of interdependence and rewiring. Sporulation, by contrast, is nearly monolithic and seems to be inherited vertically, with three weak modules constituting early and late stages of the pathway. Competence does not seem to exhibit well defined modules either at or below the pathway level. Many of the detected modules are better understood in engineering terms than in protein functional terms, as we demonstrate using a control-based ontology that classifies gene function according to roles such as 'sensor,' 'regulator,' and 'actuator.' Moreover, we show that combinations of the modules predict phenotype, yet surprisingly do not necessarily correlate with phylogenetic inheritance. The architectures of these three pathways are therefore emblematic of different modes and constraints on evolution. chemotaxis | competence | module | regulatory | sporulation

Signal processing in bacterial chemotaxis relies on large sensory complexes consisting of thousands of protein molecules. These clusters create a scaffold that increases the efficiency of pathway reactions and amplifies and integrates chemotactic signals. The cluster core in Escherichia coil comprises a ternary complex composed of receptors, kinase CheA, and adaptor protein CheW. All other chemotaxis proteins localize to clusters by binding either directly to receptors or to CheA. Here, we used fluorescence recovery after photobleaching (FRAP) to investigate the turnover of chemotaxis proteins at the cluster and their mobility in the cytoplasm. We found that cluster exchange kinetics were protein-specific and took place on several characteristic time scales that correspond to excitation, adaptation, and cell division, respectively. We further applied analytical and numerical data fitting to analyze intracellular protein diffusion and to estimate the rate constants of cluster equilibration in vivo. Our results indicate that the rates of protein turnover at the duster have evolved to ensure optimal performance of the chemotaxis pathway. bacteria | chemotaxis | diffusion | FRAP | signal transduction

Because ocean water is typically resource-poor, bacteria may gain significant growth advantages if they can exploit the ephemeral nutrient patches originating from numerous, small sources. Although this interaction has been proposed to enhance biogeochemical transformation rates in the ocean, it remains questionable whether bacteria are able to efficiently use patches before physical mechanisms dissipate them. Here we show that the rapid chemotactic response of the marine bacterium Pseudoalteromonas haloplanktis substantially enhances its ability to exploit nutrient patches before they dissipate. We investigated two types of patches important in the ocean: nutrient pulses and nutrient plumes, generated for example from lysed algae and sinking organic particles, respectively. We used microfluidic devices to create patches with environmentally realistic dimensions and dynamics. The accumulation of P. haloplanktis in response to a nutrient pulse led to formation of bacterial hot spots within tens of seconds, resulting in a 10-fold higher nutrient exposure for the fastest 20% of the population compared with nonmotile cells. Moreover, the chemotactic response of P. haloplanktis was >10 times faster than the classic chemotaxis model Escherichia coli, leading to twice the nutrient exposure. We demonstrate that such rapid response allows P. haloplanktis to colonize nutrient plumes for realistic particle sinking speeds, with up to a 4-fold nutrient exposure compared with nonmotile cells. These results suggest that chemotactic swimming strategies of marine bacteria in patchy nutrient seascapes exert strong influence on carbon turnover rates by triggering the formation of microscale hot spots of bacterial productivity. chemotaxis | marine snow | microfluidics | patchiness

Over the last decades, bacterial chemotaxis in Escherichia coli has emerged as a canonical system for the study of signal transduction. A remarkable feature of this system is the coexistence of a robust adaptive behavior observed at the population level with a large fluctuating behavior in single cells [Korobkova E, Emonet T, Vilar JMG, Shimizu TS, Cluzel P (2004) Nature 428:574-578]. Using a unified stochastic model, we demonstrate that this coexistence is not fortuitous but a direct consequence of the architecture of this adaptive system. The methylation and demethylation cycles that regulate the activity of receptor-kinase complexes are ultrasensitive because they operate outside the region of first-order kinetics. As a result, the receptor-kinase that governs cellular behavior exhibits a sigmoidal activation curve. We propose that the steepness of this kinase activation curve simultaneously controls the behavioral variability in nonstimulated individual bacteria and the duration of the adaptive response to small stimuli. We predict that the fluctuating behavior and the chemotactic response of individual cells both peak within the transition region of this sigmoidal curve. Large-scale simulations of digital bacteria suggest that the chemotaxis network is tuned to simultaneously maximize both the random spread of cells in the absence of nutrients and the cellular response to gradients of attractant. This study highlights a fundamental relation from which the behavioral variability of nonstimulated cells is used to infer the timing of the cellular response to small stimuli. agent-based | fluctuation-dissipation | noise | ultrasensitivity

Chemotaxis, a means for motile bacteria to sense the environment and achieve directed swimming, is controlled by flagellar rotation. The primary output of the chemotaxis machinery is the phosphorylated form of the response regulator CheY (P~CheY). The steady-state level of P~CheY dictates the direction of rotation of the flagellar motor. The chemotaxis signal in the form of P~CheY is terminated by the phosphatase CheZ. Efficient dephosphorylation of CheY by CheZ requires two distinct protein-protein interfaces: one involving the strongly conserved C-terminal helix of CheZ (CheZc) tethering the two proteins together and the other constituting an active site for catalytic dephosphorylation. In a previous work (J. Guhaniyogi, V. L. Robinson, and A. M. Stock, J. Mol. Biol. 359:624-645, 2006), we presented high-resolution crystal structures of CheY in complex with the [CheZ.sub.c] peptide that revealed alternate binding modes subject to the conformational state of CheY. In this study, we report biochemical and structural data that support the alternate-binding-mode hypothesis and identify key recognition elements in the CheY-[CheZ.sub.c] interaction. In addition, we present kinetic studies of the [CheZ.sub.c]-associated effect on CheY phosphorylation with its physiologically relevant phosphodonor, the histidine kinase CheA. Our results indicate mechanistic differences in phosphotransfer from the kinase CheA versus that from small-molecule phosphodonors, explaining a modest twofold increase of CheY phosphorylation with the former, observed in this study, relative to a 10-fold increase previously documented with the latter.