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 T he signaling pathway that mediates chemotaxis in Escherichia coli-like many systems-consists of transmembrane and membrane-associated proteins that function in the two-dimensional (2D) space of the lipid bilayer, where clustering, allostery, and cooperative interactions may all contribute to the regulation of signaling (1, 2). Studies of E. coli have shown that chemoreceptors, the adaptor protein, CheW (W), and the histidine kinase, CheA (A), form 2D arrays that are remarkable for the large number of receptors involved and their location at the cell poles (3-6). The ligand-binding domains of the homologous chemoreceptors endow the array with responsiveness toward specific attractant molecules; the conserved cytoplasmic domain (c-domain) conveys signals generated by ligand binding. In addition, the sensitivities of receptors toward cognate ligands are adjusted by the reversible methylation of a few conserved glutamic acid residues in the c-domain (7). Temporal comparisons are made between current and recent past chemoeffector concentrations by pathway proteins that propagate and terminate signals, which serves to bias the random-walk swimming behavior of E. coli in chemical gradients (8). Given its remarkable ...

BackgroundGeobacter species are δ-Proteobacteria and are often the predominant species in a variety of sedimentary environments where Fe(III) reduction is important. Their ability to remediate contaminated environments and produce electricity makes them attractive for further study. Cell motility, biofilm formation, and type IV pili all appear important for the growth of Geobacter in changing environments and for electricity production. Recent studies in other bacteria have demonstrated that signaling pathways homologous to the paradigm established for Escherichia coli chemotaxis can regulate type IV pili-dependent motility, the synthesis of flagella and type IV pili, the production of extracellular matrix material, and biofilm formation. The classification of these pathways by comparative genomics improves the ability to understand how Geobacter thrives in natural environments and better their use in microbial fuel cells.ResultsThe genomes of G. sulfurreducens, G. metallireducens, and G. uraniireducens contain multiple (~70) homologs of chemotaxis genes arranged in several major clusters (six, seven, and seven, respectively). Unlike the single gene cluster of E. coli, the Geobacter clusters are not all located near the flagellar genes. The probable functions of some Geobacter clusters are assignable by homology to known pathways; others appear to be unique to the Geobacter sp. and contain genes of unknown function. We identified large numbers of methyl-accepting chemotaxis protein (MCP) homologs that have diverse sensing domain architectures and generate a potential for sensing a great variety of environmental signals. We discuss mechanisms for class-specific segregation of the MCPs in the cell membrane, which serve to maintain pathway specificity and diminish crosstalk. Finally, the regulation of gene expression in Geobacter differs from E. coli. The sequences of predicted promoter elements suggest that the alternative sigma factors σ28 and σ54 play a role in regulating the Geobacter chemotaxis gene expression.ConclusionThe numerous chemoreceptors and chemotaxis-like gene clusters of Geobacter appear to be responsible for a diverse set of signaling functions in addition to chemotaxis, including gene regulation and biofilm formation, through functionally and spatially distinct signaling pathways.

Adaptation in the chemosensory pathways of bacteria like Escherichia coli is mediated by the enzymecatalyzed methylation (and demethylation) of glutamate residues in the signaling domains of methyl-accepting chemotaxis proteins (MCPs). MCPs can be methylated in trans, where the methyltransferase (CheR) molecule catalyzing methyl group transfer is tethered to the C terminus of a neighboring receptor. Here, it was shown that E. coli cells exhibited adaptation to attractant stimuli mediated through either engineered or naturally occurring MCPs that were unable to tether CheR as long as another MCP capable of tethering CheR was also present, e.g., either the full-length aspartate or serine receptor (Tar or Tsr). Methylation of isolated membrane samples in which engineered tethering and substrate receptors were coexpressed demonstrated that the truncated substrate receptors (trTsr) were efficiently methylated in the presence of tethering receptors (Tar with methylation sites blocked) relative to samples in which none of the MCPs had tethering sites. The effects of ligand binding on methylation were investigated, and an increase in rate was produced only with serine (the ligand specific for the substrate receptor trTsr); no significant change in rate was produced by aspartate (the ligand specific for the tethering receptor Tar). Although the overall efficiency of methylation was lower, receptor-specific effects were also observed in trTar-and trTsr-containing samples, where neither Tar nor Tsr possessed the CheR binding site at the C terminus. Altogether, the results are consistent with a ligand-induced conformational change that is limited to the methylated receptor dimer and does not spread to adjacent receptor dimers.Bacterial chemotaxis signaling pathways are two-component systems in which the sensors are composed of noncovalent ternary complexes of transmembrane receptor proteins, the cytoplasmic adaptor protein CheW and the histidine kinase CheA (9, 35). The transmembrane receptors, which are also known as methyl-accepting chemotaxis proteins (MCPs), are organized as homodimers to bind ligand (28), where each subunit of the dimer consists of a periplasmic ligand-binding domain (27, 49), two ␣-helical transmembrane segments (31), and a helical cytoplasmic region that contains the highly conserved signaling domain flanked by the methylation helices (16,20). Receptor dimers of different ligand specificity cluster in the membrane in synergy with CheW and CheA, frequently at the poles of the cell (26). The available evidence suggests that the receptors in these signaling patches possess the trimer-ofdimers organization, first identified in the crystal structure of the cytoplasmic domain (1, 16, 41). The complexity of the organization is further heightened by the possible involvement of interdigitating cytoplasmic domains, which have been observed by an electron microscope study of receptor arrays (46). Evidence consistent with extensive interactions among receptor dimers in the excitation and adaptation phases of sig...

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