MCP molecules typically have a periplasmic ligand-binding domain for monitoring attractant or repellent levels in the environment and a cytoplasmic signaling domain for communicating with the motor apparatus (1). The MCP signaling domain, highly conserved in structure, forms ternary complexes with two cytoplasmic proteins, CheA, a histidine kinase, and CheW, which couples CheA activity to chemoreceptor control (2, 3). Changes in receptor ligand occupancy trigger conformational changes in the signaling domain that in turn modulate the flux of phosphoryl groups from CheA to effector proteins that elicit the behavioral response (4, 5). MCPs are capable of detecting chemoeffector concentration changes of only a few parts per thousand over more than a five-log concentration range (6-9). The amplification mechanisms responsible for the high-gain signaling characteristics of bacterial chemoreceptors are still poorly understood but may rely on novel signaling principles that will prove to be widely used in biological signal transduction systems.Like many membrane receptors, MCP molecules are not uniformly distributed but rather clustered, typically at the cell poles (10). The CheA and CheW proteins also localize to these receptor clusters and are largely responsible for their integrity (10), suggesting that bacterial chemoreceptors form a two-dimensional lattice that is held together by bridging connections to CheA and CheW. Bray and colleagues (11-13) have theorized that receptors in such an array might, through conformational coupling, communicate their signaling states to neighboring receptors to produce a large gain in detection sensitivity. Experimental work, primarily with Escherichia coli, provides growing support for this notion.E. coli uses five MCP-family receptors to promote chemotactic movements toward different attractant compounds: Tar (aspartate and maltose), Tsr (serine), Tap (dipeptides), Trg (ribose and galactose), and Aer (oxygen and other electron acceptors). Tap, Trg, and, most likely, Aer are present in the cell at roughly 10% the levels of Tsr and Tar (14). Several lines of evidence indicate that high-and low-abundance receptors might signal collaboratively, and that clustering enhances their detection sensitivity. First, the ability of low-abundance receptors to mediate chemotactic responses implies that they are able to exert control over a substantial fraction of the CheA signaling molecules associated with the receptor array. Second, high-abundance receptors assist one another (15) and low-abundance receptors (16-18) in achieving the methylation changes needed to adapt to sensory stimuli. Third, a multivalent galactose ligand that promotes clustering of Trg (19) also served to recruit Tar and Tsr molecules to the cluster and greatly enhanced their detection sensitivity, implying that communication between receptors in a cluster produces signal amplification (20).In vitro studies of receptors and receptor fragments indicate that more than one receptor signaling domain is needed to form a ternar...

The team signaling model for bacterial chemoreceptors proposes that receptor dimers of different detection specificities form mixed trimers of dimers. These receptor “squads” then recruit the cytoplasmic signaling proteins CheA and CheW to form ternary signaling teams, which typically cluster at the poles of the cell. We devised cysteine-directed in vivo crosslinking approaches to ask whether mixed receptor squads could form in the absence of CheA and CheW and, if so, whether the underlying structural interactions conformed to trimer-of-dimers geometry. One approach used cysteine reporters at positions in the serine (Tsr) and aspartate (Tar) receptors that should form disulfide-linked Tsr≈Tar products when juxtaposed at the interface of a mixed trimer. Another approach used a cysteine reporter with trigonal geometry near the trimer contact region and a trifunctional maleimide reagent with a spacer length appropriate for capturing the three axial subunits in a trimer of dimers. Both approaches detected mixed receptor-crosslinking products in cells lacking CheA and CheW. Under these conditions, receptor methylation and ligand-binding state had no discernable effect on crosslinking efficiencies. Crosslinking with the trigonal reporter was rapid and did not increase with longer treatment times or higher reagent concentrations, suggesting that this method produces a short-exposure snapshot of the receptor population. The extent of crosslinking indicated that most of the cell's receptor molecules were organized in higher-order groups. Crosslinking in receptor trimer contact mutants correlated with their signaling behaviors, suggesting that trimers of dimers are both structural and functional precursors of chemoreceptor signaling teams in bacteria

The team signaling model for bacterial chemoreceptors proposes that receptor dimers of different detection specificities form mixed trimers of dimers that bind the cytoplasmic proteins CheA and CheW to form ternary signaling complexes clustered at the cell poles. We used a trifunctional crosslinking reagent targeted to cysteine residues in the aspartate (Tar) and serine (Tsr) receptors to obtain in vivo snapshots of trimer composition in the receptor population. To analyze the dynamics of trimer formation, we followed the appearance of mixed trimers when cells expressing Tar were induced for the expression of Tsr and treated with the crosslinker shortly after the onset of induction. In the absence of CheA or CheW, preformed Tar trimers exchanged partners readily with newly made Tsr. Conversely, in the presence of CheA and CheW, receptor trimers seldom exchanged partners, irrespective of the presence or absence of attractants. The C-terminal receptorcoupling domain of the CheA kinase, which contains binding determinants for the CheW protein, was essential for conferring low exchangeability to the preformed trimers of dimers. CheW also was required for this effect, but, unlike CheA, overexpression of CheW interfered with trimer formation and chemotactic behavior. The CheW effect probably occurs through binding interactions that mask the receptor sites needed for trimer formation. We propose that clustered receptors are organized in mixed trimers of dimers through binding interactions with CheA and CheW, which play distinctly different architectural roles. Moreover, once complete signaling teams have formed, they no longer undergo dynamic exchange of receptor members.chemotaxis ͉ epistasis ͉ receptor clustering ͉ signaling teams ͉ trimer of dimers E scherichia coli and other motile bacteria monitor their chemical environment with high sensitivity and broad detection ranges and use this information to seek out favorable living conditions. These chemotactic behaviors of bacteria offer tractable models for investigating the molecular basis of biological chemosensing and signal amplification. Indeed, considerable progress has been made in documenting the high-gain signaling properties of bacterial chemoreceptors, but their underlying molecular mechanisms remain elusive (recently reviewed in refs. 1 and 2).Methyl-accepting chemotaxis proteins (MCPs) are the predominant chemoreceptors in bacteria (3). E. coli possesses five MCP-like receptors with different detection specificities; its most abundant types are the serine receptor (Tsr) and the aspartate receptor (Tar) (4). MCPs are integral membrane proteins characterized by a conserved cytoplasmic domain that interacts with the coupling protein CheW and the histidine kinase CheA to form ternary signaling complexes, which communicate with the cell's flagellar motors through protein phosphorylation pathways (1, 2). Tar and Tsr have periplasmic sensing domains that monitor chemoeffector levels through high-affinity binding sites. Changes in ligand occupancy modulate MCP sig...

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A serine protease secreted by the haloalkaliphilic archaeon Natrialba magadii at the end of the exponential growth phase was isolated. This enzyme was purified 83 fold with a total yield of 25% by ethanol precipitation, affinity chromatography, and gel filtration. The native molecular mass of the enzyme determined by gel filtration was 45 kDa. Na. magadii extracellular protease was dependent on high salt concentrations for activity and stability, and it had an optimum temperature of 60 degrees C in the presence of 1.5M NaCl. The enzyme was stable and had a broad pH profile (6-12) with an optimum pH of 8-10 for azocasein hydrolysis. The protease was strongly inhibited by diisopropyl fluorophosphate (DFP), phenylmethyl sulfonylfluoride (PMSF), and chymostatin, indicating that it is a serine protease. It was sensitive to denaturing agents such as SDS, urea, and guanidine HCl and activated by thiol-containing reducing agents such as dithiotreitol (DTT) and 2-mercaptoethanol. This protease degraded casein and gelatin and showed substrate specificity for synthetic peptides containing Phe, Tyr, and Leu at the carboxyl terminus, showing that it has chymotrypsin-like activity. Na. magadii protease presented no cross-reactivity with polyclonal antibodies raised against the extracellular protease of Natronococcus occultus, suggesting that although these proteases share several biochemical traits, they might be antigenically unrelated.