Purification and characterization of the wild-type and mutant carboxy-terminal domains of the Escherichia coli Tar chemoreceptor

Abstract

The carboxy-terminal half of the Escherichia coli Tar chemoreceptor protein was cloned into an overproducing plasmid with the transcription of the insert under the control of the strong hybrid tac promoter. Two dominant mutations in the tar gene, which result in "tumble-only" (tar-526) or "swim-only" (tar-529) phenotypes and which are postulated to produce proteins locked in specific signalling modes, were introduced separately onto the overproducing plasmid. After induction with isopropyl-4-D-thiogalactopyranoside, cells containing the plasmids produced about 10% of their soluble cellular protein as the carboxy-terminal fragments. A scheme to purify the overproduced fragments was developed. Typical yields of pure fragment were 5, 30, and 20 mg per liter of induced culture for the wild type, 526 mutant, and 529 mutant, respectively. Fast-protein liquid chromatography-gel filtration analysis of the pure fragments showed that they all existed as oligomers (ca. 103,000 daltons), possibly trimers or tetramers (monomer size is 31,000 daltons). However, the 529 mutant fragment showed an additional oligomeric form (240,000 daltons) corresponding approximately to an octamer. When chromatographed in the presence of 1% octylglucoside, all three fragments showed an identical single oligomeric size of about 135,000 daltons. Further differences between the fragments such as ion-exchange behavior and susceptibility to degradation were found. Taken together, these results suggest that conformational differences between the 529 mutant fragment and the other fragments exist and that these differences may correlate with the phenotypic effects of the tar-529 mutation.Bacterial cells respond to changes in concentrations of chemicals in their environment by biasing their swimming behavior (for reviews, see references 12, 18, and 26). They can reverse the direction of rotation of their flagellar filaments, causing the cell to "tumble" and leading to a change in the direction of movement (17). Regulation of the frequency of flagellar rotation reversals affects the net movement of the cell. The frequency of reversals is controlled by a family of receptor-transducer proteins that reside in the cytoplasmic membrane (18). These proteins interact directly or via other binding proteins with a variety of ligands, and some of these act as chemoattractants. In the presence of increasing concentrations of attractant molecules, a signal is generated that results in the suppression of flagellar rotation reversal. On the other hand, when the concentration of an attractant decreases, the frequency of tumbles is increased. Four different chemotaxis receptor-transducer proteins have been identified and sequenced in Escherichia coli and Salmonella typhimurium (3,4,14,28). They vary in their specificities toward different chemoeffectors. However, they all appear to interact with the same cytoplasmic chemotaxis proteins to generate and transmit signals to change the direction of flagellar rotation.Genetic analysis of several of the E. coli chemotaxis re...

Transmembrane signal transduction in bacterial chemotaxis involves ligand-dependent activation of phosphate group transfer.

“…For example, we might imagine that sequestration or modification ofthe CheW protein or activation of the CheZ phosphatase could act to ''suppress tumbles." We have shown previously that there are structural differences between the proteins that correspond to the signaling forms of the tumbly and the smooth aspartate receptor mutants (23). These altered forms could be involved in interaction with CheW.…”

Escherichia coli aspartate receptor. Oligomerization of the cytoplasmic fragment

“…oligomer-forming c-fragments we have studied, and is consistent with the known concentration dependence of association-dissociation processes. The absolute MW of the low MW form observed in the GFC traces was found to be 31 kD by static light scattering experiments carried out with the wild-type c-fragment, which corresponded to the MW of a monomer based on the amino acid sequence (7,8). The discrepancy between the apparent MW observed by GFC (110 kD) and the true MW is probably due to a nonglobular shape of the c-fragment.…”

“…Tar is composed of a 60-kD polypeptide with two transmembrane sequences, a periplasmic ligand-binding domain and a cytoplasmic region responsible for signaling and adaptation (reviewed in references 1-4). The Tar protein is well suited for biophysical studies of the mechanism of transmembrane signaling because it can be purified in the requisite quantities (5), and because genetic methods, which are well developed in E. coli, can be used to introduce specific labeling sites in the protein (6), to generate well defined fragments (7,8) and to study the effects of mutations in vivo by gene replacement. We have studied a 31-kD cloned soluble cytoplasmic fragment of Tar (c-fragment) (see references 7 and 8).…”

Stimulus response coupling in bacterial chemotaxis: receptor dimers in signalling arrays

“…Formation of these structures was completely dependent on CheA and CheW. [There have been several previous reports that various signalling domain constructs can form trimers and tetramers in vitro (Kaplan and Simon, 1988; Long and Weis, 1992; Cochran and Kim, 1996). No higher order structures were, however, observed for the signalling domain itself, and it was shown to remain mostly monomeric under physiological conditions (Long and Weis, 1992; Surette and Stock, 1996). ]…”