No abstract

Signal transduction in Escherichia coli involves the interaction of transmembrane receptor proteins such as the aspartate receptor, Tar, and the products of four chemotaxis genes, cheA, cheY, cheW, and cheZ. It was previously shown that the cheA gene product is an autophosphorylating protein kinase that transfers phosphate to CheY, whereas the cheZ gene product acts as a specific CheY phosphatase. Here we report that the system can be reconstituted in vitro and receptor function can be coupled to CheY phosphorylation. Coupling requires the presence of the CheW protein, the appropriate form of the receptor, and the CheA and CheY proteins. Under these conditions the accumulation of CheYphosphate is enhanced %300-fold. This rate enhancement is seen in reactions using wild-type and "tumble" mutant receptors but not "smooth" mutant receptors. The increased accumulation of phosphoprotein was inhibited by micromolar concentrations of aspartate, using wild-type, but not tumble, receptors. These results provide evidence that the signal transduction pathway in bacterial chemotaxis involves receptor-mediated alteration of the levels of phosphorylated proteins. They suggest that CheW acts as the coupling factor between receptor and phosphorylation. The results also support the suggestion that CheY-phosphate is the tumble signal.Bacteria such as Escherichia coli or Salmonella typhimurium sense their environment through a series of transmembrane receptor proteins. Each of these binds a specific subset of ligands that may act as attractant or repellent. Changes in ligand concentration initiate two responses (for reviews of bacterial chemotaxis, see refs. 1 and 2). First, a rapid excitation response occurs that modulates the frequency of changes in bacterial flagellar rotation, and second, an adaptation response is initiated that presumably modifies the sensitivity of the receptor. The excitation response can be manifested in two ways. Increase -in concentration of an attractant ligand may decrease flagellar-rotation-reversal frequency, leading to "smooth" swimming of the cell. Alternatively, an increase in repellent concentration can result in a transient increase in flagellar reversal, leading to "tumbly" swimming behavior. In addition to the receptor, the excitation response requires the presence of the products of four chemotaxis genes, cheA, cheY, cheZ, and cheW (3-6). We have shown that the CheA protein is an autophosphorylating protein kinase that in the presence of ATP phosphorylates histidine residue 48 (7-10). Once CheA is phosphorylated, it is able to very rapidly transfer phosphate to the che Y gene product. CheY-phosphate or a derivative of CheY is thought to interact with proteins at the base of the flagellar motor to increase the frequency of reversal of rotation (11-13). The cheZ gene product specifically dephosphorylates CheY (7). Thus, these results suggest a plausible scheme for how the che gene products might generate a "tumble" regulator. However, little is known about how the receptor interacts wit...

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...

We have found that 1 of 20 human tumors tranplanted and passaged in nude mice was associated with a massive induction of endogenous murine leukemia virus (MuLV). Separation and growth of these viruses on various substrates indicated that both ecotropic and xenotropic MuLV were present in the induced mixture. Tryptic peptide fingerprints of the p30 and gp7O structural elements of the viruses indicated that all of the known endogenous MuLVs of BALB/c mice were present in the mixture. In addition, a new xenotropic MuLV was identified. The human tumor that induced the viruses was an oat cell carcinoma. The oat cell carcinoma possibly produced a specific hormone or factor that acts as a potent inducer of endogenous type C retroviruses.The use of the athymic nude mouse as a vehicle for the maintenance of heterotransplanted tumor tissue is an established procedure. It has been recognized that xenotropic and ecotropic murine leukemia viruses (MuLV), endogenous to nude mice, can productively infect transplanted tumor cells (1-4). The resulting addition of mouse viral proteins to the tumor cell's antigenic repertoire can complicate immunological and biochemical studies of the tumor, not to mention efforts to identify possible retroviruses endogenous to the tumor cell. In this report we describe a differential effect of various human tumors transplanted into nude mice to induce endogenous MuLV.In contrast to most human tumors established in our mouse colony, it was found that passage of an oat cell carcinoma in nude mice caused the induction of a large variety and quantity of MuLV. The viruses were grown on mouse and heterologous cells in vitro to separate host range types. Peptide mapping of viral gene products gp70 and p30 indicated that all of the known MuLVs endogenous to BALB/c mice were present in the original mixed viral population. This was surprising because some of these viruses had not been inducible by previously reported means. The superior viral inductive qualities of the oat cell carcinoma, relative to other human tumors, suggests that it possessed, induced, or produced and secreted a potent inducer of endogenous MuLV. MATERIALS AND METHODSTumor Transplantation. Oat cell carcinoma and other human tumors were serially transplanted subcutaneously in BALB/c nu/nu mice bred in our laboratory, as described (5). (6,12). Filtered supernatant fluids were concentrated and assayed for reverse transcriptase activity, as described (6). Cultures that were positive for reverse transcriptase activity were further purified according to host range by passage of cell-free virus from mouse to nonmouse cells and conversely for at least two passages.Ecotropic MuLV infectivities were tested on secondary NIH Swiss and BALB/c embryo fibroblasts by the XC or SXC assays (7,8).Virus Purification. Virus was concentrated by centrifugation of filtered supernates through 15% (wt/vol) sucrose onto a cushion of 50% sucrose. Centrifugation was for 60 min at 25,000 rpm (105,000 X g) in a Beckman SW 27 rotor in the Beckman LS-50 cent...