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J B Stock

Publications and source records attributed to J B Stock.

At least 55 records · Page 3Linked to original sources
PubMed1991

Roles of the highly conserved aspartate and lysine residues in the response regulator of bacterial chemotaxis.

The chemotactic responses of bacteria such as Escherichia coli and Salmonella typhimurium are mediated by phosphorylation of the CheY protein. Phospho-CheY interacts with the flagellar motor switch to cause tumbly behavior. CheY belongs to a large family of phosphorylated response regulators that function in bacteria to control motility and regulate gene expression. Residues corresponding to Asp57, Asp13, and Lys109 in CheY are highly conserved among all of these proteins. The site of phosphorylation in CheY is Asp57, and in the three-dimensional structure of CheY the Asp57 carboxylate side chain is in close proximity to the beta-carboxylate of Asp13 and the epsilon-amin of Lys109. To further examine the roles of these residues in response regulator function, each has been mutated to a conservative substitution. Asn for Asp and Arg for Lys. All mutations abolished CheY function in vivo. Whereas the Asp to Asn mutations dramatically reduced levels of CheY phosphorylation, the Lys to Arg mutation had the opposite effect. The high level of phosphorylation in the Lys109 mutant results from a decreased autophosphatase activity as well as a lack of phosphatase stimulation by the phosphatase activating protein, CheZ. Despite its high level of phosphorylation, the Lys109 mutant protein cannot produce tumbly behavior. Thus, Lys109 is required for an event subsequent to phosphorylation. We propose that an interaction between the epsilon-amino of Lys109 and the phosphoryl group at Asp57 is essential for the conformational switch that leads to activation of CheY.

Aspartic Acid↗
PubMed1990

Divalent metal ion binding to the CheY protein and its significance to phosphotransfer in bacterial chemotaxis.

Signal transduction in bacterial chemotaxis involves transfer of a phosphoryl group between the cytoplasmic proteins CheA and CheY. In addition to the established metal ion requirement for autophosphorylation of CheA, divalent magnesium ions are necessary for the transfer of phosphate from CheA to CheY. The work described here demonstrates via fluorescence studies that CheY contains a magnesium ion binding site. This site is a strong candidate for the metal ion site required to facilitate phosphotransfer from phospho-CheA to CheY. The diminished magnesium ion interaction with CheY mutant D13N and the lack of metal ion binding to D57N along with significant reduction in phosphotransfer to these two mutants are in direct contrast to the behavior of wild-type CheY. This supports the hypothesis that the acidic pocket formed by Asp13 and Asp57 is essential to metal binding and phosphotransfer activity. Metal ion is also required for the dephosphorylation reaction, raising the possibility that the phosphotransfer and hydrolysis reactions occur by a common metal-phosphoprotein transition-state intermediate. The highly conserved nature of the proposed metal ion binding site and site of phosphorylation within the large family of phosphorylated regulatory proteins that are homologous to CheY supports the hypothesis that all these proteins function by a similar catalytic mechanism.

Bacterial Proteins↗
PubMed1990

Signal transduction in bacteria.

Cells display a remarkable ability to respond to small fluctuations in their surroundings. In simple microbial systems, information from sensory receptors feeds into a circuitry of regulatory proteins that transfer high energy phosphoryl groups from histidine to aspartate side chains. This phosphotransfer network couples environmental signals to an array of response elements that control cell motility and regulate gene expression.

Amino Acid Sequence↗
PubMed1989

Three-dimensional structure of CheY, the response regulator of bacterial chemotaxis.

Homologies among bacterial signal transduction proteins suggest that a common mechanism mediates processes such as chemotaxis, osmoregulation, sporulation, virulence, and responses to nitrogen, phosphorous and oxygen deprivation. A common kinase-mediated phosphotransfer reaction has recently been identified in chemotaxis, nitrogen regulation, and osmoregulation. In chemotaxis, the CheA kinase passes a phosphoryl group to the cytoplasmic protein CheY, which functions as a phosphorylation-activated switch that interacts with flagellar components to regulate motility. We report here the X-ray crystal structure of the Salmonella typhimurium CheY protein. The determination of the structure was facilitated by the use of site-specific mutagenesis to engineer heavy-atom binding sites. CheY is a single-domain protein composed of a doubly wound five-stranded parallel beta-sheet. The phosphoacceptor site in CheY is probably a cluster of aspartic-acid side chains near the C-terminal edge of the beta-sheet. The pattern of sequence similarity of CheY with components of other regulatory systems can be interpreted in the light of the CheY structure and supports the view that this family of proteins have a common structural motif and active site.

Bacterial Proteins↗
PubMed1989

Phosphorylation and dephosphorylation of a bacterial transcriptional activator by a transmembrane receptor.

Signal transduction in the bacterial Omp, Che, and Ntr systems involves the phosphorylation and dephosphorylation of response regulators (OmpR, CheY and CheB, NRI) that share a homologous domain. We show that in the Omp system, the transmembrane sensor EnvZ, catalyzes both the phosphorylation of OmpR and the dephosphorylation of OmpR-P. The phosphorylation reaction proceeds by a mechanism shared with the Ntr and Che kinases, NRII, and CheA. EnvZ can phosphorylate NRI and can stimulate transcription from the glnAp2 promoter, and similarly, CheA can phosphorylate OmpR and can stimulate transcription from the ompF promoter. OmpR-P formed by either CheA or EnvZ is much more stable than CheY-P and NRI-P, but is rapidly hydrolyzed to OmpR and Pi by EnvZ in the presence of ATP, ADP, or nonhydrolyzable analogs of ATP. Because EnvZ is normally a transmembrane receptor with a periplasmic sensory domain, our results suggest that the role of EnvZ may be to control the intracellular concentration of OmpR-P in response to environmental signals.

Bacterial Outer Membrane Proteins↗
PubMed1989

Protein phosphorylation and regulation of adaptive responses in bacteria.

Bacteria continuously adapt to changes in their environment. Responses are largely controlled by signal transduction systems that contain two central enzymatic components, a protein kinase that uses adenosine triphosphate to phosphorylate itself at a histidine residue and a response regulator that accepts phosphoryl groups from the kinase. This conserved phosphotransfer chemistry is found in a wide range of bacterial species and operates in diverse systems to provide different regulatory outputs. The histidine kinases are frequently membrane receptor proteins that respond to environmental signals and phosphorylate response regulators that control transcription. Four specific regulatory systems are discussed in detail: chemotaxis in response to attractant and repellent stimuli (Che), regulation of gene expression in response to nitrogen deprivation (Ntr), control of the expression of enzymes and transport systems that assimilate phosphorus (Pho), and regulation of outer membrane porin expression in response to osmolarity and other culture conditions (Omp). Several additional systems are also examined, including systems that control complex developmental processes such as sporulation and fruiting-body formation, systems required for virulent infections of plant or animal host tissues, and systems that regulate transport and metabolism. Finally, an attempt is made to understand how cross-talk between parallel phosphotransfer pathways can provide a global regulatory curcuitry.

Amino Acid Sequence↗
PubMed1987

Purification and characterization of the S-adenosylmethionine:glutamyl methyltransferase that modifies membrane chemoreceptor proteins in bacteria.

The enzyme (EC 2.1.1.24) from Salmonella typhimurium that catalyzes the S-adenosylmethionine-dependent methyl esterification of glutamyl residues in membrane chemoreceptor proteins has been purified to homogeneity, and the nucleotide sequence of the gene coding for this protein, cheR, has been determined. The molecular weight, amino acid composition, and N-terminal amino acid sequence of the purified protein correspond to the values predicted from the sequence of the gene. The pure protein is a 33-kDa monomer. Kinetic studies indicate that, at levels of receptor and S-adenosylmethionine present in wild type cells, the transferase is nearly saturated. The enzyme has a relatively low turnover number, approximately 10 mol of methylester formed per mol of enzyme per min; and there appear to be only approximately 200 methyltransferase monomers per wild type cell.

Amino Acid Sequence↗
PubMed1987

Purification and characterization of the CheZ protein of bacterial chemotaxis.

The cheZ gene is the most distal of five genes that comprise the Meche operon of the Salmonella typhimurium chemotaxis system. We have determined the sequence of the cheZ gene along with an 800-nucleotide flanking region at its 3' end. The flanking sequence contains an open reading frame that probably corresponds to the 5' end of flaM. The cheZ coding sequence predicts an extremely acidic, hydrophilic protein with a molecular weight of 23,900. We have purified and characterized this protein. N-terminal analysis of pure CheZ yields an amino acid sequence identical to that predicted by the nucleotide sequence except that the amino-terminal methionine residue is modified by N methylation. The purified CheZ protein exhibits a native molecular weight of 115,000, but in cell extracts the majority of CheZ exists as a much larger aggregate (Mr greater than 500,000). Under these conditions, CheZ appears to be a homopolymer composed of at least 20 monomeric subunits.

Amino Acid Sequence↗
PubMed1982

Sugar transport by the bacterial phosphotransferase system. The glucose receptors of the Salmonella typhimurium phosphotransferase system.

We have previously reported that glucose can be phosphorylated by phospho-HPr and two sugar-specific pairs of proteins of the Escherichia coli and Salmonella typhimurium phosphoenolpyruvate:glycose phosphotransferase system. Each of the sugar-specific complexes comprises two proteins, lipid, and divalent cation, and each is present in membranes isolated from wild type cells. For reasons described in this report, one of the complexes is designated IIGlc and the other IIMan. The IIMan complex has previously been separated into its protein components, II-A and II-B (Kundig, W., and Roseman, S. (1971) J. Biol. Chem. 246, 1407-1418), while the accompanying reports describe dissociation of the IIGlc complex into its components, IIIGlc and II-BGlc. Curtis and Epstein (Curtis, S. J., and Epstein, W. (1975) J. Bacteriol. 122, 1189-1199) first showed that there are two phosphotransferase systems in whole cells responsible for glucose uptake and obtained the respective mutants, now designated ptsG and ptsM. The present studies provide kinetic conditions for assaying each activity separately (in vivo and in vitro), when both are present in the same membrane preparation. The IIGlc system is responsible for the uptake and phosphorylation of glucose and methyl alpha-glucoside, whereas the IIMan system is less specific and utilizes glucose, mannose, and 2-deoxyglucose. With high sugar concentrations in vitro, IIMan is also capable of phosphorylating methyl alpha-glucoside, fructose, and N-acetylmannosamine, while IIGlc phosphorylates fructose and mannose. The in vivo transport results were qualitatively consistent with the in vitro phosphorylation results, and several of the kinetic parameters also showed good quantitative agreement. The levels of the two activities depended on the growth conditions. In addition, transport studies showed that initial uptake rates of methyl alpha-glucoside and steady state levels of this analogue depended on the energy state of the cells and that these two parameters did not necessarily change in the same direction when metabolic inhibitors were used. A series of E. coli and S. typhimurium mutants were characterized both with respect to their ability to transport the glucose analogues and to phosphorylate them in vitro. The original mutants of Curtis and Epstein, ptsG and ptsM, were found to be defective in II-BGlc and the IIMan complex, respectively.

Biological Transport↗
PubMed1981

Changing reactivity of receptor carboxyl groups during bacterial sensing.

A microdistillation procedure has been developed to analyze carboxylmethylation of the Mr = 60,000 chemoreceptor proteins involved in chemotaxis of Salmonella typhimurium and Escherichia coli. Methylation levels obtained by this method are substantially higher than those reported in the literature. In highly motile strains under optimal conditions there are approximately 100,000 methylated receptor residues per cell which are entirely composed of gamma-methylglutamyl esters. Whereas with previously used methods only groups which turn over could be detected, the microdistillation assay provides absolute values. Under steady state conditions, approximately one-half the total number of methyl ester residues are continuously hydrolyzed and resynthesized, while the remainder are sequestered. A mechanism has been devised to explain the observed patterns of methyl ester synthesis and hydrolysis. According to this hypothesis, substrate glutamyl residues on the receptor are located in a restricted region near the active sites of transferase and esterase which are bound to the receptor protein. Small, stimuli-induced changes in receptor conformation effect perturbations in receptor methylation by shifting the focus of activity of the modifying enzymes from one pair of closely spaced groups to another.

Chemotactic Factors↗
PubMed1981

Bacterial chemotaxis in the absence of receptor carboxylmethylation.

Although chemoreceptor methylation in Salmonella typhimurium and Escherichia coli has been shown to be at the core of the chemotactic phenomenon, mutants in the gene (cheR) that codes for the methylating enzyme still respond and adapt to repellent stimuli. Analyses of receptor methylation and transferase activity show that this behavior is not due to residual transferase activity. That receptor methylation is not absolutely required for chemotactic sensory processing is confirmed by the isolation of cheR pseudorevertants that remain defective in receptor methylation, yet are able to respond to gradients on semi-solid tryptone agar. A detailed comparison of cheR and wild-type responses indicates that chemotaxis is mediated by at least two interdependent adaptation systems, only one of which involves receptor methylation. The interaction of these two systems produces the full response in wild-type cells. Methylation-deficient mutants exhibit a partial response and retain some chemotactic ability.

Adenosine Triphosphate↗