ABSTRACT
BACKGROUND: The skin is the first line of defence against exposure to microbial, physical, environmental and chemical insults. In mobilizing a protective response, several different cell types located in our skin release and respond to pro-inflammatory cytokines ensuring skin homeostasis and health. However, chronic activation of this response eventually causes damage resulting in premature ageing. Diosodium tetramethylhexadecenyl succinyl cysteine (TSC or SIG1273), an isoprenylcysteine small molecule, down modulates these inflammatory signalling pathways in various cell types (keratinocytes, peripheral blood mononuclear cells (PBMCs) and endothelial cells) and possesses anti-bacterial properties. Thus, TSC represents a novel cosmetic functional ingredient that provides a broad spectrum of benefits for the skin. OBJECTIVE: To assess the anti-inflammatory properties of TSC in several cutaneous cell types and further investigate its anti-microbial activity. METHODS: Cultured normal human epidermal keratinocytes were exposed to chemical irritant phorbol 12-myrisate 13-acetate (TPA) or ultraviolet-B light (UVB) to induce pro-inflammatory cytokine (IL-6, IL-8 and TNF-α) production. T-cell receptor (TCR) activation of PBMCs and nickel (Ni(2+) ) treatments of human dermal microvascular endothelial cells (HDMECs) were performed resulting in IL-4, IL-6, IL-8 and IL-17 production. Streptococcus pyogenes were cultured to determine minimal inhibitory concentration values. RESULTS: In vitro studies demonstrate TSC blocks TPA and UVB-induced cytokine production in cultured keratinocytes. Similarly, TSC inhibits overproduction of IL-4 and IL-17 in T-cell receptor (TCR)-activated PBMCs as well as nickel induction of IL-6 and IL-8 in HDMECs. Lastly, TSC demonstrated anti-microbial properties, inhibiting cell growth of S. pyogenes. CONCLUSIONS: Tetramethylhexadecenyl succinyl cysteine represents a novel cosmetic functional ingredient that provides a dual modulating benefit of skin protection to individuals by reducing inflammation in keratinocytes, endothelial and mononuclear cell types and S. pyogenes counts.
Subject(s)
Anti-Bacterial Agents/pharmacology , Anti-Inflammatory Agents/pharmacology , Cosmetics , Cysteine/analogs & derivatives , Cells, Cultured , Cysteine/chemistry , HumansABSTRACT
The x-ray crystal structure of the P1 or H domain of the Salmonella CheA protein has been solved at 2.1-A resolution. The structure is composed of an up-down up-down four-helix bundle that is typical of histidine phosphotransfer or HPt domains such as Escherichia coli ArcB(C) and Saccharomyces cerevisiae Ypd1. Loop regions and additional structural features distinguish all three proteins. The CheA domain has an additional C-terminal helix that lies over the surface formed by the C and D helices. The phosphoaccepting His-48 is located at a solvent-exposed position in the middle of the B helix where it is surrounded by several residues that are characteristic of other HPt domains. Mutagenesis studies indicate that conserved glutamate and lysine residues that are part of a hydrogen-bond network with His-48 are essential for the ATP-dependent phosphorylation reaction but not for the phosphotransfer reaction with CheY. These results suggest that the CheA-P1 domain may serve as a good model for understanding the general function of HPt domains in complex two-component phosphorelay systems.
Subject(s)
Bacterial Proteins , Chemotaxis , Histidine/metabolism , Membrane Proteins/chemistry , Adenosine Triphosphate/pharmacology , Amino Acid Sequence , Crystallization , Escherichia coli Proteins , Histidine Kinase , Membrane Proteins/physiology , Methyl-Accepting Chemotaxis Proteins , Molecular Sequence Data , Phosphorylation , Structure-Activity RelationshipABSTRACT
Phosphoprotein phosphatase 2A (PP2A) is a major phosphoserine/threonine protein phosphatase in all eukaryotes. It has been isolated as a heterotrimeric holoenzyme composed of a 65 kDa A subunit, which serves as a scaffold for the association of the 36 kDa catalytic C subunit, and a variety of B subunits that control phosphatase specificity. The C subunit is reversibly methyl esterified by specific methyltransferase and methylesterase enzymes at a completely conserved C-terminal leucine residue. Here we show that methylation plays an essential role in promoting PP2A holoenzyme assembly and that demethylation has an opposing effect. Changes in methylation indirectly regulate PP2A phosphatase activity by controlling the binding of regulatory B subunits to AC dimers.
Subject(s)
Phosphoprotein Phosphatases/chemistry , Phosphoprotein Phosphatases/metabolism , Amino Acid Sequence , Animals , Brain/enzymology , Cattle , Enzyme Stability , Holoenzymes/chemistry , Holoenzymes/metabolism , In Vitro Techniques , Methylation , Phosphoprotein Phosphatases/genetics , Protein Methyltransferases/chemistry , Protein Methyltransferases/metabolism , Protein Phosphatase 2 , Protein Structure, Quaternary , Protein Subunits , Substrate SpecificityABSTRACT
The phosphoprotein phosphatase 2A (PP2A) catalytic subunit contains a methyl ester on its C-terminus, which in mammalian cells is added by a specific carboxyl methyltransferase and removed by a specific carboxyl methylesterase. We have identified genes in yeast that show significant homology to human carboxyl methyltransferase and methylesterase. Extracts of wild-type yeast cells contain carboxyl methyltransferase activity, while extracts of strains deleted for one of the methyltransferase genes, PPM1, lack all activity. Mutation of PPM1 partially disrupts the PP2A holoenzyme in vivo and ppm1 mutations exhibit synthetic lethality with mutations in genes encoding the B or B' regulatory subunit. Inactivation of PPM1 or overexpression of PPE1, the yeast gene homologous to bovine methylesterase, yields phenotypes similar to those observed after inactivation of either regulatory subunit. These phenotypes can be reversed by overexpression of the B regulatory subunit. These results demonstrate that Ppm1 is the sole PP2A methyltransferase in yeast and that its activity is required for the integrity of the PP2A holoenzyme.
Subject(s)
Phosphoprotein Phosphatases/chemistry , Phosphoprotein Phosphatases/metabolism , Saccharomyces cerevisiae Proteins , Schizosaccharomyces pombe Proteins , Amino Acid Sequence , Animals , Cattle , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Drug Resistance, Microbial , Fungal Proteins/chemistry , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Expression , Genes, Fungal , Humans , Methylation , Molecular Sequence Data , Mutation , Phenotype , Phosphoprotein Phosphatases/genetics , Protein Methyltransferases/chemistry , Protein Methyltransferases/genetics , Protein Methyltransferases/metabolism , Protein Phosphatase 2 , Protein Subunits , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Sequence Homology, Amino Acid , Sirolimus/pharmacologyABSTRACT
The chemotaxis response regulator CheY can acquire phosphoryl groups either from its associated autophosphorylating protein kinase, CheA, or from small phosphodonor molecules such as acetyl phosphate. We report a stopped-flow kinetic analysis of CheY phosphorylation by acetyl phosphate. The results show that CheY has a very low affinity for this phosphodonor (K(s)&z.Gt;0.1 M), consistent with the conclusion that, whereas CheY provides catalytic functions for the phosphotransfer reaction, the CheA kinase may act simply to increase the effective phosphodonor concentration at the CheY active site.
Subject(s)
Amides/metabolism , Bacterial Proteins , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Organophosphates/metabolism , Phosphoric Acids/metabolism , Amides/chemistry , Biochemistry/instrumentation , Biochemistry/methods , Fluorescence , Histidine/analogs & derivatives , Histidine/metabolism , Kinetics , Methyl-Accepting Chemotaxis Proteins , Organophosphates/chemistry , Phosphoric Acids/chemistry , PhosphorylationABSTRACT
The regA and rdeA gene products of Dictyostelium are involved in the regulation of cAMP signaling. The response regulator, RegA, is composed of an N-terminal receiver domain linked to a C-terminal cAMP-phosphodiesterase domain. RdeA may be a phospho-transfer protein that supplies phosphates to RegA. We show genetically that phospho-RegA is the activated form of the enzyme in vivo, in that the predicted site of aspartate phosphorylation is required for full activity. We show biochemically that RdeA and RegA communicate, as evidenced by phospho-transfer between the two proteins in vitro. Phospho-transfer is dependent on the presumed phospho-accepting amino acids, histidine 65 of RdeA and aspartate 212 of RegA, and occurs in both directions. Phosphorylation of RegA by a heterologous phospho-donor protein activates RegA phosphodiesterase activity at least 20-fold. Our results suggest that the histidine phosphotransfer protein, RdeA, and the response regulator, RegA, constitute two essential elements in a eukaryotic His-Asp phospho-relay network that regulates Dictyostelium development and fruiting body maturation.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP/metabolism , DNA-Binding Proteins/metabolism , Protozoan Proteins/metabolism , 3',5'-Cyclic-AMP Phosphodiesterases/metabolism , Animals , Cyclic AMP-Dependent Protein Kinases/genetics , DNA-Binding Proteins/genetics , Dictyostelium/genetics , Dictyostelium/physiology , Enzyme Activation , Gene Expression Regulation, Developmental , Genes, Protozoan , Phosphorylation , Polymerase Chain Reaction , Protozoan Proteins/geneticsABSTRACT
Signal transduction in microorganisms and plants is often mediated by His-Asp phosphorelay systems. Two conserved families of proteins are centrally involved: histidine protein kinases and phospho-aspartyl response regulators. The kinases generally function in association with sensory elements that regulate their activities in response to environmental signals. A sequence analysis with 348 histidine kinase domains reveals that this family consists of distinct subgroups. A comparative sequence analysis with 298 available receiver domain sequences of cognate response regulators demonstrates a significant correlation between kinase and regulator subfamilies. These findings suggest that different subclasses of His-Asp phosphorelay systems have evolved independently of one another.
Subject(s)
Protein Kinases/physiology , Amino Acid Sequence , Archaea/enzymology , Archaea/genetics , Bacteria/enzymology , Bacteria/genetics , Evolution, Molecular , Histidine Kinase , Molecular Sequence Data , Protein Kinases/classification , Protein Kinases/genetics , Sequence Homology, Amino Acid , Signal TransductionABSTRACT
The beta-lactams are by far the most widely used and efficacious of all antibiotics. Over the past few decades, however, widespread resistance has evolved among most common pathogens. Streptococcus pneumoniae has become a paradigm for understanding the evolution of resistance mechanisms, the simplest of which, by far, is the production of beta-lactamases. As these enzymes are frequently plasmid encoded, resistance can readily be transmitted between bacteria. Despite the fact that pneumococci are naturally transformable organisms, no beta-lactamase-producing strain has yet been described. A much more complex resistance mechanism has evolved in S. pneumoniae that is mediated by a sophisticated restructuring of the targets of the beta-lactams, the penicillin-binding proteins (PBPs); however, this may not be the whole story. Recently, a third level of resistance mechanisms has been identified in laboratory mutants, wherein non-PBP genes are mutated and resistance development is accompanied by deficiency in genetic transformation. Two such non-PBP genes have been described: a putative glycosyltransferase, CpoA, and a histidine protein kinase, CiaH. We propose that these non-PBP genes are involved in the biosynthesis of cell wall components at a step prior to the biosynthetic functions of PBPs, and that the mutations selected during beta-lactam treatment counteract the effects caused by the inhibition of penicillin-binding proteins.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins , Carrier Proteins/metabolism , Hexosyltransferases , Muramoylpentapeptide Carboxypeptidase/metabolism , Peptidyl Transferases , Streptococcus pneumoniae/drug effects , beta-Lactams/pharmacology , Glycosyltransferases/genetics , Histidine Kinase , Models, Molecular , Mutation , Penicillin-Binding Proteins , Protein Kinases/genetics , Streptococcus pneumoniae/genetics , Streptococcus pneumoniae/metabolism , beta-Lactam ResistanceABSTRACT
The chemotaxis receptor for aspartate, Tar, generates responses by regulating the activity of an associated histidine kinase, CheA. Tar is composed of an extracellular sensory domain connected by a transmembrane sequence to a cytoplasmic signaling domain. The cytoplasmic domain fused to a leucine zipper dimerization domain forms soluble active ternary complexes with CheA and an adapter protein, CheW. The kinetics of kinase activity within these complexes compared to CheA alone indicate approximately a 50% decrease in the KM for ATP and a 100-fold increase in the Vmax. A truncated CheA construct that lacks the phosphoaccepting H-domain and the CheY/CheB-binding domain forms an activated ternary complex that is similar to the one formed by the full-length CheA protein. The Vmax of H-domain phosphorylation by this complex is enhanced approximately 60-fold, the KM for ATP decreased to 50%, and the KM for H-domain decreased to 20% of the values obtained with the same CheA construct in the absence of receptor and CheW. The kinetic data support a mechanism of CheA regulation that involves perturbation of an equilibrium between an inactive form where the H-domain is loosely bound and an active form where the H-domain is tightly associated with the CheA active site and properly positioned for phosphotransfer. The data are consistent with an asymmetric mechanism of CheA activation [Levit, M., Liu, I., Surette, M. G., and Stock, J. B. (1996) J. Biol. Chem. 271, 32057-32063] wherein only one phosphoaccepting domain of CheA at a time can interact with an active center within a CheA dimer.
Subject(s)
Bacterial Proteins/physiology , Chemotaxis/physiology , Escherichia coli Proteins , Membrane Proteins/metabolism , Membrane Proteins/physiology , Protein Kinases/metabolism , Receptors, Cell Surface/physiology , Signal Transduction , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Catalysis , Chemoreceptor Cells , Enzyme Activation , Histidine Kinase , Kinetics , Macromolecular Substances , Membrane Proteins/chemistry , Methyl-Accepting Chemotaxis Proteins , Models, Chemical , Phosphorylation , Protein Kinases/chemistry , Protein Structure, Tertiary , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/metabolism , Salmonella typhimuriumABSTRACT
Bacteria are able to sense a broad range of chemical and energetic stimuli and modulate their swimming behaviour to migrate to more favourable environments. Signal transduction in bacterial chemotaxis is mediated by a two-component system composed of a protein histidine kinase, CheA, and a response regulator, CheY. The phosphorylated response regulator, P approximately CheY, binds to a protein at the flagellar motor, FliM, to cause reversals in flagellar motor rotation. The level of P approximately CheY is controlled by the activity of the kinase CheA, which is in turn regulated by membrane receptors at the cell surface. Membrane receptors such as the aspartate receptor, Tar, are composed of two distinct regions: an extracellular sensing domain that binds stimulatory ligands, aspartate in the case of Tar; and an intracellular signalling domain that forms a complex with the protein kinase CheA. What is the mechanism of transmembrane signalling? How does aspartate binding to the sensing domain at the outside surface of the membrane translate into a change in kinase activity at the membrane cytosol interface? Recent results suggest that the mechanism depends on perturbations in lateral packing within an extensive array of receptors localized to patches at the cell poles. Receptor patching appears to depend on higher-order associations with the kinase CheA as well as an adaptor protein, CheW. It is difficult to assess the locus of pH effects within the context of even a simple signal transduction system like that involved in bacterial chemotaxis. Previous results with mutant strains have indicated that the serine receptor, Tsr, is critical for pH sensing, but in vitro results do not support such a straightforward interpretation of the genetic data.
Subject(s)
Bacterial Physiological Phenomena , Chemotaxis/physiology , Bacterial Proteins/metabolism , Cell Membrane/metabolism , Escherichia coli/physiology , Escherichia coli Proteins , Histidine Kinase , Humans , Hydrogen-Ion Concentration , Membrane Proteins/metabolism , Methyl-Accepting Chemotaxis Proteins , Protein Kinases/metabolism , Signal TransductionABSTRACT
The rate of protein diffusion in bacterial cytoplasm may constrain a variety of cellular functions and limit the rates of many biochemical reactions in vivo. In this paper, we report noninvasive measurements of the apparent diffusion coefficient of green fluorescent protein (GFP) in the cytoplasm of Escherichia coli. These measurements were made in two ways: by photobleaching of GFP fluorescence and by photoactivation of a red-emitting fluorescent state of GFP (M. B. Elowitz, M. G. Surette, P. E. Wolf, J. Stock, and S. Leibler, Curr. Biol. 7:809-812, 1997). The apparent diffusion coefficient, Da, of GFP in E. coli DH5alpha was found to be 7.7 +/- 2.5 microm2/s. A 72-kDa fusion protein composed of GFP and a cytoplasmically localized maltose binding protein domain moves more slowly, with Da of 2.5 +/- 0.6 microm2/s. In addition, GFP mobility can depend strongly on at least two factors: first, Da is reduced to 3.6 +/- 0.7 microm2/s at high levels of GFP expression; second, the addition to GFP of a small tag consisting of six histidine residues reduces Da to 4.0 +/- 2.0 microm2/s. Thus, a single effective cytoplasmic viscosity cannot explain all values of Da reported here. These measurements have implications for the understanding of intracellular biochemical networks.
Subject(s)
Bacterial Proteins/metabolism , Escherichia coli/metabolism , Bacterial Proteins/genetics , Base Sequence , Biological Transport, Active , Cytoplasm/metabolism , DNA Primers/genetics , Diffusion , Escherichia coli/genetics , Green Fluorescent Proteins , Kinetics , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Luminescent Proteins/radiation effects , Photochemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/radiation effectsABSTRACT
In the Escherichia coli chemotaxis system, a family of chemoreceptors in the cytoplasmic membrane binds stimulatory ligands and regulates the activity of an associated histidine kinase CheA to modulate swimming behaviour and thereby cause a net migration towards attractants and away from repellents. The chemoreceptors themselves have been shown to be predominantly dimeric, but in the presence of the kinase CheA plus an adapter protein, CheW, much higher order structures have been observed. Recent results indicate that transmembrane signalling occurs within receptor clusters rather than through isolated dimers. We propose that the mechanism involves receptor arrays where binding of ligands at the outside surface of the membrane affects lateral packing interactions that cause perturbations in the organization of the signalling array at the opposing surface of the membrane. Results with receptor chimeras as well as findings with tyrosine kinase receptors suggest that this mechanism may represent a common theme in membrane receptor function.
Subject(s)
Chemotaxis , Escherichia coli Proteins , Escherichia coli/metabolism , Receptors, Cell Surface , Bacterial Proteins/chemistry , Chemoreceptor Cells/chemistry , Dimerization , Histidine Kinase , Membrane Proteins/chemistry , Methyl-Accepting Chemotaxis Proteins , Methylation , Signal TransductionABSTRACT
Chemotaxis responses in Escherichia coli are mediated by the phosphorylated response-regulator protein P-CheY. Biochemical and genetic studies have established the mechanisms by which the various components of the chemotaxis system, the membrane receptors and Che proteins function to modulate levels of CheY phosphorylation. Detailed models have been formulated to explain chemotaxis sensing in quantitative terms; however, the models cannot be adequately tested without knowledge of the quantitative relationship between P-CheY and bacterial swimming behavior. A computerized image analysis system was developed to collect extensive statistics on freeswimming and individual tethered cells. P-CheY levels were systematically varied by controlled expression of CheY in an E.coli strain lacking the CheY phosphatase, CheZ, and the receptor demethylating enzyme CheB. Tumbling frequency was found to vary with P-CheY concentration in a weakly sigmoidal fashion (apparent Hill coefficient approximately 2.5). This indicates that the high sensitivity of the chemotaxis system is not derived from highly cooperative interactions between P-CheY and the flagellar motor, but rather depends on nonlinear effects within the chemotaxis signal transduction network. The complex relationship between single flagella rotation and free-swimming behavior was examined; our results indicate that there is an additional level of information processing associated with interactions between the individual flagella. An allosteric model of the motor switching process is proposed which gives a good fit to the observed switching induced by P-CheY. Thus the level of intracellular P-CheY can be estimated from behavior determinations: approximately 30% of the intracellular pool of CheY appears to be phosphorylated in fully adapted wild-type cells.
Subject(s)
Bacterial Proteins/physiology , Chemotaxis , Membrane Proteins/physiology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Chemotaxis/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli/physiology , Escherichia coli Proteins , Flagella/genetics , Flagella/physiology , Membrane Proteins/genetics , Membrane Proteins/metabolism , Methyl-Accepting Chemotaxis Proteins , Signal Transduction/genetics , Signal Transduction/physiology , Transformation, GeneticABSTRACT
Chemotaxis responses of Escherichia coli and Salmonella are mediated by type I membrane receptors with N-terminal extracytoplasmic sensing domains connected by transmembrane helices to C-terminal signaling domains in the cytoplasm. Receptor signaling involves regulation of an associated protein kinase, CheA. Here we show that kinase activation by a soluble signaling domain construct involves the formation of a large complex, with approximately 14 receptor signaling domains per CheA dimer. Electron microscopic examination of these active complexes indicates a well defined bundle composed of numerous receptor filaments. Our findings suggest a mechanism for transmembrane signaling whereby stimulus-induced changes in lateral packing interactions within an array of receptor-sensing domains at the cell surface perturb an equilibrium between active and inactive receptor-kinase complexes within the cytoplasm.
Subject(s)
Chemotaxis/physiology , Escherichia coli Proteins , Escherichia coli/physiology , Membrane Proteins/metabolism , Protein Kinases/metabolism , Salmonella typhimurium/physiology , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacterial Proteins/ultrastructure , Cell Membrane/physiology , Dimerization , Enzyme Activation , Histidine Kinase , Kinetics , Membrane Proteins/chemistry , Membrane Proteins/ultrastructure , Methyl-Accepting Chemotaxis Proteins , Models, Molecular , Signal TransductionABSTRACT
The aspartate receptor, Tar, is a member of a large family of signal transducing membrane receptors that interact with CheA and CheW proteins to mediate the chemotactic responses of bacteria. A highly conserved cytoplasmic region, the signaling domain, is flanked by two sequences, methylated helices 1 and 2 (MH1 and MH2), that are predicted to form alpha-helical coiled-coils. MH1 and MH2 contain glutamine and glutamate residues that are subject to deamidation, methylation, and demethylation. We show that the signaling domain is an independently folding unit that binds CheW. When expressed in vivo the signaling domain inhibits CheA kinase activity, but if MH1 or an unrelated leucine zipper coiled-coil sequence is attached to the signaling domain, CheA is activated. A construct that contains a leucine zipper fused to MH1-signaling domain-MH2 also activates the kinase, both in vivo and in vitro, and this activation is regulated by the level of glutamate modification. These findings support a model for receptor signaling where aspartate binding controls the relative orientation of receptor monomers to favor the formation of coiled-coils between MH1 and/or MH2 between subunits. Glutamate modification may stabilize these coiled-coils by reducing electrostatic repulsion between helices.
Subject(s)
Bacterial Physiological Phenomena , Bacterial Proteins/metabolism , Chemotaxis/physiology , Escherichia coli Proteins , Membrane Proteins/metabolism , Protein Structure, Tertiary , Receptors, Cell Surface/metabolism , Adaptation, Physiological , Bacterial Proteins/chemistry , Chemoreceptor Cells , Enzyme Activation , Histidine Kinase , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/chemistry , Methyl-Accepting Chemotaxis Proteins , Protein Binding , Protein Folding , Protein Kinase Inhibitors , Receptors, Cell Surface/chemistry , Signal TransductionABSTRACT
Laboratory mutants of Streptococcus pneumoniae resistant to either cefotaxime or piperacillin reveal defects in competence development independent of the selective beta-lactam. A resistance determinant ciaH encoding a putative histidine kinase of a two-component signal-transducing system that is also involved in competence regulation was recently identified in cefotaxime-resistant mutants. We show now that the CiaH protein can be phosphorylated by ATP in vitro, and that it also phosphorylates the cognate response regulator CiaR. The mutant C306 containing the CiaH mutation Thr-230-Pro is completely noncompetent. It does not release competence-inducing activity (competence factor) into the medium nor can such an activity be released from the cells. Competence in C306 cannot be induced upon addition of external competence factor, in contrast to the competence-defective piperacillin-resistant mutants P506 and P408. A novel resistance determinant cpoA specific for piperacillin was identified in piperacillin-resistant mutants. CpoA is responsible for the competence defect in P506 but not in P408. The results document a tight link between the action of beta-lactams and competence development in the pneumococcus and confirm that the two beta-lactams piperacillin and cefotaxime act via different primary targets.
Subject(s)
Anti-Bacterial Agents/pharmacology , Streptococcus pneumoniae/drug effects , Streptococcus pneumoniae/genetics , beta-Lactam Resistance/genetics , Cloning, Molecular , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Enzymologic , Histidine Kinase , Phosphorylation , Protein Kinases/genetics , Protein Kinases/metabolism , Streptococcus pneumoniae/enzymology , beta-LactamsABSTRACT
Escherichia coli is attracted by pyrroloquinoline quinone (PQQ), and chemotaxis toward glucose is enhanced by the presence of PQQ. A ptsI mutant showed no chemotactic response to either glucose or PQQ alone but did show a chemotactic response to a mixture of glucose and PQQ. A strain lacking the methylated chemotaxis receptor protein Tar showed no response to PQQ.
Subject(s)
Chemotactic Factors/pharmacology , Chemotaxis/physiology , Escherichia coli Proteins , Escherichia coli/physiology , Quinolones/pharmacology , Receptors, Cell Surface , Bacterial Proteins/genetics , Chemoreceptor Cells , Coenzymes/pharmacology , Drug Interactions , Escherichia coli/drug effects , Glucose/pharmacology , Glucose Dehydrogenases/metabolism , Membrane Proteins/genetics , Mutation , PQQ Cofactor , Phosphoenolpyruvate Sugar Phosphotransferase System/genetics , Phosphotransferases (Nitrogenous Group Acceptor)/genetics , Signal TransductionABSTRACT
The histidine protein kinase CheA plays an essential role in stimulus-response coupling during bacterial chemotaxis. The kinase is a homodimer that catalyzes the reversible transfer of a gamma-phosphoryl group from ATP to the N-3 position of one of its own histidine residues. Kinetic studies of rates of autophosphorylation show a second order dependence on CheA concentrations at submicromolar levels that is consistent with dissociation of the homodimer into inactive monomers. The dissociation was confirmed by chemical cross-linking studies. The dissociation constant (CheA2<==>2CheA; KD = 0.2-0.4 microM) was not affected by nucleotide binding, histidine phosphorylation, or binding of the response regulator, CheY. The turnover number per active site within a dimer (assuming 2 independent sites/dimer) at saturating ATP was approximately 10/min. The kinetics of autophosphorylation and ATP/ADP exchange indicated that the dissociation constants of ATP and ADP bound to CheA were similar (KD values approximately 0.2-0.3 mM), whereas ATP had a reduced affinity for CheA approximately P (KD approximately 0.8 mM) compared with ADP (KD approximately 0.3 mM). The rates of phosphotransfer from bound ATP to the phosphoaccepting histidine and from the phosphohistidine back to ADP seem to be essentially equal (kcat approximately 10 min-1).
Subject(s)
Bacterial Proteins , Membrane Proteins/metabolism , Signal Transduction , Chemotaxis , Escherichia coli , Escherichia coli Proteins , Histidine Kinase , Membrane Proteins/chemistry , Methyl-Accepting Chemotaxis Proteins , Protein Conformation , Protein Kinases/metabolism , Salmonella typhimuriumSubject(s)
Bacterial Physiological Phenomena , Bacterial Proteins/physiology , Gene Expression Regulation, Bacterial , Protein Kinases/physiology , Signal Transduction/physiology , Transcription, Genetic , Adaptation, Physiological , Animals , Bacteria/genetics , Bacterial Outer Membrane Proteins/physiology , Escherichia coli/genetics , Escherichia coli/physiology , Histidine Kinase , Models, Biological , Phosphorylation , Protein Processing, Post-Translational , Receptors, Cell Surface/physiologyABSTRACT
Most guanine nucleotide binding proteins (G-proteins) possess an S-prenylated C-terminal cysteine whose carboxyl group can be reversibly methylated. The prenylcysteine analog N-acetyl-S-geranylgeranyl-cysteine (AGGC) (50 microM), a competitive inhibitor of prenylcysteine methyl transferases, introduced into streptolysin-O permeabilized HIT-T15 cells doubled the rate of basal (0.1 microM Ca2+) and of stimulated (10 microM Ca2+ or 100 microM GTP gamma S) insulin secretion in a reversible and ATP-dependent manner. N-acetyl-S-farnesylcysteine (AFC) was less potent while N-acetyl-S-geranyl-cysteine was inactive. Prenylcysteine action on exocytosis did not involve inhibition of G-protein methylation, since (1) the methyl ester derivative of AFC, an inefficient inhibitor of methyltransferases in HIT-T15 cell fractions, was as potent as AGGC in stimulating exocytosis; (2) S-adenosyl-homocysteine, a general inhibitor of methylation reactions, did not alter basal or GTP gamma S-triggered secretion while inhibiting Ca(2+)-induced insulin release. The binding of G-proteins to Rab/GDP-dissociation inhibitor, Rab3A/GTPase activating protein or rabphilin-3A was not affected by the prenylcysteine analogs. AGGC or AFC had the same effect on insulin release as a synthetic peptide mimicking the amino acid residues 52-67 of the G-protein Rab3A (Rab3AL). Moreover, the action on secretion of the combination of Rab3AL and prenylcysteines was not additive. We propose that the prenylcysteines and the Rab3AL peptide influence exocytosis by affecting the association of Rab3A with different proteins of the exocytotic machinery of insulin-secreting cells.