ABSTRACT
The WalK (a histidine kinase)/WalR (a response regulator, aka YycG/YycF) two-component system is indispensable in the signal transduction pathway for the cell-wall metabolism of Bacillus subtilis and Staphylococcus aureus. The inhibitors directed against WalK would be expected to have a bactericidal effect. After we screened 1368 culture broths of Streptomyces sp. by a differential growth assay, walkmycin A, B and C, which were produced by strain MK632-100F11, were purified using silica-gel column chromatography and HPLC. In this paper, the chemical structure of the major product (walkmycin B) was determined to be di-anthracenone (C(44)H(44)Cl(2)O(14)), which was very similar to BE40665A. MICs of walkmycin B against B. subtilis and S. aureus were 0.39 and 0.20 microg ml(-1), and IC(50) measurements against WalK were 1.6 and 5.7 microM, respectively. To clarify the affinity between WalK and walkmycin B, surface plasmon resonance was measured to obtain the equilibrium dissociation constant, K(D1), of 7.63 microM at the higher affinity site of B. subtilis WalK. These results suggest that walkmycin B inhibits WalK autophosphorylation by binding to the WalK cytoplasmic domain.
Subject(s)
Anthracenes/pharmacology , Anti-Bacterial Agents/pharmacology , Protein Kinases/metabolism , Anthracenes/chemistry , Anti-Bacterial Agents/chemistry , Bacteria/drug effects , Molecular Structure , Streptomyces/enzymologyABSTRACT
A two-component signal transduction system (TCS) is an attractive target for antibacterial agents. In this chapter, we review the TCS inhibitors developed during the past decade and introduce novel drug discovery systems to isolate the inhibitors of the YycG/YycF system, an essential TCS for bacterial growth, in an effort to develop a new class of antibacterial agents.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Signal Transduction/drug effects , Bacteria/genetics , Bacterial Physiological Phenomena/drug effects , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/genetics , Bacterial Proteins/physiology , Drug Design , Drug Resistance, Multiple, Bacterial/genetics , Drug Resistance, Multiple, Bacterial/physiology , Histidine Kinase , Microbial Sensitivity Tests , Protein Kinase Inhibitors/pharmacology , Protein Kinases/genetics , Protein Kinases/physiology , Signal Transduction/genetics , Signal Transduction/physiology , User-Computer InterfaceABSTRACT
A response regulator YycF and its cognate sensor kinase YycG constitute the two-component signal transduction system essential for growth of Gram-positive bacteria with a low GC content. We have determined the X-ray crystal structure of the effector domain of Bacillus subtilis YycF involved in DNA binding. The structure, containing a winged helix-turn-helix motif, was found to be very similar to that of the response regulator PhoB from Escherichia coli. Specific binding of YycF to the PhoB-regulated alkaline phosphatase promoter was also demonstrated.
Subject(s)
Bacillus subtilis/growth & development , Bacterial Proteins/chemistry , DNA/chemistry , Gene Expression Regulation, Bacterial , Helix-Turn-Helix Motifs , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , Crystallography, X-Ray , DNA/metabolism , Helix-Turn-Helix Motifs/genetics , Molecular Sequence Data , Promoter Regions, Genetic , Protein Structure, TertiaryABSTRACT
Ascochytatin, a new spirodioxynaphthalene metabolite produced by a marine-derived fungus, was found from a screening program focused on the bacterial two-component regulatory system. The structure of ascochytatin was determined by spectroscopic methods, including NMR and MS. The relative stereochemistry was determined by an X-ray crystallographic analysis, and the absolute stereochemistry was determined by the modified Mosher's method.
Subject(s)
Anti-Bacterial Agents/pharmacology , Ascomycota/chemistry , Naphthalenes/pharmacology , Spiro Compounds/pharmacology , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/isolation & purification , Antibiotics, Antineoplastic/isolation & purification , Antibiotics, Antineoplastic/pharmacology , Ascomycota/classification , Bacillus subtilis/drug effects , Bacteria/drug effects , Cell Line, Tumor , Chemical Phenomena , Chemistry, Physical , Chromatography, Thin Layer , Crystallography, X-Ray , Culture Media , Humans , Magnetic Resonance Spectroscopy , Microbial Sensitivity Tests , Models, Molecular , Naphthalenes/chemistry , Naphthalenes/isolation & purification , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Fast Atom Bombardment , Spectrophotometry, Infrared , Spectrophotometry, Ultraviolet , Spiro Compounds/chemistry , Spiro Compounds/isolation & purificationABSTRACT
Two-component signal-transduction systems (TCSs) of bacteria are considered to form an intricate signal network to cope with various environmental stresses. One example of such a network in Escherichia coli is the signal transduction cascade from the EvgS/EvgA system to the PhoQ/PhoP system, where activation of the EvgS/EvgA system promotes expression of PhoP-activated genes. As a factor connecting this signal transduction cascade, we have identified a small inner membrane protein (65 aa), B1500. Expression of the b1500 gene is directly regulated by the EvgS/EvgA system, and b1500 expression from a heterologous promoter simultaneously activated the expression of mgtA and other PhoP regulon genes. This activation was PhoQ/PhoP-dependent and EvgS/EvgA-independent. Furthermore, deletion of b1500 from an EvgS-activated strain suppressed mgtA expression. B1500 is localized in the inner membrane, and bacterial two-hybrid data showed that B1500 formed a complex with the sensor PhoQ. These results indicate that the small membrane protein, B1500, connected the signal transduction between EvgS/EvgA and PhoQ/PhoP systems by directly interacting with PhoQ, thus activating the PhoQ/PhoP system.
Subject(s)
Bacterial Proteins/metabolism , Cell Membrane/metabolism , Escherichia coli Proteins/metabolism , Membrane Proteins/metabolism , Protein Kinases/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Base Sequence , Cloning, Molecular , Escherichia coli , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial , Membrane Proteins/genetics , Molecular Sequence Data , Protein Binding , Protein Kinases/genetics , Signal Transduction , Transcription Factors/geneticsABSTRACT
We have developed two screening systems for isolating inhibitors that target bacterial two-component signal transduction: (1) a differential growth assay using a temperature-sensitive yycF mutant (CNM2000) of Bacillus subtilis, which is supersensitive to histidine kinase inhibitors, and (2) a high-throughput genetic system for targeting the homodimerization of histidine kinases essential for the bacterial two-component signal transduction. By using these methods, we have been able to identify various types of inhibitors that block the autophosphorylation of histidine kinases with different modes of actions.
