Multiple signals modulate the activity of the complex sensor kinase TodS

The reason for the existence of complex sensor kinases is little understood but thought to lie in the capacity to respond to multiple signals. The complex, seven‐domain sensor kinase TodS controls in concert with the TodT response regulator the expression of the toluene dioxygenase pathway in Pseudomonas putida F1 and DOT‐T1E. We have previously shown that some aromatic hydrocarbons stimulate TodS activity whereas others behave as antagonists. We show here that TodS responds in addition to the oxidative agent menadione. Menadione but no other oxidative agent tested inhibited TodS activity in vitro and reduced P_todX expression in vivo. The menadione signal is incorporated by a cysteine‐dependent mechanism. The mutation of the sole conserved cysteine of TodS (C320) rendered the protein insensitive to menadione. We evaluated the mutual opposing effects of toluene and menadione on TodS autophosphorylation. In the presence of toluene, menadione reduced TodS activity whereas toluene did not stimulate activity in the presence of menadione. It was shown by others that menadione increases expression of glucose metabolism genes. The opposing effects of menadione on glucose and toluene metabolism may be partially responsible for the interwoven regulation of both catabolic pathways. This work provides mechanistic detail on how complex sensor kinases integrate different types of signal molecules.     

Solvent tolerance in Gram-negative bacteria

Bacteria have been found in all niches explored on Earth, their ubiquity derives from their enormous metabolic diversity and their capacity to adapt to changes in the environment. Some bacterial strains are able to thrive in the presence of high concentrations of toxic organic chemicals, such as aromatic compounds, aliphatic alcohols and solvents. The extrusion of these toxic compounds from the cell to the external medium represents the most relevant aspect in the solvent tolerance of bacteria, however, solvent tolerance is a multifactorial process that involves a wide range of genetic and physiological changes to overcome solvent damage. These additional elements include reduced membrane permeabilization, implementation of a stress response programme, and in some cases degradation of the toxic compound. We discuss the recent advances in our understanding of the mechanisms involved in solvent tolerance.     

Effector-repressor interactions, binding of a single effector molecule to the operator-bound TtgR homodimer mediates derepression

The RND family transporter TtgABC and its cognate repressor TtgR from Pseudomonas putida DOT-T1E were both shown to possess multidrug recognition properties. Structurally unrelated molecules such as chloramphenicol, butyl paraben, 1,3-dihydroxynaphthalene, and several flavonoids are substrates of TtgABC and activate pump expression by binding to the TtgR-operator complex. Isothermal titration calorimetry was employed to determine the thermodynamic parameters for the binding of these molecules to TtgR. Dissociation constants were in the range from 1 to 150 microm, the binding stoichiometry was one effector molecule per dimer of TtgR, and the process was driven by favorable enthalpy changes. Although TtgR exhibits a large multidrug binding profile, the plant-derived compounds phloretin and quercetin were shown to bind with the highest affinity (K(D) of around 1 microm), in contrast to other effectors (chloramphenicol and aromatic solvents) for which exhibited a more reduced affinity. Structure-function studies of effectors indicate that the presence of aromatic rings as well as hydroxyl groups are determinants for TtgR binding. The binding of TtgR to its operator DNA does not alter the protein effector profile nor the effector binding stoichiometry. Moreover, we demonstrate here for the first time that the binding of a single effector molecule to the DNA-bound TtgR homodimer induces the dissociation of the repressor-operator complex. This provides important insight into the molecular mechanism of effector-mediated derepression.     

On the mechanism of apoplastic H<Subscript>2</Subscript>O<Subscript>2</Subscript> production during lignin formation and elicitation in cultured spruce cells—peroxidases after elicitation

A cell culture of Picea abies (L.) Karst. was used for studies of H₂O₂ generation during constitutive extracellular lignin formation and after elicitation by cell wall fragments of a pathogenic fungus, Heterobasidium parviporum. Stable, micromolar levels of H₂O₂ were present in the culture medium during lignin formation. Elicitation induced a burst of H₂O₂, peaking at ca. 90 min after elicitation. Of exogenous reducing substrates that may be responsible for the synthesis of H₂O₂ from O₂, NADH stimulated H₂O₂ production irrespective of elicitation. Cysteine (Cys) and glutathione (GSH) partially scavenged the constitutive H₂O₂, but usually increased or prolonged elicitor-induced H₂O₂ formation. Culture medium peroxidases were not able to generate H₂O₂ in vitro with Cys or GSH as reductants. These thiols, however, generated H₂O₂ non-enzymically at pH 4.5. [³⁵S]Sulphate feeding to spruce cells showed that endogenous sulphur-containing compounds (including GSH, GSSG and cysteic acid) existed in the culture medium. The apoplastic levels of these were, however, undetectable by the monobromobimane method suggesting that their contribution to apoplastic H₂O₂ formation is probably minor. Azide, an inhibitor of haem-containing enzymes, slightly inhibited constitutive H₂O₂ generation but strongly delayed the elicitor-induced H₂O₂ accumulation. Diphenylene iodonium, an inhibitor of flavin-containing enzymes, efficiently inhibited H₂O₂ production irrespective of elicitation. Elicitation led to downregulation of the expression of several peroxidase genes, and peroxidase activity in the culture medium was slightly reduced. Expression of three other peroxidase genes and a respiratory burst oxidase homologue (rboh) gene were upregulated. These data suggest that both peroxidases and rboh may contribute to H₂O₂ generation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Design and synthesis of novel nitrogen-containing polyhydroxylated aromatics as HIV-1 integrase inhibitors from caffeic acid phenethyl ester

A series of nitrogen-containing polyhydroxylated aromatics from caffeic acid phenethyl ester were designed and synthesized as HIV-1 integrase inhibitors. Most of these compounds exhibited potent inhibitory activities at micromolar concentrations against HIV-1 integrase in the 3′-end processing and the strand transfer. Their key structure–activity relationship was also discussed.     

Receptor assay guided structure‐activity studies of helicokinin insect neuropeptides and peptidomimetic analogues

Neuropeptides control numerous physiological processes in insects. The regulation of water balance is a crucial aspect of homeostasis in terrestrial insects and has been shown to be under endocrine control, primarily by corticotrophin releasing factor (CRF)‐related peptides and kinins. For helicokinin I, a diuretic neuropeptide from the economically important insect pest Heliothis virescens, detailed structure‐activity relationships have been established based on truncated structures, diverse amino acid scans and peptidomimetic analogues. The activities of selected compounds on functional expressed helicokinin receptors are compared with the results of a Malphigian tubule assay. Implications for further peptidomimetic variations are provided. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Autophagy of HSP70 and chelation of lysosomal iron in a non-redox-active form

Lysosomes contain most of the cell's supply of labile iron, which makes them sensitive to oxidative stress. To keep lysosomal labile iron at a minimum, a cellular strategy might be to autophagocytose iron binding proteins that temporarily would chelate iron in a non-redox-active form. Previously we have shown that autophagy of metallothioneins, as well as of non-Fe-saturated ferritin, meets this goal. Here we add another stress-regulated protein to the list, namely HSP70.