Study of the TmoS/TmoT two‐component system: towards the functional characterization of the family of TodS/TodT like systems

The two‐component system TmoS/TmoT controls the expression of the toluene‐4‐monooxygenase pathway in Pseudomonas mendocina RK1 via modulation of P_tmoX activity. The TmoS/TmoT system belongs to the family of TodS/TodT like proteins. The sensor kinase TmoS is a 108 kDa protein composed of seven different domains. Using isothermal titration calorimetry we show that purified TmoS binds a wide range of aromatic compounds with high affinities. Tightest ligand binding was observed for toluene (K_D = 150 nM), which corresponds to the highest affinity measured between an effector and a sensor kinase. Other compounds with affinities in the nanomolar range include benzene, the 3 xylene isomers, styrene, nitrobenzene or p‐chlorotoluene. We demonstrate that only part of the ligands that bind to TmoS increase protein autophosphorylation in vitro and consequently pathway expression in vivo. These compounds are referred to as agonists. Other TmoS ligands, termed antagonists, failed to increase TmoS autophosphorylation, which resulted in their incapacity to stimulate gene expression in vivo. We also show that TmoS saturated with different agonists differs in their autokinase activities. The effector screening of gene expression showed that promoter activity of P_tmoX and P_todX (controlled by the TodS/TodT system) is mediated by the same set of 22 compounds. The common structural feature of these compounds is the presence of a single aromatic ring. Among these ligands, toluene was the most potent inducer of both promoter activities. Information on the TmoS/TmoT and TodS/TodT system combined with a sequence analysis of family members permits to identify distinct features that define this protein family.     

The two‐component histidine kinase Fhk1 controls stress adaptation and virulence of Fusarium oxysporum

Fungal histidine kinases (HKs) have been implicated in different processes, such as the osmostress response, hyphal development, sensitivity to fungicides and virulence. Members of HK class III are known to signal through the HOG mitogen‐activated protein kinase (MAPK), but possible interactions with other MAPKs have not been explored. In this study, we have characterized fhk1, encoding a putative class III HK from the soil‐borne vascular wilt pathogen Fusarium oxysporum. Inactivation of fhk1 resulted in resistance to phenylpyrrole and dicarboximide fungicides, as well as increased sensitivity to hyperosmotic stress and menadione‐induced oxidative stress. The osmosensitivity of Δfhk1 mutants was associated with a striking and previously unreported change in colony morphology. The Δfhk1 strains showed a significant decrease in virulence on tomato plants. Epistatic analysis between Fhk1 and the Fmk1 MAPK cascade indicated that Fhk1 does not function upstream of Fmk1, but that the two pathways may interact to control the response to menadione‐induced oxidative stress.     

Distinct functions of JNK and c-Jun in oxidant-induced hepatocyte death

Overactivation of c‐Jun N‐terminal kinase (JNK)/c‐Jun signaling is a central mechanism of hepatocyte injury and death including that from oxidative stress. However, the functions of JNK and c‐Jun are still unclear, and this pathway also inhibits hepatocyte death. Previous studies of menadione‐induced oxidant stress demonstrated that toxicity resulted from sustained JNK/c‐Jun activation as death was blocked by the c‐Jun dominant negative TAM67. To further delineate the function of JNK/c‐Jun signaling in hepatocyte injury from oxidant stress, the effects of direct JNK inhibition on menadione‐induced death were examined. In contrast to the inhibitory effect of TAM67, pharmacological JNK inhibition by SP600125 sensitized the rat hepatocyte cell line RALA255‐10G to death from menadione. SP600125 similarly sensitized mouse primary hepatocytes to menadione toxicity. Death from SP600125/menadione was c‐Jun dependent as it was blocked by TAM67, but independent of c‐Jun phosphorylation. Death occurred by apoptosis and necrosis and activation of the mitochondrial death pathway. Short hairpin RNA knockdowns of total JNK or JNK2 sensitized to death from menadione, whereas a jnk1 knockdown was protective. Jnk2 null mouse primary hepatocytes were also sensitized to menadione death. JNK inhibition magnified decreases in cellular ATP content and β‐oxidation induced by menadione. This effect mediated cell death as chemical inhibition of β‐oxidation also sensitized cells to death from menadione, and supplementation with the β‐oxidation substrate oleate blocked death. Components of the JNK/c‐Jun signaling pathway have opposing functions in hepatocyte oxidant stress with JNK2 mediating resistance to cell death and c‐Jun promoting death. J. Cell. Biochem. 113: 3254–3265, 2012. © 2012 Wiley Periodicals, Inc.     

Heat Shock Inhibits the Induced Expression of the SOS and SoxRS Regulons in Escherichia coli

Oxidative stress formed in Escherichia coli cells is known to bring about a complex induction of alternative DNA repair processes, including SOS, SoxRS, and heat-shock response (HSR). The modification by heat shock of the expression ofsfiA and soxS genes induced by oxidative agents H₂O₂, menadione and 4-nitroquinoline-1-oxide (4NQO) was studied for the first time. Quantitative parameters of gene expression were examined inE. coli strains with fused genes (promoters) sfiA::lacZ and soxS::lacZ.The expression of these genes induced by cell treatment with H₂O₂, but not menadione or 4NQO, was shown to decrease selectively after exposure to heat shock. Since genetic activity of menadione and 4NQO depends mainly on the formation of superoxide anion ,O^¯ ₂ it is assumed that the effect of selective inhibition by heat-shock of sfiA and soxS gene expression in experiments with H₂O₂ is connected with activity of DnaK heat shock protein, which, unlike other heat-shock proteins, cannot be induced by superoxide anion O^¯ ₂.     

The detection of possible sensor histidine kinases regulating citrate/malate metabolism from the bovine rumen microbial ecosystem

Aims: To detect sensor histidine protein kinases (HPKs) similar to accessory gene regulator C (AgrC) from the rumen microbial ecosystem. Methods and the Results: Genes related to sensor HPKs were amplified by PCR using two pairs of agrC‐specfic primers from DNA extracted from bovine rumen contents. The PCR products were cloned, sequenced and phylogenetically analysed. It appeared that two sequences were HPKs. Conclusions: Although amino acid sequences deduced from the nucleotide sequences obtained in this study showed high similarities with sensor HPKs responding to citrate or C₄‐dicarboxylates, they did not show high similarities with AgrC. Significance and Impact of the Study: This study revealed the presence in the rumen of sensor HPKs responding to citrate or C₄‐dicarboxylates, which could stimulate rumen fermentation. Therefore, it has been shown that citrate or C₄‐dicarboxylate metabolism is partially regulated by a two‐component regulatory system in some rumen bacteria.     

MYC‐driven inhibition of the glutamate‐cysteine ligase promotes glutathione depletion in liver cancer

How MYC reprograms metabolism in primary tumors remains poorly understood. Using integrated gene expression and metabolite profiling, we identify six pathways that are coordinately deregulated in primary MYC‐driven liver tumors: glutathione metabolism; glycine, serine, and threonine metabolism; aminoacyl‐tRNA biosynthesis; cysteine and methionine metabolism; ABC transporters; and mineral absorption. We then focus our attention on glutathione (GSH) and glutathione disulfide (GSSG), as they are markedly decreased in MYC‐driven tumors. We find that fewer glutamine‐derived carbons are incorporated into GSH in tumor tissue relative to non‐tumor tissue. Expression of GCLC, the rate‐limiting enzyme of GSH synthesis, is attenuated by the MYC‐induced microRNA miR‐18a. Inhibition of miR‐18a in vivo leads to increased GCLC protein expression and GSH abundance in tumor tissue. Finally, MYC‐driven liver tumors exhibit increased sensitivity to acute oxidative stress. In summary, MYC‐dependent attenuation of GCLC by miR‐18a contributes to GSH depletion in vivo, and low GSH corresponds with increased sensitivity to oxidative stress in tumors. Our results identify new metabolic pathways deregulated in primary MYC tumors and implicate a role for MYC in regulating a major antioxidant pathway downstream of glutamine.     

A molecular approach to drought‐induced reduction in leaf CO2 exchange in drought‐resistant Quercus ilex

Drought‐induced reduction of leaf gas exchange entails a complex regulation of the plant leaf metabolism. We used a combined molecular and physiological approach to understand leaf photosynthetic and respiratory responses of 2‐year‐old Quercus ilex seedlings to drought. Mild drought stress resulted in glucose accumulation while net photosynthetic CO₂ uptake (P_n) remained unchanged, suggesting a role of glucose in stress signaling and/or osmoregulation. Simple sugars and sugar alcohols increased throughout moderate‐to‐very severe drought stress conditions, in parallel to a progressive decline in P_n and the quantum efficiency of photosystem II; by contrast, minor changes occurred in respiration rates until drought stress was very severe. At very severe drought stress, 2‐oxoglutarate dehydrogenase complex gene expression significantly decreased, and the abundance of most amino acids dramatically increased, especially that of proline and γ‐aminobutyric acid (GABA) suggesting enhanced protection against oxidative damage and a reorganization of the tricarboxylic cycle acid cycle via the GABA shunt. Altogether, our results point to Q. ilex drought tolerance being linked to signaling and osmoregulation by hexoses during early stages of drought stress, and enhanced protection against oxidative damage by polyols and amino acids under severe drought stress.     

Ligand and antagonist driven regulation of the Vibrio cholerae quorum-sensing receptor CqsS

Quorum sensing, a bacterial cell–cell communication process, controls biofilm formation and virulence factor production in Vibrio cholerae, a human pathogen that causes the disease cholera. The major V. cholerae autoinducer is (S)‐3‐hydroxytridecan‐4‐one (CAI‐1). A membrane bound two‐component sensor histidine kinase called CqsS detects CAI‐1, and the CqsS → LuxU → LuxO phosphorelay cascade transduces the information encoded in CAI‐1 into the cell. Because the CAI‐1 ligand is known and because the signalling circuit is simple, consisting of only three proteins, this system is ideal for analysing ligand regulation of a sensor histidine kinase. Here we reconstitute the CqsS → LuxU → LuxO phosphorylation cascade in vitro. We find that CAI‐1 inhibits the initial auto‐phosphorylation of CqsS whereas subsequent phosphotransfer steps and CqsS phosphatase activity are not CAI‐1‐controlled. CAI‐1 binding to CqsS causes a conformational change that renders His194 in CqsS inaccessible to the CqsS catalytic domain. CqsS mutants with altered ligand detection specificities are faithfully controlled by their corresponding modified ligands in vitro. Likewise, pairing of agonists and antagonists allows in vitro assessment of their opposing activities. Our data are consistent with a two‐state model for ligand control of histidine kinases.     

5‐Aminoimidazole‐4‐carboxamide ribonucleoside‐mediated adenosine monophosphate‐activated protein kinase activation induces protective innate responses in bacterial endophthalmitis

The retina is considered to be the most metabolically active tissue in the body. However, the link between energy metabolism and retinal inflammation, as incited by microbial infection such as endophthalmitis, remains unexplored. In this study, using a mouse model of Staphylococcus aureus (SA) endophthalmitis, we demonstrate that the activity (phosphorylation) of 5' adenosine monophosphate‐activated protein kinase alpha (AMPKα), a cellular energy sensor and its endogenous substrate; acetyl‐CoA carboxylase is down‐regulated in the SA‐infected retina. Intravitreal administration of an AMPK activator, 5‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR), restored AMPKα and acetyl‐CoA carboxylase phosphorylation. AICAR treatment reduced both the bacterial burden and intraocular inflammation in SA‐infected eyes by inhibiting NF‐kB and MAP kinases (p38 and JNK) signalling. The anti‐inflammatory effects of AICAR were diminished in eyes pretreated with AMPK inhibitor, Compound C. The bioenergetics (Seahorse) analysis of SA‐infected microglia and bone marrow‐derived macrophages revealed an increase in glycolysis, which was reinstated by AICAR treatment. AICAR also reduced the expression of SA‐induced glycolytic genes, including hexokinase 2 and glucose transporter 1 in microglia, bone marrow‐derived macrophages and the mouse retina. Interestingly, AICAR treatment enhanced the bacterial phagocytic and intracellular killing activities of cultured microglia, macrophages and neutrophils. Furthermore, AMPKα1 global knockout mice exhibited increased susceptibility towards SA endophthalmitis, as evidenced by increased inflammatory mediators and bacterial burden and reduced retinal function. Together, these findings provide the first evidence that AMPK activation promotes retinal innate defence in endophthalmitis by modulating energy metabolism and that it can be targeted therapeutically to treat ocular infections.     

Rac1 Is a Possible Link Between Obesity and Oxidative Stress in Chinese Overweight Adolescents

Enhanced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in the monocytes occurred in metabolic syndrome, hypertension, diabetes and obese patients in adults. However, whether NADPH oxidase is involved in the oxidative stress of overweight adolescents without comorbidities is still unclear. This study aimed to identify whether and how NADPH oxidase plays a crucial role in overweight adolescents. The study was performed in 93 overweight adolescents and 31 normal weight controls. Moreover, 87 overweight adolescents were enrolled in weight‐loss program. Demographics characteristics, anthropometrics, composition and clinical characteristics were analyzed. Oxidative stress indexes including the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in plasma and the expression of NADPH oxidase in the monocytes were examined. Overweight adolescents showed a higher oxidative stress state, as indicated by decreased SOD activity and elevated MDA level (P < 0.01). Furthermore, increased NADPH oxidase activity in the monocytes was accompanied by Rac1 upregulation. A significant positive bivariate correlation was found between Rac1 expression and MDA (r = 0.289). There also was a significant positive bivariate correlation between Rac1 expression and obesity‐related indexes including BMI (r = 0.227) and percentage of trunk fat (r = 0.233). Data from weight‐loss program reinforced the results. Partial correlation analysis indicated that obesity‐induced oxidative stress and Rac1 expression is a consequence of aberrant glucose‐lipid metabolism in overweight adolescents. In conclusion, we provided novel data showing that NADPH oxidase in the monocytes was highly activated by enhancing Rac1 expression in Chinese overweight adolescents and Rac1 may act as a link between obesity and oxidative stress in overweight adolescents.     

Molecular mechanisms controlling fructose‐specific memory and catabolite repression in lactose metabolism by Streptococcus mutans

Lactose is an abundant dietary carbohydrate metabolized by the dental pathogen Streptococcus mutans. Lactose metabolism presents both classic diauxic behaviors and long‐term memory, where the bacteria can pause for >11 h before initiating growth on lactose. Here, we explored mechanisms contributing to unusual aspects of regulation of the lac operon. The fructose‐phosphate metabolites, F‐1‐P and F‐6‐P, could modulate the DNA‐binding activities of the lactose repressor. Recombinant LacR proteins bound upstream of lacA and Gal‐6‐P induced the formation of different LacR‐DNA complexes. Deletion of lacR resulted in strain‐specific growth phenotypes on lactose, but also on a number of mono‐ and di‐saccharides that involve the glucose‐PTS or glucokinase in their catabolism. The phenotypes were consistent with the novel findings that loss of LacR altered glucose‐PTS activity and expression of the gene for glucokinase. CcpA was also shown to affect lactose metabolism in vivo and to bind to the lacA promoter region in vitro. Collectively, our study reveals complex molecular circuits controlling lactose metabolism in S. mutans, where LacR and CcpA integrate cellular and environmental cues to regulate metabolism of a variety of carbohydrates that are critical to persistence and pathogenicity of S. mutans.     

A selective and sensitive fluorescent sensor for cysteine detection based on bi‐8‐carboxamidoquinoline derivative and Cu2+ complex

In this paper, a novel fluorescent sensor 1 for selective and sensitive detection of cysteine was developed based on a complex between bi‐8‐carboxamidoquinoline derivative ligand ( L ) and Cu²⁺. The interaction of Cu²⁺ with the ligand causes a dramatic fluorescence quenching most likely due to its high affinity towards Cu²⁺ and a ligand–metal charge transfer (LMCT) process. The in situ generated L–Cu ² complex was utilized as a chemosensing ensemble for cysteine. In the presence of cysteine, the fluorophore, L , was released from L–Cu ² complex because of the strong affinity of cysteine to Cu²⁺ via the Cu–S bond, leading to the fluorescence recovery of the ligand. The proposed displacement mechanism was confirmed by the results of mass spectrometry (MS) study. Under optimized conditions, the recovered fluorescence intensity is linear with cysteine concentrations in the range 1 × 10^?6 mol/l to 8 × 10^?6 mol/l. The detection limit for cysteine is 1.92 × 10^?7 mol/l. Furthermore, the established method showed a highly sensitive and selective response to cysteine among the 20 fundamental α‐amino acids used as the building blocks of proteins, after Ni²⁺ was used as a masking agent to eliminate the interference of His. The proposed sensor is applicable in monitoring cysteine in practical samples with good recovery rate.