Trehalose metabolism in Escherichia coli: stress protection and stress regulation of gene expression

Endogenously synthesized trehalose is a stress protectant in Escherichia coli. Externally supplied trehalose does not serve as a stress protectant, but it can be utilized as the sole source of carbon and energy. Mutants defective in trehalose synthesis display an impaired osmotic tolerance in minimal growth media without glycine betaine, and an impaired stationary-phaseinduced heat tolerance. Mechanisms for stress protection by trehalose are discussed. The genes for trehalose-6-phosphate synthase (otsA) and anabolic trehalose-6-phosphate phosphatase (otsB) constitute an operon. Their expression is induced both by osmotic stress and by growth into the stationary phase and depend on the sigma factor encoded by rpoS (katF). rpoS is amber-mutated in E. coli K-12 and its DNA sequence varies among K-12 strains. For trehalose catabolism under osmotic stress E. coli depends on the osmoticcally inducible periplasmic trehalase (TreA). In the absence of osmotic stress, trehalose induces the formation of an enzyme II^Tre (TreB) of the group translocation system, a catabolic trehalose-6-phosphate phosphatase (TreE), and an amylotrehalase (TreC) which converts trehalose to free glucose and a glucose polymer.     

The role of Corynebacterium glutamicum spiA gene in whcA-mediated oxidative stress gene regulation

The Corynebacterium glutamicum WhcA protein, which inhibits the expression of oxidative stress response genes, is known to interact with the SpiA protein. In this study, we constructed and analyzed spiA mutant cells with the goal of better understanding the function of the spiA gene. A C. glutamicum strain overexpressing the spiA gene showed retarded cell growth, which was caused by an increased sensitivity to oxidants. Expression of the spiA and whcA genes was repressed by oxidant diamide, indicating coordinate regulation and dispensability of the genes in cells under oxidative stress. In the spiA-overexpressing cells, the trx gene, which encodes thioredoxin reductase, was severely repressed. Deletion of whcA in spiA-overexpressing cells (or vice versa) produced phenotypes similar to the wild-type strain. Collectively, these data demonstrate a negative regulatory role of the spiA gene in whcA-mediated oxidative stress response and provide additional clues on the mechanism by which the whcA gene is regulated.     

A nuclear gene encoding mitochondrial Delta-pyrroline-5-carboxylate dehydrogenase and its potential role in protection from proline toxicity

Delta1-pyrroline-5-carboxylate (P5C), an intermediate in biosynthesis and degradation of proline (Pro), is assumed to play a role in cell death in plants and animals. Toxicity of external Pro and P5C supply to Arabidopsis suggested that P5C dehydrogenase (P5CDH; EC 1.2.1.12) plays a crucial role in this process by degrading the toxic Pro catabolism intermediate P5C. Also in a Deltaput2 yeast mutant, lacking P5CDH, Pro led to growth inhibition and formation of reactive oxygen species (ROS). Complementation of the Deltaput2 mutant allowed identification of the Arabidopsis P5CDH gene. AtP5CDH is a single-copy gene and the encoded protein was localized to the mitochondria. High homology of AtP5CDH to LuFIS1, an mRNA up-regulated during susceptible pathogen attack in flax, suggested a role for P5CDH in inhibition of hypersensitive reactions. An Arabidopsis mutant (cpr5) displaying a constitutive pathogen response was found to be hypersensitive to external Pro. In agreement with a role in prevention of cell death, AtP5CDH was expressed at a basal level in all tissues analysed. The highest expression was found in flowers that are known to contain the highest Pro levels under normal conditions. External supply of Pro induced AtP5CDH expression, but much more slowly than Pro dehydrogenase (AtProDH) expression. Uncoupled induction of the AtProDH and AtP5CDH genes further supports the hypothesis that P5C levels have to be tightly controlled. These results indicate that, in addition to the well-studied functions of Pro, for example in osmoregulation, the Pro metabolism intermediate P5C also serves as a regulator of cellular stress responses.     

Inhibition of aldehyde dehydrogenase 2 by oxidative stress is associated with cardiac dysfunction in diabetic rats

Left ventricular (LV) dysfunction is a common comorbidity in diabetic patients, although the molecular mechanisms underlying this cardiomyopathic feature are not completely understood. Aldehyde dehydrogenase 2 (ALDH2) has been considered a key cardioprotective enzyme susceptible to oxidative inactivation. We hypothesized that hyperglycemia-induced oxidative stress would influence ALDH2 activity, and ALDH2 inhibition would lead to cardiac functional alterations in diabetic rats. Diabetes was induced by intraperitoneal (i.p.) injection of 60 mg/kg streptozotocin. Rats were divided randomly into four groups: control, untreated diabetic, diabetic treated with N-acetylcysteine (NAC) and diabetic treated with α-lipoic acid (α-LA). Cardiac contractile function, oxidative stress markers and reactive oxygen species (ROS) levels were assessed. ALDH2 activity and expression also were determined. The role of ALDH2 activity in change in hyperglycemia-induced mitochondrial membrane potential (Δψ) was tested in cultured neonatal cardiomyocytes. Myocardial MDA content and ROS were significantly higher in diabetic rats than in controls, whereas GSH content and Mn-SOD activity were decreased in diabetic rats. Compared with controls, diabetic rats exhibited significant reduction in LV ejection fraction and fractional shortening, accompanied by decreases in ALDH2 activity and expression. NAC and α-LA attenuated these changes. Mitochondrial Δψ was decreased greatly with hyperglycemia treatment, and high glucose combined with ALDH2 inhibition with daidzin further decreased Δψ. The ALDH2 activity can be regulated by oxidative stress in the diabetic rat heart. ALDH2 inhibition may be associated with LV reduced contractility, and mitochondrial impairment aggravated by ALDH2 inhibition might reflect an underlying mechanism which causes cardiac dysfunction in diabetic rats.     

The role of iron in Campylobacter gene regulation,metabolism and oxidative stress defense

Enteric Campylobacter species cause gastrointestinal diseases in humans. Like almost all organisms, campylobacters have an absolute requirement for iron, but are faced with variable availability of iron in the environment and host tissues. Campylobacters have developed mechanisms to scavenge sufficient iron for metabolism and growth. However, iron also participates in the formation of reactive oxygen species, and this forces pathogens to maintain intracellular iron homeostasis and to cope with oxidative stresses. The presence of two separate, but possibly overlapping iron-responsive regulatory systems, which regulate iron acquisition and oxidative stress defense, and the presence of genes encoding multiple iron acquisition and detoxification systems in Campylobacter indicate the central role that iron plays in Campylobacter gene regulation and virulence.     

A role for SPINDLY gene in the regulation of oxidative stress response in Arabidopsis

SPINDLY (SPY) gene encodes a putative O-linked N-acetyl-glucosamine transferase, and yeast two-hybrid assay identified GIGANTEA (GI) as a SPY-interacting partner in Arabidopsis. GIGANTEA gene was previously shown to be involved in the regulation of oxidative stress response; however, it is unclear whether SPY gene is also involved in oxidative stress response. Here we showed that SPY plays a role in the regulation of the oxidative stress response. The spy-1 mutant was more tolerant to paraquat (PQ)-or hydrogen peroxide (H₂O₂)-mediated oxidative stress than wild-type plants. Analyses of endogenous H₂O₂ and superoxide anion radicals as well as lipid peroxidation revealed that enhanced tolerance of the spy-1 mutant to PQ-stress was not due to defects in the PQ uptake or the PQ sequestration from its site of action but rather the spy-1 mutation alleviated oxidative damage of plant cells upon PQ stress. Higher constitutive activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) in spy-1 are more likely to be due to activation of both CSD2 gene encoding chloroplast Cu/Zn SOD and APX1 gene. Taken together, these results suggest that enhanced tolerance of the spy-1 mutant to oxidative stress is associated, at least in part, with constitutive activation of CSD2 and APX1. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 4, pp. 604–611. The text was submitted by the authors in English.     

Bilirubin: its role in cytoprotection against oxidative stress

Bilirubin, the end product of heme catabolism in mammals, is generally regarded as a potentially cytotoxic, lipid-soluble waste product that needs to be excreted. However, in the last 10 years, in vitro and in vivo studies, have demonstrated that bilirubin exhibits potent anti-oxidant properties preventing the oxidative damage triggered by a wide range of oxidant-related stimuli. Therefore, the idea of a beneficial and physiological role for bilirubin in cytoprotection against short and long-lasting oxidant-mediated cell injury is highlighted here.     

The role of putrescine in of oxidative stress defense genes expression regulation in Escherichia coli

The role of putrescine in the adaptive response of Escherichia coli grown aerobically in synthetic M9 medium with glucose to the H2O2-induced oxidative stress was studied. Under oxidative stress, the expression of the single-copy reporter gene fusions oxyR::lacZ and katG::lacZ was found to undergo biphasic changes, which were most pronounced in glucose-starved E. coli cells. The concentration-dependent activating effect of putrescine on the expression of the oxyR regulon genes was maximum when the oxyR gene was inhibited by high concentrations of hydrogen peroxide.     

Deficiency in a mitochondrial aldehyde dehydrogenase increases vulnerability to oxidative stress in PC12 cells

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) plays a major role in acetaldehyde detoxification. The alcohol sensitivity is associated with a genetic deficiency of ALDH2. We have previously reported that this deficiency influences the risk for late-onset Alzheimer's disease. However, the biological effects of the deficiency on neuronal cells are poorly understood. Thus, we obtained ALDH2-deficient cell lines by introducing mouse mutant Aldh2 cDNA into PC12 cells. The mutant ALDH2 repressed mitochondrial ALDH activity in a dominant negative fashion, but not cytosolic activity. The resultant ALDH2-deficient transfectants were highly vulnerable to exogenous 4-hydroxy-2-nonenal, an aldehyde derivative generated by the reaction of superoxide with unsaturated fatty acid. In addition, the ALDH2-deficient transfectants were sensitive to oxidative insult induced by antimycin A, accompanied by an accumulation of proteins modified with 4-hydroxy-2-nonenal. Thus, these findings suggest that mitochondrial ALDH2 functions as a protector against oxidative stress.     

RNA interference and its role in the regulation of eucaryotic gene expression

Several years ago it was discovered that plant transformation with a transcribed sense transgene could shut down the expression of a homologous endogenous gene. Moreover, it was shown that the introduction into the cell of dsRNA (double-stranded RNA) containing nucleotide sequence complementary to an mRNA sequence causes selective degradation of the latter and thus silencing of a specific gene. This phenomenon, called RNA interference (RNAi) was demonstrated to be present in almost all eukaryotic organisms. RNAi is also capable of silencing transposons in germ line cells and fighting RNA virus infection. Enzymes involved in this process exhibit high homology across species. Some of these enzymes are involved in other cellular processes, for instance developmental timing, suggesting strong interconnections between RNAi and other metabolic pathways. RNAi is probably an ancient mechanism that evolved to protect eukaryotic cells against invasive forms of nucleic acids.     

An overlap between osmotic and anaerobic stress responses: a potential role for DNA supercoiling in the coordinate regulation of gene expression

The regulation of several genes in response to osmotic and anaerobic stress has been examined. We have demonstrated a clear overlap between these two regulatory signals. Thus, the osmotically induced proU and ompC genes require anaerobic growth for optimum induction while the anaerobically induced tppB gene is also regulated by osmolarity. Furthermore, normal expression of tppB and ompC requires the positive regulatory protein OmpR, yet this requirement can be partially, or even fully, overcome by altering the growth conditions. Finally, the pleiotropic, anaerobic regulatory locus, oxrC, is also shown to affect expression of the osmotically regulated proU gene. The oxrC mutation is shown to affect the level of negative supercoiling of plasmid DNA and its effects on gene expression can be explained as secondary consequences of altered DNA topology. We suggest that there is a class of 'stress-regulated' genes that are regulated by a common mechanism in response to different environmental signals. Furthermore, our data are consistent with the notion that this regulatory overlap is mediated by changes in DNA supercoiling in response to these environmental stresses.     

Mitochondrial gene expression and increased oxidative metabolism: role in increased lifespan of fat-specific insulin receptor knock-out mice

Caloric restriction, leanness and decreased activity of insulin/insulin-like growth factor 1 (IGF-1) receptor signaling are associated with increased longevity in a wide range of organisms from Caenorhabditis elegans to humans. Fat-specific insulin receptor knock-out (FIRKO) mice represent an interesting dichotomy, with leanness and increased lifespan, despite normal or increased food intake. To determine the mechanisms by which a lack of insulin signaling in adipose tissue might exert this effect, we performed physiological and gene expression studies in FIRKO and control mice as they aged. At the whole body level, FIRKO mice demonstrated an increase in basal metabolic rate and respiratory exchange ratio. Analysis of gene expression in white adipose tissue (WAT) of FIRKO mice from 6 to 36 months of age revealed persistently high expression of the nuclear-encoded mitochondrial genes involved in glycolysis, tricarboxylic acid cycle, β-oxidation and oxidative phosphorylation as compared to expression of the same genes in WAT from controls that showed a tendency to decline in expression with age. These changes in gene expression were correlated with increased cytochrome c and cytochrome c oxidase subunit IV at the protein level, increased citrate synthase activity, increased expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and PGC-1β, and an increase in mitochondrial DNA in WAT of FIRKO mice. Together, these data suggest that maintenance of mitochondrial activity and metabolic rates in adipose tissue may be important contributors to the increased lifespan of the FIRKO mouse.