Bacillus subtilis MrgA is a Dps(PexB) homologue: evidence for metalloregulation of an oxidative-stress gene

Upon the cessation of exponential growth, Bacillus subtilis enters a transition phase leading to either sporulation or a non-sporulating stationary phase. During this transition period, cells secrete degradative enzymes, become competent for DNA transformation, are motile and acquire resistance to oxidative killing. We now report that mrgA , originally identified as a gene repressed by metal ions, encodes a member of the Dps/PexB family of general stress proteins. Like Escherichia coli Dps(PexB), MrgA forms highly stable, multimeric protein-DNA complexes which accumulate in stationary-phase cells and protect against oxidative killing. MrgA is part of an inducible oxidative stress response in B. subtilis : mrgA is induced by hydrogen peroxide, and a strain lacking MrgA displays increased sensitivity to oxidative killing. In addition, a hydrogen peroxide-resistant mutant, which constitutively overproduces catalase and alkyl hydroperoxide reductase, also overproduces MrgA. These results indicate a complex interplay between metal ions and the expression of the B. subtilis oxidative stress response.     

Salt stress adaptation of Bacillus subtilis: a physiological proteomics approach

The adaptation to osmotic stress is crucial for growth and survival of Bacillus subtilis in its natural ecosystem. Dual channel imaging and warping of 2-D protein gels were used to visualize global changes in the protein synthesis pattern of cells in response to osmotic stress (6% NaCl). Many vegetative enzymes were repressed in response to salt stress and derepressed after resumption of growth. The enzymes catalyzing the metabolic steps from glucose to 2-oxoglutarate, however, were almost constantly synthesized during salt stress despite the growth arrest. This indicates an enhanced need for the proline precursor glutamate. The synthesis of enzymes involved in sulfate assimilation and in the formation of Fe-S clusters was also induced, suggesting an enhanced need for the formation or repair of Fe-S clusters in response to salt stress. One of the most obvious changes in the protein synthesis profile can be followed by the very strong induction of the SigB regulon. Furthermore, members of the SigW regulon and of the PerR regulon, indicating oxidative stress after salt challenge, were also induced. This proteomic approach provides an overview of cell adaptation to an osmotic upshift in B. subtilis visualizing the most dramatic changes in the protein synthesis pattern.     

A comparative proteomic analysis for capsaicin-induced apoptosis between human hepatocarcinoma (HepG2) and human neuroblastoma (SK-N-SH) cells

The endogenous ROS levels were increased during HepG2 apoptosis, whereas they were decreased during SK-N-SH apoptosis in response to capsaicin treatments. We used 2-DE-based proteomics to analyze the altered protein levels in both cells, with special attention on oxidative stress proteins before and after capsaicin treatments. The 2-DE analysis demonstrated that 23 proteins were increased and 26 proteins were decreased significantly (fold change>1.4) in capsaicin-treated apoptotic HepG2 and SK-N-SH cells, respectively. The distinct effect of capsaicin-induced apoptosis on the expression pattern of HepG2 proteins includes the downregulation of some antioxidant enzymes including aldose reductase (AR), catalase, enolase 1, peroxiredoxin 1, but upregulation of peroxiredoxin 6, cytochrome c oxidase, and SOD2. In contrast, most antioxidant enzymes were increased in SK-N-SH cells in response to capsaicin, where catalase might play a pivotal role in maintenance of low ROS levels in the course of apoptosis. The global gene expression for oxidative stress and antioxidant defense genes revealed that 84 gene expressions were not significantly different in HepG2 cells between control and capsaicin-treated cells. In contrast, a number of oxidative genes were downregulated in SK-N-SH cells, supporting the evidence of low ROS environment in apoptotic SK-N-SH cells after capsaicin treatment. It was concluded that the different relationship between endogenous ROS levels and apoptosis of two cancer cells presumably resulted from complicated expression patterns of many oxidative stress and antioxidant genes, rather than the individual role of some classical antioxidant enzymes such as SOD and catalase.     

Endogenous antioxidant defence system in rat liver following mercury chloride oral intoxication

Mercury is a highly toxic metal which induces oxidative stress. Superoxide dismutases, catalase, and glutathion peroxidase are proteins involved in the endogenous antioxidant defence system. In the present study rats were administered orally, by gavage, a single daily dose of HgCl2 for three consecutive days. In order to find a relation between the proteins involved in the antioxidant defence and mercury intoxication, parameters of liver injury, redox state of the cells, as well as intracellular protein levels and enzyme activities of Mn-dependent superoxide dismutase (MnSOD), Cu-Zn-dependent superoxide dismutase (CuZnSOD), catalase, and glutathione peroxidase (GPx) were assayed both in blood and in liver homogenates. HgCl2 at the doses of 0.1 mg/kg produced liver damage which that was detected by a slight increase in serum alanine aminotransferase and gamma glutamyl transferase. Hepatic GSH/GSSG ratio was assayed as a parameter of oxidative stress and a significant decrease was detected, as well as significant increases in enzyme activities and protein levels of hepatic antioxidant defence systems. Changes in both MnSOD and CuZnSOD were parallel to those of liver injury and oxidative stress, while the changes detected in catalase and GPx activities were progressively increased along with the mercury intoxication. Other enzyme activities related to the glutathione redox cycle, such as glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PDH), also increased progressively. We conclude that against low doses of mercury that produce a slight oxidative stress and liver injury, the response of the liver was to induce the synthesis and activity of the enzymes involved in the endogenous antioxidant system. The activities of all the enzymes assayed showed a rapidly induced coordinated response.     

Bacillus subtilis tolerance of moderate concentrations of rifampin involves the sigma(B)-dependent general and multiple stress response.

Low concentrations of the RNA polymerase inhibitor rifampin added to an exponentially growing culture of Bacillus subtilis led to an instant inhibition of growth. Survival experiments revealed that during the growth arrest the cells became tolerant to the antibiotic and the culture was able to resume growth some time after rifampin treatment. L-[(35)S]methionine pulse-labeled protein extracts were separated by two-dimensional polyacrylamide gel electrophoresis to investigate the change in the protein synthesis pattern in response to rifampin. The sigma(B)-dependent general stress proteins were found to be induced after treatment with the antibiotic. Part of the oxidative stress signature was induced as indicated by the catalase KatA and MrgA. The target protein of rifampin, the beta subunit (RpoB) of the DNA-dependent RNA polymerase, and the flagellin protein Hag belonging to the sigma(D) regulon were also induced. The rifampin-triggered growth arrest was extended in a sigB mutant in comparison to the wild-type strain, and the higher the concentration, the more pronounced this effect was. Activity of the RsbP energy-signaling phosphatase in the sigma(B) signal transduction network was also important for this protection against rifampin, but the RsbU environmental signaling phosphatase was not required. The sigB mutant strain was less capable of growing on rifampin-containing agar plates. When plated from a culture that had already reached stationary phase without previous exposure to the antibiotic during growth, the survival rate of the wild type exceeded that of the sigB mutant by a factor of 100. We conclude that the general stress response of B. subtilis is induced by rifampin depending on RsbP activity and that loss of SigB function causes increased sensitivity to the antibiotic.     

Survival and antioxidant defence of the yeast Saccharomyces cerevisiae during starvation and oxidative stress

The role of catalase in response of the yeast Saccharomyces cerevisiae to oxidative stress induced by hydrogen peroxide under starvation was investigated. It was shown that under conditions used in this study 0.5 mM H2O2 did not change the number of viable cells in the wild strain YPH250, but this parameter was decreased by 15% in the acatalsaemic strain YWT1. Cells treatment with 0.5 mM H2O2 for 30 min did not modify the levels of carbonyl proteins in the parental strain, but caused its 1.4-fold increase in the defective strain. The observed 1.5-fold activation of catalase in the wild strain cells in response to H2O2-stress suggests that under starvation conditions catalase can be involved in the yeast cell protection, particularly they can prevent oxidative modification of some antioxidant and associated enzymes.     

Bacillus subtilis Vegetative Catalase Is an Extracellular Enzyme

Strong catalase activity was secreted by Bacillus subtilis cells during stationary growth phase in rich medium but not in sporulation-inducing medium. N-terminal sequencing indicated that the secreted activity was due to the vegetative catalase KatA, previously considered an endocellular enzyme. Extracellular catalase protected B. subtilis cells from oxidative assault.     

Characterization of an inducible oxidative stress system in Bacillus subtilis.

Exponentially growing cells of Bacillus subtilis demonstrated inducible protection against killing by hydrogen peroxide when prechallenged with a nonlethal dose of this oxidative agent. Cells deficient in a functional recE+ gene product were as much as 100 times more sensitive to the H2O2 but still exhibited an inducible protective response. Exposure to hydrogen peroxide also induced the recE(+)-dependent DNA damage-inducible (din) genes, the resident prophage, and the product of the recE+ gene itself. Thus hydrogen peroxide is capable of inducing the SOS-like or SOB system of B. subtilis. However, the induction of this DNA repair system by other DNA-damaging agents is not sufficient to activate the protective response to hydrogen peroxide. Therefore, at least one more regulatory network (besides the SOB system) that responds to oxidative stress must exist. Furthermore, the data presented indicate that a functional catalase gene is necessary for this protective response.     

RpoS involvement and requirement for exogenous nutrient for osmotically induced cross protection in Vibrio vulnificus

Vibrio vulnificus is an opportunistic human pathogen which is the causative agent of food-borne disease and wound infections. V. vulnificus is able to adapt to a variety of potentially stressful environmental changes, such as osmotic, nutrient, and temperature variations in estuarine environments, as well as oxidative, osmotic, and acidity differences following infection of a human host. After exposure to sub-lethal levels of a particular environmental stress, many bacteria become resistant to unrelated stresses, a phenomenon termed cross protection. In this study, we examined the ability of osmotic shock to cross protect V. vulnificus to high temperature as well as oxidative stress. Log phase cells of V. vulnificus strain C7184o were cross protected by prior osmotic shock to both heat and oxidative challenge, but only when exogenous nutrient was present during the osmotic upshift. Further, and unlike other bacteria, nutrient starvation alone did not result in cross protection against either stress. When small amounts of nutrient were present during osmotic shock, cross protection to an otherwise lethal heat challenge developed extremely rapidly, with significant protection seen within 10 min. Cross protection to oxidative stress was slower to develop, requiring several hours. Although stationary phase alone conferred some cross protection to heat and oxidative stress, the alternate sigma factor RpoS was required for complete cross protection of log phase cells to oxidative stress but not for resistance to heat challenge. Together these findings suggest that the cross protective response in V. vulnificus is complex and appears to involve multiple mechanisms.     

Estrogen down-regulates uncoupling proteins and increases oxidative stress in breast cancer

Oxidative stress has been postulated as one of the mechanisms underlying the estrogen carcinogenic effect in breast cancer. Estrogens are known to increase mitochondrial-derived reactive oxygen species (ROS) by an unknown mechanism. Given that uncoupling proteins (UCPs) are key regulators of mitochondrial energy efficiency and ROS production, our aim was to check the presence and activity of UCPs in estrogen receptor (ER)-positive and ER-negative breast cancer cells and tumors, as well as their relation to oxidative stress. Estrogen (1 nM) induced higher oxidative stress in the ER-positive MCF-7 cell line, showing increased mitochondrial membrane potential, H₂O₂ levels, and DNA and protein damage compared to ER-negative MDA-MB-231 cells. All isoforms of uncoupling proteins were highly expressed in ER-positive breast cancer cells and tumors compared to negative ones. ROS production in mitochondria isolated from MCF-7 was increased by inhibition of UCPs with GDP, but not in mitochondria from MDA-MB-231. Estrogen treatment decreased uncoupling protein and catalase levels in MCF-7 and decreased GDP-dependent ROS production in isolated mitochondria. On the whole, these results suggest that estrogens, through an ER-dependent mechanism, may increase mitochondrial ROS production by repressing uncoupling proteins, which offers a new perspective on the understanding of why estrogens are a risk factor for breast cancer.     

Role of the antioxidant system in response of Escherichia coli bacteria to cold stress

The response of aerobically grown Escherichia coli cells to the cold shock induced by the rapid lowering of growth temperature from 37 to 20 degrees C was found to be basically the same as the oxidative stress response. The enhanced sensitivity of cells deficient in two superoxide dismutases, Mn-SOD and Fe-SOD, and the increased expression of the Mn-SOD gene, sodA, in response to cold stress were interpreted as both oxidative and cold stresses are due to a rise in the intracellular level of superoxide anion. The long-term cultivation of E. coli at 20 degrees C was also accompanied by the typical oxidative stress response reactions--an enhanced expression of the Mn-SOD and catalase HPI genes and a decrease in the intracellular level of reduced glutathione (GSH) and in the GSH/GSSG ratio.     

Stress-induced proteins of Agrobacterium tumefaciens

The pattern of proteins produced by bacteria represents the physiological state of the organism as well as the environmental conditions encountered. Environmental stress induces the expression of several regulons encoding stress proteins. Extensive information about the proteins which constitute these regulons (or stimulons) and their control is available for very few bacteria, such as the Gram-positive Bacillus subtilis and the Gram-negative Escherichia coli (gamma-proteobacteria) and is minimal for all other bacteria. Agrobacterium tumefaciens is a Gram-negative plant pathogen of the alpha-proteobacteria, which constitutes the main tool for plant recombinant genetics. Our previous studies on the control of chaperone-coding operons indicated that A. tumefaciens has unique features and combines regulatory elements from both B. subtilis and E. coli. Therefore, we examined the patterns of proteins induced in A. tumefaciens by environmental changes using two-dimensional gel electrophoresis and dual-channel image analysis. Shifts to high temperature, oxidative and mild acid stresses stimulated the expression of 97 proteins. The results indicate that most of these stress-induced proteins (80/97) were specific to one stress stimulon. Only 10 proteins appear to belong to a general stress regulon.     

Not changes in membrane fluidity but proteotoxic stress triggers heat shock protein expression in Chlamydomonas reinhardtii

A conserved reaction of all organisms exposed to heat stress is an increased expression of heat shock proteins (HSPs). Several studies have proposed that HSP expression in heat‐stressed plant cells is triggered by an increased fluidity of the plasma membrane. Among the main lines of evidence in support of this model are as follows: (a) the degree of membrane lipid saturation was higher in cells grown at elevated temperatures and correlated with a lower amplitude of HSP expression upon a temperature upshift, (b) membrane fluidizers induce HSP expression at physiological temperatures, and (c) membrane rigidifier dimethylsulfoxide dampens heat‐induced HSP expression. Here, we tested whether this holds also for Chlamydomonas reinhardtii. We show that heat‐induced HSP expression in cells grown at elevated temperatures was reduced because they already contained elevated levels of cytosolic HSP70A/90A that apparently act as negative regulators of heat shock factor 1. We find that membrane rigidifier dimethylsulfoxide impaired translation under heat stress conditions and that membrane fluidizer benzyl alcohol not only induced HSP expression but also caused protein aggregation. These findings support the classical model for the cytosolic unfolded protein response, according to which HSP expression is induced by the accumulation of unfolded proteins. Hence, the membrane fluidity model should be reconsidered.     

Nitric oxide stress induces different responses but mediates comparable protein thiol protection in Bacillus subtilis and Staphylococcus aureus

The nonpathogenic Bacillus subtilis and the pathogen Staphylococcus aureus are gram-positive model organisms that have to cope with the radical nitric oxide (NO) generated by nitrite reductases of denitrifying bacteria and by the inducible NO synthases of immune cells of the host, respectively. The response of both microorganisms to NO was analyzed by using a two-dimensional gel approach. Metabolic labeling of the proteins revealed major changes in the synthesis pattern of cytosolic proteins after the addition of the NO donor MAHMA NONOate. Whereas B. subtilis induced several oxidative stress-responsive regulons controlled by Fur, PerR, OhrR, and Spx, as well as the general stress response controlled by the alternative sigma factor SigB, the more resistant S. aureus showed an increased synthesis rate of proteins involved in anaerobic metabolism. These data were confirmed by nuclear magnetic resonance analyses indicating that NO causes a drastically higher increase in the formation of lactate and butanediol in S. aureus than in B. subtilis. Monitoring the intracellular protein thiol state, we observed no increase in reversible or irreversible protein thiol modifications after NO stress in either organism. Obviously, NO itself does not cause general protein thiol oxidations. In contrast, exposure of cells to NO prior to peroxide stress diminished the irreversible thiol oxidation caused by hydrogen peroxide.     

Reactive oxygen species differentially affect T cell receptor-signaling pathways

Oxidative stress plays an important role in the induction of T lymphocyte hyporesponsiveness observed in several human pathologies including cancer, rheumatoid arthritis, leprosy, and AIDS. To investigate the molecular basis of oxidative stress-induced T cell hyporesponsiveness, we have developed an in vitro system in which T lymphocytes are rendered hyporesponsive by co-culture with oxygen radical-producing activated neutrophils. We have observed a direct correlation between the level of T cell hyporesponsiveness induced and the concentration of reactive oxygen species produced. Moreover, induction of T cell hyporesponsiveness is blocked by addition of N-acetyl cysteine, Mn(III)tetrakis(4-benzoic acid)porphyrin chloride, and catalase, confirming the critical role of oxidative stress in this system. The pattern of tyrosine-phosphorylated proteins was profoundly altered in hyporesponsive as compared with normal T cells. In hyporesponsive T cells, T cell receptor (TCR) ligation no longer induced phospholipase C-gamma1 activation and caused reduced Ca(2+) flux. In contrast, despite increased levels of ERK1/2 phosphorylation, TCR-dependent activation of mitogen-activated protein kinase ERK1/2 was unaltered in hyporesponsive T lymphocytes. A late TCR-signaling event such as caspase 3 activation was as well unaffected in hyporesponsive T lymphocytes. Our data indicate that TCR-signaling pathways are differentially affected by physiological levels of oxidative stress and would suggest that although "hyporesponsive" T cells have lost certain effector functions, they may have maintained or gained others.     

Kinetic studies of temperature-induced helix hand inversion in Bacillus subtilis macrofibers.

The inversion of Bacillus subtilis macrofibers from right to left handedness induced by a temperature upshift was compared with inversion from left to right handedness induced by a temperature downshift. Following an upshift the new steady-state growth rate was achieved prior to inversion of helix orientation. There was no discernible perturbation of growth rate at the time of inversion. The time required after a temperature shift up or down for fiber rotation in the original sense to cease was dependent on the temperature to which the fibers were transferred and was always shortest when this temperature was highest. The results suggest a basic asymmetry in the two inversion processes. Cessation of rotation in the right-to-left inversion appeared to reflect contributions of the old and new wall materials that depended on their twist values, whereas the left-to-right inversion appeared to require that a specific amount of newly made wall material be inserted into the cell surface. The degree of twist of the newly inserted right-handed material appeared not to influence the timing of inversion.     

Mitochondrial O2*- and H2O2 mediate glucose deprivation-induced stress in human cancer cells

The hypothesis that glucose deprivation-induced cytotoxicity in transformed human cells is mediated by mitochondrial O2*- and H2O2 was first tested by exposing glucose-deprived SV40-transformed human fibroblasts (GM00637G) to electron transport chain blockers (ETCBs) known to increase mitochondrial O2*- and H2O2 production (antimycin A (AntA), myxothiazol (Myx), or rotenone (Rot)). Glucose deprivation (2-8 h) in the presence of ETCBs enhanced parameters indicative of oxidative stress (i.e. GSSG and steady-state levels of oxygen-centered radicals) as well as cytotoxicity. Glucose deprivation in the presence of AntA also significantly enhanced cytotoxicity and parameters indicative of oxidative stress in several different human cancer cell lines (PC-3, DU145, MDA-MB231, and HT-29). In addition, human osteosarcoma cells lacking functional mitochondrial electron transport chains (rho0) were resistant to glucose deprivation-induced cytotoxicity and oxidative stress in the presence of AntA. In the absence of ETCBs, aminotriazole-mediated inactivation of catalase in PC-3 cells demonstrated increases in intracellular steady-state levels of H2O2 during glucose deprivation. Finally, in the absence of ETCBs, overexpression of manganese containing superoxide dismutase and/or mitochondrial targeted catalase using adenoviral vectors significantly protected PC-3 cells from toxicity and oxidative stress induced by glucose deprivation with expression of both enzymes providing greater protection than was seen with either alone. Overall, these findings strongly support the hypothesis that mitochondrial O2*- and H2O2 significantly contribute to glucose deprivation-induced cytotoxicity and metabolic oxidative stress in human cancer cells.     

Stress- and Growth Phase-Associated Proteins of Clostridium acetobutylicum

The response of Clostridium acetobutylicum ATCC 4259 to the stresses produced by a temperature upshift from 28°C to 45°C and by exposure of the organisms to 0.1% n-butanol or to air was examined by analysis of pulse-labeled proteins. The stress response was the induction of the synthesis of a number of proteins, some of which were elicited by the three forms of stress. Eleven heat shock proteins were identified by two-dimensional electrophoresis, as were two proteins whose synthesis was heat sensitive. In the absence of applied stress, the synthesis of four proteins was found to be associated with the growth phase in batch culture; three of these proteins had a higher rate of de novo synthesis when the cells entered the solvent production phase. One of the stress-induced proteins, hsp74, was partially purified an found to be immunologically related to Escherichia coli heat shock protein Dnak. The similarities of the proteins induced at the onset of solventogenesis and by stress suggest a relationship between the two processes.