The whcA gene plays a negative role in oxidative stress response of Corynebacterium glutamicum

In this study, we analyzed the whcA gene from Corynebacterium glutamicum , which codes for a homologue of the WhiB-family of proteins. Deletion of the gene did not affect the growth of the mutant cells, indicating that the whcA gene was not essential under ordinary growth conditions. However, cells overexpressing the protein not only showed retarded growth as compared with the wild-type or the Δ whcA mutant cells but also showed increased sensitivity to a variety of oxidants, such as diamide, menadione, and hydrogen peroxide. Thioredoxin reductase activity was repressed in the whcA -overexpressing cells, whereas its activity in the Δ whcA mutant strain was derepressed regardless of the presence of oxidative stress. The whcA gene was constitutively expressed throughout the growth phase and its expression level was not affected by oxidative stress. A set of proteins under the control of whcA were identified by two-dimensional polyacrylamide gel electrophoresis and they were annotated as NADH oxidase, alcohol dehydrogenase, quinone reductase, and cysteine desulfurase. The corresponding genes encoding the identified proteins were not transcribed in Δ sigH mutant cells. Collectively, these data suggest that the whcA gene of C. glutamicum plays a negative role in the sigH -mediated stress response pathway.     

The alternative sigma factor SigH regulates major components of oxidative and heat stress responses in Mycobacterium tuberculosis

Mycobacterium tuberculosis is a specialized intracellular pathogen that must regulate gene expression to overcome stresses produced by host defenses during infection. SigH is an alternative sigma factor that we have previously shown plays a role in the response to stress of the saprophyte Mycobacterium smegmatis. In this work we investigated the role of sigH in the M. tuberculosis response to heat and oxidative stress. We determined that a M. tuberculosis sigH mutant is more susceptible to oxidative stresses and that the inducible expression of the thioredoxin reductase/thioredoxin genes trxB2/trxC and a gene of unknown function, Rv2466c, is regulated by sigH via expression from promoters directly recognized by SigH. We also determined that the sigH mutant is more susceptible to heat stress and that inducible expression of the heat shock genes dnaK and clpB is positively regulated by sigH. The induction of these heat shock gene promoters but not of other SigH-dependent promoters was markedly greater in response to heat versus oxidative stress, consistent with their additional regulation by a heat-labile repressor. To further understand the role of sigH in the M. tuberculosis stress response, we investigated the regulation of the stress-responsive sigma factor genes sigE and sigB. We determined that inducible expression of sigE is regulated by sigH and that basal and inducible expression of sigB is dependent on sigE and sigH. These data indicate that sigH plays a central role in a network that regulates heat and oxidative-stress responses that are likely to be important in M. tuberculosis pathogenesis.     

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.     

The high-affinity cAMP phosphodiesterase of Saccharomyces cerevisiae is the major determinant of cAMP levels in stationary phase: involvement of different branches of the Ras-cyclic AMP pathway in stress responses

The Ras-cyclic AMP (cAMP) pathway is a major determinant of intrinsic stress resistance of the yeast Saccharomyces cerevisiae. Here, we isolated IRA2, encoding the Ras GTPase activator, as a global stress response gene. Subsequently, we studied the other negative regulators on the separate branch of the Ras-cAMP pathway, the low- or high-affinity cAMP phosphodiesterase encoded by PDE1 or PDE2, respectively. Deletion of PDE2, similar to ira2 deletion, rendered cells sensitive to freeze-thawing, peroxides, paraquat, cycloheximide, heavy metals, NaCl, heat, or cold shock. However, deletion of PDE1 did not affect stress tolerance, although it exacerbated stress sensitivity caused by the pde2 deletion, indicating that PDE1 can partly compensate for PDE2. Deletion of IRA2 uniquely led to high sensitivity to cumene hydroperoxide, suggesting that IRA2 may have a distinct role for the response to this stress. Stress sensitivity of yeast cells in general correlated with the basal level of cAMP. Interestingly, yeast cells lacking PDE2 maintained higher cAMP levels in stationary phase than exponential growth phase, suggesting that Pde2p is the major regulator of cAMP levels in stationary phase. Depletion of Ras activity could not effectively suppress stress sensitivity caused by lack of cAMP phosphodiesterases although it could suppress stress sensitivity caused by lack of IRA2, indicating that cAMP accumulation in stationary phase can be mediated by other signaling proteins in addition to Ras. Our study shows that control of cAMP basal levels is important for determining intrinsic stress tolerance of yeast, and that the cAMP level during stationary phase is a result of a dynamic balance between its rates of synthesis and degradation.     

Proteomic analysis of the oxidative stress response in Candida albicans

An efficient oxidative stress response (OSR) is important for the facultative pathogenic yeast Candida albicans to survive within the human host. We used a large scale 2-D protein gel electrophoresis approach to analyze the stress response mechanisms of C. albicans after treatment with hydrogen peroxide and the thiol oxidizing agent, diamide. Quantitation of in vivo protein synthesis after pulse labeling of the proteins with radioactive L-[35S]-methionine resulted in characteristic proteome signatures for hydrogen peroxide and diamide with significant overlap of 21 up-regulated proteins for both stressors. Among the induced proteins were enzymes with known antioxidant functions like catalase or thioredoxin reductase and a set of oxidoreductases. 2-D gel analysis of mutants in the CAP1 gene revealed that the synthesis of 12 proteins is controlled by the oxidative stress regulator Cap1p. Stressing its importance for the C. albicans OSR, all 12 proteins were also induced after oxidative challenge by hydrogen peroxide or diamide.     

Protection against oxidative stress in Escherichia coli stationary phase by a phosphate concentration-dependent genes expression

Escherichia coli gradually decline the capacity to resist oxidative stress during stationary phase. Besides the aerobic electron transport chain components are down-regulated in response to growth arrest. However, we have previously reported that E. coli cells grown in media containing at least 37 mM phosphate maintained ndh expression in stationary phase, having high viability and low NADH/NAD⁺ ratio. Here we demonstrated that, in the former condition, other aerobic respiratory genes (nuoAB, sdhC, cydA, and ubiC) expression was maintained. In addition, reactive oxygen species production was minimal and consequently the levels of thiobarbituric acid-reactive substances and protein carbonylation were lower than the expected for stationary cells. Interestingly, defense genes (katG and ahpC) expression was also maintained during this phase. Our results indicate that cells grown in high phosphate media exhibit advantages to resist endogenous and exogenous oxidative stress in stationary phase.     

Transaldolase exhibits a protective role against menadione toxicity in Xanthomonas campestris pv. phaseoli

A talA gene encoded transaldolase, a rate-limiting enzyme in the non-oxidative branch of the pentose-phosphate pathway, was cloned from Xanthomonas campestris pv. phaseoli. talA located in a region of the bacterial genome rich in genes involved in oxidative stress protection and regulation. TalA from X. campestris pv. phaseoli showed a high degree of homology to many previously reported transaldolases from both prokaryotic and eukaryotic sources. The expression of X. campestris pv. phaseoli talA was high at log-phase of growth, then declined at stationary phase, and could not be induced by oxidants. A talA mutant constructed by insertional inactivation did not possess any detectable transaldolase activity. Lack of a functional talA gene did not affect bacterial growth in a rich medium containing glucose or sucrose as a carbon source. However, the talA knockout mutant showed increased sensitivity to the superoxide generator menadione, but not to other oxidants. This increased menadione sensitivity phenotype could be complemented by expression of talA in a plasmid vector. The data demonstrated a novel and essential role of transaldolase in protection against menadione toxicity in X. campestris.     

Antioxidant defence system during exponential and stationary growth phases of Phycomyces blakesleeanus: Response to oxidative stress by hydrogen peroxide

An analysis of the components of the antioxidant defence system in exponential and stationary growth phases of filamentous fungus Phycomyces blakesleeanus and the response to the oxidative stress hydrogen peroxide were performed. There is a strong positive correlation between mycelial antioxidant capacity and the contents of gallic acid, d-erythroascorbate (d-EAA) or d-erythroascorbate monoglucoside (d-EAAG). These secondary metabolites are specifically synthesized by this fungus and reach maximal values in the stationary growth phase, suggesting that they can play some role in the antioxidant defence system of this fungus. There is a differential expression of the two more notable antioxidant activities, catalase (CAT) and superoxide dismutase (SOD), depending of the growth stage of P. blakesleeanus, CAT being expressed in the exponential and SOD in the stationary phase. Phycomyces blakesleeanus showed a high resistance to the oxidative stress caused by H₂O₂ (50 and 200 mM) which was higher in exponential phase. This higher resistance can be explained by the presence of CAT, glutathione peroxidase (GPx), and the probable contribution of glutathione-S-transferase (GST) and high levels of reduced form of glutathione (GSH). The transition to stationary phase was accompanied with a higher physiological oxidative damage illustrated by the higher protein carbonylation. In this growth stage the resistance of the fungus to the oxidative stress caused by H₂O₂ could be explained by the presence of SOD, GPx, and the probable contribution of GST as well as of secondary metabolites, mainly d-EAA and d-EAAG. These results highlight a specific response to oxidative stress by H₂O₂ depending on the growth phase of P. blakesleeanus.