Differential regulation of glutaredoxin gene expression in response to stress conditions in the yeast Saccharomyces cerevisiae

Glutaredoxins are small heat-stable proteins that are active as glutathione-dependent oxidoreductases and are encoded by two genes, designated GRX1 and GRX2, in the yeast Saccharomyces cerevisiae. We report here that the expression of both genes is induced in response to various stress conditions including oxidative, osmotic, and heat stress and in response to stationary phase growth and growth on non-fermentable carbon sources. Furthermore, both genes are activated by the high-osmolarity glycerol pathway and negatively regulated by the Ras-protein kinase A pathway via stress-responsive STRE elements. GRX1 contains a single STRE element and is induced to significantly higher levels compared to GRX2 following heat and osmotic shock. GRX2 contains two STRE elements, and is rapidly induced in response to reactive oxygen species and upon entry into stationary phase growth. Thus, these data support the idea that the two glutaredoxin isoforms in yeast play distinct roles during normal cellular growth and in response to stress conditions.     

Induction of a growth-phase-dependent promoter triggers transcription of bolA, an Escherichia coli morphogene.

The bolA gene, which is involved in the morphogenetic pathways of Escherichia coli, was sequenced and two potential promoters were identified. Expression from promoter P1, proximal to the bolA structural gene is specifically induced during the transition to the stationary phase of growth. This promoter contains an unusual--10 region (CGGCTAGTA), which defines a new class of E. coli promoters necessary for the dramatic increase in the rate of synthesis of a large set of proteins during the cessation of logarithmic growth. This conclusion was confirmed by identifying two additional E. coli promoters and one plasmid promoter, which also were induced during the transition to the stationary phase of growth. Analysis of proteins produced during the exponential and stationary phases of growth in a bolA null mutant suggest a possible role for the BolA protein in the induction of the expression of penicillin-binding protein 6 (PBP6) in the transition to the stationary phase. Supporting this hypothesis is the presence of a putative DNA-binding domain within the bolA coding sequence.     

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.     

Activation of the expression of the microcin C51 operon upon glucose starvation of cells at the exponential growth phase

It was earlier shown that expression of the microcin C51 operon in Escherichia coli cells is activated upon decelerated growth of cells during their transition to the stationary growth phase and depends on the sigmaS subunit of RNA polymerase. Using a single-copy construct containing the cloned promoter region of the microcin C51 operon and a promoterless lac operon (P(mcc)-lac), it was shown that the promoter of the microcin operon was also induced by stress caused by the transition of cells at the exponential growth phase into the medium without glucose as a sole carbon source. Activation of P(mcc)-lac expression upon severe glucose starvation occurred in rpoS+ and rpoS- strains. In cells carrying the rpoD800 mutation that renders the sigma70 subunit of RNA polymerase temperature-sensitive, an activation of P(mcc)-lac expression was observed at nonpermissive temperature, in contrast to its complete inhibition in E. coli cells at the phase of delayed growth. Other stressors-nitrogen starvation, high temperatures, osmotic shock, tetracycline and chloramphenicol-did not activate P(mcc)-lac expression in cells at the exponential growth phase.     

Effect of Escherichia coli morphogene bolA on biofilms

Biofilm physiology is established under a low growth rate. The morphogene bolA is mostly expressed under stress conditions or in stationary phase, suggesting that bolA could be implicated in biofilm development. In order to verify this hypothesis, we tested the effect of bolA on biofilm formation. Overexpression of bolA induces biofilm development, while bolA deletion decreases biofilms.     

Growth phase-regulated expression of bolA and morphology of stationary-phase Escherichia coli cells are controlled by the novel sigma factor sigma S.

The novel sigma factor (sigma S) encoded by rpoS (katF) is required for induction of many growth phase-regulated genes and expression of a variety of stationary-phase phenotypes in Escherichia coli. Here we demonstrate that wild-type cells exhibit spherical morphology in stationary phase, whereas rpoS mutant cells remain rod shaped and are generally larger. Size reduction of E. coli cells along the growth curve is a continuous and at least biphasic process, the second phase of which is absent in rpoS-deficient cells and correlates with induction of the morphogene bolA in wild-type cells. Stationary-phase induction of bolA is dependent on sigma S. The "gearbox" a characteristic sequence motif present in the sigma S-dependent growth phase- and growth rate-regulated bolAp1 promoter, is not recognized by sigma S, since stationary-phase induction of the mcbA promoter, which also contains a gearbox, does not require sigma S, and other sigma S-controlled promoters do not contain gearboxes. However, good homology to the potential -35 and -10 consensus sequences for sigma S regulation is found in the bolAp1 promoter.     

MiR-34a inhibits lymphatic metastasis potential of mouse hepatoma cells

Genetic adaptation is one of the key features of Escherichia coli (E. coli) that ensure its survival in different hostile environments. E. coli seems to initiate biofilm development in response to specific environmental cues. A number of properties inherent within bacterial biofilms indicate that their gene expression is different from that of planktonic bacteria. Two of the possible important genes are rpoS and bolA. The rpoS gene has been known as the alternative sigma (σ) factor, which controls the expression of a large number of genes, which are involved in responses to a varied number of stresses, as well as transition to stationary phase from exponential form of growth. Morphogene bolA response to stress environment leads to round morphology of E. coli cells, but little is known about its involvement in biofilms and its development or maintenance. The purpose of this study was to understand and analyse the responses of rpoS and bolA gene to sudden change in the environment. In this study, E. coli K-12 MG1655, rpoS, and bolA mutant strains were used and gene expression was studied. Results show that both genes contribute to the ability to respond and adapt in response to various types of stresses. RpoS response to various stress environments was somehow constant in both the planktonic and biofilm phases, whereas bolA responded well under various stress conditions, in both planktonic and biofilm mode, up to 5-6-fold change in the expression was noticed in the case of pH variation and hydrogen peroxide stress (H(2)O(2)) as compared with rpoS.     

Delayed Turnover of Unphosphorylated Ssk1 during Carbon Stress Activates the Yeast Hog1 Map Kinase Pathway

In Saccharomyces cerevisiae, the Hog1 mitogen-activated protein kinase (MAPK) pathway coordinates the adaptation to osmotic stress and was recently reported to respond to acute changes in glucose levels. Similarly as in osmotic stress, glucose starvation leads to a transient accumulation of Hog1 in the nucleus. However, the kinetics and the mechanism of Hog1 activation are different for these stress conditions. During osmotic shock the activation of Hog1 can be transduced by either the Sho1 or the Sln1/Ypd1/Ssk1 branch. During glucose starvation the phosphorylation of Hog1 is slower and is completely dependent on Ssk1, but independent of Sho1. To characterize the mechanism of activation of Hog1 during carbon stress, we examined the turnover of Ssk1 protein levels upon glucose starvation in the presence of cycloheximide and monitored protein levels by western blotting. Our data demonstrate that unphosphorylated Ssk1 was quickly degraded during exponential growth and after osmotic stress but remained remarkably stable during glucose limitation. We conclude that glucose starvation induces a delay in the turnover of unphosphorylated Ssk1, which is sufficient to activate the Hog1 MAPK pathway. Although unphosphorylated Ssk1 is known to be degraded by the proteasome, its stabilization is apparently not due to changes in cellular localization or decrease in ubiquitination levels during glucose limitation.     

Cooperative regulation of DOG2, encoding 2-deoxyglucose-6-phosphate phosphatase, by Snf1 kinase and the high-osmolarity glycerol-mitogen-activated protein kinase cascade in stress responses of Saccharomyces cerevisiae

We screened the genome of Saccharomyces cerevisiae for the genes responsive to oxidative stress by using the lacZ transposon-insertion library. As a result, we found that expression of the DOG2 gene coding for 2-deoxyglucose-6-phosphate phosphatase was induced by oxidative stress. The expression of DOG2 was also induced by osmotic stress. We found a putative cis element (STRE, a stress response element) in the DOG2 promoter adjacent to a consensus sequence to which the Mig1p repressor is known to bind. The basal levels of DOG2 gene expression were increased in a mig1Delta mutant, while the derepression of DOG2 was not observed in a snf1Delta mutant under glucose-deprived conditions. Induction of the DOG2 gene expression by osmotic stress was observed in any of the three disruptants pbs2Delta, hog1Delta, and snf1Delta. However, the osmotic induction was completely abolished in both the snf1Delta pbs2Delta mutant and the snf1Delta hog1Delta mutant. Additionally, these single mutants as well as double mutants failed to induce DOG2 expression by oxidative stress. These results suggest that Snf1p kinase and the high-osmolarity glycerol-mitogen-activated protein kinase cascade are likely to be involved in the signaling pathway of oxidative stress and osmotic stress in regulation of DOG2.