Mitogen-activated protein kinase Hog1 mediates adaptation to G1 checkpoint arrest during arsenite and hyperosmotic stress

Cells slow down cell cycle progression in order to adapt to unfavorable stress conditions. Yeast (Saccharomyces cerevisiae) responds to osmotic stress by triggering G₁ and G₂ checkpoint delays that are dependent on the mitogen-activated protein kinase (MAPK) Hog1. The high-osmolarity glycerol (HOG) pathway is also activated by arsenite, and the hog1Δ mutant is highly sensitive to arsenite, partly due to increased arsenite influx into hog1Δ cells. Yeast cell cycle regulation in response to arsenite and the role of Hog1 in this process have not yet been analyzed. Here, we found that long-term exposure to arsenite led to transient G₁ and G₂ delays in wild-type cells, whereas cells that lack the HOG1 gene or are defective in Hog1 kinase activity displayed persistent G₁ cell cycle arrest. Elevated levels of intracellular arsenite and “cross talk” between the HOG and pheromone response pathways, observed in arsenite-treated hog1Δ cells, prolonged the G₁ delay but did not cause a persistent G₁ arrest. In contrast, deletion of the SIC1 gene encoding a cyclin-dependent kinase inhibitor fully suppressed the observed block of G₁ exit in hog1Δ cells. Moreover, the Sic1 protein was stabilized in arsenite-treated hog1Δ cells. Interestingly, Sic1-dependent persistent G₁ arrest was also observed in hog1Δ cells during hyperosmotic stress. Taken together, our data point to an important role of the Hog1 kinase in adaptation to stress-induced G₁ cell cycle arrest.     

MAP KINASE PHOSPHATASE1 promotes osmotolerance by suppressing PHYTOALEXIN DEFICIENT4-independent immunity

Initial exposure of plants to osmotic stress caused by drought, cold, or salinity leads to acclimation, termed acquired tolerance, to subsequent severe stresses. Acquired osmotolerance induced by salt stress is widespread across Arabidopsis (Arabidopsis thaliana) accessions and is conferred by disruption of a nucleotide-binding leucine-rich repeat gene, designated ACQUIRED OSMOTOLERANCE. De-repression of this gene under osmotic stress causes detrimental autoimmunity via ENHANCED DISEASE SUSCEPTIBILITY1 and PHYTOALEXIN DEFICIENT4 (PAD4). However, the mechanism underlying acquired osmotolerance remains poorly understood. Here, we isolated an acquired osmotolerance-defective mutant (aod13) by screening 30,000 seedlings of an ion beam-mutagenized M2 population of Bu-5, an accession with acquired osmotolerance. We found that AOD13 encodes the dual-specificity phosphatase MAP KINASE PHOSPHATASE1 (MKP1), which negatively regulates MITOGEN-ACTIVATED PROTEIN KINASE3/6 (MPK3/6). Consistently, MPK3/6 activation was greater in aod13 than in the Bu-5 wild-type (WT). The aod13 mutant was sensitive to osmotic stress but tolerant to salt stress. Under osmotic stress, pathogenesis-related genes were strongly induced in aod13 but not in the Bu-5 WT. Loss of PAD4 in pad4 aod13 plants did not restore acquired osmotolerance, implying that activation of immunity independent of PAD4 renders aod13 sensitive to osmotic stress. These findings suggest that AOD13 (i.e. MKP1) promotes osmotolerance by suppressing the PAD4-independent immune response activated by MPK3/6.

Under osmotic stress, MAP KINASE PHOSPHATASE1 represses the MITOGEN-ACTIVATED PROTEIN KINASE3/6-dependent immune response that impairs osmotolerance of Arabidopsis thaliana.     

Evolutionary engineering of a glycerol‐3‐phosphate dehydrogenase‐negative,acetate‐reducing Saccharomyces cerevisiae strain enables anaerobic growth at high glucose concentrations

Glycerol production by Saccharomyces cerevisiae, which is required for redox-cofactor balancing in anaerobic cultures, causes yield reduction in industrial bioethanol production. Recently, glycerol formation in anaerobic S. cerevisiae cultures was eliminated by expressing Escherichia coli (acetylating) acetaldehyde dehydrogenase (encoded by mhpF) and simultaneously deleting the GPD1 and GPD2 genes encoding glycerol-3-phosphate dehydrogenase, thus coupling NADH reoxidation to reduction of acetate to ethanol. Gpd^– strains are, however, sensitive to high sugar concentrations, which complicates industrial implementation of this metabolic engineering concept. In this study, laboratory evolution was used to improve osmotolerance of a Gpd^– mhpF-expressing S. cerevisiae strain. Serial batch cultivation at increasing osmotic pressure enabled isolation of an evolved strain that grew anaerobically at 1 M glucose, at a specific growth rate of 0.12 h⁻¹. The evolved strain produced glycerol at low concentrations (0.64 ± 0.33 g l⁻¹). However, these glycerol concentrations were below 10% of those observed with a Gpd⁺ reference strain. Consequently, the ethanol yield on sugar increased from 79% of the theoretical maximum in the reference strain to 92% for the evolved strains. Genetic analysis indicated that osmotolerance under aerobic conditions required a single dominant chromosomal mutation, and one further mutation in the plasmid-borne mhpF gene for anaerobic growth.     

Heat Stress Activates Fission Yeast Spc1/StyI MAPK by a MEKK-Independent Mechanism

Fission yeast Spc1/StyI MAPK is activated by many environmental insults including high osmolarity, oxidative stress, and heat shock. Spc1/StyI is activated by Wis1, a MAPK kinase (MEK), which is itself activated by Wik1/Wak1/Wis4, a MEK kinase (MEKK). Spc1/StyI is inactivated by the tyrosine phosphatases Pyp1 and Pyp2. Inhibition of Pyp1 was recently reported to play a crucial role in the oxidative stress and heat shock responses. These conclusions were based on three findings: 1) osmotic, oxidative, and heat stresses activate Spc1/StyI in wis4 cells; 2) oxidative stress and heat shock activate Spc1/StyI in cells that express Wis1AA, in which MEKK consensus phosphorylation sites were replaced with alanine; and 3) Spc1/StyI is maximally activated in Δpyp1 cells. Contrary to these findings, we report: 1) Spc1/StyI activation by osmotic stress is greatly reduced in wis4 cells; 2) wis1-AA and Δwis1 cells have identical phenotypes; and 3) all forms of stress activate Spc1/StyI in Δpyp1 cells. We also report that heat shock, but not osmotic or oxidative stress, activate Spc1 in wis1-DD cells, which express Wis1 protein that has the MEKK consensus phosphorylation sites replaced with aspartic acid. Thus osmotic and oxidative stress activate Spc1/StyI by a MEKK-dependent process, whereas heat shock activates Spc1/StyI by a novel mechanism that does not require MEKK activation or Pyp1 inhibition.     

Cellular Responses of Candida albicans to Phagocytosis and the Extracellular Activities of Neutrophils Are Critical to Counteract Carbohydrate Starvation,Oxidative and Nitrosative Stress

Neutrophils are key players during Candida albicans infection. However, the relative contributions of neutrophil activities to fungal clearance and the relative importance of the fungal responses that counteract these activities remain unclear. We studied the contributions of the intra- and extracellular antifungal activities of human neutrophils using diagnostic Green Fluorescent Protein (GFP)-marked C. albicans strains. We found that a carbohydrate starvation response, as indicated by up-regulation of glyoxylate cycle genes, was only induced upon phagocytosis of the fungus. Similarly, the nitrosative stress response was only observed in internalised fungal cells. In contrast, the response to oxidative stress was observed in both phagocytosed and non-phagocytosed fungal cells, indicating that oxidative stress is imposed both intra- and extracellularly. We assessed the contributions of carbohydrate starvation, oxidative and nitrosative stress as antifungal activities by analysing the resistance to neutrophil killing of C. albicans mutants lacking key glyoxylate cycle, oxidative and nitrosative stress genes. We found that the glyoxylate cycle plays a crucial role in fungal resistance against neutrophils. The inability to respond to oxidative stress (in cells lacking superoxide dismutase 5 or glutathione reductase 2) renders C. albicans susceptible to neutrophil killing, due to the accumulation of reactive oxygen species (ROS). We also show that neutrophil-derived nitric oxide is crucial for the killing of C. albicans: a yhb1Δ/Δ mutant, unable to detoxify NO^•, was more susceptible to neutrophils, and this phenotype was rescued by the nitric oxide scavenger carboxy-PTIO. The stress responses of C. albicans to neutrophils are partially regulated via the stress regulator Hog1 since a hog1Δ/Δ mutant was clearly less resistant to neutrophils and unable to respond properly to neutrophil-derived attack. Our data indicate that an appropriate fungal response to all three antifungal activities, carbohydrate starvation, nitrosative stress and oxidative stress, is essential for full wild type resistance to neutrophils.     

The Cch1-Mid1 High-Affinity Calcium Channel Contributes to the Virulence of Cryptococcus neoformans by Mitigating Oxidative Stress

Pathogenic fungi have developed mechanisms to cope with stresses imposed by hosts. For Cryptococcus spp., this implies active defense mechanisms that attenuate and ultimately overcome the onslaught of oxidative stresses in macrophages. Among cellular pathways within Cryptococcus neoformans'' arsenal is the plasma membrane high-affinity Cch1-Mid1 calcium (Ca²⁺) channel (CMC). Here we show that CMC has an unexpectedly complex and disparate role in mitigating oxidative stress. Upon inhibiting the Ccp1-mediated oxidative response pathway with antimycin, strains of C. neoformans expressing only Mid1 displayed enhanced growth, but this was significantly attenuated upon H₂O₂ exposure in the absence of Mid1, suggesting a regulatory role for Mid1 acting through the Ccp1-mediated oxidative stress response. This notion is further supported by the interaction detected between Mid1 and Ccp1 (cytochrome c peroxidase). In contrast, Cch1 appears to have a more general role in promoting cryptococci survival during oxidative stress. A strain lacking Cch1 displayed a growth defect in the presence of H₂O₂ without BAPTA [(1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, cesium salt] or additional stressors such as antimycin. Consistent with a greater contribution of Cch1 to oxidative stress tolerance, an intracellular growth defect was observed for the cch1Δ strain in the macrophage cell line J774A.1. Interestingly, while the absence of either Mid1 or Cch1 significantly compromises the ability of C. neoformans to tolerate oxidative stress, the absence of both Mid1 and Cch1 has a negligible effect on C. neoformans growth during H₂O₂ stress, suggesting the existence of a compensatory mechanism that becomes active in the absence of CMC.     

Tor1/Sch9-Regulated Carbon Source Substitution Is as Effective as Calorie Restriction in Life Span Extension

The effect of calorie restriction (CR) on life span extension, demonstrated in organisms ranging from yeast to mice, may involve the down-regulation of pathways, including Tor, Akt, and Ras. Here, we present data suggesting that yeast Tor1 and Sch9 (a homolog of the mammalian kinases Akt and S6K) is a central component of a network that controls a common set of genes implicated in a metabolic switch from the TCA cycle and respiration to glycolysis and glycerol biosynthesis. During chronological survival, mutants lacking SCH9 depleted extracellular ethanol and reduced stored lipids, but synthesized and released glycerol. Deletion of the glycerol biosynthesis genes GPD1, GPD2, or RHR2, among the most up-regulated in long-lived sch9Δ, tor1Δ, and ras2Δ mutants, was sufficient to reverse chronological life span extension in sch9Δ mutants, suggesting that glycerol production, in addition to the regulation of stress resistance systems, optimizes life span extension. Glycerol, unlike glucose or ethanol, did not adversely affect the life span extension induced by calorie restriction or starvation, suggesting that carbon source substitution may represent an alternative to calorie restriction as a strategy to delay aging.     

Differential Responses of Nitrogen-Fixing Cyanobacteria to Salinity and Osmotic Stresses

Two nitrogen-fixing Anabaena strains were found to be differentially tolerant to salinity and osmotic stresses. Anabaena torulosa, a brackish-water, salt-tolerant strain, was relatively osmosensitive. Anabaena sp. strain L-31, a freshwater, salt-sensitive strain, on the other hand, displayed significant osmotolerance. Salinity and osmotic stresses affected nitrogenase activity differently. Nitrogen fixation in both of the strains was severely inhibited by the ionic, but not by the osmotic, component of salinity stress. Such differential sensitivity of diazotrophy to salinity-osmotic stresses was observed irrespective of the inherent tolerance of the two strains to salt-osmotic stress. Exogenously added ammonium conferred significant protection against salinity stress but was ineffective against osmotic stress. Salinity and osmotic stresses also affected stress-induced gene expression differently. Synthesis of several proteins was repressed by salinity stress but not by equivalent or higher osmotic stress. Salinity and osmotic stresses induced many common proteins. In addition, unique salt stress- or osmotic stress-specific proteins were also induced in both strains, indicating differential regulation of protein synthesis by the two stresses. These data show that cyanobacterial sensitivity and responses to salinity and osmotic stresses are distinct, independent phenomena.     

Overexpression of fetA (ybbL) and fetB (ybbM), Encoding an Iron Exporter,Enhances Resistance to Oxidative Stress in Escherichia coli

Reactive oxygen species are generated by redox reactions and the Fenton reaction of H₂O₂ and iron that generates the hydroxyl radical that causes severe DNA, protein, and lipid damage. We screened Escherichia coli genomic libraries to identify a fragment, containing cueR, ybbJ, qmcA, ybbL, and ybbM, which enhanced resistance to H₂O₂ stress. We report that the ΔybbL and ΔybbM strains are more susceptible to H₂O₂ stress than the parent strain and that ybbL and ybbM overexpression overcomes H₂O₂ sensitivity. The ybbL and ybbM genes are predicted to code for an ATP-binding cassette metal transporter, and we demonstrate that YbbM is a membrane protein. We investigated various metals to identify iron as the likely substrate of this transporter. We propose the gene names fetA and fetB (for Fe transport) and the gene product names FetA and FetB. FetAB allows for increased resistance to oxidative stress in the presence of iron, revealing a role in iron homeostasis. We show that iron overload coupled with H₂O₂ stress is abrogated by fetA and fetB overexpression in the parent strain and in the Δfur strain, where iron uptake is deregulated. Furthermore, we utilized whole-cell electron paramagnetic resonance to show that intracellular iron levels in the Δfur strain are decreased by 37% by fetA and fetB overexpression. Combined, these findings show that fetA and fetB encode an iron exporter that has a role in enhancing resistance to H₂O₂-mediated oxidative stress and can minimize oxidative stress under conditions of iron overload and suggest that FetAB facilitates iron homeostasis to decrease oxidative stress.     

Characterization of the kinetic and thermodynamic landscape of RNA folding using a novel application of isothermal titration calorimetry

A novel isothermal titration calorimetry (ITC) method was applied to investigate RNA helical packing driven by the GAAA tetraloop–receptor interaction in magnesium and potassium solutions. Both the kinetics and thermodynamics were obtained in individual ITC experiments, and analysis of the kinetic data over a range of temperatures provided Arrhenius activation energies (ΔH^‡) and Eyring transition state entropies (ΔS^‡). The resulting rich dataset reveals strongly contrasting kinetic and thermodynamic profiles for this RNA folding system when stabilized by potassium versus magnesium. In potassium, association is highly exothermic (ΔH_25°C = −41.6 ± 1.2 kcal/mol in 150 mM KCl) and the transition state is enthalpically barrierless (ΔH^‡ = −0.6 ± 0.5). These parameters are sigificantly positively shifted in magnesium (ΔH_25°C = −20.5 ± 2.1 kcal/mol, ΔH^‡ = 7.3 ± 2.2 kcal/mol in 0.5 mM MgCl₂). Mixed salt solutions approximating physiological conditions exhibit an intermediate thermodynamic character. The cation-dependent thermodynamic landscape may reflect either a salt-dependent unbound receptor conformation, or alternatively and more generally, it may reflect a small per-cation enthalpic penalty associated with folding-coupled magnesium uptake.     

Prion formation and polyglutamine aggregation are controlled by two classes of genes

Prions are self-perpetuating aggregated proteins that are not limited to mammalian systems but also exist in lower eukaryotes including yeast. While much work has focused around chaperones involved in prion maintenance, including Hsp104, little is known about factors involved in the appearance of prions. De novo appearance of the [PSI ⁺] prion, which is the aggregated form of the Sup35 protein, is dramatically enhanced by transient overexpression of SUP35 in the presence of the prion form of the Rnq1 protein, [PIN ⁺]. When fused to GFP and overexpressed in [ps⁻] [PIN ⁺] cells, Sup35 forms fluorescent rings, and cells with these rings bud off [PSI ⁺] daughters. We investigated the effects of over 400 gene deletions on this de novo induction of [PSI ⁺]. Two classes of gene deletions were identified. Class I deletions (bug1Δ, bem1Δ, arf1Δ, and hog1Δ) reduced the efficiency of [PSI ⁺] induction, but formed rings normally. Class II deletions (las17Δ, vps5Δ, and sac6Δ) inhibited both [PSI⁺] induction and ring formation. Furthermore, class II deletions reduced, while class I deletions enhanced, toxicity associated with the expanded glutamine repeats of the huntingtin protein exon 1 that causes Huntington''s disease. This suggests that prion formation and polyglutamine aggregation involve a multi-phase process that can be inhibited at different steps.     

Germination variation in Arabidopsis thaliana accessions under moderate osmotic and salt stresses

Background

Water and salt stresses are two important environmental factors that limit the germination of seeds in most ecological environments. Most studies conducted so far to address the genetic basis of the above phenomenon have used stress conditions that are much more extreme than those found in natural environments. Furthermore, although an excess of ions and water restrictions have similar osmotic effects on germination, the common and divergent signalling components mediating the effects of both factors remain unknown.

Methods

The germination of seeds was compared under solutions of NaCl (50 mm) and polyethylene glycol (PEG, −0·6 MPa), that establish mild stress conditions, in 28 Arabidopsis thaliana accessions. Because Bayreuth (Bay) and Shadara (Sha) accessions showed contrasting sensitivity responses to both stresses, a quantitative trait locus (QTL) analysis was carried out using Bay × Sha recombinant inbred lines (RILs) to identify loci involved in the control of germination under mild salt and osmotic stresses.

Key Results

Two loci associated with the salt sensitivity response, named SSR1 and SSR2 QTLs, and four loci for the osmotic sensitivity response, named OSR1–OSR4 QTLs, were mapped. The effects of the SSR1 QTL on toxic salt sensitivity, and the osmotic contribution of OSR1, were confirmed by heterogeneous inbred families (HIFs). Whilst the SSR1 QTL had a significant effect under a wide range of NaCl concentrations, the OSR1 QTL was confirmed only under moderate drought stress. Interestingly the OSR1 QTL also showed pleiotropic effects on biomass accumulation in response to water deficit.

Conclusions

The regulation of germination under moderate salt and osmotic stresses involves the action of independent major loci, revealing the existence of loci specifically associated with the toxic component of salt and not just its osmotic effect. Furthermore, this work demonstrates that novel loci control germination under osmotic stress conditions simulating more realistic ecological environments as found by populations of seeds in nature.     

Evaluation of the importance of ionic and osmotic components of salt stress on the photosynthetic efficiency of epiphytic lichens

Salt stress can significantly disrupt the functioning of lichens which are self-sufficient symbiotic organisms inhabiting various severe environments. The aim was to test the effect of salt and sucrose on the photosynthetic efficiency of two selected epiphytic lichens inhabiting the interior of the land. Firstly, we compared the effect of salt and sucrose solutions of different concentrations. Secondly, the effect of salt and sucrose solutions with identical osmotic pressures was compared. The results showed that short-term salt stress leads to a significant reduction of F_V/F_M, greater changes in chlorophyll fluorescence parameters and OJIP transients compared to the osmotic effects induced by sucrose. This proved that the negative impact of salt stress is associated primarily with ionic effects. The most symptomatic effect of the ionic stress was a significant reduction of the utilisation of trapped energy in electron transport and thereby down-regulation of electron transfer. Since lichens are resistant to a temporary lack of water, ionic stress could have more serious consequences than osmotic stress itself. Hypogymnia physodes was more sensitive to salt stress than Pseudevernia furfuracea, but the reduction of photosynthetic efficiency was not permanent since after 24 h F_V/F_M returned to the level characteristic for healthy lichens. Nevertheless, repeated exposure to salt may reduce the vitality of lichens growing along communication routes sprinkled with salt in the winter season. Finally, the changes in certain JIP-test parameters were stronger than F_V/F_M, thus they could be better indicators of salt stress in lichens.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-022-01134-2.     

Membrane topology of Salmonella invasion protein SipB confers osmotolerance

Salmonella enterica serovar Typhimurium is a major cause of human gastrointestinal illness worldwide. This pathogen can persist in a wide range of environments, making it of great concern to public health. Here, we report that the salmonella pathogenicity island (SPI)-1 effector protein SipB exhibits a membrane topology that confers bacterial osmotolerance. Disruption of the sipB gene or the invG gene (SPI-1 component) significantly reduced the osmotolerance of S. Typhimurium LT2. Biochemical assays showed that NaCl osmolarity increased the membrane topology of SipB, and a neutralising antibody against SipB reduced osmotolerance in the WT strain. The WT strain, but not the sipB mutant, exhibited elevated cyclopropane fatty acid C19:0 during conditions of osmotic stress, correlating with the observed levels of survival and membrane integrity. This result suggests a link between SipB and the altered fatty acid composition induced upon exposure to osmotic stress. Overall, our findings provide the first evidence that the Salmonella virulence translocon SipB affects membrane fluidity and alters bacterial osmotolerance.