The action of ultraviolet radiation on yeast catalase

The effect of prolonged UV irradiation (mostly 2537 A) on the catalase activity of an aqueous yeast suspension was divisible into 4 periods. First, the period during which the cells lost their ability to form colonies, but during which no change in catalase activity was noted. Second, the period during which a considerable rise in catalase activity (Euler effect) occurred. The Euler effect was accompanied by enzyme alteration as shown by the simultaneous decrease in the activation energy of the enzyme-substrate system. However, during the initial phase of this period, as the catalase activity of the suspension began to increase, the activation energy rose to a transient level higher even than that characterizing the unaltered enzyme. Heat accelerated the rate of alteration when applied either during or after the irradiation; the activation energy for the over-all alteration reaction was 24 kcal., a value close to that recorded previously for alteration induced by chemical agents. Nevertheless, the rate-limiting step appeared to be different in the two cases. A model of these events was presented in which the primary photochemical action was on the site at which catalase is located within the cell. Third, a rather long period during which irradiation led to no diminution in the catalase activity of the maximally active suspension. This protection effect was duplicated in intro by a model crystalline catalase-KNA system, or by adding either ribonuclease digestion products of RNA or adenine to a catalase solution prior to irradiation. Evidence was adduced that the protection effect was not a simple screening, but involved some sort of interaction between the enzyme and the nitrogenous components of RNA, an interaction which must likewise occur within the cell. Alteration induced by CHCl₃ did not eliminate the protection effect, but that by butanol did. The onset of photoinactivation was due to modification of protein structure, not of RNA. Fourth, the period of photoinactivation of the intracellular enzyme, which was quite similar to that of the crystalline enzyme in vitro.     

The alteration of intracellular enzymes. II. The relation between the surface and the biological activities of altering agents

1. The ability of homologous series of alcohols, ketones, and aldehydes to cause alteration of intracellular catalase increases approximately threefold for each methylene group added, thus following Traube's rule. Equiactive concentrations of alcohols (methanol to octanol) varied over a 4,000-fold range, yet the average corresponding surface tension was 42 ± 2 dynes/cm., that for ketones 43 ± 2, and for aldehydes (above C₁) 41 ± 3. 2. Above C₈ the altering activity of alcohols ceased to follow Traube's rule, and at C₁₈ was nil. Yet the surface activities of alcohols from nonanol to dodecanol did follow Traube's rule. These two facts show that the interface which is being affected by these agents is not the cell surface, for if it were, altering activity should not fall off between C₉ and C₁₂ where surface activity is undiminished; they show also that micelle formation by short range association of hydrocarbon "tails," usually invoked to explain decrease in biological activity of compounds above C₈, is not responsible for this effect in these experiments, in which permeability of the cell membrane probably is involved. 3. The most soluble alcohols and aldehydes (alcohols C₁ to C₈; aldehydes C₁, C₂), but not ketones, cause, above optimal concentration, an irreversible inhibition of yeast catalase. 4. The critical concentration of altering agent (i.e., that concentration just sufficient to cause doubling of the catalase activity of the yeast suspension) was independent of the concentration of the yeast cells. 5. Viability studies show that the number of yeast cells killed by the altering agents was not related to the degree of activation of the catalase produced. While all the cells were invariably killed by concentrations of altering agent which produced complete activation, all the cells had been killed by concentrations which were insufficient to cause more than 50 per cent maximal activation. Further, the evidence suggested that the catalase may be partially activated by concentrations of altering agent which cause no decrease in viability at all. Hence alteration, unlike death, may not be all-or-none per cell. 6. The fact that the biological criterion being examined was the activation of a water-soluble enzyme rules out the possibility that the reason for the logarithmic increase in altering activity with chain length was increase in concentration of the altering agent in some intracellular fat phase. It is concluded that these surface-active agents cause enzyme alteration by becoming adsorbed at some intracellular interface and thus causing, directly or indirectly, the modification of catalase properties. 7. It is considered that these data support, but do not provide critical proof for, the interfacial hypothesis, which states that catalase is present at the intracellular interface in question, but is desorbed into solution as a consequence of the alteration process.     

The energetic barrier to single-file water flow through narrow channels

Various nanoscopic channels of roughly equal diameter and length facilitate single-file diffusion at vastly different rates. The underlying variance of the energetic barriers to transport is poorly understood. First, water partitioning into channels so narrow that individual molecules cannot overtake each other incurs an energetic penalty. Corresponding estimates vary widely depending on how the sacrifice of two out of four hydrogen bonds is accounted for. Second, entropy differences between luminal and bulk water may arise: additional degrees of freedom caused by dangling OH-bonds increase entropy. At the same time, long-range dipolar water interactions decrease entropy. Here, we dissect different contributions to Gibbs free energy of activation, ΔG^‡, for single-file water transport through narrow channels by analyzing experimental results from water permeability measurements on both bare lipid bilayers and biological water channels that (i) consider unstirred layer effects and (ii) adequately count the channels in reconstitution experiments. First, the functional relationship between water permeabilities and Arrhenius activation energies indicates negligible differences between the entropies of intraluminal water and bulk water. Second, we calculate ΔG^‡ from unitary water channel permeabilities using transition state theory. Plotting ΔG^‡ as a function of the number of H-bond donating or accepting pore-lining residues results in a 0.1 kcal/mol contribution per residue. The resulting upper limit for partial water dehydration amounts to 2 kcal/mol. In the framework of biomimicry, our analysis provides valuable insights for the design of synthetic water channels. It thus may aid in the urgent endeavor towards combating global water scarcity.     

Temperature Dependence of Nonelectrolyte Permeation across Red Cell Membranes

The temperature dependence of permeation across human red cell membranes has been determined for a series of hydrophilic and lipophilic solutes, including urea and two methyl substituted derivatives, all the straight-chain amides from formamide through valeramide and the two isomers, isobutyramide and isovaleramide. The temperature coefficient for permeation by all the hydrophilic solutes is 12 kcal mol^-1 or less, whereas that for all the lipophilic solutes is 19 kcal mol^-1 or greater. This difference is consonant with the view that hydrophilic molecules cross the membrane by a path different from that taken by the lipophilic ones. The thermodynamic parameters associated with lipophile permeation have been studied in detail. ΔG is negative for adsorption of lipophilic amides onto an oil-water interface, whereas it is positive for transfer of the polar head from the aqueous medium to bulk lipid solvent. Application of absolute reaction rate theory makes it possible to make a clear distinction between diffusion across the water-red cell membrane interface and diffusion within the membrane. Diffusion coefficients and apparent activation enthalpies and entropies have been computed for each process. Transfer of the polar head from the solvent into the interface is characterized by ΔG^‡ = 0 kcal mol^-1 and ΔS^‡ negative, whereas both of these parameters have large positive values for diffusion within the membrane. Diffusion within the membrane is similar to what is expected for diffusion through a highly associated viscous fluid.     

Characterization of the glutathione‐dependent reduction of the peroxiredoxin 5 homolog PfAOP from Plasmodium falciparum

Peroxiredoxins use a variety of thiols to rapidly reduce hydroperoxides and peroxynitrite. While the oxidation kinetics of peroxiredoxins have been studied in great detail, enzyme‐specific differences regarding peroxiredoxin reduction and the overall rate‐limiting step under physiological conditions often remain to be deciphered. The 1‐Cys peroxiredoxin 5 homolog PfAOP from the malaria parasite Plasmodium falciparum is an established model enzyme for glutathione/glutaredoxin‐dependent peroxiredoxins. Here, we reconstituted the catalytic cycle of PfAOP in vitro and analyzed the reaction between oxidized PfAOP and reduced glutathione (GSH) using molecular docking and stopped‐flow measurements. Molecular docking revealed that oxidized PfAOP has to adopt a locally unfolded conformation to react with GSH. Furthermore, we determined a second‐order rate constant of 6 × 10⁵ M⁻¹ s⁻¹ at 25°C and thermodynamic activation parameters ΔH ^‡, ΔS ^‡, and ΔG ^‡ of 39.8 kJ/mol, −0.8 J/mol, and 40.0 kJ/mol, respectively. The gain‐of‐function mutant PfAOP^L109M had almost identical reaction parameters. Taking into account physiological hydroperoxide and GSH concentrations, we suggest (a) that the reaction between oxidized PfAOP and GSH might be even faster than the formation of the sulfenic acid in vivo, and (b) that conformational changes are likely rate limiting for PfAOP catalysis. In summary, we characterized and quantified the reaction between GSH and the model enzyme PfAOP, thus providing detailed insights regarding the reactivity of its sulfenic acid and the versatile chemistry of peroxiredoxins.     

Permeability of the Ehrlich Ascites Tumor Cell to Water

The osmotic permeability coefficient for water has been measured for the Ehrlich mouse ascites tumor cell. Measurements were made of the rate of cell shrinkage in hyperosmotic solutions of NaCI, a functionally impermeable solute. During the first 9 months of weekly serial transplantation the mean was 6.4 µ³/µ³/atm. ± 0.8 (S.E.). By the end of the 2nd year the permeability coefficient was much lower and averaged 1.6 ± 0.09. There were no significant differences in the volume of the tumor cells which could explain the discrepancy on the basis of a change in the volume to surface area ratio. Studies of the effect of temperature were done and Eyring's theory of absolute reaction rates was applied to the data. The apparent energy of activation was 9.6 kcal./mol and ΔS‡ was 39.1 entropy units. The thermodynamic data are twice as high as data reported by Wang for self-diffusion and viscous properties of water. Two alternate explanations have been advanced based on the pore hypothesis of membrane permeability. One explains the thermodynamic data from a change in the A'/Δx available for water movement; the other assumes A'/Δx constant and bases the results on the interaction of water dipoles with each other and the membrane.     

Yeast Pah1p Phosphatidate Phosphatase Is Regulated by Proteasome-mediated Degradation

Yeast PAH1-encoded phosphatidate phosphatase is the enzyme responsible for the production of the diacylglycerol used for the synthesis of triacylglycerol that accumulates in the stationary phase of growth. Paradoxically, the growth phase-mediated inductions of PAH1 and phosphatidate phosphatase activity do not correlate with the amount of Pah1p; enzyme abundance declined in a growth phase-dependent manner. Pah1p from exponential phase cells was a relatively stable protein, and its abundance was not affected by incubation with an extract from stationary phase cells. Recombinant Pah1p was degraded upon incubation with the 100,000 × g pellet fraction of stationary phase cells, although the enzyme was stable when incubated with the same fraction of exponential phase cells. MG132, an inhibitor of proteasome function, prevented degradation of the recombinant enzyme. Endogenously expressed and plasmid-mediated overexpressed levels of Pah1p were more abundant in the stationary phase of cells treated with MG132. Pah1p was stabilized in mutants with impaired proteasome (rpn4Δ, blm10Δ, ump1Δ, and pre1 pre2) and ubiquitination (hrd1Δ, ubc4Δ, ubc7Δ, ubc8Δ, and doa4Δ) functions. The pre1 pre2 mutations that eliminate nearly all chymotrypsin-like activity of the 20 S proteasome had the greatest stabilizing effect on enzyme levels. Taken together, these results supported the conclusion that Pah1p is subject to proteasome-mediated degradation in the stationary phase. That Pah1p abundance was stabilized in pah1Δ mutant cells expressing catalytically inactive forms of Pah1p and dgk1Δ mutant cells with induced expression of DGK1-encoded diacylglycerol kinase indicated that alteration in phosphatidate and/or diacylglycerol levels might be the signal that triggers Pah1p degradation.     

Ligand-Induced Changes of the Apparent Transition-State Position in?Mechanical Protein Unfolding

Force-spectroscopic measurements of ligand-receptor systems and the unfolding/folding of nucleic acids or proteins reveal information on the underlying energy landscape along the pulling coordinate. The slope Δx^‡ of the force-dependent unfolding/unbinding rates is interpreted as the distance from the folded/bound state to the transition state for unfolding/unbinding and, hence, often related to the mechanical compliance of the sample molecule. Here we show that in ligand-binding proteins, the experimentally inferred Δx^‡ can depend on the ligand concentration, unrelated to changes in mechanical compliance. We describe the effect in single-molecule, force-spectroscopy experiments of the calcium-binding protein calmodulin and explain it in a simple model where mechanical unfolding and ligand binding occur on orthogonal reaction coordinates. This model predicts changes in the experimentally inferred Δx^‡, depending on ligand concentration and the associated shift of the dominant barrier between the two reaction coordinates. We demonstrate quantitative agreement between experiments and simulations using a realistic six-state kinetic scheme using literature values for calcium-binding kinetics and affinities. Our results have important consequences for the interpretation of force-spectroscopic data of ligand-binding proteins.     

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.     

Integration of Metabolic Modeling and Phenotypic Data in Evaluation and Improvement of Ethanol Production Using Respiration-Deficient Mutants of Saccharomyces cerevisiae

Flux balance analysis and phenotypic data were used to provide clues to the relationships between the activities of gene products and the phenotypes resulting from the deletion of genes involved in respiratory function in Saccharomyces cerevisiae. The effect of partial or complete respiratory deficiency on the ethanol production and growth characteristics of hap4Δ/hap4Δ, mig1Δ/mig1Δ, qdr3Δ/qdr3Δ, pdr3Δ/pdr3Δ, qcr7Δ/qcr7Δ, cyt1Δ/cyt1Δ, and rip1Δ/rip1Δ mutants grown in microaerated chemostats was investigated. The study provided additional evidence for the importance of the selection of a physiologically relevant objective function, and it may improve quantitative predictions of exchange fluxes, as well as qualitative estimations of changes in intracellular fluxes. Ethanol production was successfully predicted by flux balance analysis in the case of the qdr3Δ/qdr3Δ mutant, with maximization of ethanol production as the objective function, suggesting an additional role for Qdr3p in respiration. The absence of similar changes in estimated intracellular fluxes in the qcr7Δ/qcr7Δ mutant compared to the rip1Δ/rip1Δ and cyt1Δ/cyt1Δ mutants indicated that the effect of the deletion of this subunit of complex III was somehow compensated for. Analysis of predicted flux distributions indicated self-organization of intracellular fluxes to avoid NAD⁺/NADH imbalance in rip1Δ/rip1Δ and cyt1Δ/cyt1Δ mutants, but not the qcr7Δ/qcr7Δ mutant. The flux through the glycerol efflux channel, Fps1p, was estimated to be zero in all strains under the investigated conditions. This indicates that previous strategies for improving ethanol production, such as the overexpression of the glutamate synthase gene GLT1 in a GDH1 deletion background or deletion of the glycerol efflux channel gene FPS1 and overexpression of GLT1, are unnecessary in a respiration-deficient background.     

The Saccharomyces cerevisiae Actin Patch Protein App1p Is a Phosphatidate Phosphatase Enzyme

Phosphatidate phosphatase (PAP) catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol. In the yeast Saccharomyces cerevisiae, PAP is encoded by PAH1, DPP1, and LPP1. The presence of PAP activity in the pah1Δ dpp1Δ lpp1Δ triple mutant indicated another gene(s) encoding the enzyme. We purified PAP from the pah1Δ dpp1Δ lpp1Δ triple mutant by salt extraction of mitochondria followed by chromatography with DE52, Affi-Gel Blue, phenyl-Sepharose, MonoQ, and Superdex 200. Liquid chromatography/tandem mass spectrometry analysis of a PAP-enriched sample revealed multiple putative phosphatases. By analysis of PAP activity in mutants lacking each of the proteins, we found that APP1, a gene whose molecular function has been unknown, confers ∼30% PAP activity of wild type cells. The overexpression of APP1 in the pah1Δ dpp1Δ lpp1Δ mutant exhibited a 10-fold increase in PAP activity. The PAP activity shown by App1p heterologously expressed in Escherichia coli confirmed that APP1 is the structural gene for the enzyme. Introduction of the app1Δ mutation into the pah1Δ dpp1Δ lpp1Δ triple mutant resulted in a complete loss of PAP activity, indicating that distinct PAP enzymes in S. cerevisiae are encoded by APP1, PAH1, DPP1, and LPP1. Lipid analysis of cells lacking the PAP genes, singly or in combination, showed that Pah1p is the only PAP involved in the synthesis of triacylglycerol as well as in the regulation of phospholipid synthesis. App1p, which shows interactions with endocytic proteins, may play a role in vesicular trafficking through its PAP activity.     

Effects of Disruption of Homocitrate Synthase Genes on Nostoc sp. Strain PCC 7120 Photobiological Hydrogen Production and Nitrogenase

In the case of nitrogenase-based photobiological hydrogen production systems of cyanobacteria, the inactivation of uptake hydrogenase (Hup) leads to significant increases in hydrogen production activity. However, the high-level-activity stage of the Hup mutants lasts only a few tens of hours under air, a circumstance which seems to be caused by sufficient amounts of combined nitrogen supplied by active nitrogenase. The catalytic FeMo cofactor of nitrogenase binds homocitrate, which is required for efficient nitrogen fixation. It was reported previously that the nitrogenase from the homocitrate synthase gene (nifV) disruption mutant of Klebsiella pneumoniae shows decreased nitrogen fixation activity and increased hydrogen production activity under N₂. The cyanobacterium Nostoc sp. strain PCC 7120 has two homocitrate synthase genes, nifV1 and nifV2, and with the ΔhupL variant of Nostoc sp. strain PCC 7120 as the parental strain, we have constructed two single mutants, the ΔhupL ΔnifV1 strain (with the hupL and nifV1 genes disrupted) and the ΔhupL ΔnifV2 strain, and a double mutant, the ΔhupL ΔnifV1 ΔnifV2 strain. Diazotrophic growth rates of the two nifV single mutants and the double mutant were decreased moderately and severely, respectively, compared with the rates of the parent ΔhupL strain. The hydrogen production activity of the ΔhupL ΔnifV1 mutant was sustained at higher levels than the activity of the parent ΔhupL strain after about 2 days of combined-nitrogen step down, and the activity in the culture of the former became higher than that in the culture of the latter. The presence of N₂ gas inhibited hydrogen production in the ΔhupL ΔnifV1 ΔnifV2 mutant less strongly than in the parent ΔhupL strain and the ΔhupL ΔnifV1 and ΔhupL ΔnifV2 mutants. The alteration of homocitrate synthase activity can be a useful strategy for improving sustained photobiological hydrogen production in cyanobacteria.     

Broad sensitivity of Saccharomyces cerevisiae lacking ribosome-associated chaperone ssb or zuo1 to cations, including aminoglycosides

The Hsp70 Ssb and J protein Zuo1 of Saccharomyces cerevisiae are ribosome-associated molecular chaperones, proposed to be involved in the folding of newly synthesized polypeptide chains. Cells lacking Ssb and/or Zuo1 have been reported to be hypersensitive to cationic aminoglycoside protein synthesis inhibitors that affect translational fidelity and to NaCl. Since we found that Δssb1 Δssb2 (Δssb1,2), Δzuo1, and wild-type cells have very similar levels of translational misreading in the absence of aminoglycosides, we asked whether the sensitivities to aminoglycosides and NaCl represent a general increase in sensitivity to cations. We found that Δssb1,2 and Δzuo1 cells are hypersensitive to a wide range of cations. This broad sensitivity is similar to that of cells having lowered activity of major plasma membrane transporters, such as the major K⁺ transporters Trk1 and Trk2 or their regulators Hal4 and Hal5. Like Δhal4,5 cells, Δssb1,2 and Δzuo1 cells have increased intracellular levels of Na⁺ and Li⁺ upon challenge with higher-than-normal levels of these cations, due to an increased rate of influx. In the presence of aminoglycosides, Δssb1,2, Δzuo1, and Δhal 4,5 cells have similarly increased levels of translational misreading. We conclude that, in vivo, the major cause of the aminoglycoside sensitivity of cells lacking ribosome-associated molecular chaperones is a general increase in cation influx, perhaps due to altered maturation of membrane proteins.     

Mitochondrial Efficiency-Dependent Viability of Saccharomyces cerevisiae Mutants Carrying Individual Electron Transport Chain Component Deletions

Mitochondria play a crucial role in eukaryotic cells; the mitochondrial electron transport chain (ETC) generates adenosine triphosphate (ATP), which serves as an energy source for numerous critical cellular activities. However, the ETC also generates deleterious reactive oxygen species (ROS) as a natural byproduct of oxidative phosphorylation. ROS are considered the major cause of aging because they damage proteins, lipids, and DNA by oxidation. We analyzed the chronological life span, growth phenotype, mitochondrial membrane potential (MMP), and intracellular ATP and mitochondrial superoxide levels of 33 single ETC component-deleted strains during the chronological aging process. Among the ETC mutant strains, 14 (sdh1Δ, sdh2Δ, sdh4Δ, cor1Δ, cyt1Δ, qcr7Δ, qcr8Δ, rip1Δ, cox6Δ, cox7Δ, cox9Δ, atp4Δ, atp7Δ, and atp17Δ) showed a significantly shorter life span. The deleted genes encode important elements of the ETC components succinate dehydrogenase (complex II) and cytochrome c oxidase (complex IV), and some of the deletions lead to structural instability of the membrane-F₁F₀-ATP synthase due to mutations in the stator stalk (complex V). These short-lived strains generated higher superoxide levels and produced lower ATP levels without alteration of MMP. In summary, ETC mutations decreased the life span of yeast due to impaired mitochondrial efficiency.     

The conserved GTPase LepA contributes mainly to translation initiation in Escherichia coli

LepA is a paralog of EF-G found in all bacteria. Deletion of lepA confers no obvious growth defect in Escherichia coli, and the physiological role of LepA remains unknown. Here, we identify nine strains (ΔdksA, ΔmolR1, ΔrsgA, ΔtatB, ΔtonB, ΔtolR, ΔubiF, ΔubiG or ΔubiH) in which ΔlepA confers a synthetic growth phenotype. These strains are compromised for gene regulation, ribosome assembly, transport and/or respiration, indicating that LepA contributes to these functions in some way. We also use ribosome profiling to deduce the effects of LepA on translation. We find that loss of LepA alters the average ribosome density (ARD) for hundreds of mRNA coding regions in the cell, substantially reducing ARD in many cases. By contrast, only subtle and codon-specific changes in ribosome distribution along mRNA are seen. These data suggest that LepA contributes mainly to the initiation phase of translation. Consistent with this interpretation, the effect of LepA on ARD is related to the sequence of the Shine–Dalgarno region. Global perturbation of gene expression in the ΔlepA mutant likely explains most of its phenotypes.     

Expression of the Bacterial Type III Effector DspA/E in Saccharomyces cerevisiae Down-regulates the Sphingolipid Biosynthetic Pathway Leading to Growth Arrest

Erwinia amylovora, the bacterium responsible for fire blight, relies on a type III secretion system and a single injected effector, DspA/E, to induce disease in host plants. DspA/E belongs to the widespread AvrE family of type III effectors that suppress plant defense responses and promote bacterial growth following infection. Ectopic expression of DspA/E in plant or in Saccharomyces cerevisiae is toxic, indicating that DspA/E likely targets a cellular process conserved between yeast and plant. To unravel the mode of action of DspA/E, we screened the Euroscarf S. cerevisiae library for mutants resistant to DspA/E-induced growth arrest. The most resistant mutants (Δsur4, Δfen1, Δipt1, Δskn1, Δcsg1, Δcsg2, Δorm1, and Δorm2) were impaired in the sphingolipid biosynthetic pathway. Exogenously supplied sphingolipid precursors such as the long chain bases (LCBs) phytosphingosine and dihydrosphingosine also suppressed the DspA/E-induced yeast growth defect. Expression of DspA/E in yeast down-regulated LCB biosynthesis and induced a rapid decrease in LCB levels, indicating that serine palmitoyltransferase (SPT), the first and rate-limiting enzyme of the sphingolipid biosynthetic pathway, was repressed. SPT down-regulation was mediated by dephosphorylation and activation of Orm proteins that negatively regulate SPT. A Δcdc55 mutation affecting Cdc55-PP2A protein phosphatase activity prevented Orm dephosphorylation and suppressed DspA/E-induced growth arrest.     

Tricarboxylic Acid Cycle and One-Carbon Metabolism Pathways Are Important in Edwardsiella ictaluri Virulence

Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia of channel catfish (ESC). The disease causes considerable economic losses in the commercial catfish industry in the United States. Although antibiotics are used as feed additive, vaccination is a better alternative for prevention of the disease. Here we report the development and characterization of novel live attenuated E. ictaluri mutants. To accomplish this, several tricarboxylic acid cycle (sdhC, mdh, and frdA) and one-carbon metabolism genes (gcvP and glyA) were deleted in wild type E. ictaluri strain 93-146 by allelic exchange. Following bioluminescence tagging of the E. ictaluri ΔsdhC, Δmdh, ΔfrdA, ΔgcvP, and ΔglyA mutants, their dissemination, attenuation, and vaccine efficacy were determined in catfish fingerlings by in vivo imaging technology. Immunogenicity of each mutant was also determined in catfish fingerlings. Results indicated that all of the E. ictaluri mutants were attenuated significantly in catfish compared to the parent strain as evidenced by 2,265-fold average reduction in bioluminescence signal from all the mutants at 144 h post-infection. Catfish immunized with the E. ictaluri ΔsdhC, Δmdh, ΔfrdA, and ΔglyA mutants had 100% relative percent survival (RPS), while E. ictaluri ΔgcvP vaccinated catfish had 31.23% RPS after re-challenge with the wild type E. ictaluri.     

Affinity comparison of different THCA synthase to CBGA using modeling computational approaches

The Δ^9-Tetrahydrocannabinol (THCA) is the primary psychoactive compound of Cannabis Sativa. It is produced by Δ^1- Tetrahydrocannabinolic acid synthase (THCA) which catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) the precursor of the THCA. In this study, we were interested by the three dimensional structure of THCA synthase protein. Generation of models were done by MODELLER v9.11 and homology modeling with Δ1-tetrahydrocannabinolic acid (THCA) synthase X ray structure (PDB code 3VTE) on the basis of sequences retrieved from GenBank. Procheck, Errat, and Verify 3D tools were used to verify the reliability of the six 3D models obtained, the overall quality factor and the Prosa Z-score were also used to check the quality of the six modeled proteins. The RMSDs for C-alpha atoms, main-chain atoms, side-chain atoms and all atoms between the modeled structures and the corresponding template ranged between 0.290 Å-1.252 Å, reflecting the good quality of the obtained models. Our study of the CBGA-THCA synthase docking demonstrated that the active site pocket was successfully recognized using computational approach. The interaction energy of CBGA computed in ‘fiber types’ proteins ranged between -4.1 95 kcal/mol and -5.95 kcal/mol whereas in the ‘drug type’ was about -7.02 kcal/mol to -7.16 kcal/mol, which maybe indicate the important role played by the interaction energy of CBGA in the determination of the THCA level in Cannabis Sativa L. varieties. Finally, we have proposed an experimental design in order to explore the binding energy source of ligand-enzyme in Cannabis Sativa and the production level of the THCA in the absence of any information regarding the correlation between the enzyme affinity and THCA level production. This report opens the doors to more studies predicting the binding site pocket with accuracy from the perspective of the protein affinity and THCA level produced in Cannabis Sativa.