Environmental factors modulating antibiotic and siderophore biosynthesis by Pseudomonas fluorescens biocontrol strains.

Understanding the environmental factors that regulate the biosynthesis of antimicrobial compounds by disease-suppressive strains of Pseudomonas fluorescens is an essential step toward improving the level and reliability of their biocontrol activity. We used liquid culture assays to identify several minerals and carbon sources which had a differential influence on the production of the antibiotics 2,4-diacetylphloroglucinol (PHL), pyoluteorin (PLT), and pyrrolnitrin and the siderophores salicylic acid and pyochelin by the model strain CHA0, which was isolated from a natural disease-suppressive soil in Switzerland. Production of PHL was stimulated by Zn2+, NH4Mo2+, and glucose; the precursor compound mono-acetylphloroglucinol was stimulated by the same factors as PHL. Production of PLT was stimulated by Zn2+, Co2+, and glycerol but was repressed by glucose. Pyrrolnitrin production was increased by fructose, mannitol, and a mixture of Zn2+ and NH4Mo2+. Pyochelin production was increased by Co2+, fructose, mannitol, and glucose. Interestingly, production of its precursor salicylic acid was increased by different factors, i.e., NH4Mo2+, glycerol, and glucose. The mixture of Zn2+ and NH4Mo2+ with fructose, mannitol, or glycerol further enhanced the production of PHL and PLT compared with either the minerals or the carbon sources used alone, but it did not improve siderophore production. Extending fermentation time from 2 to 5 days increased the accumulation of PLT, pyrrolnitrin, and pyochelin but not of PHL. When findings with CHA0 were extended to an ecologically and genetically diverse collection of 41 P. fluorescens biocontrol strains, the effect of certain factors was strain dependent, while others had a general effect. Stimulation of PHL by Zn2+ and glucose was strain dependent, whereas PLT production by all strains that can produce this compound was stimulated by Zn2+ and transiently repressed by glucose. Inorganic phosphate reduced PHL production by CHA0 and seven other strains tested but to various degrees. Production of PLT but not pyrrolnitrin by CHA0 was also reduced by 100 mM phosphate. The use of 1/10-strength nutrient broth-yeast extract, compared with standard nutrient broth-yeast extract, amended with glucose and/or glycerol resulted in dramatically increased accumulations of PHL (but not PLT), pyochelin, and salicylic acid, indicating that the ratio of carbon source to nutrient concentration played a key role in the metabolic flow. The results of this study (i) provide insight into the biosynthetic regulation of antimicrobial compounds, (ii) limit the number of factors for intensive study in situ, and (iii) indicate factors that can be manipulated to improve bacterial inoculants.     

A KATP Channel-Dependent Pathway within α Cells Regulates Glucagon Release from Both Rodent and Human Islets of Langerhans

Glucagon, secreted from pancreatic islet α cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring β cells, or to an intrinsic glucose sensing by the α cells themselves. We examined hormone secretion and Ca²⁺ responses of α and β cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn²⁺ signalling was blocked, but was reversed by low concentrations (1–20 μM) of the ATP-sensitive K⁺ (K_ATP) channel opener diazoxide, which had no effect on insulin release or β cell responses. This effect was prevented by the K_ATP channel blocker tolbutamide (100 μM). Higher diazoxide concentrations (≥30 μM) decreased glucagon and insulin secretion, and α- and β-cell Ca²⁺ responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 μM) stimulated glucagon secretion, whereas high concentrations (>10 μM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the K_ATP channel, inhibition of voltage-gated Na⁺ (TTX) and N-type Ca²⁺ channels (ω-conotoxin), but not L-type Ca²⁺ channels (nifedipine), prevented glucagon secretion. Both the N-type Ca²⁺ channels and α-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an α-cell K_ATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.     

Evaluation of complexation of metal-mediated DNA-binding drugs to oligonucleotides via electrospray ionization mass spectrometry

The interactions of self-complementary oligonucleotides with a group of metal-mediated DNA-binding drugs, including chromomycin A₃, mithramycin and the novel compound UK-1, were examined via electrospray ionization quadrupole ion trap mass spectrometry. Both chromomycin and mithramycin were shown to bind preferentially to GC-rich oligonucleotide duplexes in a 2:1 drug:metal ratio, while UK-1 was shown to bind in a 1:1 drug:metal stoichiometric ratio without a strong sequence preference. These trends were observed in the presence of Co²⁺, Ni²⁺ and Zn²⁺, with the exception that chromomycin–Zn²⁺ complexes were not readily observed. The binding stoichiometries as well as the sequence specificities are in agreement with literature reports for solution studies. Binding selectivities and stabilities of the complexes were also probed using electrospray ionization mass spectrometry. Both of the GC-rich oligomers 5′-GCGCGC-3′ and 5′-GCGCATGCGC-3′ exhibited a binding preference for chromomycin over mithramycin in the presence of Co²⁺ and Ni²⁺. Energy-variable collisionally activated dissociation of the complexes was employed to determine the stabilities of the complexes. The relative metal-dependent binding energies were Ni²⁺ > Zn²⁺ > Co²⁺ for UK-1–oligomer complexes and Ni²⁺ > Co²⁺ for both mithramycin and chromomycin complexes.     

The Adhesion-Associated sca Operon in Streptococcus gordonii Encodes an Inducible High-Affinity ABC Transporter for Mn2+ Uptake

ScaA lipoprotein in Streptococcus gordonii is a member of the LraI family of homologous polypeptides found among streptococci, pneumococci, and enterococci. It is the product of the third gene within the scaCBA operon encoding the components of an ATP-binding cassette (ABC) transporter system. Inactivation of scaC (ATP-binding protein) or scaA (substrate-binding protein) genes resulted in both impaired growth of cells and >70% inhibition of ⁵⁴Mn²⁺ uptake in media containing <0.5 μM Mn²⁺. In wild-type and scaC mutant cells, production of ScaA was induced at low concentrations of extracellular Mn²⁺ (<0.5 μM) and by the addition of ≥20 μM Zn²⁺. Sca permease-mediated uptake of ⁵⁴Mn²⁺ was inhibited by Zn²⁺ but not by Ca²⁺, Mg²⁺, Fe²⁺, or Cu²⁺. Reduced uptake of ⁵⁴Mn²⁺ by sca mutants and by wild-type cells in the presence of Zn²⁺ was abrogated by the uncoupler carbonylcyanide m-chlorophenylhydrazone, suggesting that Mn²⁺ uptake under these conditions was proton motive force dependent. The frequency of DNA-mediated transformation was reduced >20-fold in sca mutants. The addition of 0.1 mM Mn²⁺ to the transformation medium restored only partly the transformability of mutant cells, implying an alternate role for Sca proteins in the transformation process. Cells of sca mutants were unaffected in other binding properties tested and were unaffected in sensitivity to oxidants. The results show that Sca permease is a high-affinity mechanism for the acquisition of Mn²⁺ and is essential for growth of streptococci under Mn²⁺-limiting conditions.     

The external K+ concentration and mutations in the outer pore mouth affect the inhibition of kv1.5 current by Ni2+

By examining the consequences both of changes of [K⁺]_o and of point mutations in the outer pore mouth, our goal was to determine if the mechanism of the block of Kv1.5 ionic currents by external Ni²⁺ is similar to that for proton block. Ni²⁺ block is inhibited by increasing [K⁺]_o, by mutating a histidine residue in the pore turret (H463Q) or by mutating a residue near the pore mouth (R487V) that is the homolog of Shaker T449. Aside from a slight rightward shift of the Q-V curve, Ni²⁺ had no effect on gating currents. We propose that, as with H_o⁺, Ni²⁺ binding to H463 facilitates an outer pore inactivation process that is antagonized by K_o⁺ and that requires R487. However, whereas H_o⁺ substantially accelerates inactivation of residual currents, Ni²⁺ is much less potent, indicating incomplete overlap of the profiles of these two metal ions. Analyses with Co²⁺ and Mn²⁺, together with previous results, indicate that for the first-row transition metals the rank order for the inhibition of Kv1.5 in 0 mM K_o⁺ is Zn²⁺ (K_D ~ 0.07 mM) ≥ Ni²⁺ (K_D ~ 0.15 mM) > Co²⁺ (K_D ~ 1.4 mM) > Mn²⁺ (K_D > 10 mM).     

Bioinformatic and Expression Analyses of Genes Mediating Zinc Homeostasis in Nostoc punctiforme

Zinc homeostasis was investigated in Nostoc punctiforme. Cell tolerance to Zn²⁺ over 14 days showed that ZnCl₂ levels above 22 μM significantly reduced cell viability. After 3 days in 22 μM ZnCl₂, ca. 12% of the Zn²⁺ was in an EDTA-resistant component, suggesting an intracellular localization. Zinquin fluorescence was detected within cells exposed to concentrations up to 37 μM relative to 0 μM treatment. Radiolabeled ⁶⁵Zn showed Zn²⁺ uptake increased over a 3-day period, while efflux occurred more rapidly within a 3-h time period. Four putative genes involved in Zn²⁺ uptake and efflux in N. punctiforme were identified: (i) the predicted Co/Zn/Cd cation transporter, putative CDF; (ii) the predicted divalent heavy-metal cation transporter, putative Zip; (iii) the ATPase component and Fe/Zn uptake regulation protein, putative Fur; and (iv) an ABC-type Mn/Zn transport system, putative zinc ZnuC, ZnuABC system component. Quantitative real-time PCR indicated the responsiveness of all four genes to 22 μM ZnCl₂ within 3 h, followed by a reduction to below basal levels after 24 h by putative ZIP, ZnuC, and Fur and a reduction to below basal level after 72 h by putative CDF efflux gene. These results demonstrate differential regulation of zinc transporters over time, indicating a role for them in zinc homeostasis in N. punctiforme.     

cadDX Operon of Streptococcus salivarius 57.I

A CadDX system that confers resistance to Cd²⁺ and Zn²⁺ was identified in Streptococcus salivarius 57.I. Unlike with other CadDX systems, the expression of the cad promoter was negatively regulated by CadX, and the repression was inducible by Cd²⁺ and Zn²⁺, similar to what was found for CadCA systems. The lower G+C content of the S. salivarius cadDX genes suggests acquisition by horizontal gene transfer.     

Characterization of Cadmium Uptake in Lactobacillus plantarum and Isolation of Cadmium and Manganese Uptake Mutants

Two different Cd²⁺ uptake systems were identified in Lactobacillus plantarum. One is a high-affinity, high-velocity Mn²⁺ uptake system which also takes up Cd²⁺ and is induced by Mn²⁺ starvation. The calculated K_m and V_max are 0.26 μM and 3.6 μmol g of dry cell⁻¹ min⁻¹, respectively. Unlike Mn²⁺ uptake, which is facilitated by citrate and related tricarboxylic acids, Cd²⁺ uptake is weakly inhibited by citrate. Cd²⁺ and Mn²⁺ are competitive inhibitors of each other, and the affinity of the system for Cd²⁺ is higher than that for Mn²⁺. The other Cd²⁺ uptake system is expressed in Mn²⁺-sufficient cells, and no K_m can be calculated for it because uptake is nonsaturable. Mn²⁺ does not compete for transport through this system, nor does any other tested cation, i.e., Zn²⁺, Cu²⁺, Co²⁺, Mg²⁺, Ca²⁺, Fe²⁺, or Ni²⁺. Both systems require energy, since uncouplers completely inhibit their activities. Two Mn²⁺-dependent L. plantarum mutants were isolated by chemical mutagenesis and ampicillin enrichment. They required more than 5,000 times as much Mn²⁺ for growth as the parental strain. Mn²⁺ starvation-induced Cd²⁺ uptake in both mutants was less than 5% the wild-type rate. The low level of long-term Mn²⁺ or Cd²⁺ accumulation by the mutant strains also shows that the mutations eliminate the high-affinity Mn²⁺ and Cd²⁺ uptake system.     

A crystallographic study of the binding of 13 metal ions to two related RNA duplexes

Metal ions, and magnesium in particular, are known to be involved in RNA folding by stabilizing secondary and tertiary structures, and, as cofactors, in RNA enzymatic activity. We have conducted a systematic crystallographic analysis of cation binding to the duplex form of the HIV-1 RNA dimerization initiation site for the subtype-A and -B natural sequences. Eleven ions (K⁺, Pb²⁺, Mn²⁺, Ba²⁺, Ca²⁺, Cd²⁺, Sr²⁺, Zn²⁺, Co²⁺, Au³⁺ and Pt⁴⁺) and two hexammines [Co (NH₃)₆]³⁺ and [Ru (NH₃)₆]³⁺ were found to bind to the DIS duplex structure. Although the two sequences are very similar, strong differences were found in their cation binding properties. Divalent cations bind almost exclusively, as Mg²⁺, at ‘Hoogsteen’ sites of guanine residues, with a cation-dependent affinity for each site. Notably, a given cation can have very different affinities for a priori equivalent sites within the same molecule. Surprisingly, none of the two hexammines used were able to efficiently replace hexahydrated magnesium. Instead, [Co (NH₃)₄]³⁺ was seen bound by inner-sphere coordination to the RNA. This raises some questions about the practical use of [Co (NH₃)₆]³⁺ as a [Mg (H₂O)₆]²⁺ mimetic. Also very unexpected was the binding of the small Au³⁺ cation exactly between the Watson–Crick sites of a G-C base pair after an obligatory deprotonation of N1 of the guanine base. This extensive study of metal ion binding using X-ray crystallography significantly enriches our knowledge on the binding of middleweight or heavy metal ions to RNA, particularly compared with magnesium.     

Expression of the Escherichia coli pntA and pntB Genes, Encoding Nicotinamide Nucleotide Transhydrogenase, in Saccharomyces cerevisiae and Its Effect on Product Formation during Anaerobic Glucose Fermentation

We studied the physiological effect of the interconversion between the NAD(H) and NADP(H) coenzyme systems in recombinant Saccharomyces cerevisiae expressing the membrane-bound transhydrogenase from Escherichia coli. Our objective was to determine if the membrane-bound transhydrogenase could work in reoxidation of NADH to NAD⁺ in S. cerevisiae and thereby reduce glycerol formation during anaerobic fermentation. Membranes isolated from the recombinant strains exhibited reduction of 3-acetylpyridine-NAD⁺ by NADPH and by NADH in the presence of NADP⁺, which demonstrated that an active enzyme was present. Unlike the situation in E. coli, however, most of the transhydrogenase activity was not present in the yeast plasma membrane; rather, the enzyme appeared to remain localized in the membrane of the endoplasmic reticulum. During anaerobic glucose fermentation we observed an increase in the formation of 2-oxoglutarate, glycerol, and acetic acid in a strain expressing a high level of transhydrogenase, which indicated that increased NADPH consumption and NADH production occurred. The intracellular concentrations of NADH, NAD⁺, NADPH, and NADP⁺ were measured in cells expressing transhydrogenase. The reduction of the NADPH pool indicated that the transhydrogenase transferred reducing equivalents from NADPH to NAD⁺.     

Elimination of Glycerol Production in Anaerobic Cultures of a Saccharomyces cerevisiae Strain Engineered To Use Acetic Acid as an Electron Acceptor

In anaerobic cultures of wild-type Saccharomyces cerevisiae, glycerol production is essential to reoxidize NADH produced in biosynthetic processes. Consequently, glycerol is a major by-product during anaerobic production of ethanol by S. cerevisiae, the single largest fermentation process in industrial biotechnology. The present study investigates the possibility of completely eliminating glycerol production by engineering S. cerevisiae such that it can reoxidize NADH by the reduction of acetic acid to ethanol via NADH-dependent reactions. Acetic acid is available at significant amounts in lignocellulosic hydrolysates of agricultural residues. Consistent with earlier studies, deletion of the two genes encoding NAD-dependent glycerol-3-phosphate dehydrogenase (GPD1 and GPD2) led to elimination of glycerol production and an inability to grow anaerobically. However, when the E. coli mhpF gene, encoding the acetylating NAD-dependent acetaldehyde dehydrogenase (EC 1.2.1.10; acetaldehyde + NAD⁺ + coenzyme A ↔ acetyl coenzyme A + NADH + H⁺), was expressed in the gpd1Δ gpd2Δ strain, anaerobic growth was restored by supplementation with 2.0 g liter⁻¹ acetic acid. The stoichiometry of acetate consumption and growth was consistent with the complete replacement of glycerol formation by acetate reduction to ethanol as the mechanism for NADH reoxidation. This study provides a proof of principle for the potential of this metabolic engineering strategy to improve ethanol yields, eliminate glycerol production, and partially convert acetate, which is a well-known inhibitor of yeast performance in lignocellulosic hydrolysates, to ethanol. Further research should address the kinetic aspects of acetate reduction and the effect of the elimination of glycerol production on cellular robustness (e.g., osmotolerance).Bioethanol production by Saccharomyces cerevisiae is currently, by volume, the single largest fermentation process in industrial biotechnology. A global research effort is under way to expand the substrate range of S. cerevisiae to include lignocellulosic hydrolysates of nonfood feedstocks (e.g., energy crops and agricultural residues) and to increase productivity, robustness, and product yield (for reviews see references 20 and 35). A major challenge relating to the stoichiometry of yeast-based ethanol production is that substantial amounts of glycerol are invariably formed as a by-product (24). It has been estimated that, in typical industrial ethanol processes, up to 4% of the sugar feedstock is converted into glycerol (24). Although glycerol also serves as a compatible solute at high extracellular osmolarity (10), glycerol production under anaerobic conditions is primarily linked to redox metabolism (34).During anaerobic growth of S. cerevisiae, sugar dissimilation occurs via alcoholic fermentation. In this process, the NADH formed in the glycolytic glyceraldehyde-3-phosphate dehydrogenase reaction is reoxidized by converting acetaldehyde, formed by decarboxylation of pyruvate to ethanol via NAD⁺-dependent alcohol dehydrogenase. The fixed stoichiometry of this redox-neutral dissimilatory pathway causes problems when a net reduction of NAD⁺ to NADH occurs elsewhere in the metabolism. Such a net production of NADH occurs in assimilation when yeast biomass is synthesized from glucose and ammonia (34). Under anaerobic conditions, NADH reoxidation in S. cerevisiae is strictly dependent on reduction of sugar to glycerol (34). Glycerol formation is initiated by reduction of the glycolytic intermediate dihydroxyacetone phosphate to glycerol-3-phosphate, a reaction catalyzed by NAD⁺-dependent glycerol-3-phosphate dehydrogenase. Subsequently, the glycerol-3-phosphate formed in this reaction is hydrolyzed by glycerol-3-phosphatase to yield glycerol and inorganic phosphate.The importance of glycerol production for fermentative growth of yeasts was already observed in the 1960s during studies of non-Saccharomyces yeasts that exhibit a so-called “Custers effect.” In such yeast species, which are naturally unable to produce glycerol, fermentative growth on glucose is possible only in the presence of an external electron acceptor that can be reduced via an NADH-dependent reaction (e.g., the reduction of acetoin to butanediol via NAD⁺-dependent butanediol dehydrogenase) (29). It was later shown that gpd1Δ gpd2Δ strains of S. cerevisiae, which are also unable to produce glycerol, are similarly unable to grow under anaerobic conditions unless provided with acetoin as an external electron acceptor (8).In view of its large economic significance, several metabolic engineering strategies have been explored to reduce or eliminate glycerol production in anaerobic cultures of S. cerevisiae. Nissen et al. (25) changed the cofactor specificity of glutamate dehydrogenase, the major ammonia-fixing enzyme of S. cerevisiae, thereby increasing NADH consumption in biosynthesis. This approach significantly reduced glycerol production in anaerobic cultures grown with ammonia as the nitrogen source. Attempts to further reduce glycerol production by expression of a heterologous transhydrogenase, with the aim to convert NADH and NADP⁺ into NAD⁺ and NADPH, were unsuccessful (24) because intracellular concentrations of these pyridine nucleotide cofactor couples favor the reverse reaction (23).The goal of the present study was to investigate whether the engineering of a linear pathway for the NADH-dependent reduction of acetic acid to ethanol can replace glycerol formation as a redox sink in anaerobic, glucose-grown cultures of S. cerevisiae and thus provide a stoichiometric basis for elimination of glycerol production during industrial ethanol production. Significant amounts of acetic acid are released upon hydrolysis of lignocellulosic biomass, and, in fact, acetic acid is studied as an inhibitor of yeast metabolism in lignocellulosic hydrolysates (5, 7, 26). The S. cerevisiae genome already contains genes encoding acetyl coenzyme A (acetyl-CoA) synthetase (32) and NAD⁺-dependent alcohol dehydrogenases (ADH1-5 [12]). To complete the linear pathway for acetic acid reduction, we expressed an NAD⁺-dependent, acetylating acetaldehyde dehydrogenase (EC 1.2.1.10) from Escherichia coli into a gpd1Δ gpd2Δ strain of S. cerevisiae. This enzyme, encoded by the E. coli mhpF gene (15), catalyzes the reaction acetaldehyde + NAD⁺ + coenzyme A ↔ acetyl coenzyme A + NADH + H⁺. Growth and product formation of the engineered strain were then compared in the presence and absence of acetic acid and compared to those of a congenic reference strain.     

Metal Ions May Suppress or Enhance Cellular Differentiation in Candida albicans and Candida tropicalis Biofilms

Candida albicans and Candida tropicalis are polymorphic fungi that develop antimicrobial-resistant biofilm communities that are characterized by multiple cell morphotypes. This study investigated cell type interconversion and drug and metal resistance as well as community organization in biofilms of these microorganisms that were exposed to metal ions. To study this, Candida biofilms were grown either in microtiter plates containing gradient arrays of metal ions or in the Calgary Biofilm Device for high-throughput susceptibility testing. Biofilm formation and antifungal resistance were evaluated by viable cell counts, tetrazolium salt reduction, light microscopy, and confocal laser scanning microscopy in conjunction with three-dimensional visualization. We discovered that subinhibitory concentrations of certain metal ions (CrO₄²⁻, Co²⁺, Cu²⁺, Ag⁺, Zn²⁺, Cd²⁺, Hg²⁺, Pb²⁺, AsO₂⁻, and SeO₃²⁻) caused changes in biofilm structure by blocking or eliciting the transition between yeast and hyphal cell types. Four distinct biofilm community structure types were discerned from these data, which were designated “domed,” “layer cake,” “flat,” and “mycelial.” This study suggests that Candida biofilm populations may respond to metal ions to form cell-cell and solid-surface-attached assemblages with distinct patterns of cellular differentiation.     

Divalent metal-dependent catalysis and cleavage specificity of CSP41, a chloroplast endoribonuclease belonging to the short chain dehydrogenase/reductase superfamily

CSP41 is a ubiquitous chloroplast endoribonuclease belonging to the short chain dehydrogenase/reductase (SDR) superfamily. To help elucidate the role of CSP41 in chloroplast gene regulation, the mechanisms that determine its substrate recognition and catalytic activity were investigated. A divalent metal is required for catalysis, most probably to provide a nucleophile for cleavage 5′ to the phosphodiester bond, and may also participate in cleavage site selection. This requirement distinguishes CSP41 from other Rossman fold-containing proteins from the SDR superfamily, including several RNA-binding proteins and endonucleases. CSP41 is active only in the presence of MgCl₂ and CaCl₂. Although Mg²⁺- and Ca²⁺-activated CSP41 cleave at identical sites in the single-stranded regions of a stem–loop-containing substrate, Mg²⁺-activated CSP41 was also able to cleave within the double-stranded region of the stem–loop. Mixed metal experiments with Mg²⁺ and Ca²⁺ suggest that CSP41 contains a single divalent metal-binding site which is non-selective, since Mn²⁺, Co²⁺ and Zn²⁺ compete with Mg²⁺ for binding, although there is no activity in their presence. Using site-directed mutagenesis, we identified three residues, Asn71, Asp89 and Asp103, which may form the divalent metal-binding pocket. The activation constant for Mg²⁺ (K_A,Mg = 2.1 ± 0.4 mM) is of the same order of magnitude as the stromal Mg²⁺ concentrations, which fluctuate between 0.5 and 10 mM as a function of light and of leaf development. These changes in stromal Mg²⁺ concentration may regulate CSP41 activity, and thus cpRNA stability, during plant development.     

Minimization of Glycerol Production during the High-Performance Fed-Batch Ethanolic Fermentation Process in Saccharomyces cerevisiae, Using a Metabolic Model as a Prediction Tool

On the basis of knowledge of the biological role of glycerol in the redox balance of Saccharomyces cerevisiae, a fermentation strategy was defined to reduce the surplus formation of NADH, responsible for glycerol synthesis. A metabolic model was used to predict the operating conditions that would reduce glycerol production during ethanol fermentation. Experimental validation of the simulation results was done by monitoring the inlet substrate feeding during fed-batch S. cerevisiae cultivation in order to maintain the respiratory quotient (RQ) (defined as the CO₂ production to O₂ consumption ratio) value between 4 and 5. Compared to previous fermentations without glucose monitoring, the final glycerol concentration was successfully decreased. Although RQ-controlled fermentation led to a lower maximum specific ethanol production rate, it was possible to reach a high level of ethanol production: 85 g · liter⁻¹ with 1.7 g · liter⁻¹ glycerol in 30 h. We showed here that by using a metabolic model as a tool in prediction, it was possible to reduce glycerol production in a very high-performance ethanolic fermentation process.     

New Expression System Tightly Controlled by Zinc Availability in Lactococcus lactis

Here we developed the new expression system P_Zn zitR, based on the regulatory signals (P_Zn promoter and zitR repressor) of the Lactococcus lactis zit operon, involved in Zn²⁺ high-affinity uptake and regulation. A P_Zn zitR-controlled expression vector was constructed, and expression regulation was studied with two reporter genes, uspnuc and lacLM; these genes encode, respectively, a protein derived from Staphylococcus aureus secreted nuclease and Leuconostoc mesenteroides cytoplasmic β-galactosidase. Nuclease and β-galactosidase activities of L. lactis MG1363 cells expressing either uspnuc or lacLM under the control of P_Zn zitR were evaluated on plates and quantified from liquid cultures as a function of divalent metal ion, particularly Zn²⁺, availability in the environment. Our results demonstrate that P_Zn zitR is highly inducible upon divalent cation starvation, obtained either through EDTA addition or during growth in chemically defined medium, and is strongly repressed in the presence of excess Zn²⁺. The efficiency of the P_Zn zitR expression system was compared to that of the well-known nisin-controlled expression (NICE) system with the same reporter genes cloned under either P_Zn zitR or P_nisA nisRK control. lacLM induction levels reached with both systems were on the same order of magnitude, even though the NICE system is fivefold more efficient than the P_Zn zitR system. An even smaller difference or no difference was observed after 3 h of induction when nuclease was used as a reporter for Western blotting detection. P_Zn zitR proved to be a powerful expression system for L. lactis, as it is tightly controlled by the zinc concentration in the medium.     

Strontium-Induced Repetitive Calcium Spikes in a Unicellular Green Alga

The divalent cation Sr²⁺ induced repetitive transient spikes of the cytosolic Ca²⁺ activity [Ca²⁺]_cy and parallel repetitive transient hyperpolarizations of the plasma membrane in the unicellular green alga Eremosphaera viridis. [Ca²⁺]_cy measurements, membrane potential measurements, and cation analysis of the cells were used to elucidate the mechanism of Sr²⁺-induced [Ca²⁺]_cy oscillations. Sr²⁺ was effectively and rapidly compartmentalized within the cell, probably into the vacuole. The [Ca²⁺]_cy oscillations cause membrane potential oscillations, and not the reverse. The endoplasmic reticulum (ER) Ca²⁺-ATPase blockers 2,5-di-tert-butylhydroquinone and cyclopiazonic acid inhibited Sr²⁺-induced repetitive [Ca²⁺]_cy spikes, whereas the compartmentalization of Sr²⁺ was not influenced. A repetitive Ca²⁺ release and Ca²⁺ re-uptake by the ER probably generated repetitive [Ca²⁺]_cy spikes in E. viridis in the presence of Sr²⁺. The inhibitory effect of ruthenium red and ryanodine indicated that the Sr²⁺-induced Ca²⁺ release from the ER was mediated by a ryanodine/cyclic ADP-ribose type of Ca²⁺ channel. The blockage of Sr²⁺-induced repetitive [Ca²⁺]_cy spikes by La³⁺ or Gd³⁺ indicated the necessity of a certain influx of divalent cations for sustained [Ca²⁺]_cy oscillations. Based on these data we present a mathematical model that describes the baseline spiking [Ca²⁺]_cy oscillations in E. viridis.     

Catalytic cleavage of cis- and trans-acting antigenomic delta ribozymes in the presence of various divalent metal ions

Catalytic activity of four structural variants of the antigenomic delta ribozyme, two cis- and two trans-acting, has been compared in the presence of selected divalent metal ions that effectively support catalysis. The ribozymes differ in regions that are not directly involved in formation of the ribozyme active site: the region immediately preceding the catalytic cleavage site, the P4 stem and a stretch of the viral RNA sequence extending the minimal ribozyme sequence at its 3′-terminus. The variants show high cleavage activity in the presence of Mg²⁺, Ca²⁺ and Mn²⁺, lower with Co²⁺ and Sr²⁺ and some variants are also active with Cd²⁺ and Zn²⁺ ions. In the presence of a particular metal ion the ribozymes cleave, however with different initial rates, according to pseudo-first or higher order kinetics and to different final cleavage extents. On the other hand, relatively small differences are observed in the reactions induced by various metal ions. The cleavage of trans-acting ribozymes induced by Mg²⁺ is partially inhibited in the presence of Na⁺, spermidine and some other divalent metal ions. The inert Co(NH₃)₆³⁺ complex is unable to support catalysis, as reported earlier for the genomic ribozyme. The results are discussed in terms of the influence of structural elements peripheral to the ribozyme active site on its cleavage rate and efficiency as well as the role of metal ions in the cleavage mechanism. Some implications concerning further studies and possible applications of delta ribozymes are also considered.     

Enhancing the Production of Hydroxyl Radicals by Pleurotus eryngii via Quinone Redox Cycling for Pollutant Removal

The induction of hydroxyl radical (OH) production via quinone redox cycling in white-rot fungi was investigated to improve pollutant degradation. In particular, we examined the influence of 4-methoxybenzaldehyde (anisaldehyde), Mn²⁺, and oxalate on Pleurotus eryngii OH generation. Our standard quinone redox cycling conditions combined mycelium from laccase-producing cultures with 2,6-dimethoxy-1,4-benzoquinone (DBQ) and Fe³⁺-EDTA. The main reactions involved in OH production under these conditions have been shown to be (i) DBQ reduction to hydroquinone (DBQH₂) by cell-bound dehydrogenase activities; (ii) DBQH₂ oxidation to semiquinone (DBQ⁻) by laccase; (iii) DBQ⁻ autoxidation, catalyzed by Fe³⁺-EDTA, producing superoxide (O₂⁻) and Fe²⁺-EDTA; (iv) O₂⁻ dismutation, generating H₂O₂; and (v) the Fenton reaction. Compared to standard quinone redox cycling conditions, OH production was increased 1.2- and 3.0-fold by the presence of anisaldehyde and Mn²⁺, respectively, and 3.1-fold by substituting Fe³⁺-EDTA with Fe³⁺-oxalate. A 6.3-fold increase was obtained by combining Mn²⁺ and Fe³⁺-oxalate. These increases were due to enhanced production of H₂O₂ via anisaldehyde redox cycling and O₂⁻ reduction by Mn²⁺. They were also caused by the acceleration of the DBQ redox cycle as a consequence of DBQH₂ oxidation by both Fe³⁺-oxalate and the Mn³⁺ generated during O₂⁻ reduction. Finally, induction of OH production through quinone redox cycling enabled P. eryngii to oxidize phenol and the dye reactive black 5, obtaining a high correlation between the rates of OH production and pollutant oxidation.The degradation of lignin and pollutants by white-rot fungi is an oxidative and rather nonspecific process based on the production of substrate free radicals (36). These radicals are produced by ligninolytic enzymes, including laccase and three kinds of peroxidases: lignin peroxidase, manganese peroxidase, and versatile peroxidase (VP) (23). The H₂O₂ required for peroxidase activities is provided by several oxidases, such as glyoxal oxidase and aryl-alcohol oxidase (AAO) (9, 18). This free-radical-based degradative mechanism leads to the production of a broad variety of oxidized compounds. Common lignin depolymerization products are aromatic aldehydes and acids, and quinones (34). In addition to their high extracellular oxidation potential, white-rot fungi show strong ability to reduce these lignin depolymerization products, using different intracellular and membrane-bound systems (4, 25, 39). Since reduced electron acceptors of oxidized compounds are donor substrates for the above-mentioned oxidative enzymes, the simultaneous actions of both systems lead to the establishment of redox cycles (35). Although the function of these redox cycles is not fully understood, they have been hypothesized to be related to further metabolism of lignin depolymerization products that require reduction to be converted in substrates of the ligninolytic enzymes (34). A second function attributed to these redox cycles is the production of reactive oxygen species, i.e., superoxide anion radicals (O₂⁻), H₂O₂, and hydroxyl radicals (OH), where lignin depolymerization products and fungal metabolites act as electron carriers between intracellular reducing equivalents and extracellular oxygen. This function has been studied in Pleurotus eryngii, whose ligninolytic system is composed of laccase (26), VP (24), and AAO (9). Incubation of this fungus with different aromatic aldehydes has been shown to provide extracellular H₂O₂ on a constant basis, due to the establishment of a redox cycle catalyzed by an intracellular aryl-alcohol dehydrogenase (AAD) and the extracellular AAO (7, 10). The process was termed aromatic aldehyde redox cycling, and 4-methoxybenzaldehyde (anisaldehyde) serves as the main Pleurotus metabolite acting as a cycle electron carrier (13). A second cyclic system, involving a cell-bound quinone reductase activity (QR) and laccase, was found to produce O₂⁻ and H₂O₂ during incubation of P. eryngii with different quinones (11). The process was described as the cell-bound divalent reduction of quinones (Q) by QR, followed by extracellular laccase oxidation of hydroquinones (QH₂) into semiquinones (Q⁻), which autoxidized to some extent, producing O₂⁻ (Q⁻ + O₂ ⇆ Q + O₂⁻). H₂O₂ was formed by O₂⁻ dismutation (O₂⁻ + HO₂ + H⁺ → O₂ + H₂O₂). In an accompanying paper, we describe the extension of this O₂⁻ and H₂O₂ generation mechanism to OH radical production by the addition of Fe³⁺-EDTA to incubation mixtures of several white-rot fungi with different quinones (6). Among them, those derived from 4-hydroxyphenyl, guaiacyl, and syringyl lignin units were used: 1,4-benzoquinone (BQ), 2-methoxy-1,4-benzoquinone (MBQ), and 2,6-dimethoxy-1,4-benzoquinone (DBQ), respectively. Semiquinone autoxidation under these conditions was catalyzed by Fe³⁺-EDTA instead of being a direct electron transfer to O₂. The intermediate Fe²⁺-EDTA reduced not only O₂, but also H₂O₂, leading to OH radical production by the Fenton reaction (H₂O₂ + Fe²⁺ → OH + OH⁻ + Fe³⁺).Although OH radicals are the strongest oxidants produced by white-rot fungi (2, 14), studies of their involvement in pollutant degradation are quite scarce. In this context, the objectives of this study were to (i) determine possible factors enhancing the production of OH radicals by P. eryngii via quinone redox cycling and (ii) test the validity of this inducible OH production mechanism as a strategy for pollutant degradation. Our selection of possible OH production promoters was guided by two observations (6). First, the redox cycle of benzoquinones working with washed P. eryngii mycelium is rate limited by hydroquinone oxidation, since the amounts of the ligninolytic enzymes that remained bound to the fungus under these conditions were not large. Second, H₂O₂ is the limiting reagent for OH production by the Fenton reaction.With these considerations in mind, anisaldehyde and Mn²⁺ were selected to increase H₂O₂ production. As mentioned above, anisaldehyde induces H₂O₂ production in P. eryngii via aromatic aldehyde redox cycling (7). Mn²⁺ has been shown to enhance H₂O₂ production during the oxidation of QH₂ by P. eryngii laccase by reducing the O₂⁻ produced in the semiquinone autoxidation reaction (Mn²⁺ + O₂⁻ → Mn³⁺ + H₂O₂ + 2 H⁺) (26). Mn²⁺ was also selected to increase the hydroquinone oxidation rate, since this reaction has been shown to be propagated by the Mn³⁺ generated in the latter reaction (QH₂ + Mn³⁺ → Q⁻ + Mn²⁺ + 2 H⁺). The replacement of Fe³⁺-EDTA by Fe³⁺-oxalate was also planned in order to increase the QH₂ oxidation rate above that resulting from the action of laccase. Oxalate is a common extracellular metabolite of wood-rotting fungi to which the function of chelating iron and manganese has been attributed (16, 45). The use of Fe³⁺-oxalate and nonchelated Fe³⁺, both QH₂ oxidants, has been proven to enable quinone redox cycling in fungi that do not produce ligninolytic enzymes, such as the brown-rot fungus Gloeophyllum trabeum (17, 40, 41). Finally, phenol and the azo dye reactive black 5 (RB5) were selected as model pollutants.     

Modulation of the Local SR Ca2+ Release by Intracellular Mg2+ in Cardiac Myocytes

In cardiac muscle, Ca²⁺-induced Ca²⁺ release (CICR) from the sarcoplasmic reticulum (SR) defines the amplitude and time course of the Ca²⁺ transient. The global elevation of the intracellular Ca²⁺ concentration arises from the spatial and temporal summation of elementary Ca²⁺ release events, Ca²⁺ sparks. Ca²⁺ sparks represent the concerted opening of a group of ryanodine receptors (RYRs), which are under the control of several modulatory proteins and diffusible cytoplasmic factors (e.g., Ca²⁺, Mg²⁺, and ATP). Here, we examined by which mechanism the free intracellular Mg²⁺ ([Mg²⁺]_free) affects various Ca²⁺ spark parameters in permeabilized mouse ventricular myocytes, such as spark frequency, duration, rise time, and full width, at half magnitude and half maximal duration. Varying the levels of free ATP and Mg²⁺ in specifically designed solutions allowed us to separate the inhibition of RYRs by Mg²⁺ from the possible activation by ATP and Mg²⁺-ATP via the adenine binding site of the channel. Changes in [Mg²⁺]_free generally led to biphasic alterations of the Ca²⁺ spark frequency. For example, lowering [Mg²⁺]_free resulted in an abrupt increase of spark frequency, which slowly recovered toward the initial level, presumably as a result of SR Ca²⁺ depletion. Fitting the Ca²⁺ spark inhibition by [Mg²⁺]_free with a Hill equation revealed a K_i of 0.1 mM. In conclusion, our results support the notion that local Ca²⁺ release and Ca²⁺ sparks are modulated by Mg²⁺ in the intracellular environment. This seems to occur predominantly by hindering Ca²⁺-dependent activation of the RYRs through competitive Mg²⁺ occupancy of the high-affinity activation site of the channels. These findings help to characterize CICR in cardiac muscle under normal and pathological conditions, where the levels of Mg²⁺ and ATP can change.     

Mitochondrial Contribution to the Anoxic Ca2+ Signal in Maize Suspension-Cultured Cells

Anoxia induces a rapid elevation of the cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in maize (Zea mays L.) cells, which is caused by the release of the ion from intracellular stores. This anoxic Ca²⁺ release is important for gene activation and survival in O₂-deprived maize seedlings and cells. In this study we examined the contribution of mitochondrial Ca²⁺ to the anoxic [Ca²⁺]_cyt elevation in maize cells. Imaging of intramitochondrial Ca²⁺ levels showed that a majority of mitochondria released their Ca²⁺ in response to anoxia and took up Ca²⁺ upon reoxygenation. We also investigated whether the mitochondrial Ca²⁺ release contributed to the increase in [Ca²⁺]_cyt under anoxia. Analysis of the spatial association between anoxic [Ca²⁺]_cyt changes and the distribution of mitochondrial and other intracellular Ca²⁺ stores revealed that the largest [Ca²⁺]_cyt increases occurred close to mitochondria and away from the tonoplast. In addition, carbonylcyanide p-trifluoromethoxyphenyl hydrazone treatment depolarized mitochondria and caused a mild elevation of [Ca²⁺]_cyt under aerobic conditions but prevented a [Ca²⁺]_cyt increase in response to a subsequent anoxic pulse. These results suggest that mitochondria play an important role in the anoxic elevation of [Ca²⁺]_cyt and participate in the signaling of O₂ deprivation.