Relationship between intracellular pH and antibiotic biosynthesis in Fusidium coccineum

A correlation between the value of the intracellular pH and the biosynthesis of fusidic acid was studied by ³¹P-NMR spectroscopy in two strains of the fungus Fusidium coccineum. One of the strains was highly active and the other strain had low activity with respect to the antibiotic production. The position of the orthophosphate resonance in the ³¹P-NMR spectra was considered as a measure of the intracellular pH. In the cells of the highly active strain pH was in the range 7.0–7.5 in the cytoplasm and 6.1–6.25 in the vacuoles. In the cells of the strain with low activity was in the range 7.3–7.9 in the cytoplasm and 6.0–6.2 in the vacuoles. During high antibiotic productivity, the intracellular pH in the highly active strain full sharply, while in the less active strain it effectively did not change. This suggested that the change in the intracellular pH was responsible for the action of the enzymes in the cells and could be a factor defining the function of the cyanide-resistant respiration pathway and consequently the synthesis of fusidic acid in F. coccineum. *** DIRECT SUPPORT *** AG903062 00009     

Mechanism of nigericin bioconversion by the ciliateTetrahymena pyriformis GL

Summary Nigericin, an ionophorous antibiotic, is converted to epinigericin by the ciliateTetrahymena pyriformis. Bioconversion occurs in the presence of intact cells but not with broken cells (sonicated ciliates). However, broken cells obtained from intact ciliates pretreated with 1 mg/l of nigericin, before sonication, converted nigericin to epinigericin. Non-impairment after adding actinomycin D and cycloheximide to the ciliate culture suggests that the induction mechanism involves no mRNA synthesis, nor any de novo synthesis of protein. Cell fractionation ofTetrahymena demonstrates that the cytosolic compartment is implicated in this bioconversion.     

The species-specific mode of action of the antimicrobial peptide subtilosin against Listeria monocytogenes Scott A

Aims: To elucidate the molecular mechanism of action of the antimicrobial peptide subtilosin against the foodborne pathogen Listeria monocytogenes Scott A. Methods and Results: Subtilosin was purified from a culture of Bacillus amyloliquefaciens. The minimal inhibitory concentration of subtilosin against L. monocytogenes Scott A was determined by broth microdilution method. The effect of subtilosin on the transmembrane electrical potential (ΔΨ) and pH gradient (ΔpH), and its ability to induce efflux of intracellular ATP, was investigated. Subtilosin fully inhibited L. monocytogenes growth at a concentration of 19 μg ml^?1. Subtilosin caused a partial depletion of the ΔΨ and had a similar minor effect on the ΔpH. There was no significant efflux of intracellular ATP. Conclusion: Subtilosin likely acts upon L. monocytogenes Scott A by perturbing the lipid bilayer of the cellular membrane and causing intracellular damage, leading to eventual cell death. Subtilosin’s mode of action against L. monocytogenes Scott A differs from the one previously described for another human pathogen, Gardnerella vaginalis. Significance and Impact of the Study: This is the first report on the specific mode of action of subtilosin against L. monocytogenes and the first report of a bacteriocin with a species‐specific mode of action.     

Effect of ethanol and fatty acids on malolactic activity ofLeuconostoc oenos

Medium-chain fatty acids (C₆ to C₁₂), produced by yeast metabolism during alcoholic fermentation, are known to be inhibitory to lactic acid bacteria. The purpose of this work was to clarify the effect of both ethanol and decanoic and dodecanoic acids on the growth and malolactic activity of aLeuconostoc oenos strain isolated from Portuguese red wine. Ethanol in concentrations up to 12% had no significant effect on malolactic activity but strongly inhibited cell growth. The fatty acids decanoic acid, in concentrations up to 12.5 mg l^–1, and, dodecanoic acid up to 2.5 mg l^–1 seemed to act as growth factors stimulating also malolactic activity; at higher concentrations they exerted an inhibitory effect. We found clear pH dependence between pH 3.0 and pH 6.0, between decanoic acid concentration and its effect on malolactic activity, indicating that the undissociated molecule is the active form. At pH 3.0 the results can be explained by considering that fatty acids enter the cell as protonated molecules and dissociate in the cytoplasm due to the higher internal pH, leading to increased intracellular hydrogenous concentration. This may be the basis of two different effects that contribute to the observed inhibition: decrease in the intracellular pH and dissipation of the transmembrane proton gradient, thus inhibiting intracellular enzymes and ApH-dependent transport systems.     

A non-polyene antifungal antibiotic fromStreptomyces albidoflavus PU 23

In all 312 actinomycete strains were isolated from water and soil samples from different regions. All these isolates were purified and screened for their antifungal activity against pathogenic fungi. Out of these, 22% of the isolates exhibited activity against fungi. One promising strain,Streptomyces albidoflavus PU 23 with strong antifungal activity against pathogenic fungi was selected for further studies. Antibiotic was extracted and purified from the isolate.Aspergillus spp. was most sensitive to the antibiotic followed by other molds and yeasts. The antibiotic was stable at different temperatures and pH tested and there was no significant loss of the antifungal activity after treatment with various detergents and enzymes. Synergistic effect was observed when the antibiotic was used in combination with hamycin. The antibiotic was fairly stable for a period of 12 months at 4°C. The mode of action of the antibiotic seems to be by binding to the ergosterol present in the fungal cell membrane resulting in the leakage of intracellular material and eventually death of the cell. The structure of the antibiotic was determined by elemental analysis and by ultraviolet (UV), Fourier transform infrared (FTIR), nuclear magnetic resonance (NMR) and liquid chromatography mass spectra (LCMS). The antibiotic was found to be a straight chain polyhydroxy, polyether, non-proteinic compound with a single double bond, indicating a nonpolyene antifungal antibiotic     

Structure–Depolarizing Activity Relationship for Achatin-I,a Tetrapeptide with a d-Phe Residue,and Its Derivatives toward the Crayfish Giant Axon

Achatin-I (Gly-d-Phe-Ala-Asp) is a tetrapeptide isolated from the ganglia of an African giant snail (Achatina fulica Férussac). The compound and its derivatives were synthesized, and their effects on the action potential of the crayfish giant axon elicited by electrical stimulation were examined by an intracellular recording method. The membrane potential after the action potential had fallen was elevated by some compounds at an acidic but not neutral pH level. An analysis of the structure-activity relationship for the activity to elevate the afterpotential at an acidic pH level, suggests that the factors favorable for activity were the β-turn conformation, a β- or γ-carboxyl group in the C-terminal residue, appropriate hydrophobicity and/or bulkiness in the protecting groups for the α-amino group at the N-terminus, and the number of negative charges in the entire molecule.     

One‐step magnetic modification of yeast cells by microwave‐synthesized iron oxide microparticles

Baker's yeast (Saccharomyces cerevisiae) cells were magnetically modified with magnetic iron oxide particles prepared by microwave irradiation of iron(II) sulfate at high pH. The modification procedure was very simple and fast. Both non‐cross‐linked and glutaraldehyde cross‐linked magnetic cells enabled efficient sucrose conversion into glucose and fructose, due to the presence of active intracellular invertase. The prepared magnetic whole‐cell biocatalyst was stable; almost the same catalytic activity was observed after 1‐month storage at 4°C. Simple magnetic separation and stability of the developed biocatalyst enabled its reusability without significant loss of enzyme activity.

Significance and Impact of the Study

Magnetic whole yeast cell biocatalyst containing intracellular invertase in its natural environment has been prepared. Magnetic properties enable its easy separation from reaction mixture. Magnetically modified Saccharomyces cerevisiae cells have been used for invert sugar production, hydrolysing sucrose into glucose and fructose. The described magnetization procedure employing microwave‐synthesized iron oxide microparticles is a low‐cost and easy‐to‐perform alternative to already existing magnetization techniques.     

The protective effect of EGF-activated ROS in human corneal epithelial cells by inducing mitochondrial autophagy via activation TRPM2

Oxidative stress is a major pathogenesis of some ocular surface diseases. Our previous study demonstrated that epidermal growth factor (EGF)-activated reactive oxygen species (ROS) could protect against human corneal epithelial cell (HCE) injury. In the present study, we aimed to explore the role and mechanisms of oxidative stress and mitochondrial autophagy in HCE cells subjected to scratch injury. CCK-8 assays, EdU assays, Western blot analysis, wound-healing assays, and flow cytometry were conducted to determine cell viability, proliferation, protein expression, cell apoptosis, and intracellular ROS levels, respectively. The results showed that EGF could promote damage repair and inhibit cell apoptosis in scratch injured HCE cells by upregulating ROS (**p < .01, ***p < .001). EGF also induced mitochondrial autophagy and alleviated mitochondrial damage. Interestingly, the combination of the mitochondrial autophagy inhibitor and mitochondrial division inhibitor 1 (MDIVI-1) with EGF could reduce cell proliferation, viability, and the ROS level (*p < .05, **p < .01, ***p < .001). Treatment using the ROS inhibitor N-acetyl- l -cysteine abrogated the increase in mitochondrial membrane potential after EGF treatment. (*p < .05). Taken together, these findings indicated that EGF plays an important role in HCE damage repair and could activate ROS to protect against HCE injury by inducing mitochondrial autophagy via activation of TRPM2.     

Properties and biological activity of a new peptide antibiotic (Colisan)

Colisan has the formula Lys₃Val₄Leu₃IleTyr and a molecular weight of approximately 1500. Neither N-terminal nor C-terminal group have been found. The peptide is highly resistant to hydrolysis by proteolytic enzymes (pepsin and trypsin). The ?-amino groups of lysine are indispensable for biological activity, since either deamination or acetylation completely inactivates Colisan. A comparative study with basic peptides like polylysine demonstrated their similarity of action, on one hand, and an antagonistic effect to Colisan, on the other. The fast action of the antibiotic and the specific effect on the cell membranes of many different biological systems (Entameba, Paramecium, erythrocytes, smooth muscle, and others) support the hypothesis that the primary damage occurs in the membrane with consequent alteration of permeability. The leakage of intracellular material (histamine) from mast cells after application of Colisan is in accord with this hypothesis. The results strongly suggest that the biological activity of the peptide is based essentially, but not exclusively, on its basic character.     

Flow cytometric method for quantifying viable Mycoplasma agassizii,an agent of upper respiratory tract disease in the desert tortoise (Gopherus agassizii)

Aims: Mycoplasma agassizii can cause upper respiratory tract disease in the threatened desert tortoise of the Southwestern United States. Two technical challenges have impeded critical microbiological studies of this microorganism: (i) its small size limits the use of light microscopy for cell counting and (ii) its extremely slow growth in broth and agar cultures impedes colony counting. Our aim was to develop a rapid and sensitive flow cytometric method using a vital fluorescent dye to enumerate viable M. agassizii cells. Methods and Results: Here, we demonstrate that the nonfluorescent molecule 5‐carboxyfluorescein (5‐CF) diacetate acetoxymethyl ester penetrates M. agassizii cell membranes and it is converted in the cytoplasm to the fluorescent molecule 5‐CF by the action of intracellular esterases. Labelled mycoplasma cells can be easily detected by flow cytometry, and cultures with as few as 100 viable mycoplasma cells ml^?1 can be labelled and counted in less than 1 h. Experiments using temperature‐induced cell death demonstrated that only viable M. agassizii cells are labelled with this procedure. Conclusions: A rapid and sensitive flow cytometric technique has been developed for enumerating viable M. agassizii cells. Significance and Impact of the Study: This technique should facilitate basic immunological, biochemical and pharmacological studies of this important pathogen which may lead to new diagnostic and therapeutic methods.     

Dextran dextrinase and dextran of Gluconobacter oxydans

Certain strains of Gluconobacter oxydans have been known since the 1940s to produce the enzyme dextran dextrinase (DDase; EC2.4.1.2)—a transglucosidase converting maltodextrins into (oligo)dextran. The enzyme catalyses the transfer of an α1,4 linked glucosyl unit from a donor to an acceptor molecule, forming an α1,6 linkage: consecutive glucosyl transfers result in the formation of high molecular weight dextran from maltodextrins. In the early 1990s, the group of K. Yamamoto in Japan revived research on DDase, focussing on the purification and characterisation of the intracellular DDase produced by G. oxydans ATCC 11894. More recently, this was taken further by Y. Suzuki and coworkers, who investigated the properties and kinetics of the extracellular DDase formed by the same strain. Our group further elaborated on fermentation processes to optimise DDase production and dextran formation, DDase characterisation and its use as a biocatalyst, and the physiological link between intracellular and extracellular DDase. Here, we present a condensed overview of the current scientific status and the application potential of G. oxydans DDase and its products, (oligo)dextrans. The production of DDase as well as of dextran is first described via optimised fermentation processes. Specific assays for measuring DDase activity are also outlined. The general characteristics, substrate specificity, and mode of action of DDase as a transglucosidase are described in detail. Two forms of DDase are produced by G. oxydans depending on nutritional fermentation conditions: an intracellular and an extracellular form. The relationship between the two enzyme forms is also discussed. Furthermore, applications of DDase, e.g. production of (oligo)dextran, transglucosylated products and speciality oligosaccharides, are summarized.     

Dxr is essential in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>and fosmidomycin resistance is due to a lack of uptake

   

Fosmidomycin is a phosphonic antibiotic which inhibits 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr), the first committed step of the non-mevalonate pathway of isoprenoid biosynthesis. In Mycobacterium tuberculosis Dxr is encoded by Rv2870c, and although the antibiotic has been shown to inhibit the recombinant enzyme [1], mycobacteria are intrinsically resistant to fosmidomycin at the whole cell level. Fosmidomycin is a hydrophilic molecule and in many bacteria its uptake is an active process involving a cAMP dependent glycerol-3-phosphate transporter (GlpT). The fact that there is no glpT homologue in the M. tuberculosis genome and the highly impervious nature of the hydrophobic mycobacterial cell wall suggests that resistance may be due to a lack of cellular penetration.     

How do antibiotic‐producing bacteria ensure their self‐resistance before antibiotic biosynthesis incapacitates them?

Acquired antibiotic resistance among dangerous bacterial pathogens is an increasing medical problem. While in Mycobacterium tuberculosis this occurs by mutation in the genes encoding the targets for antibiotic action, other pathogens have generally gained their resistance genes by horizontal gene transfer from non‐pathogenic bacteria. The ultimate source of many of these genes is almost certainly the actinomycetes that make the antibiotics and therefore need self‐protective mechanisms to avoid suicide. How do they ensure that they are resistant at the time when intracellular antibiotic concentrations reach potentially lethal levels? In this issue of Molecular Microbiology, Tahlan et al. describe a solution to this problem in which an antibiotically inactive precursor of a Streptomyces coelicolor antibiotic induces resistance – in this example by means of a trans‐membrane export pump – so that the organism is already primed for resistance at the time when it is needed. The authors generalize their interpretation to other cases where antibiotic resistance depends on export, but it will be interesting to find out whether it could in fact apply more widely, to include the other major mechanisms of resistance: target modification and the synthesis of antibiotics via a series of chemically modified intermediates, with removal of the protective group at the time of secretion into the outside medium.     

Suitability of Arabidopsis for studies on intracellular pH regulation: correlation between H+ pump activity, cytosolic pH and malate level in wild-type and in a mutant partially insensitive to fusicoccin

Data are presented on the suitability of Arabidopsis thaliana seedlings for studies on intracellular pH regulation. In this material, grown in the dark in liquid medium, the determination of weak acid distribution at equilibrium provides an adequate method for calculating cytosolic pH values, in spite of the failure of benzylamine as a vacuolar pH probe. The stimulation of the H⁺ pump by K⁺ or K⁺ and fusicoccin (FC) is associated with a marked alkalinization of both cytosol and cell sap, and with a strong increase in malate level, whereas its inhibition by erythrosin B (EB) leads to the opposite effects. A good quantitative correlation is evident between the changes in net H⁺ extrusion and those in intracellular pH and malate content, in particular, with FC+K⁺. Cell sap buffer capacity is strongly influenced by the different treatments, its changes being substantially accounted for by changes in malate level. A comparison between the values of intracellular pH and malate level in wt and in the 5-2 mutant shows that in the mutant the cytosolic pH is always more acidic, and the intracellular alkalinization induced by FC+K⁺ and also by K⁺ alone is significatively lower. These results support the view that the partial insensitivity of 5-2 to FC is due to a reduced functionality of the H⁺-extruding system on which FC acts, and that the depression of the H⁺ pump activity in the mutant does not depend on a possible regulation by constitutively higher cytosolic pH values.     

A stringent role of F-actin in the ascogonial differentiation ofNeurospora crassa Fluffy mutant

Cytochalasin D, an inhibitor of actin polymerization, interferes with ascogonial differentiation in the fertilefluffy mutant ofNeurospora crassa. As the total level of actin and its mRNA remain unchanged, this suggests that it is in its microfilamentous form (F-form) that actin is stringently required for female differentiation. *** DIRECT SUPPORT *** A00EN042 00011     

Flow cytometric analysis of intracellular pH in cultured opossum kidney (OK) cells

Summary Suspensions of OK cells (a continuous renal epithelial cell line originating from the opossum kidney) were examined by flow cytometry. Three parameters were evaluated simultaneously; cell integrity as assayed by propidium iodide fluorescence, cell size as measured by time-of-flight, and intracellular pH as measured by fluorescence of 2,7-bis-(2-carboxyethyl)-5,6 carboxyfluorescein (BCECF). The suspension was shown to be composed of both intact singlets and doublets of cells, and no difference was noted in the behavior of these two populations with respect to the resting intracellular pH, or of the response of intracellular BCECF to changes in pH. Evidence suggests that using NH₄ prepulses to create an acid load broadens the intracellular pH distribution. The population of OK cells demonstrates a recovery from this acid load which is very homogeneous with respect to its sensitivity to Na⁺ removal or EIPA (ethylisopropyl-amiloride), suggesting that virtually all cells utilize Na⁺/H⁺ exchange for this recovery. The data also suggest heterogeneity in the cellular pH recovery from an acid load with respect to the observed rates of Na⁺/H⁺ exchange. Despite this heterogeneity, the Na⁺/H⁺ exchanger is observed to focus the resting intracellular pH of the population to approximately pH 7.4–7.5. The response of the population to PTH suggests that the majority of cells respond to the hormone, and that the total Na⁺/H⁺ exchange in individual cells is only partially inhibited even in the presence of saturating PTH concentrations.     

Evidence for a peptidoglycan‐like structure in Orientia tsutsugamushi

Bacterial cell walls are composed of the large cross‐linked macromolecule peptidoglycan, which maintains cell shape and is responsible for resisting osmotic stresses. This is a highly conserved structure and the target of numerous antibiotics. Obligate intracellular bacteria are an unusual group of organisms that have evolved to replicate exclusively within the cytoplasm or vacuole of a eukaryotic cell. They tend to have reduced amounts of peptidoglycan, likely due to the fact that their growth and division takes place within an osmotically protected environment, and also due to a drive to reduce activation of the host immune response. Of the two major groups of obligate intracellular bacteria, the cell wall has been much more extensively studied in the Chlamydiales than the Rickettsiales. Here, we present the first detailed analysis of the cell envelope of an important but neglected member of the Rickettsiales, Orientia tsutsugamushi. This bacterium was previously reported to completely lack peptidoglycan, but here we present evidence supporting the existence of a peptidoglycan‐like structure in Orientia, as well as an outer membrane containing a network of cross‐linked proteins, which together confer cell envelope stability. We find striking similarities to the unrelated Chlamydiales, suggesting convergent adaptation to an obligate intracellular lifestyle.     

Solubilization capacity of nonionic surfactant micelles exhibiting strong influence on export of intracellular pigments in Monascus fermentation

In this study, perstractive fermentation of intracellular Monascus pigments in nonionic surfactant micelle aqueous solution had been studied. The permeability of cell membrane modified by nonionic surfactant might have influence on the rate of export of intracellular pigments into its extracellular broth while nearly no effect on the final extracellular pigment concentration. However, the solubilization of pigments in nonionic surfactant micelles strongly affected the final extracellular pigment concentration. The solubilization capacity of micelles depended on the kind of nonionic surfactant, the super‐molecule assembly structure of nonionic surfactant in an aqueous solution, and the nonionic surfactant concentration. Elimination of pigment degradation by export of intracellular Monascus pigments and solubilizing them into nonionic surfactant micelles was also confirmed experimentally. Thus, nonionic surfactant micelle aqueous solution is potential for replacement of organic solvent for perstractive fermentation of intracellular product.     

Action Mechanisms of the Secondary Metabolite Euplotin C: Signaling and Functional Role in Euplotes

ABSTRACT. Among secondary metabolites, the acetylated hemiacetal sesquiterpene euplotin C has been isolated from the marine, ciliated protist Euplotes crassus, and provides an effective mechanism for reducing populations of potential competitors through its cytotoxic properties. However, intracellular signaling mechanisms and their functional correlates mediating the ecological role of euplotin C are largely unknown. We report here that, in E. vannus (an Euplotes morphospecies that does not produce euplotin C and shares with E. crasssus the same interstitial habitat), euplotin C rapidly increases the intracellular concentration of both Ca²⁺ and Na⁺, suggesting a generalized effect of this metabolite on cation transport systems. In addition, euplotin C does not induce oxidative stress, but modulates the electrical properties of E. vannus through an increase of the amplitude of graded action potentials. These events parallel the disassembling of the ciliary structures, the inhibition of cell motility, the occurrence of aberrant cytoplasmic vacuoles, and the rapid inhibition of phagocytic activity. Euplotin C also increases lysosomal pH and decreases lysosomal membrane stability of E. vannus. These results suggest that euplotin C exerts a marked disruption of those homeostatic mechanisms whose efficiency represents the essential prerequisite to face the challenges of the interstitial environment.