Interaction of aminoglycosides and ionophores in the killing of Crithidia fasciculata

Crithidia fasciculata was utilized as a prescreen to determine the antiprotozoal action of aminoglycoside antibiotics alone and in combination with surface-altering agents. Paromomycin was tested with the carrier ionophores nigericin and valinomycin, the channel ionophore gramicidin and the polyene antibiotics amphotericin B and nystatin. After exposure to the drugs in suspension, organisms were plated out to determine the survival of C. fasciculata. Killing was time dependent for both the antibiotic and the ionophore. Paromomycin action was found to be potentiated by all the surface altering agents. The aminoglycosides kanamycin, gentamycin and streptomycin were studied alone and in combination with nigericin. Synergistic effects were demonstrated both with kanamycin and gentamycin in combination with nigericin. Streptomycin was ineffective both alone and with surface-altering agents.     

In vitro and in vivo antagonism of pathogenic turfgrass fungi by Streptomyces hygroscopicus strains YCED9 and WYE53

Disease prevention is a current practice used to minimize fungal diseases of turfgrasses in lawns and golf greens. Prevention is accomplished through fungicide applications, and by periodic thatch removal. During the development of a microbial biodethatch product utilizing the lignocellulose-degrading Streptomyces hygroscopicus strains YCED9 and WYE53, we demonstrated using in vitro plate antagonism bioassays that both strains are antagonists of various turfgrass fungal pathogens. These activities were present when the cultures were growing on thatch, as demonstrated by antifungal antagonism bioassays with culture filtrates. Experiments conducted using a growth chamber demonstrated that a bio-dethatch formulation containing spores of strains YCED9 and WYE53 in a zeolite carrier, provided protection for Kentucky bluegrass seedlings against turfgrass pathogens, including Pythium ultimum, Fusarium oxysporum, Rhizoctonia solani, Sclerotinia homeocarpa, Gaeumannomyces graminis and Microdochium nivale. Results showed that by integrating the use of the S. hygroscopicus YCED9/WYE53 bio-dethatch formulation into routine turf management practices, it should be possible to both minimize thatch build-up while also controlling fungal turfgrass diseases by way of the antifungal biocontrol activity of these strains. This in turn would help control fungal pathogens in turfgrass while minimizing the need for routine chemical fungicide applications. Received 19 October 1998/ Accepted in revised form 19 March 1999     

The action of certain antibiotics on mitochondrial, erythrocyte and artificial phospholipid membranes. The role of induced proton permeability

1. The action of the antibiotics enniatin A, valinomycin, the actin homologues, gramicidin, nigericin and dianemycin on mitochondria, erythrocytes and smectic mesophases of lecithin–dicetyl hydrogen phosphate was studied. 2. These antibiotics induced permeability to alkali-metal cations on all three membrane systems. 3. The ion specificity on each membrane system was the same. 4. Enniatin A, valinomycin and the actins did not induce permeability to protons, whereas nigericin and dianemycin rendered all three membrane systems freely permeable to protons. 5. Several differences were noted between permeability induced by nigericin and that induced by gramicidin. 6. The action of all these antibiotics on mitochondrial respiration could be accounted for by changes in passive ion permeability of the mitochondrial membrane similar to those induced in erythrocytes and phospholipid membranes, if it is assumed that a membrane potential is present in respiring mitochondria.     

An Investigation of the Sustained Component of Nonphotochemical Quenching of Chlorophyll Fluorescence in Isolated Chloroplasts and Leaves of Spinach

The slowly reversible component of nonphotochemical quenching of Chl fluorescence, ql, has been investigated in intact leaves and chloroplasts of spinach (Spinacia oleracea). In leaves, between 50 and 100% of ql (defined as the quenching that remained after at least 10 min of dark adaptation of a previously illuminated leaf) is instantly reversible when leaves were infiltrated with nigericin. Chloroplasts isolated from leaves in which ql had been induced by prior illumination retained the same level of quenching. No pH gradient, as measured by quenching of 9-aminoacridine fluorescence, was present. However, addition of nigericin caused a partial removal of ql, as observed in whole leaves. It is concluded that ql is not related to a persistence of a bulk phase pH gradient in darkness but to a structural change in the thylakoid that can be reversed by addition of nigericin. The relationship between these observations and the hypothesis that nonphotochemical quenching of chlorophyll fluorescence results from protonation of light-harvesting complex of photosystem II components is discussed.     

Antibiotics and enzymes produced by the biocontrol agent Streptomyces violaceusniger YCED-9

Streptomyces violaceusniger strain YCED-9 is an antifungal biocontrol agent antagonistic to many different classes of plant pathogenic fungi. We discovered that strain YCED-9 produces three antimicrobial compounds with antifungal activity. These compounds were purified and identified, and included: AFA (Anti-Fusarium Activity), a fungicidal complex of polyene-like compounds similar to guanidylfungin A and active against most fungi except oomycetes; nigericin, a fungistatic polyether; and geldanamycin, a benzoquinoid polyketide highly inhibitory of mycelial growth of Pythium and Phytophthora spp. Antimicrobial assays were developed to estimate the production of each antibiotic independently. Medium composition had differential effects on the production of each metabolite. The hydrolytic enzymes chitinase and β-1,3-glucanase are also produced under induction by colloidal chitin and laminarin, respectively. Fungal cell walls induced the production of both enzymes. A potential for biological control of diseases caused by P. infestans was also suggested by strain YCED-9’s strong in vitro antagonism towards pathogenic isolates of this fungus. Received 27 October 1997/ Accepted in revised form 8 June 1998     

Ionophores and intact cells. I. Valinomycin and nigericin act preferentially on mitochondria and not on the plasma membrane of Saccharomyces cerevisiae

Valinomycin and nigericin prevented growth of 13 strains of the yeast Saccharomyces cerevisiae on non-fermentable substrate glycerol without affecting much fermentative growth on glucose. The two antibiotics did not induce swelling and lysis of yeast protoplasts in potassium acetate and did not modify uptake and release of Rb+ by the yeast cells. Both antibiotics were taken up by yeast cells at a relatively low rate. Nigericin accelerated the glucose-induced changes of fluorescence of a cyanine dye absorbed by yeast cells, which had been previously ascribed to a depolarization-repolarization cycle of the mitochondrial membrane. The data suggest that valinomycin and nigericin act as ionophores in the inner mitochondrial membrane and not in the plasma membrane of intact yeast cells.     

Relationship of the effects of nigericin on the aggregation and cytoplasmic pH of bovine platelets in the presence of different cations

The effect of nigericin on aggregation of bovine platelets was investigated in media containing the chloride salts of various alkali metal cations of quaternary ammonium cations. In medium with K+, which has the highest permeability with the ionophore among the cations tested, nigericin slightly enhanced both ADP- and thrombin-induced aggregation. In medium with Na+, nigericin scarcely affected ADP-induced aggregation, and slightly inhibited thrombin-induced aggregation. In media with Cs+, choline and tetramethylammonium, it inhibited the aggregations induced by both ADP and thrombin. Measurement of the cytoplasmic pH with the fluorescent probe 2',7'-bis(carboxyethyl)5,6-carboxyfluorescein showed that nigericin increased the intracellular pH in K+ medium and caused its stable decrease (of about 0.6) in Cs+, choline and tetramethylammonium media, but caused only a small transient decrease in medium with Na+. These results suggest that the effects of nigericin on platelet aggregation are mainly due to its effects on the cytoplasmic pH. This conclusion is supported by the findings that the effects on platelet aggregation of other types of ionophore tested were also proportional to their effects on the cytoplasmic pH.     

The effect of ionophores on growth and glycosyltransferase production of Streptococcus sanguis

Abstract Batch cultures of Streptococcus sanguis NCTC7865 were grown in complex medium in the presence and absence of the ionophores gramicidin, valinomycin and nigericin, to study their effects on growth and glycosyltransferase production. Growth of S. sanguis was markedly inhibited by nigericin or gramicidin, whereas valinomycin had no significant effect. The presence of ionophores caused only slight decreases in glucosyltransferase activity. Fructosyltransferase activity was however reduced by at least 90%. The results indicate that ΔpH rather than ΔΨ is essential for maintaining normal growth in S. sanguis . However, both ΔpH and ΔΨ are necessary for fructosyltransferase synthesis and secretion, but are not apparently involved in the synthesis and secretion of glucosyltransferase.     

Ionophores and intact cells I. Valinomycin and nigericin act preferentially on mitochondria and not on the plasma membrane of Saccharomyces cerevisiae

Valinomycin and nigericin prevented growth of 13 strains of the yeast Saccharomyces cerevisiae on non-fermentable substrate glycerol without affecting much fermentative growth on glucose. The two antibiotics did not induce swelling and lysis of yeast protoplasts in potassium acetate and did not modify uptake and release of Rb⁺ by the yeast cells. Both antibiotics were taken up by yeast cells at a relatively low rate. Nigericin accelerated the glucose-induced changes of fluorescence of a cyanine dye absorbed by yeast cells, which had been previously ascribed to a depolarization-repolarization cycle of the mitochondrial membrane. The data suggest that valinomycin and nigericin act as ionophores in the inner mitochondrial membrane and not in the plasma membrane of intact yeast cells.     

Characterization of a mitochondrial inorganic pyrophosphatase in Saccharomyces cerevisiae.

We have studied a mitochondrial inorganic pyrophosphatase (PPase) in the yeast Saccharomyces cerevisiae. The uncoupler FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and the ionophores valinomycin and nigericin stimulate the PPase activity of repeatedly washed yeast mitochondria 2-3-fold. We have previously cloned a yeast gene, PPA2, encoding the catalytic subunit of a mitochondrial PPase. Uncouplers stimulate the PPase activity several-fold in mitochondria from both cells that overexpress PPA2 from a high copy number plasmid and cells with normal expression. These results indicate that the PPA2 polypeptide functions as an energy linked and membrane associated PPase. The stimulation of mitochondrial PPase activity by FCCP, but not by valinomycin and nigericin, was greatly enhanced by the presence of DTT. The antibiotics Dio-9, equisetin and the F0F1-ATPase inhibitor oligomycin also increase mitochondrial PPase activity several fold. This stimulation is much higher, whereas basal PPase activity is lower, in isotonic than in hypotonic solution, which indicates that intact membranes are a prerequisite for maximal effects.     

Effects of nigericin and monactin on cation permeability of Streptococcus faecalis and metabolic capacities of potassium-depleted cells

At a concentration of 10(-6)m, nigericin and monactin inhibited growth of Streptococcus faecalis, and the inhibition was reversed by addition of excess K(+). In the presence of certain antibiotics, the cells exhibited increased permeability to certain cations; internal Rb(+) was rapidly lost by exchange with external H(+), K(+) Rb(+), and, more slowly, with Na(+) and Li(+). No effect was observed on the penetration of other small molecules. Cation exchanges induced by nigericin and monactin were metabolically passive and apparently did not involve the energy-dependent K(+) pump. When the cells were washed, the cytoplasmic membrane recovered its original impermeability to cations. By use of monactin, we prepared cells whose K(+) content had been completely replaced by other cations, and the metabolic characteristics of K(+)-depleted cells were studied. Cells containing only Na(+) glycolyzed almost as well as did normal ones and, under proper conditions, could accumulate amino acids and orthophosphate. These cells also incorporated (14)C-uracil into ribonucleic acid but incorporation of (14)C-leucine into protein was strictly dependent upon the addition of K(+). When K(+) or Rb(+) was added to sodium-loaded cells undergoing glycolysis, these ions were accumulated by stoichiometric exchange for Na(+). From concurrent measurements of the rate of glycolysis, it was calculated that one mole-pair of cations was exchanged for each mole of adenosine triphosphate produced.     

Distribution of psychrophilic microorganisms in soils of Terra Nova Bay and Edmonson Point, Victoria Land and their biosynthetic capabilities

Microbiota of soil samples from Terra Nova Bay and Edmonson Point, Antarctica was observed by dilutions spread plate method. Variety of mesophilic and psychrophilic microorganisms was detected and isolated. Bacteria, actinomycetes, fungi, and microalgae occurred. Fungi genera Penicillium, Aspergillus, Paecilomyces, Cladosporium, Mortierella, Candida, Rhodotorula were found. By morphology and cell wall aminoacid composition the actinomycete genus Streptomyces was characterized. The bacteria and actinomycetes were screened for biologically active products. Some cultures formed enzymes, glycolipids and antibiotics. Psychrophilic strain Streptomyces sp. no. 8 was studied more detail and was established that it produced following antibiotics: azalomycin B, nigericin and non-polyenic macrolide antibiotic composed from two components that inhibited the growth of Gram-positive bacteria, yeasts, and phytopathogenic fungi.     

Effect of solvent adaptation on the antibiotic resistance in Pseudomonas putida S12

The effect of the adaptation to toluene on the␣resistance to different antibiotics was investigated in the␣solvent-resistant strain Pseudomonas putida S12. We␣followed the process of the solvent adaptation of P.␣putida S12 by cultivating the strain in the presence␣of␣increasing concentrations of toluene and studied␣the correlation of this gradual adaptation to the resistance towards antibiotics. It was shown that the tolerance to various chemically and structurally unrelated antibiotics, with different targets in the cell, increased during this gradual adaptation. The survival of P. putida S12 in the presence of antibiotics like tetracycline, nigericin, polymyxin B, piperacillin or chloramphenicol increased 30- to and 1000-fold after adaptation to 600 mg/l toluene. However, cells grown in the absence of any solvents lost their adaptation to toluene even when grown in the presence of antibiotics. Results are discussed in terms of the physico-chemical properties of membranes as affected by the observed cis/trans isomerization of unsaturated fatty acids, as well as in terms of the active efflux of molecules from the cytoplasmic membrane. Received: 9 May 1997 / Received revision: 4 July 1997 / Accepted: 4 July 1997     

Preliminary Investigations about the Mode of Transmission and Spread of Xanthomonas campestris pv. graminis on Forage Grasses

Extensive studies showed that no disease was caused when seeds of different forage grasses were inoculated with Xanthomonas campestris pv. graminis. The disease could easily be induced by infecting the plants in the root system, leaves or flower. The inoculation site in the leaf proved to be of vital importance for the development of the disease. Wilting symptoms were quickly induced when the pathogen was inoculated near the leaf base. Plants in root-contact with diseased plants showed disease symptoms. It is not known whether these symptoms were caused by the bacteria or by toxins released by nearby diseased plants. Cross inoculation trials on different grass varieties revealed that different pathovars exist in the group of xanthomonads, pathogenic to forage grasses. Some have a broad host range whereas others are more limited to a single plant genus. Field trials suggest that in Belgian climatic conditions, the losses caused by bacterial wilt are rather limited.     

The effect of valinomycin and nigericin on the efficacy of bacteriophage infection of staphylococcal cells

The effect of ionophore antibiotics, valinomycin and nigericin, on the generation of the membrane potential, the pH gradient and the efficacy of phage infection in tetracycline-resistant staphylococci has been studied. Valinomycin at a concentration of 0.5 microM induces the dissipation of the membrane potential, and nigericin at a concentration of 12.0 microM decreases the value of the pH gradient on the membrane of staphylococci. The separate use of antibiotics has no essential influence on the efficacy of phage infection. The combined use of valinomycin and nigerimycin produces the maximum inhibition of phage infection (64.5%) at the stage of the introduction of DNA into the bacterial cell, which is indicative of a definite role played by the membrane potential and the pH gradient in the transport of phage DNA into staphylococcal cells.     

Etiology and control of durian foliar blight and dieback caused by Rhizoctonia solani

Foliar blight and dieback of durian seedlings and trees in Peninsular Malaysia was found to be caused by Rhizoctonia solani (teleomorph - Thanatephoms cucumeris) The fungus grew well and produced abundant sclerotia at temperatures higher than 24°C with an optimum at 28°C. It grew poorly at 35°C and did not grow at 10°C. The strains studied were found to belong to the anastomosis group AG-1. They were pathogenic on durian, papaya, cucumber, long bean, Mikania weed, padi, musk melon, mung bean, Zoysia grass, Bermuda grass, and St Augustine grass. They were mildly pathogenic on groundnut, and non-pathogenic on maize, guava and Brassica‘pak choy’. The disease was effectively controlled by foliar sprays of pencycuron and benomyl; triadimefon and an antagonistic bacterium suspension treatment were less effective and quintozene-etridiazole mixture gave poor disease control.     

Regulation of the phosphoinositide cycle by Na+/H+ exchange and intracellular pH in human platelets.

We have found that thrombin-induced activation of protein kinase C (PKC) in platelets, measured by phosphorylation of the 47 kDa protein, is synergistically enhanced by the amiloride analogue ethylisopropylamiloride (EIA), a specific inhibitor of Na+/H+ exchange. This EIA effect was further synergistically enhanced by lowering intracellular pH (pHi) with either nigericin or sodium propionate, and reversed by raising pHi with monensin or ammonium chloride. The synergistic enhancement of thrombin-activated PKC by EIA plus nigericin was not observed when PKC was directly activated by phorbol esters. EIA and EIA plus nigericin caused a 3- to 6-fold increase in thrombin-induced diacylglycerol (DAG), but not phosphatidic acid (PA), production. EIA and nigericin also caused a marked increase in thrombin-induced breakdown and inhibition of resynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2). In summary, we have presented evidence that inhibition of Na+/H+ exchange causes primarily a H(+)-mediated interruption of the phosphoinositide cycle in activated platelets, including the accumulation of DAG associated with the enhancement of PKC activation, the inhibition of conversion of DAG to PA, and increased PIP2 breakdown. These data suggest a model in which Na+/H+ and pHi play an important regulatory role in permitting the phosphoinositide cycle to proceed in thrombin-activated platelets.     

Clinical streptococci and their sensitivity to enterocins produced by different strains of the species Enterococcus faecium (short communication)

In children, acute otitis media (AOM) is one of the most frequently occurring infections caused by Streptococcus pneumoniae and Str. pyogenes. The standard treatment of AOMis provided by antibiotics; however, an increased resistance of the causative agents to antibiotics requires the need to search for innovations. This study was focused on in vitro testing sensitivity of streptococci isolated from AOM to enterocins produced by 9 different origin strains of E. faecium. Enterocins (Ent) represent ribosomally synthesized proteinaceous substances with antimicrobial activity against Gram-positive and/or Gram-negative bacteria which are produced mostly by strains of the species Enterococcus faecium. Str. pneumoniae were sensitive at least to 1 Ent. Str. pneumoniae SPn 754 was sensitive to 5 Ent. Five Str. pyogenes were sensitive to enterocins. Ent A (P) inhibited the growth of 3 Str. pneumoniae, and 4 Str. pyogenes (activity between 100 and 3,200 AU/ml). Most of Ent inhibited the growth of streptococci tested (100-3,200 AU/ml). Str. pyogenes were more sensitive to Ent than Str. pneumoniae. Although more detailed further studies are required, our results indicate a new possibility for enterocin use.     

Calcium ions and the differentiation of Streptomyces hygroscopicus 155, a producer of an antibiotic complex]

The effect of Ca2+ on differentiation of Streptomyces hygroscopicus 155 and its inactive variant 155-0 was studied. Addition of Ca2+ to the medium induced formation of the aerial mycelium in the inactive variant and accelerated formation of the aerial mycelium in the parent strain. The inhibitory effect of EGTA, verapamil, nifedipin, chlorpromazine and dilthiazeme on the aerial mycelium formation demonstrated the physiological role of Ca2+ in the process. Addition of pandavir (nigericin) and azalomycin B, the antibiotics produced by the streptomycete, induced formation of the aerial mycelium in the inactive variant. The effect was higher in the presence of Ca2+. Streptomyces hygroscopicus 155 and its inactive variant synthesized a proteolytic complex containing metalloproteases and trypsin-like proteases. The total proteolytic activity of the inactive variant was lower than that of the parent strain. Addition of Ca2+ to the medium stimulated their proteolytic activity. The inducing action of the antibiotics produced by the parent strain on differentiation of S.hygroscopicus 155-0 and the increase of their action in the presence of Ca2+ suggested that they controlled the differentiation and that such a function of the antibiotics expressed itself through the Ca2+ signal system.     

Monoxenic in vitro production and colonization potential of AM fungus Glomus intraradices

The paper reports the establishment of mycorrhizal infection of a non-mycorrhizal Ri-T-DNA transformed carrot root when co-cultured with a surface sterilized sweet potato root segment colonized by arbuscular mycorrhizal (AM) fungus G. intraradices on minimal M medium. Extensive fungal hyphal emergence from each cut end of the mycorrhizal sweet potato root piece was observed in one week old cultures. These hyphae caused infection on contacting the transformed-carrot- root segment and produced many hyphae and spores both inside and outside the zone of the root after 6 week of growth. Axenically produced fungal propagules proliferated on the surface of fresh minimal M medium when sub-cultured without any root segment. On repeated sub-culturing, these propagules did not lose their ability to grow and produced many juvenile small spore-like vesicles during the non-symbiotic phase. Although these spores were morphologically and anatomically similar to their soil borne counter parts, they were much smaller. When placed in the vicinity of a fresh hairy root on the minimal medium or a Sudan grass seedling in sand culture, the axenically produced AM fungal propagules caused root infection, but the infection characteristics were significantly different to the original culture in terms of shape (spherical vs oval) and size (20 microm vs 45 microm) of the intraradical vesicles, and absence of 'H' branches. Sudan grass seedlings inoculated with the axenically cultured fungus showed significantly (P < 0.05) higher dry weights plant'. When compared to the plants inoculated with sand cultures, the growth parameters and the percentage infection were not significantly different. However, when both sources of inocula were used together, a synergistic effect on plant growth as well as root infection was observed.