Inhibition of Three Potato Pathogens by Phenazine-Producing Pseudomonas spp. Is Associated with Multiple Biocontrol-Related Traits.

Phenazine-producing Pseudomonas spp. are effective biocontrol agents that aggressively colonize the rhizosphere and suppress numerous plant diseases. In this study, we compared the ability of 63 plant-beneficial phenazine-producing Pseudomonas strains representative of the worldwide diversity to inhibit the growth of three major potato pathogens: the oomycete Phytophthora infestans, the Gram-positive bacterium Streptomyces scabies, and the ascomycete Verticillium dahliae. The 63 Pseudomonas strains are distributed among four different subgroups within the P. fluorescens species complex and produce different phenazine compounds, namely, phenazine-1-carboxylic acid (PCA), phenazine-1-carboxamide (PCN), 2-hydroxyphenazine-1-carboxylic acid, and 2-hydroxphenazine. Overall, the 63 strains exhibited contrasted levels of pathogen inhibition. Strains from the P. chlororaphis subgroup inhibited the growth of P. infestans more effectively than strains from the P. fluorescens subgroup. Higher inhibition was not associated with differential levels of phenazine production nor with specific phenazine compounds. The presence of additional biocontrol-related traits found in P. chlororaphis was instead associated with higher P. infestans inhibition. Inhibition of S. scabies by the 63 strains was more variable, with no clear taxonomic segregation pattern. Inhibition values did not correlate with phenazine production nor with specific phenazine compounds. No additional synergistic biocontrol-related traits were found. Against V. dahliae, PCN producers from the P. chlororaphis subgroup and PCA producers from the P. fluorescens subgroup exhibited greater inhibition. Additional biocontrol-related traits potentially involved in V. dahliae inhibition were identified. This study represents a first step toward harnessing the vast genomic diversity of phenazine-producing Pseudomonas spp. to achieve better biological control of potato pathogens. IMPORTANCE Plant-beneficial phenazine-producing Pseudomonas spp. are effective biocontrol agents, thanks to the broad-spectrum antibiotic activity of the phenazine antibiotics they produce. These bacteria have received considerable attention over the last 20 years, but most studies have focused only on the ability of a few genotypes to inhibit the growth of a limited number of plant pathogens. In this study, we investigated the ability of 63 phenazine-producing strains, isolated from a wide diversity of host plants on four continents, to inhibit the growth of three major potato pathogens: Phytophthora infestans, Streptomyces scabies, and Verticillium dahliae. We found that the 63 strains differentially inhibited the three potato pathogens. These differences are in part associated with the nature and the quantity of the phenazine compounds being produced but also with the presence of additional biocontrol-related traits. These results will facilitate the selection of versatile biocontrol agents against pathogens.

Identification of Phenazine-Based MEMO1 Small-Molecule Inhibitors: Virtual Screening, Fluorescence Polarization Validation, and Inhibition of Breast Cancer Migration.

Phosphorylation-dependent protein-protein interactions play a significant role in biological signaling pathways; therefore, small molecules that are capable of influencing these interactions can be valuable research tools and have potential as pharmaceutical agents. MEMO1 (mediator of ErbB2-cell driven motility) is a phosphotyrosine-binding protein that interacts with a variety of protein partners and has been found to be upregulated in breast cancer patients. Herein, we report the first small-molecule inhibitors of MEMO1 interactions identified through a virtual screening platform and validated in a competitive fluorescence polarization assay. Initial structure-activity relationships have been investigated for these phenazine-core inhibitors and the binding sites have been postulated using molecular dynamics simulations. The most potent biochemical inhibitor is capable of disrupting the large protein interface with a KI of 2.7 µm. In addition, the most promising phenazine core compounds slow the migration of breast cancer cell lines in a scratch assay.

Bioactive phenazines from an earwig-associated Streptomyces sp.

Three new phenazine-type compounds, named phenazines SA-SC (1-3), together with four new natural products (4-7), were isolated from the fermentation broth of an earwig-associated Streptomyces sp. NA04227. The structures of these compounds were determined by extensive analyses of NMR, high resolution mass spectroscopic data, as well as single-crystal X-ray diffraction measurement. Sequencing and analysis of the genome data allowed us to identify the gene cluster (spz) and propose a biosynthetic pathway for these phenazine-type compounds. Additionally, compounds 1-5 exhibited moderate inhibitory activity against acetylcholinesterase (AChE), and compound 3 showed antimicrobial activities against Micrococcus luteus.

Investigating the antifungal activity and mechanism of a microbial pesticide Shenqinmycin against Phoma sp.

Tea white scab (TWS) is a major disease affecting tea trees in mid-elevation regions and often occurs during rainy seasons with low temperatures. This disease is caused by the fungal pathogen Phoma sp. TWS can infect young stems, tender leaves, and tender shoots and lead to the production of low-quality tea. Owing to the absence of an effective control, TWS can result in substantial loss in tea production. In this study, we isolated and identified the pathogen from tea leaves infected by TWS and then evaluated in vitro the antifungal activity of Shenqinmycin, polyoxin, azoxystrobin, oligosaccharins, and tebuconazole against Phoma sp. Our results indicated that Shenqinmycin can inhibit the growth of Phoma sp. mycelia, with the EC50 value of 0.74µg/mL. After Phoma sp. being incubated in PDB liquid medium with Shenqinmycin, its mycelia were distorted and distended at 1.56µg/mL of minimum inhibitory concentration for 6h. Crucial genes associated with cell redox homeostasis, proteins synthesis, energy metabolism, and cytoskeleton were studied at mRNA and protein levels through RT-qPCR and Nano-LC-MS/MS. The results showed that the genes of 3-phosphate-glyceraldehyde dehydrogenase, citrate synthase, NADH-ubiquinone oxidoreductase subunit (NADH-subunit), ribosomal protein, eukaryotic initiation factor 4A-I, ß-tubulin, and α-tubulin were up-regulated. Meanwhile, the genes of formate dehydrogenase (FDH), malate dehydrogenase, mitochondrial heat shock protein, and protein disulfide-isomerase (PDI) were up-regulated at mRNA level but down-regulated at protein level. These results indicated that Shenqinmycin contribute to cell redox homeostasis by up- or down-regulating NADH-subunit, FDH, and PDI.

Differential expression of selected candidate genes in bovine embryos produced in vitro and cultured with chemicals modulating lipid metabolism.

Lipid accumulated in embryos produced in vitro has been linked to reductions in both quality and postcryopreservation viability. Therefore, the objective of the present study was to investigate the influence of lipid-reducing chemicals on embryo development, quality, and postcryopreservation viability, in addition to expression profiles of selected lipid metabolism-regulating genes. Bovine cumulus-oocyte complexes were matured and fertilized in vitro; eight-cell stage embryos were cultured in IVC medium supplemented with phenazine ethosulfate (PES), L-carnitine (LC), PES + LC, or no supplementation (control). Culturing embryos in medium with LC increased (P < 0.05) blastocyst rate (38.8%) compared with the other groups (control = 28.1%, PES = 27.1%, PES + LC = 26.3%). Embryos cultured with supplements had greater total cell number and fewer apoptotic cells than the control. Cytoplasmic lipid content was reduced, whereas mitochondria density was increased in embryos treated with culture supplements; this was linked to altered expression profiles of selected genes regulating lipid metabolism. For example, transcript abundance of transmembrane lipid gene (SGPP1) was greater in LC- and PES-treated embryos, and they had increased postcryopreservation hatching ability (indicative of embryo cryotolerance). In conclusion, the two lipid metabolism regulators added to the culture media had improved embryo quality and cryotolerance, but embryo development rate and downstream lipid metabolism-regulating genes were more influenced with LC supplementation.

Plant chemical genomics: gravity sensing and response.

The gene families that encode the vesicle trafficking machinery in plants are highly expanded compared to those from protists and animals. As such, classical genetic screens for mutants with lesions in these genes are fraught with issues of redundancy and lethality. A chemical genomics approach can, in theory, circumvent these issues because inhibitory or stimulatory molecules may be applied at any point in development at sublethal concentrations. This chapter describes the protocols for a chemical genomics screen designed to identify components of the plant cell vesicle trafficking machinery. A two-tiered screen was designed where the primary screen assayed for chemicals that modified the gravitropic response, a process that in plant cells is intimately tied to vesicle trafficking; the secondary screen employed fluorescent marker lines that were treated with gravitropic inhibitors or inducers to assay for changes in endomembrane system morphology. We thus identified four compounds by which we can further explore the relationship between gravitropic signal transduction and vesicle trafficking.

Antibacterial activity of extracellular compounds produced by a Pseudomonas strain against methicillin-resistant Staphylococcus aureus (MRSA) strains.

BACKGROUND: The emergence of multidrug-resistant bacteria is a world health problem. Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA) strains, is one of the most important human pathogens associated with hospital and community-acquired infections. The aim of this work was to evaluate the antibacterial activity of a Pseudomonas aeruginosa-derived compound against MRSA strains. METHODS: Thirty clinical MRSA strains were isolated, and three standard MRSA strains were evaluated. The extracellular compounds were purified by vacuum liquid chromatography. Evaluation of antibacterial activity was performed by agar diffusion technique, determination of the minimal inhibitory concentration, curve of growth and viability and scanning electron microscopy. Interaction of an extracellular compound with silver nanoparticle was studied to evaluate antibacterial effect. RESULTS: The F3 (ethyl acetate) and F3d (dichloromethane- ethyl acetate) fractions demonstrated antibacterial activity against the MRSA strains. Phenazine-1-carboxamide was identified and purified from the F3d fraction and demonstrated slight antibacterial activity against MRSA, and synergic effect when combined with silver nanoparticles produced by Fusarium oxysporum. Organohalogen compound was purified from this fraction showing high antibacterial effect. Using scanning electron microscopy, we show that the F3d fraction caused morphological changes to the cell wall of the MRSA strains. CONCLUSIONS: These results suggest that P. aeruginosa-produced compounds such as phenazines have inhibitory effects against MRSA and may be a good alternative treatment to control infections caused by MRSA.

Lipid content in pig blastocysts cultured in the presence or absence of protein and vitamin E or phenazine ethosulfate.

In the present study, total lipid content and content of triglycerides, phospholipids and cholesterol were determined in pig blastocysts cultured in medium without protein, supplemented with bovine serum albumin (BSA), with fetal calf serum (FCS), vitamin E or phenazine ethosulfate (PES). In comparison to blastocysts cultured in NCSU-23 with BSA, we observed a decrease of the total lipid content in PES-treated embryos. Triglyceride content in FCS-, vitamin E- and PES-treated embryos as well as in blastocysts cultured without protein was 81.9%, 70.2%, 57.2% and 74.8% of that found in the blastocysts cultured in NCSU-23 with BSA, respectively. Nevertheless the content of phospholipids remained unchanged. This decrease of triglyceride content in the porcine blastocyst after in vitro culture may be explained by altered lipid metabolism in embryos.

In vitro assessment of novel transcription inhibitors and topoisomerase poisons in rhabdomyosarcoma cell lines.

PURPOSE: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Current chemotherapy regimes include the topoisomerase II poison etoposide and the transcription inhibitor actinomycin D. Poor clinical response necessitate identification of new agents to improve patient outcomes. METHODS: We assessed the in vitro cytotoxicity (MTT assay) of DNA intercalating agents in five established human RMS cell lines. These include novel classes of transcription inhibitors and topoisomerase poisons, previously shown to have potential as anti-cancer agents. RESULTS: Amongst the former agents, bisintercalating bis(9-aminoacridine-4-carboxamides) linked through the 9-position, and bis(phenazine-1-carboxamides) linked via their side chains, are compared with established transcription inhibitors. Amongst the latter, monofunctional acridine-4-carboxamides related to N-[2-(dimethylamino)ethyl]acridine-4-carboxamide, DACA, are compared with established topoisomerase poisons. CONCLUSIONS: Our findings specifically highlight the topoisomerase poison 9-amino-DACA, its 5-methylsulphone derivative, AS-DACA, and the bis(phenazine-1-carboxamide) transcription inhibitor MLN944/XR5944, currently in phase I trial, as candidates for further research into new agents for the treatment of RMS.

Novel N-aryl- and N-heteryl phenazine-1-carboxamides as potential agents for the treatment of infections sustained by drug-resistant and multidrug-resistant Mycobacterium tuberculosis.

We investigated the in vitro activity of a new class of N-aryl and N-heteryl phenazine-1-carboxamide derivatives against Mycobacterium tuberculosis H37Rv and against drug-resistant ATCC M. tuberculosis strains. The activity against M. tuberculosis in J774 macrophage cells was also investigated. In most cases, minimum inhibitory concentrations (MICs) ranging between 0.19 mg/L and 0.79 mg/L were found, and comparable MIC values were obtained against 26 susceptible and 5 drug-resistant clinical isolates. Several derivatives were shown to be effective in inhibiting the growth both of susceptible and resistant strains at comparable concentrations. Results obtained indicate that these compounds could represent a promising class of agents useful for the treatment of M. tuberculosis infections caused by drug-resistant strains.

Effects of protein source, vitamin E and phenazine ethosulfate on developmental competence and quality of porcine embryos cultured in vitro.

Effects of fetal calf serum (FCS) or bovine serum albumin (BSA), with or without vitamin E (vit. E) or phenazine ethosulfate (PES) supplementation on developmental competence and quality of cultured porcine embryos were examined. The experiment was done on zygotes and 2-cell embryos obtained from superovulated gilts. Morphologically normal zygotes were cultured in vitro in NCSU-23 medium supplemented with: experiment 1-0.004 g/ml BSA, 10% FCS, protein-free (control); experiment 2-0 (control), 25, 50 or 100 microM vit. E; experiment 3-0 (control), 0.025, 0.05 or 0.075 microM PES. Embryo quality criteria were developmental competence (cleavage, morula and blastocyst rates), total cell number per blastocyst and degree of apoptosis as assessed by TUNEL staining. Presence of BSA in culture medium increased significantly morula and blastocysts production as compared to FCS (P < 0.001) and protein-free group (P < 0.05 and P < 0.001, respectively). The blastocysts cultured in protein-free medium had higher average number of apoptotic nuclei and DNA fragmented nucleus index as compared to the BSA (P < 0.05 and P < 0.01, respectively) and FCS (P < 0.5) group. Supplementation in culture medium of 100 microM vit. E increased blastocyst production as compared to control and 50 microM vit-E (P < 0.05). Both the number of cells per and percentage of TUNEL positive nuclei per blastocyst were slightly lower in PES treated than control groups.

[Lectins from Sambucus nigra L inflorescences: isolation and investigation of biological activity using procaryotic test-systems].

Isolation of lectins from extracts of the Sambucus nigra inflorescences and of pollen material have been performed using isoelectric focusing without carrier ampholytes (autofocusing). Fractions active in agglutination tests with different carbohydrate specificity were subjected to SDS-PAGE. The major lectin found in whole inflores-cences was GalNAc specific and is proposed to be a heterotetramer with subunits of about 30 and 33 kDa. It was called SNAflu-I. At least two other lectins were present in the pollen material and supposed to consist of identical subunits. Major positively charged lectin was Glc/Man specific with subunit of 26 kDa and called SNApol-I. Other pollen component (SNApol-II) was Gal specific with subunit of about 20 kDa. In order to elucidate cell targets sensitive for the S. nigra lectin's activity the combined effects of the lectins and transcriptional of phenazine origin on B. subtilis cells growth have been studied. Only SNApol-I demonstrated the antagonistic activity against these inhibitors in vivo. This lectin but not the SNAflu-I can also inhibit transcription in vitro. It is supposed that lectins from the same source may act in different directions on cell metabolism. Particularly one of the common targets may be the DNA-dependent synthesis of RNA.

Phenazine 5,10-dioxide derivatives as hypoxic selective cytotoxins.

The synthesis and evaluation as hypoxic selective cytotoxins of 2-amino- or 2-hydroxyphenazine 5,10-dioxide derivatives and reduced analogues are reported. In vitro cytotoxicities on V79 cells under hypoxic and aerobic conditions were determined. Some derivatives, such as 7(8)-bromo-2-hydroxyphenazine 5,10-dioxide, showed selective toxicity toward hypoxic cells and along with derivatives 7(8)-bromo-2-aminophenazine 5,10-dioxide and 7(8)-chloro-2-aminophenazine 5,10-dioxide behave as hypoxic trigger cytotoxins. These compounds represent interesting leads for further chemical modifications and biological studies.

[Studies on control of root rot on Panax notoginseng].

Chemical Control tests of pot, plot and field for Panax notoginseng root rot were conducted during 1995-1996. The results indicated that the chemical control is a effect measure to control rapidly occurring and spreading of Panax notoginseng root rot. It was the best treatment to coordinate use of bactericide and fungicide, obviously better than alone or mixed use of fungicide and also better than alone use of bactericide. In the pot and plot tests, the best coordinate treatment was the treatment of 10% phenazine plus 70% dexon plus 50% bavistin and plus water (1:1:500), the control effect was 70%; in the field test, the control effect of over 70% was also get with the treatment of 10% phenazine plus 70% dexon and plus small soil (1 Kg:1 Kg:150 Kg) per mu.

The effect of bacterial toxins on levels of intracellular adenosine nucleotides and human ciliary beat frequency.

Toxins that slow ciliary beat are virulence determinants of bacteria that infect or invade ciliated epithelial surfaces. We have previously shown that the effect of the Pseudomonas aeruginosa toxin pyocyanin on ciliary beat is associated with a fall in intracellular cAMP and ATP. We have now investigated whether reduction in intracellular adenosine nucleotides might be a common mechanism of action of other bacterial toxins which slow ciliary beat. Two other P. aeruginosa toxins, 1-hydroxyphenazine (1-HP) and rhamnolipid, and two Haemophilus influenzae fractions produced by gel filtration of broth cultures were tested. The effect on human nasal epithelium ciliary beat frequency (CBF), and intracellular cAMP and ATP were measured, and the effect of two pharmacological agents, dibutyryl cAMP and salmeterol, on these changes was assessed. 1-HP, rhamnolipid and the two H. influenzae fractions slowed CBF before there was significant release of lactate dehydrogenase from the cells. The toxins also caused a fall in intracellular cAMP and ATP. Dibutyryl cAMP and salmeterol at the concentrations used do not increase baseline CBF, but diminished the fall in CBF and intracellular adenosine nucleotides. The cAMP and ATP levels in these studies were combined with those previously obtained with pyocyanin. there was a good correlation between cAMP and ATP levels and CBF. Bacterial toxins which slow CBF may act by causing a fall in intracellular adenosine nucleotides, and agents which stimulate cAMP may prevent toxin-induced slowing of ciliary beat.

Extracellular metabolites of Pseudomonas aeruginosa produce bronchoconstriction by different mechanisms.

Bacterial supernatants (BS) obtained from broth cultures of Pseudomonas aeruginosa cause bronchoconstriction in sheep, suggesting that BS contain proinflammatory metabolites. In this study we investigated the mechanism(s) responsible for this bronchial effect. BS were obtained from 48 h cultures and sterilized by filtration. Sheep (n = 6) were intubated and swallowed an esophageal balloon for the measurement of specific lung resistance (SRL). Aerosols of BS (3 ml total) immediately increased SRL (541%). Neither aerosolized broth (control) nor inhaled endotoxin in excess of that contained in the BS had an effect. BS challenges were repeated on separate occasions except that the sheep were treated 30 min before challenge with the anticholinergic agent atropine (0.2 mg/kg, intravenously); the anti-allergic agent nedocromil (1 mg/kg, aerosol); the histamine H1 antagonist chlorpheniramine (2 mg/kg); or the bradykinin (BK) B2 receptor antagonists NPC-567 (5 mg/ml, aerosol) or NPC-17761 (1 mg/ml aerosol). The results showed that greater than 90% protection (p < 0.05) was achieved when the animals were pretreated with atropine, nedocromil sodium, or either of the two BK antagonists, but only 27 +/- 21% protection was seen with chlorpheniramine pretreatment. These findings are characteristic of a BK-mediated response. Analysis of bronchoalveolar lavage fluid obtained before and after BS challenge confirmed that i-kinins, but not histamine, increased (p < 0.05) from 61 +/- 7 to 304 +/- 55 pg/ml. Control (broth) challenges produced no such change. To identify the metabolites involved, we tested the effects of aerosolizing two suspected components of BS, 1-hydroxyphenazine (1-HP) and pyocyanine (PYO) in five sheep.(ABSTRACT TRUNCATED AT 250 WORDS)

Activity of phenazine analogs against Mycobacterium leprae infections in mice.

Twenty-five compounds structurally related to clofazimine were tested for their ability to inhibit the growth of Mycobacterium leprae using the kinetic method of drug evaluation in the mouse foot pad model of leprosy. Seven of the phenazine derivatives displayed anti-M. leprae activity comparable to that of clofazimine when administered at a concentration of 0.01% (w/w) in the diet. Three of the compounds, B746, B4087, and B4101, were active when administered at 0.001% in the diet. At a dietary concentration of 0.0001%, B4087 and B4101 were slightly more active than clofazimine, while B746 was less active. In the kinetic method of drug evaluation, greater anti-M. leprae activity of phenazine derivatives was generally associated with greater pigmentation of abdominal fat. Of the compounds which did not cause pigmentation when fed at a concentration of 0.01% in the diet B4090 was the most active. This compound also inhibits the growth of a clofazimine-resistant M. smegmatis strain.

Studies on cell growth stimulating substances of low molecular weight. Part 2. Exfoliazone and lavanducyanin, potent growth promoting substances of rat liver cell line, RLN-8, produced by Streptomyces exfoliatus and Streptomyces aeriouvifer.

Exfoliazone and lavanducyanin isolated from Streptomyces exfoliatus BT-38 and Streptomyces aeriouvifer CL-190, respectively, showed strong growth promoting activities to liver cell RLN-8 established from normal Donryu rat. When RLN-8 cells were cultured in Eagle's minimal essential medium containing 1% fetal bovine serum, exfoliazone significantly stimulated the growth of RLN-8 cells. However, no effect was observed under serum-free conditions. Effective dose of exfoliazone was at the range of 0.004-0.1 microgram/ml. Cell proliferation was confirmed by MTT assay and by the increases of cell number and DNA synthesis. Lavanducyanin also stimulated the growth of RLN-8 cells in the same medium. It showed growth promoting activity at lower concentrations than exfoliazone and the effective dose was at the range of 0.0001-0.06 microgram/ml. Analogous compounds of exfoliazone and lavanducyanin also promoted the growth of RLN-8 cells. In addition, exfoliazone and lavanducyanin enhanced the growth of NIH 3T3 and T601 cells. These results indicate that exfoliazone, lavanducyanin and their related compounds seem to be a new type of growth promoting substances with low molecular weight produced by microorganisms, and that they can partially substitute for functions of serum. Since 12-O-tetradecanoylphorbol-13-acetate (TPA) did not show the growth promoting activities under the same conditions, the action mechanism(s) of exfoliazone and lavanducyanin are different from that of TPA.

Isolation of a new phenazine antibiotic, DOB-41, from Pseudomonas species.

A new phenazine antibiotic, DOB-41, was isolated from the culture broth of a Pseudomonas strain. The antibiotic obtained as yellow crystals showed UV maxima at 255 nm and 370 nm. A molecular formula, C19H18N2O6, was indicated by elemental analysis and mass spectrometry. The structure was elucidated by X-ray diffraction analysis. The antibiotic exhibited inhibitory activity against Gram-positive bacteria, and antitumor effect against leukemia P388 in mice.