Natural Products Biosynthesis by Streptomyces netropsis IMV Ac-5025 under Exogenous Sterol Action.

Streptomycetes are known as producers of bioactive substances, particularly antibiotics. Streptomyces netropsis IMV Ac-5025 simultaneously produces different classes of antibiotics, including polyene compounds, phytohormones, and sterols, but the metabolic pathways involved in their biosynthesis are largely understudied. The aim of this work was to explore the biosynthesis of polyene antibiotics, sterols, and phytohormones when the producer is cultivated in a nutrient medium supplemented with exogenous ß-sitosterol. Gas chromatography and high-performance liquid chromatography were applied to analyze the spectrum of bioactive compounds. The obtained results demonstrated not only an increase in the accumulation of biomass but also polyene antibiotics, intracellular sterols, auxins, and cytokinins, when cultivating S. netropsis IMV Ac-5025 in a liquid medium with the addition of ß-sitosterol. The amount of biomass raised 1.5-2-fold, whilst the sum of polyene antibiotics increased 4.5-fold, sterols' sum (ergosterol, cholesterol, stigmasterol, ß-sitosterol, and 24-epibrassinolide) by 2.9-fold, auxins' sum (indole-3-acetic acid, indole-3-acetic acid hydrazide, indole-3-carbinol, indole-3-butyric acid, indole-3-carboxaldehyde, and indole-3-carboxylic acid) by 6-fold, and cytokinins' sum (zeatin, isopentyladenine, zeatin riboside, and isopentenyladenosine) by 11-fold. Thus, we put forward the hypothesis that ß-sitosterol plays a regulatory role in the network of biosynthetic reactions of S. netropsis IMV Ac-5025.

Molecular Research of Lipid Peroxidation and Antioxidant Enzyme Activity of Comamonas testosteroni Bacterial Cells under the Hexachlorobenzene Impact.

The species of Comamonas testosteroni is the most common human pathogen of the genus, which can be associated with acute appendicitis, infections of the bloodstream, the peritoneal cavity, cerebrospinal fluid, inflammatory bowel disease, and in general, bacteremia. According to the literature, Comamonas testosteroni has destructive activity to a wide range of toxic chemical compounds, including chlorobenzenes. The specified strains were isolated from the soil of the organochlorine waste landfill, where hexachlorobenzene (HCB) was predominant. These strains were expected to be capable of degrading HCB. Microbiological (bacterial enrichment and cultivating, bacterial biomass obtaining), molecular biology, biochemical (enzymatic activities, malondialdehyde measuring, peroxidation lipid products measuring), and statistical methods were carried out in this research. The reaction of both strains (UCM B-400 and UCM B-401) to the hexachlorobenzene presence differed in the content of diene and triene conjugates and malondialdehyde, as well as different catalase and peroxidase activity levels. In terms of primary peroxidation products, diene conjugates were lower, except conditions with 20 mg/L HCB, where these were higher up to two times, than the pure control. Malondialdehyde in strain B-400 cells decreased up to five times, in B-401, but increased up to two times, compared to the pure control. Schiff bases in strain B-400 cells were 2-3 times lower than the pure control. However, in B-401 cells Schiff bases under higher HCB dose were in the same level with the pure control. Catalase activity was 1.5 times higher in all experimental variants, compared to the pure control (in the strain B-401 cells), but in the B-400 strain, cells were 2 times lower, compared to the pure control. The response of the two strains to hexachlorobenzene was similar only in peroxidase activity terms, which was slightly higher compared to the pure control. The physiological response of Comamonas testosteroni strains to hexachlorobenzene has a typical strain reaction. The physiological response level of these strains to hexachlorobenzene confirms its tolerance, and indirectly, the ability to destroy the specified toxic compound.

Stenotrophomonas maltophilia IMV B-7288, Pseudomonas putida IMV B-7289 and Bacillus megaterium IMV B-7287 - new selected destructors of organochlorine pesticide hexachlorocyclohexane.

Microbial destruction of organochlorine pollutants is the one of the most effective approaches, the safest and promising methods for remediation the environment from pollution. This study presents strains of microorganisms that destroy hexachlorocyclohexane: Stenotrophomonas maltophilia IMV B-7288, Pseudomonas putida IMV B-7289 and Bacillus megaterium IMV B-7287-newly selected destructors of organochlorine pesticide hexachlorocyclohexane. Their advantages and features are considered, namely-exclusively of natural origin-microorganisms are isolated from places of total pollution. The studied strains are characterized by high resistance to the HCH contaminant (in the range of 100-1000 mg/l) and the ability to decompose in soil and liquid medium. We have found that strains B. megaterium IMV B-7287 and P. putida IMV B-7289 showed a high efficiency of destruction of HCH in laboratory studies when cultivated on a chlorine-free MM medium at 70.4-89.3% from initial content. For S. maltophilia IMV B-7288 has been found that the ability to degrade HCH-isomers depends on the season a little and it was at maximum in the summer for every studied HCH-isomer: 61.6-82.1% from initial amount. The investigated strains are promising for further work to create microbial compositions with the aim to provide an effictive destruction of HCH-isomers complex.

Adenosine-5'-Phosphosulfate- and Sulfite Reductases Activities of Sulfate-Reducing Bacteria from Various Environments.

A comparative study of the kinetic characteristics (specific activity, initial and maximum rate, and affinity for substrates) of key enzymes of assimilatory sulfate reduction (APS reductase and dissimilatory sulfite reductase) in cell-free extracts of sulphate-reducing bacteria (SRB) from various biotopes was performed. The material for the study represented different strains of SRB from various ecotopes. Microbiological (isolation and cultivation), biochemical (free cell extract preparation) and chemical (enzyme activity determination) methods served in defining kinetic characteristics of SRB enzymes. The determined affinity data for substrates (i.e., sulfite) were 10 times higher for SRB strains isolated from environmental (soil) ecotopes than for strains from the human intestine. The maximum rate of APS reductase reached 0.282-0.862 µmol/min×mg-1 of protein that is only 10 to 28% higher than similar initial values. The maximum rate of sulfite reductase for corrosive relevant collection strains and SRB strains isolated from heating systems were increased by 3 to 10 times. A completely different picture was found for the intestinal SRB Vmax in the strains Desulfovibrio piger Vib-7 (0.67 µmol/min × mg-1 protein) and Desulfomicrobium orale Rod-9 (0.45 µmol/min × mg-1 protein). The determinant in the cluster distribution of SRB strains is the activity of the terminal enzyme of dissimilatory sulfate reduction-sulfite reductase, but not APS reductase. The data obtained from the activity of sulfate reduction enzymes indicated the adaptive plasticity of SRB strains that is manifested in the change in enzymatic activity.

ATP sulfurylase activity of sulfate-reducing bacteria from various ecotopes.

Sulfate-reducing bacteria (SRB) are widespread in various ecotopes despite their growth and enzymatic features not compared. In this study, the enzymatic parameters of ATP sulfurylase in cell-free extracts of sulfate-reducing bacteria isolated from various ecotopes such as soils, corrosion products and human large intestine were determined. Comparative analysis of both enzyme characteristics and growth parameters were carried out and similar research has not been reported yet. The initial and maximum rates of enzymatic reaction catalyzed by ATP sulfurylase were significantly different (p < 0.05) in the bacterial strains isolated from various environmental ecotopes. The specific activity of this enzyme in sulfate-reducing bacteria was determined for corrosive and intestinal strains 0.98-1.56 and 0.98-2.26 U × mg-1 protein, respectively. The Michaelis constants were 1.55-2.29 mM for corrosive and 2.93-3.13 mM for intestinal strains and the affinity range were demonstrated. Based on cluster analysis, the parameters of physiological and biochemical characteristics of sulfate-reducing bacteria from different ecotopes are divided into 3 clusters corresponding to the location of their isolation (soils, heating systems and human intestine). Understanding the enzymatic parameters of the initial stages of sulfate consumption in the process of dissimilatory sulfate reduction will allow the development of effective methods for controlling the production of toxic metabolites, including hydrogen sulfide.

RNAi-Based Biocontrol of Wheat Nematodes Using Natural Poly-Component Biostimulants.

With the growing global demands on sustainable food production, one of the biggest challenges to agriculture is associated with crop losses due to parasitic nematodes. While chemical pesticides have been quite successful in crop protection and mitigation of damage from parasites, their potential harm to humans and environment, as well as the emergence of nematode resistance, have necessitated the development of viable alternatives to chemical pesticides. One of the most promising and targeted approaches to biocontrol of parasitic nematodes in crops is that of RNA interference (RNAi). In this study we explore the possibility of using biostimulants obtained from metabolites of soil streptomycetes to protect wheat (Triticum aestivum L.) against the cereal cyst nematode Heterodera avenae by means of inducing RNAi in wheat plants. Theoretical models of uptake of organic compounds by plants, and within-plant RNAi dynamics, have provided us with useful insights regarding the choice of routes for delivery of RNAi-inducing biostimulants into plants. We then conducted in planta experiments with several streptomycete-derived biostimulants, which have demonstrated the efficiency of these biostimulants at improving plant growth and development, as well as in providing resistance against the cereal cyst nematode. Using dot blot hybridization we demonstrate that biostimulants trigger a significant increase of the production in plant cells of si/miRNA complementary with plant and nematode mRNA. Wheat germ cell-free experiments show that these si/miRNAs are indeed very effective at silencing the translation of nematode mRNA having complementary sequences, thus reducing the level of nematode infestation and improving plant resistance to nematodes. Thus, we conclude that natural biostimulants produced from metabolites of soil streptomycetes provide an effective tool for biocontrol of wheat nematode.