SECONDARY METABOLITES WITH ANTIBIOTIC ACTIVITY OF MARINE ACTINOBACTERIA

Marine actinobacteria are active producers and an unused rich source of various biologically active secondary metabolites, such as antibiotics, antitumor, antiviral and antiinflammatory compounds, biopesticides, plant growth hormones, pigments, enzymes, enzyme inhibitors.In this review describes data from current literature sources for the period from 2017 to 2021 about various bioactive compounds that produce marine actinobacteria, their antibiotic activity and biotechnological potential, the main groups of secondary metabolites and their producers.

CHARACTERISTICS OF ACTINOBACTERIA FROM MYTILUS GALLOPROVINCIALIS OF ODESSA GULF OF THE BLACK SEA

Today both academic and commercial interests in marine actinobacteria are growing. As they live in a unique environment that promotes the synthesis of new biologically active metabolites. The aim of the work is isolation, primary identification and study of morphological, cultural, physiological and biochemical properties of actinobacteria, separate from mussels (Mytilus galloprovincialis) of the Odessa Bay of the Black Sea. Methods. Samples of mussels collected in the coastal zone of Odessa Bay were used as material for the isolation of actinobacteria. Isolation of actinobacteria and the study of their morphological, cultural, physiological and biochemical properties was carried out by traditional microbiological methods. The fatty acid composition was determined on an Agilent 7890 semi-ionization gas chromatograph (Agilent Technologies, USA) to identify test strains using the Sherlock Microbial Identification System library. Results. From samples of mussels collected in 2020 in the area of the Hydrobiological Station of Odessa National I.I. Mechnikov University were isolated 14 strains of actinobacteria. They were identified by fatty acid analysis to Streptomyces genus. Strains of Streptomyces sp. Myt2, Myt6 and Myt7ch synthesized melanoid pigments. Strains of actinobacteria are well absorbed by most of the studied sources of carbon, except strains of Streptomyces sp. Myt12a, Myt12b. Almost half of the strains have oxidase activity and coagulate milk. Conclusions. As a result of microbiological studies for the first time were characterized morphological, cultural and physiological and biochemical properties and determined taxonomic composition by fatty acid spectra for actinobacteria isolated from the mussels of the Odessa Bay of the Black Sea.

BIOSURFACTANTS FROM MARINE MICROORGANISMS: I. STRUCTURE AND FUNCTIONS

Marine microorganisms have unique metabolic and physiological characteristics and are an important source of new biomolecules such as biosurfactants. Low molecular weight surfactants are glycolipids, phospholipids, fatty acids, lipopeptides and lipoproteins, and high molecular weight surfactants are mixtures of heteropolysaccharides, lipopolysaccharides, lipoproteins and proteins. The main general of bacteria that synthesize biosurfactants are Pseudomonas, Bacillus, Acinetobacter, Antarctobacter, Rhodococcus, Halomonas, Alcanivorax, Pseudoalteromonas and Marinobacter. This review examines the structure and function of biosurfactants isolated from marine microorganisms.

THE COMPOSITION OF CELLULAR FATTY ACIDS OF ACTINOBACTERIA FROM THE SURFACES OF BIOLOGICAL GROWTH OF THE ODESA GULF OF THE BLACK SEA

Aim. Determination of fatty acid composition of actinobacteria isolated from the surfaces of biological growth of the Odesa gulf of the Black Sea, and their identification. Methods. The 31 isolated strains of actinobacteria were grown in TSB at 28 ° C and 150 rpm for 72 hours. Fatty acid methyl esters of the studied strains were determined according to the MIS Operating Manual on a gas chromatograph Agilent 7890, identification was performed using the identification system of microorganisms MIDI Sherlock. Results. Using chromatographic analysis of fatty acids, it was found that of the 27 studied strains of actinobacteria were identified to the genus Streptomyces, and the 4 strains - to the genus Nocardiopsis. It was found that the fatty acid profiles of the studied actinobacteria of the genus Nocardiopsis were dominated by fatty acids: 15:0 ANTEISO, 16:0 ISO, 17:0 ANTEISO, 18:1 CIS 9, and the fatty acid profiles of bacteria of the genus Streptomyces - 14:0 ISO, 15:0 ANTEISO, 16:0 ISO, 17:0 ANTEISO. Conclusions. Actinobacteria the surfaces of biological growth of the Odesa gulf of the Black Sea belong to the genera Streptomyces and Nocardiopsis, and their fatty acid profiles are characterized by the dominance of isomers of branched saturated fatty acids.

BIOSURFACTANTS SYNTHESIS BY PSEUDOMONAS AERUGINOSA BACTERIA ISOLATED FROM THE SURFACE OF MUSSELS OF THE BLACK SEA

Aim. Establishing of the ability to synthesize surface-active compounds by Pseudomonas aeruginosa bacteria isolated from the surface of Black Sea mussels. Methods. During the research several marine Pseudomonas spp strains isolated from petroleum hydrocarbon contaminated areas of Black Sea wereused: P. aeruginosa M1, P. aeruginosa M4 and P. aeruginosa PA01 as reference strain in suspension and biofilm cultures (LB and Giss media). Cultivation of Pseudomonas aeruginosa strains was performed at 37 °C for 120 and 168 hours. Planktonic culture growth was determined spectrophotometrically on the wave length 600 nm. Biofilm mass was determined spectrophotometrically on the wave length 592 nm by CV-test. The presence of surface-active compounds was determined in a drop test. The quantitative content of rhamnolipids was evaluated by the color reaction of rhamnose with orcin. Results. P. aeruginosa strains M1 and M4 isolated from Black Sea mussel’s surfaces synthesize 25% and 66% more surfactants than the reference strain PA01. All strains in Giss medium synthesized 10–20 times less rhamnolipids than in LB medium. In biofilm cultures the same biosurfactant synthesis dependence on the composition of the nutrient medium is observed as in suspension cultures. According to the intensity of rhamnolipid production in biofilm cultures, the studied strains can be arranged in the following row: P. aeruginosa M4 > P. aeruginosa M1 >> P. aeruginosa PA01.Conclusions. The strains of P. aeruginosa isolated from the Black Sea are more efficient producers of rhamnolipids than the reference strain of P. aeruginosa PA01; the intensity of biosynthesis of surfactants significantly depends on the composition of the nutrient medium and the method of cultivation.

ISOLATION OF BACTERIA FROM THE SITES OF FEED AND NESTING ACTIVITY OF LARUS DOMINICANUS (GALINDEZISLAND, THE MARITIMEANTARCTIC) AND THEIR CHARACTERISTICS

The aim of the study was to investigate the number of different groups of microorganisms in soil samples from sites of feed and nesting activity of Larus dominicanus (Galindez Island, the maritime Antarctic) and to characterize the physiological and biochemical properties of isolated microorganisms. Methods. In the work standard microbiological and biochemical research methods were used (cultural, microscopy methods, determination of enzymatic activity). Genomic DNA was isolated by soft lysis. The 16 S rRNA gene was amplified using universal primers 27F and 1492R. Identification of isolates was performed based on the determination of the 16 S rRNA gene sequence, physiological and biochemical properties. Results. The number of microorganisms of different groups in soil samples from sites of food and nesting activity of Larus dominicanus (Galindez Island, the maritime Antarctic) was established. 74 isolates of bacteria were isolated. Isolate 2U-K-37, that was isolated from upper layer of soil, and isolate 2B-K-54, that was isolated from a depth of 2–5 cm, were characterized by amylase, lipase, phospholipase, protease activities, the ability to form exopolysaccharides.They were identified by sequencing of the 16 S rRNA gene, physiological and biochemical properties as Pedobacter sp. 2U-K-37 and Pseudarthrobacter sp. 2B-K-54. In the soil samples from sites of feed and nesting activity of Larus dominicanus microorganisms that metabolize nitrogen of organic compounds were the most numerous. Oligotrophic microorganisms and microorganisms that metabolize nitrogen of inorganic compounds were less numerous. The number of microorganisms of groups in the samples from the soil surface and lower layers differed statistically significant. Isolated and identified obligate psychrophilic strain Pedobacter sp. 2U-K-37 and psychrotolerant strain Pseudarthrobacter sp. 2B-K-54 are moderate halophiles that are able to hydrolyze starch, gelatin, tween-20 and synthesize exopolysaccharides.

EFFECT OF BACILLUS MEGATERIUM ONU 500 ON THE GERMINATION AND GROWTH OF SUNFLOWER SEEDLINGS

The aim of the study was to evaluate the effect of Bacillus megaterium ONU 500 on the germination and growth of sunflower seedlings. Materials and methods. Sunflower seeds were sown in containers with soil inoculated with a suspension of bacteria B. megaterium ONU 500 in concentration of 106 CFU/ml. After 21 days growth characteristics of seedlings were compared: germination, average height, root length, leaf area, dry weight. Effect of these microorganisms on biofilm formation on roots was investigated with light microscopy. Results. Inoculation of soil with B. megaterium ONU 500 resulted in the positive effect on roots length (increase by 21%), leaf area (increase by 22%) and dry weight of seedlings (increase by 17.8%). No significant effect on germination and plant height was observed. On the roots of plants grown in soil treated with B. megaterium ONU 500 more developed biofilms with well formed matrix were observed, which indicates the stimulation of ability of plant and soil microbiota to form biofilms in presence of bacilli from the studied strain. Conclusion. The investigations demonstrated that bacteria of B. megaterium ONU 500 strain caused a positive effect on development of sunflower seedlings and formation of biofilms on their roots.

ISOLATION AND STRUCTURE ANALYSIS OF THE MUTANT FORM OF N-TERMINAL CATALYTIC MODULE OF BOS TAURUS TYROSYL-tRNA SYNTHETASE WITH REPLACEMENT OF Trp 40 AND Trp 87 BY ALANINE

Aim. Isolation and analysis of the structure of the mutant monotryptophan protein mini BtTyrRS for study of conformational changes of the enzyme at the stage of interaction with tRNA using fluorescence spectroscopy and determination of the effect of tryptophan residues in position 40 and 87 in its structure on the functional properties of the enzyme. Methods. Electrophoresis, metal-chelating affinity chromatography, fluorescence spectroscopy, spatial structure modeling. Results. It was found that the replacement of two codons of Trp by codons of Ala in the cloned cDNA mini BtTyrRS does not affect the synthesis of the mutant form of the enzyme in E. coli strain BL21 (DE3) pLysE. The yield of affinity purified protein on Ni-NTA agarose is on average 3.5 mg per 100 ml of culture medium. Computer modeling of the structure and fluorescence spectroscopy of the monotryptophan form of mini BtTyrRS indicates a compact structure of the mutant enzyme, in which Trp 283 is in an immobilized microenvironment. Conclusions. Affinity purified on Ni-NTA agarose mutant monotryptophan protein mini TyrRS have been obtained which is suitable for fluorescent studies of structural-dynamic and functional properties of the enzyme.

MICROBIOLOGICAL AND SANITARY - CHEMICAL CHARACTERISTICS OF WASTEWATER FROM PHARMACEUTICAL COMPANY

Aim. Оssessment of wastewater from a pharmaceutical company according to sanitary-microbiological and chemical indicators to determine the method of treatment and remediation of them from pollutants and pathogens. Methods. In sanitary and bacteriological studies, classical microbiological methods were used to determine the total microbial number of pathogenic bacteria. Determination of the concentration of ammonium, nitrite and nitrate in wastewater samples from a pharmaceutical company was carried out spectrophotometrically using the chemical reaction of ions to Nessler's reagent, Griss reagent, and phenol sulfide acid. The content of heavy metal ions was determined by the method of electrothermal AAS using the device "Saturn-2", by the photometric method using 4-aminoantipyrine – phenol, by the extraction-photometric method – by surfactants, by the method of infrared spectrometry – by petroleum hydrocarbons. Results. It was experimentally confirmed that the total microbial count in wastewater samples from a pharmaceutical company slightly exceeded the standard value. The BGKP index and the E. coli index were within the normal range. The pathogenic bacteria Salmonella moscow and Klebsiella pneumoniae were found in the wastewater of pharmaceutical company. The contamination of pharmaceutical stock can be judged by the 8.5 times excess of the concentration of ammonium ions in them compared to the MPC. The results of chemical analysis of wastewater samples from a pharmaceutical company indicate their multicomponent composition.Of the heavy metal ions, ions of zinc, copper, lead, hexavalent chromium and cadmium were mainly detected. With the exception of cadmium ions, the concentration of heavy metal ions was 1.4–7.2 times higher than the MPC norm. Anionic surfactants predominated in pharmaceutical stocks – their concentration was 8.2 ± 0.7 mg/dm3. The level of phenolic and oil pollution of wastewater was minimal in comparison with the content of anionic surfactants, which could be caused by the activity of aboriginal strains of bacteria-destructors with a high phenolic capacity. Conclusion. The results of sanitary-microbiological and chemical analysis of wastewaters of a pharmaceutical company indicate their multicomponent nature and environmental hazard – they contain pathogenic bacteria Salmonella moscow and Klebsiella pneumoniae and pollutants of various nature: heavy metal ions Cu (II), Cr (VI) and Pb (II) at a concentration of 36.0 ± 14 μg/dm3, 3.0 ± 0.4 μg/dm3 and 14.0 ± 0.7 μg/dm3, respectively, and organic pollutants – phenol, oil products and anionic surfactants at a concentration of 0.003 ± 0.0006 mg/dm3, 0.81 ± 0.05 mg/dm3 and 8.2 ± 0.7 mg/dm3, respectively. Werecommend to carry out a comprehensive purification of wastewater from a pharmaceutical company from phenol, heavy metal ions [Cu (II), Cr (VI), Zn (II)], oil products, anionic surfactants and pathogens.

THE EFFECT OF BACTERIOCIN ENTEROCOCCUS ITALICUS ONU 547 AND ESSENTIAL OILS ON THE GROWTH OF OPPORTUNISTIC MICROORGANISMS

Purpose. To evaluate the effectiveness of bacteriocin of Enterococcus italicusONU 547 and essential oils individually and in combination against indicatorstrains of opportunistic pathogens. Methods. The antagonistic activity of essential oils and bacteriocin was determined by the optical density of the bacterial suspension of test strains (Salmonella enterica NCTC 6017, Escherichia coli АТСС 25922, Pseudomonas aeruginosa АТСС 27853, Pseudomonas putida KT 2440, Enterococcus faecalis АТСС 29212, Klebsiella pneumoniae АТСС 10031,Staphylococcus aureus АТСС 25923). The index of fractional inhibitory activity(FIC) was calculated by adding the FIC values of bacteriocin E. italicus ONU 547and the studied essential oils. The results. The maximum antimicrobial effect was caused by the essential oils of Anisum officinalis and Melissa officinalis, which suppressed the growth of test strains more than 50.0%. When using bacteriocin of E. italicus ONU 547 suppression of viability of indicator strains did not exceed 30.0%. Experimental combinations of essential oils and bacteriocin caused 4 types of consequences: synergy effect; additive effect; antagonistic effect; interactiveeffect (no interaction effect). In most cases, the combined action of bacteriocinand essential oils caused an additive effect. Conclusions. This study demonstrated the potential of using different combinations of natural antimicrobial compounds. The obtained results provide grounds for further development and optimization ofcombinations of essential oils and probiotic strains of microorganisms for use in food and pharmaceutical biotechnology.