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Vol 61, No 1 (2025)
Articles
Practical Successes of Laboratory Evolution
Abstract
Adaptive Laboratory Evolution (ALE) represents a novel methodology for the generation of microbial strains with desired characteristics and the production of value-added products. Additionally, ALE is employed as a means of enhancing comprehension of the genetic and/or metabolic pathways of evolution. The objective of this review is to analyze the results of studies that elucidate and demonstrate the potential of microorganisms as model objects for laboratory evolutionary experiments. These experiments are becoming increasingly prevalent in the study of adaptation, the evaluation of evolutionary dynamics, and the testing of various evolutionary hypotheses. Concurrently, ALE has demonstrated itself to be a promising and efficacious methodology, which, when employed for biotechnological applications, has already resulted in the generation of novel and useful microbial strains. It is important to note that the current successes not only demonstrate the power and versatility of this approach but also highlight a number of unanswered questions. The conclusions drawn on the basis of ALE require a cautious interpretation of the results obtained.
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Rhodococcus erythropolis A-27 as a Biocatalyst for Enantioselective Reduction of Ketones in the Presence of High Concentrations of Isopropanol
Abstract
It has been shown that in the presence of the cells of five strains of Rhodococcus erythropolis, isolated from various anthropogenically polluted ecosystems, and an exogenous reducing agent (isopropanol), enantioselective reduction of ketones (acetophenone and 6-methyl-5-hepten-2-one) occurs with the formation of the corresponding S-configuration alcohols with high enantiomeric excess. Using the most active strain of R. erythropolis A-27 at the optimal concentration of isopropanol, products (S-1-phenylethanol and S-6-methyl-5-hepten-2-ol) were obtained with an enantiomeric excess of at least 99.9 % and a yield of 92 and 93% respectively. This strain was found to be tolerant to isopropanol and could effectively reduce 6-methyl-5-hepten-2-one to S-6-methyl-5-hepten-2-ol in a buffer containing 50% isopropanol.
16-24
Method for Analyzing the Antimicrobial Activity of Peptides via Escherichia coli Expression System
Abstract
The test system for an assay of new potential antimicrobial peptides (AMP) based on the expression of recombinant AMP-encoding genes in Escherichia coli cells has been proposed. This method has a number of advantages over the use of chemically synthesized peptides and both approaches effectively complement each other. Our approach does not impose limitations on the AMP size, facilitates high-throughput screening of mutant plasmid libraries, and has lower cost and complexity compared to the use of synthetic peptides. The core of our methodology involves transformation of the model gram-negative bacterium E. coli with plasmids carrying a recombinant AMP-encoding gene regulated by an inducible promoter. Following transcription induction, bacteria synthesize the AMP, which ultimately leads to cell death. The assessment of bacterial growth is carried out either by measuring the optical density of a bacterial culture grown in liquid media in a microplate or by drip seeding of serial culture dilutions on an agar-based nutrient medium.
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Catalytic Properties of Immobilized Phytase of Silvania hatchlandensis FG 3.9.1
Abstract
A phytase preparation was obtained from the cells of haloalkalitolerant bacteria identified as Silvania hatchlandensis isolated from the soil of a soda sludge storage facility. When the enzyme was immobilized in barium alginate gel and cross-linked with activated chitosan, 97 and 95% of the native protein activity, respectively, was retained. It was shown that 70% of the phytase activity was retained when using the enzyme immobilized in alginate and bound to chitosan over 6 consecutive reaction cycles. Immobilization resulted in an insignificant decrease in the maximum reaction rate and a decrease in the Michaelis constant. Immobilized phytase was more thermally stable compared to the free form of the enzyme: the thermal inactivation constant of the immobilized enzyme at 70°C decreased by 1.1–1.2 times. The immobilized enzyme retained activity at pH 3–12; the pH optimum of the enzyme after immobilization did not change and was equal to 5.0. The specific activity of the enzyme covalently attached to activated chitosan is higher than that of the native enzyme in low and high pH environments. Immobilized phytase of haloalkalitolerant S. hatchlandensis can be used in feed production and other areas of agriculture.
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Bioavailable Nanocomposition of Chitosan-copper Nanoparticles as an Alternative to Antibiotics in BroilerFeed
Abstract
Pathogens pose a serious threat to agriculture as they reduce the growth rate and efficiency of farm birds, animals and cause economic losses. Therefore, there is a need for their use despite all the negative effects of antibiotics and bacterial resistance to them, and therefore, there is a need for effective alternatives that exclude the use of vaccines and drugs. An aggregatively stable nanocomposition of chitosan-nanoparticles of copper with an average size of the latter 25–30 nm was developed. The bactericidal effect of the nanocomposition was shown in vitro on pathogenic bacteria Enterococcus faecalis and investigated in vivo in the composition of broiler chickens’ drink and feed in comparison with antibiotic “Maxus”, used in their diet, on a wide range of pathogenic microorganisms. It was shown that the number of bacteria in broilers was 1.88% when the nanocomposition was administered, which is more than two times less compared to the group where the antibiotic was used.
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Biologically Active Quinolinone Alkaloid from Marine Fungus Penicillium polonicum KMM 4719
Abstract
The marine fungal strain KMM 4719 was isolated from the sea cucumber Apostichopus japonicus and identified as Penicillium polonicum based on three molecular genetic markers: ITS, BenA, and CaM. 3-O-methylviridicatin was isolated from the ethyl acetate extract of the strain culture. 3-O-methylviridicatin demonstrated cardioprotective activity for the first time, as well as urease inhibitory activity (IC50 97.3 μM). In addition, 3-O-methylviridicatin at a concentration of 100 μM (25.1 μg/ml) inhibited the growth of the yeast-like fungus Candida albicans at 23.2%.
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Identification of the Causal Agent of Downy Mildew of Plasmopara viticola Grapes by Quantitative PCR
Abstract
A new method is proposed for the early diagnosis of the causal agent of grapes downy mildew, Plasmopara viticola, based on the method of quantitative real-time PCR (qPCR RT) using SYBR Green I fluorescence. Six pairs primers were developed for the diagnosis of P. viticola, among the designed primers, PvITS1_2-real-s/a demonstrated the highest effectiveness for early detection of grapevine downy mildew with a strong positive correlation with the metagenomic data of P. viticola distribution in Far Eastern grape species and varieties, where a linear dependence was found (R2 = 0.86). Thus, qPCR RT of PvITS1_2 can be used for early detection and monitoring of asymptomatic P. viticola infections. The developed method can be used as a basis for predicting epidemics of downy mildew of grapes and for its control in vineyards.
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Phytostimulation Activity of Methylobacterium dichloromethanicum subsp. dichloromethanicum DM4 and Its groEL2 Gene Knockout Mutant
Abstract
For the first time, the genome of the dichloromethane destructor Methylobacterium dichloromethanicum subsp. dichloromethanicum DM4 was analyzed for the presence of genetic determinants indicating its potential as a plant growth stimulator, and the ability of this strain and its groEL2 gene mutant for the to improve plant growth was determined. The genome of strain DM4 contains genes involved in the biosynthesis of phytohormones (indolyl-3-acetic acid and cytokinins), siderophores, carotenoids, poly-β-hydroxybutyrate, hydrolytic enzymes, as well as enzymes involved in the degradation of D-cysteine, protection from UV-damage and phosphate solubilization. Inoculation of lettuce sprouts by strain DM4 had a positive effect on plant growth and development, and increased adaptive defense and resistance to short-term temperature stress in plant growth experiments. Comparative analysis of the production of auxins, siderophores, hydrolytic enzymes, D-cysteine desulfohydrase activity, and the ability to solubilize insoluble phosphates in strains DM4 and DM4 ΔgroEL2 showed that disruption of the groEL2 gene led to a decrease in the synthesis of indole derivatives and phosphate solubilizing ability in the mutant strain. Assessment of the impact of inoculation of lettuce plants by these strains also demonstrated a decrease in the phytostimulation potential of DM4 ΔgroEL2 compared to the original strain. The data obtained indicate that the chaperonin GroEL2 in M. dichloromethanicum subsp. dichloromethanicum DM4 indirectly affects its phytostimulation activity.
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New Agronomically Valuable Strains of the Genus Streptomyces and Their Biochemical Characteristics
Abstract
Organic farming is a global trend that increases the demand for biological preparations for use in agricultural production. The paper characterizes new actinomycete isolates from soil samples taken in various agrocenoses of the Vyatka-Kama Urals. As a result of preliminary testing (about 350 strains), strains 8Al3, N27-25 and P15-2 were isolated on the basis of antifungal activity. The cultures were identified by 16S rRNA as bacteria of the genus Streptomyces. The active substance with antifungal effect — scopafungin was identified using HPLC-MS/MS. Along with the inhibitory effect on phytopathogenic fungi, these strains produce auxins in the presence of 100 mcg/ml of L-tryptophan (17.4–20.8 mcg/ml), have cellulolytic activity and have a stimulating effect on germination and accumulation of dry biomass by wheat, clover and mustard seedlings. Possessing a complex of beneficial properties for plants, Streptomyces strains 8Al3, N27-25 and P15-2 can be used as candidates for the creation of biological preparations with fungicidal and phytostimulating effects.
89-99
New Features of Pectobacterium atrosepticum Virulence Factors
Abstract
The work analyzed nine strains of Pectobacterium atrosepticum (Pca) and obtained new information about the properties of the pathogen and the regulation of its main virulence factors. Anionic groups (PO34–) were identified in exopolysaccharides (EPS) of Pca, which differ in quantity among different strains and may contribute to the formation of denser biofilms. Most Pca strains inhibited the growth of potato plants in vitro to varying degrees, but one of them (strain 426) significantly activated this indicator in plants of the resistant potato variety Lugovskoy. Based on the totality of symptoms of the disease (chlorosis, necrosis, wilt, inhibition of growth rate), all strains of P. atrosepticum were divided according to the degree of virulence. In addition, it was shown that incubation of bacteria with a homogenate from potato plants in vitro modulated the activity of pectinase Pca: a homogenate from plants of the resistant potato variety Lugovskoy inhibited the activity of exopectinase, and from the susceptible variety Lugovskoy activated it. At the same time, the exopectinase activity of the avirulent strain 426 was inhibited to the greatest extent. It is assumed that this was the reason for stimulating the growth of potato plants in vitro of a resistant variety.
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