Applied Biochemistry and Microbiology最新文献

Nine strains of Pectobacterium atrosepticum (Pca) were analyzed in this work, and new information about the pathogen properties and regulation of its main virulence factors was obtained. In exopolysaccharides (EPS) of Pca, anionic groups (({text{PO}}_{4}^{{3 - }})) were detected, the amount of which varied in different strains and probably contributed to the formation of denser biofilms. Most Pca strains inhibited to varying degrees the growth of potato plants in vitro; however, one of them (426) activated significantly the growth of the plants of the resistant potato variety Lugovskoi. Based on the totality of the disease symptoms (chlorosis, necrosis, wilt, inhibition of growth rate), all P. atrosepticum strains were divided by the degree of virulence. In addition, it was demonstrated that the incubation of bacteria with homogenate from potato plants in vitro modulated the activity of pectinase in Pca homogenate from the plants of the resistant potato variety Lugovskoi inhibited the activity of exopectinase, while that from the susceptible variety Lukyanovskii activated it. At the same time, the activity of exopectinase from the avirulent strain 426 was inhibited to the greatest extent. It is assumed that this was a reason for the stimulation of the growth of the resistant potato variety plants in vitro.

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 six 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 media. Immobilized phytase of haloalkalitolerant Silvania hatchlandensis can be used in feed production and other areas of agriculture.

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 approach has a number of advantages over the use of chemically synthesized peptides, and both proposed methods 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 spot plating of bacterial culture serial dilutions on an solid agar medium.

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.

A new method is proposed for early identification of the causal agent of grapevine downy mildew Plasmopara viticola, based on a real-time quantitative PCR (real-time PCR) method using the fluorescent dye SYBR Green I. Six pairs of primers were developed for real-time PCR for identification of P. viticola; among them, the primer pair PvITS1_2-real-s/a demonstrated the highest efficiency for early detection of grapevine downy mildew. Moreover, a positive correlation (R = 0.86) was demonstrated when comparing the results of real-time PCR with the primers PvITS1_2-real-s/a with data on metataxonomic analysis of the distribution of P. viticola among plants of Far Eastern grape species and varieties. Thus, real-time PCR with the primer pair PvITS1_2-real-s/a is a cheap and efficient method for early detection and monitoring of asymptomatic P. viticola infections. The developed method can be a basis for predicting epidemics of grapevine downy mildew and its control in vineyards.

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 has 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%.

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 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. Assessment of the impact of inoculation of lettuce plants by these strains also demonstrated a decrease in the phytostimulating 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 phytostimulating activity.

Organic farming is a global trend that increases the demand for biological preparations for use in agricultural production. This 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. An 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 μg/mL of L-tryptophan (17.4–20.8 μg/mL), have cellulolytic activity, and have a stimulating effect on germination and accumulation of the dry biomass by wheat, clover, and mustard seedlings. In possessing a complex of properties beneficial 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.

Pathogens pose a serious threat to agriculture as they cause diseases, reduce the growth rate and efficiency of farm birds and animals and causing economic losses. Therefore, antibiotics continue to be used to combat pathogens and for prevention, despite all their negative effects and the bacterial resistance to them. Effective alternatives are needed that exclude the use of vaccines and drugs. An aggregatively stable nanocomposition of chitosan and nanoparticles of copper with an average size of the latter of 25–30 nm was developed. The bactericidal effect of the nanocomposition was shown in vitro on the pathogenic bacteria Enterococcus faecalis and investigated in vivo in the composition of the drink and feed of the broiler chickens in comparison with the antibiotic “Maxus,” which is 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 in which the antibiotic was used.

It is known that sorbitol metabolism in the yeast K. kurtzmanii and K. phaffii is limited by the level of sorbitol dehydrogenase expression. In the present work, we obtained transformants of K. kurtzmanii and K. phaffii that synthesized the thermostable endoglucanase Egh12 of Thielavia terrestris, xyloglucanase Xgh12b of Aspergillus cervinus, and cathepsin-L CathL of Tribolium castaneum under the control of the methanol-inducible promoter P_AOX1 and had different rates of sorbitol metabolism due to additional expression of the sorbitol dehydrogenase gene. Synthesis and secretion of the target enzymes were investigated on media containing methanol and sorbitol in different ratios. The results obtained showed that an increase in the rate of sorbitol metabolism in the producers had a negative effect on the synthesis of target proteins up to complete inhibition of their production. At the same time, low-efficiency sorbitol assimilation by these strains, limited by the expression of sorbitol dehydrogenase or by the concentration of sorbitol in the medium, promoted an increase in the secretion of Egh12 and increased enzyme production up to 30% by stimulating the growth of the cell biomass.