Volume 60, Nº 5 (2024)

Mycoplasma contamination of continuous cell cultures and collection viral strains remains a serious problem in the biotechnology industry and experimental research. The frequency of mycoplasma contamination of cultured cell lines and viruses is 15–35%, in some cases up to 80%. Mycoplasmas cause various changes in cultures contaminated by them, up to cell death, have immunomodulatory properties, and affect the yield of certain viruses propagated in cell culture. Mycoplasmas do not have a cell wall, are able to pass through a bacterial filter, have the smallest genome (≈580 kb) among bacteria, and are capable of independent reproduction and existence. These microorganisms are resistant to most antibiotics commonly used in cell culture. Derivative groups of tetracyclines and fluoroquinolones (BM-Cyclin®, Ciprobay®, Baytril®, Plasmocin®, MRA) have shown certain effectiveness in decontaminating viral strains and cell cultures from mycoplasmas. Timely, highly sensitive detection and prevention of mycoplasma infection is of great importance. For routine scanning of mycoplasma infection of continuous cell cultures and viral strains, the methods of indicator cell culture (cytochemical) and polymerase chain reaction (PCR) are recommended, for more accurate – microbiological analysis of mycoplasma colonies on a special medium.

The potential of new genetically modified recombinant endolysins as antimicrobial agents against Gram-negative bacteria was investigated. A series of enzymes based on LysSi3 lysozyme-like muramidase were obtained by modifying its sequence with antimicrobial peptides of different families and recombinant expression in E. coli was demonstrated. Modification of LysSi3 resulted in increased bacteriolytic activity against the model isolate of A. baumannii and higher kinetics rate compared to the native enzyme. The cytotoxic properties of new engineered lysins were investigated with the HEK293 and HaCaT cell lines and it was shown that modification of LysSi3 with antimicrobial peptides does not significantly increase the toxic properties in vitro.

Bacteria of the genus Azotobacter sp. produce two classes of biologically important biocompatible and biodegradable polymers – polyoxyalkanoates, which are the bacterial reserve, and alginates (ALG), which perform the function of protecting nitrogenase from oxygen. Both polymers are becoming increasingly important for use in bioengineering, pharmaceuticals and medicine, so studies of their biosynthesis and properties are currently highly relevant. The present work shows the possibility of regulating alginate and poly-3-hydroxybutyrate (PHB) synthesis by A. vinelandii 12 culture depending on the increase of sucrose concentration in the medium under different aeration conditions. At high aeration and high sucrose concentration in the medium (50 g/L), the maximum yield of free (1.08 g/L) and capsular ALG (2.26 g/L) in the medium was obtained. Under low aeration conditions, the synthesis of free ALG was completely inhibited. The maximum value of РНB synthesis was observed at medium aeration and high concentration of sucrose (50 g/l) in the medium. The maximum molecular weight (MW) of ALG was 477 kDa, while the maximum MW of PHB was much higher, reaching 1479 kDa. At low sucrose concentrations in the medium (5 to 20 g/l), capsular ALG is predominantly synthesized (up to 100% of the sum of all polymers) at all aeration levels. With increasing sucrose concentration, PHB is predominantly synthesized (68%) under low aeration conditions, an equal ratio of PHB and capsular ALG synthesis is observed under medium aeration conditions, and free ALG is actively synthesized under high aeration conditions. This work demonstrates the possibility of obtaining a selective synthesis of ALG or PHB by A. vinelandii 12 by modifying its cultivation conditions. The results obtained can be used for the development of directed biosynthesis of target products (PHB and ALG) in biotechnology.

For the first time, the interaction between bacteria and bacteriophage was studied under space conditions. The model system of E. coli and bacteriophage T7 was used. The results of the interaction depended on the duration of exposure of the system to space flight factors. During the first 2 days of microgravity exposure the virus replication rate in Space was higher than on Earth. The bacteria then have adapted to space conditions and acquired resistance to the bacteriophage, which persisted for 2 days after return to Earth. Over the next three days, the sensitivity of the E. coli to the T7 bacteriophage returned to its original level.

Polyextremophilic red microalgae of the genus Galdieria, which inhabit hot sulphur springs under conditions unusual for eukaryotes, are capable of heterotrophy. Among the dozens of exogenous organic substrates identified for Galdieria, ethanol is not mentioned as a possible energy source. As it turned out that ethanol did not alter the growth of the model species Galdieria sulphuraria when grown in the dark. In contrast, the growth of microalgae is activated in the light, despite the known cell stressor effect of ethanol. The effect of ethanol as an oxidative stress factor may be indicated by the increase in cellular respiration observed in the dark and also in the light even before the activation of photosynthesis. The marked acceleration of growth of G. sulphuraria culture in the light is most likely due to the stimulation of respiration by ethanol with generation of CO₂ and its use by chloroplasts as an additional carbon substrate during the photosynthetic process. Compared to the classical organic substrate glucose, the light-induced growth of G. sulphuraria cultures in the presence of ethanol is less intense. It can be speculated that ethanol stress in light induces the system of two consecutive key enzymes in the primary alcohol metabolism chain (alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase), which then leads to the eventual complete oxidation of ethanol, resulting in accelerated growth of G. sulphuraria.

Actinobacteria cells Rhodococcus erythropolis 4-1 and Rhodococcus erythropolis 11-2 and Proteobacteria Alcaligenes faecalis 2, which have amidase activity, were immobilized by entrapping barium alginate and agarose into the gel structure, as well as by obtaining biofilms on thermally expanded graphite (TEG). The operational stability of such immobilized biocatalysts after storage in frozen and dehydrated form was determined, and a prototype of a conductometric acrylamide biosensor based on such a bioselective agent was developed. The most preferred method for storing immobilized cells was freezing at temperatures from –20 to –80°C; long-term storage is also possible wet at 4–25°C. It was shown that these cells were most preferable for the biodetection of acrylamide A. faecalis 2, immobilized in an agarose gel structure. An agarose gel with bacterial cells immobilized in its structure had greater mechanical strength and stability during successive cycles of conversion of acrylamide into acrylic acid compared to barium alginate gel. The mechanical strength of barium alginate gel can be enhanced by the addition of carbon nanomaterials during cell immobilization. Growing biofilms on carbon materials used for manufacturing electrodes is also promising. Biofilms of R. erythropolis 11-2 on TEG are capable of converting acrylamide into acrylic acid in more than 20 reaction cycles while maintaining at least 50% amidase activity.

The work implements a method for specific in vivo biotinylation of recombinant proteins M1 and B7 of the variola virus during biosynthesis in CHO-K1 cells. To do this, co-expression of the biotin ligase BirA and target genes encoding the ectodomains of the M1 and B7 proteins with a C-terminal avi-tag was carried out in CHO-K1 cells in the presence of biotin in the culture medium. The optimal biotin concentration for the expression of M1 and B7 proteins was 125 μM. The production of biotinylated recombinant proteins has been complicated by low yields. To increase the production of target proteins, low molecular weight enhancers were added to the culture medium: lithium acetate, sodium valproate and caffeine. The enhancers increased the yield of the target protein by 1.3–4.9 times and did not affect the efficiency of biotinylation. The highest yield of biotinylated protein was achieved with the simultaneous addition of a concentration of 10 mM lithium acetate and 2.5 mM sodium valproate.