N. G. Belogurova

Quantitative Turbidimetric Assay of Enzymatic Gram-Negative Bacteria Lysis

In this Technical Note, the quantitative turbidimetric assay for determination of the bacteriolytic activity of enzymes with gram-negative bacteria is proposed. The reactivity of hen white-egg lysozyme toward gram-negative E. coli intact cells was studied. It was found that the highest lysis rate occurred at pH 8.9 in the system containing 0.03 M NaCl. The mechanism of the reaction is discussed and applied for the quantitative evaluation of the reaction rate. The proposed method enables fast, reliable, and reproducible analysis of bacteriolytic activity of lysozyme with gram-negative bacteria.

Lysis of Escherichia coli cells by lysozyme: discrimination between adsorption and enzyme action.

The key factors of enzymatic lysis of cells are the interaction between the enzyme and the cell - catalytic and non-catalytic adsorption of enzyme on cell surface. Here, the studies of lysis of intact Escherichia coli cells by chicken egg white lysozyme were performed. It was found that the ionic strength has a dual effect onto the system. On the one hand, the desorption constant of the enzyme increases with the increase of the solution ionic strength, which results in a better enzyme performance. On the other hand, due to the higher osmosis, the cell lysis rate decreases with the increasing of ionic strength of the system. It was found that pH 8.6 and 30 mM NaCl are optimal conditions for lysis of E. coli cells by lysozyme.

Bacteriolytic activity of human interleukin-2

In this paper we report the discovery of bacteriolytic activity of an immune system cytokine mediator, interleukin-2. Bacteriolytic activity of interleukin-2 was compared with a well-known bacteriolytic enzyme — chicken egg white lysozyme — by monitoring the lysis of the Gram-negative bacterium Escherichia coli, the Gram-positive coccus Micrococcusluteus, and the Gram-positive spore-forming bacillus Bacillus subtilis. It was found that interleukin-2 has greater specificity to the Gram-negative bacterium E. coli than does lysozyme. In contrast to chicken egg white lysozyme, interleukin-2 does not lyse the Gram-positive coccus M. luteus and the Gram-positive spore-forming bacillus B. subtilis. These results give a new understanding of the biological functions of interleukin-2, a regulatory protein that plays a role in oncological and infectious diseases.

Kinetics of growth and metabolism of Clostridium thermosaccharolyticum culture. Isolation and characteristics of its plasmids.

The kinetics of growth and metabolism ofClostridium thermo saccharolyticum DSM 571 has been studied at varying initial pH and glu cose concentration. A weak inhibitory effect of excess glucose on the specific growth rate has been shown. The effect of antibiotics of various classes on culture growth and hydrogen evolution has been studied. Streptomycin and kanamycin resistance of this culture has been re vealed as well as the phenomenon of increased hydrogen production in the presence of the above antibiotics. New plasmids, pNBl (4.9 kb) and pNB2 (2.0 kb), were isolated fromC. thermosaccharolyticum DSM 571. The restriction analysis of pNBl and pNB2 has been performed.The kinetics of growth and metabolism of Clostridium thermosaccharolyticum DSM 571 has been studied at varying initial pH and glucose concentration. A weak inhibitory effect of excess glucose on the specific growth rate has been shown. The effect of antibiotics of various classes on culture growth and hydrogen evolution has been studied. Streptomycin and kanamycin resistance of this culture has been revealed as well as the phenomenon of increased hydrogen production in the presence of the above antibiotics. New plasmids, pNB1 (4.9 kb) and pNB2 (2.0 kb), were isolated from C. thermosaccharolyticum DSM 571. The restriction analysis of pNB1 and pNB2 has been performed.

Bacteriolytic enzymes from the blood plasma of the sheep

Bacteriolytic factors from the blood plasma of healthy sheep have been studied. Three enzymes not described earlier in the literature have been identified and characterized. Two enzymes exhibit activity toward Escherichia coli and have molecular weights of 15 ± 2 kDa. The third enzyme that exhibits activity toward E. coli and Micrococcus luteus has a molecular weight of 34 ± 4 kDa. The kinetic parameters of bacterial lysis for all enzymes have been determined; in particular, optimal pH values for each of the substrates used have been found. For the identification of the enzymes, trypsinolysis and a mass-spectroscopic study of fragments have been carried out. The results were compared with the data on sheep proteins available in the Swiss-Prot, NCBI, and MSDB databases.

Enzymes of SPZ7 Phage: Isolation and Properties

Bacteriophage enzyme preparations exolysin and endolysin were studied. Exolysin (a phage-associated enzyme) was obtained from tail fraction and endolysin from phage-free cytoplasmic fraction of disintegrated Salmonella enteritidis cells. A new method for purification of these enzymes was developed, and their molecular masses were determined. The main catalytic properties of the studied enzymes (pH optimum and specificity to bacterial substrates) were found to be similar. Both enzymes lyse Escherichia coli cells like chicken egg lysozyme, but more efficiently lyse S. enteritidis cells and cannot lyse Micrococcus luteus, a good substrate for chicken egg lysozyme. Similar properties of exolysin and endolysin suggest that these enzymes are structurally similar or even identical.

Comparison of bacteriolytic activity of human interleukin-2 and chicken egg lysozyme on Lactobacillus plantarum and Escherichia coli cells

The article continues studies of the recently discovered bacteriolytic activity of interleukin-2. It was detected earlier that interleukin (IL-2) possesses greater substrate specificity in comparison with chicken egg lysozyme. IL-2 disrupted the cell wall of Escherichia coli but did not lyse lysozyme substrates such as the cell walls of Micrococcus luteus and Bacillus subtilis. In the present study it is demonstrated for the first time that both IL-2 and chicken egg lysozyme are capable of lysing Lactobacillus plantarum. The effects of IL-2 and chicken egg lysozyme on Lactobacillus plantarum are compared with those on Escherichia coli. The dependences of the rate of lysis on the concentration of bacteriolytic factors and pH are studied.

Enzymatic Lysis of Living Microbial Cells: A Universal Approach to Calculating the Rate of Cell Lysis in Turbidimetric Measurements

The data of the turbidimetric measurement for the enzymatic lysis of various living bacterial cells are analyzed. A method for the correct recalculation of the turbidimetric data (–ΔA/Δt) into absolute values of the change in the concentration of living cells (–ΔCFU/Δt) is proposed. The dimensionless efficiency of cell lysis–(1/CFU0) · ΔCFU/Δt for various bacterial cells is calculated to correctly compare the efficiency of the action of different bacteriolytic factors on various bacterial cells.

Determination of the activity of bacteriolytic enzymes and measurement of their sorption in the system of living cells of Lactobacillus plantarum

A turbidimetric method for determining the activity of bacteriolytic enzymes against living Lactobacillus plantarum cells as a substrate is developed. A technique for measuring the sorption of an enzyme on living cells in an experiment for activity determination is described, with chicken egg lysozyme being used as a standard model enzyme. The correctness of the calculations of the kinetic parameters is proved by counting the colony-forming units (CFUs). The physical and chemical parameters of the sorption of the enzyme on the cells are calculated and the correctness of the obtained equilibrium desorption constants is confirmed.

Bacteriophage SPZ7 endolysin: The influence of effectors on the lytic activity of the enzyme on the lysis of gram-negative microorganisms

The possibilities of bacteriophage SPZ7 endolysin functioning in the lysis of gram-negative bacteria “from without” were studied. A significant 1.5–3-fold increase in bacteriophage SPZ7 endolysin activity during the lysis of S. enteritidis N60 and E. coli TG1 cells in the presence of high-molecular surfactants, pluronics with a large hydrophobic block, hen’s egg lysozyme, and low concentrations of a peptide antibiotic (colistin), was shown. The developed approach may be promising for improving the efficiency of pharmaceutical bacteriophage enzyme-based antibacterials working against gram-negative microorganisms.