Formation of recombinants of F' plasmids in recombination-defective E. coli cells and their properties]

As a result of mating of cells carrying plasmid F' lac with cells carrying plasmid F'his merodiploids carrying plasmid complex F'lacF'his were isolated. The plasmid genes of the isolated merodiploids are donated together into the recipient cells and are eliminated jointly from host-cells both spontaneously and by acridine orange. As the formation of the plasmid complex took place in E. coli cells carrying mutation in rec A gene the recombination of plasmids F' is supposed to take place in the absence of the product of gene rec. A.     

Rifampicin action on the ultrastructure of E. coli cells]

The ultrastructure of Coli bacteria exposed to rifampicin was studied. Dependence of the level of the ultrastructural changes on the antibiotic concentration and the time of incubation with the antibiotic was shown. After exclusion of the antibiotic from the medium the organism growth with normal ultrastructure was observed.     

Makeup and properties of E. coli cells with a varying level of resistance to tetracycline]

Lipopolysaccharide composition of tetracycline sensitive and resistant strains of E. coli was studied comparatively. It was shown that that resistance of E. coli to tetracycline was probably due to the differences in the lipopolysaccharide component composition of the outer membrane. On the basis of the activity comparison of the Mg2+- and Ca2+-activated ATP-ase of the membrane fraction of the tetracycline sensitive and resistance strains of E. coli it was concluded that the resistance development in the strains tested to tetracycline was not associated with the changes in the ATP-ase activity.     

alpha-Hemolysin of E. coli]

The activity of alpha-hemolysin increased at the log growth phase in the culture of E. coli P678 Hly+ hemolytic strain; this activity diminished with the change into the stationary phase, and then fell sharply. Replacement of the culture medium in the stationary growth phase by fresh one led to restoration of the hemolytic activity of the culture. The culture fluid separated from the cells at the stationary growth phase produced an inhibitory action on the alpha-hemolysin Ca ions activated and stabilized the alpha-hemolysin. Sodium citrate and sucrose served as hemolysis inhibitors. The action of alpha-hemolysin was maximal against human erythrocytes at pH 6.5. Hemolytic activity was characterized in time by a distinct lag-phase and the phase of the greatest rate of reaction. The duration of the lag-phase and also the rate of hemolysis depended on the concentration of alpha-hemolysin (with the increase of the hemolysin concentration lag-phase was shortened and the reaction was accelerated). There proved to be a linear relationship between the amount of erythrocytes taken into the reaction and the rate of hemoglobin release, and also there was noted a temperature activation of the hemolytic reaction.     

Lighting E. coli cells as biological sensors for Cd2+

Whole cells of E. coli expressing a chimeric cadmium-binding peptide fused to green fluorescent protein (CdBP-GFP) were prepared and applied for the determination of cadmium. Construction of the structural gene was performed by inserting two synthetic oligonucleotides coding for four repeats of a Cd-binding peptide (His-Ser-Gln-Lys-Val-Phe) into the 5-end of the GFPuv gene. Similarly, a hexahistidine-green fluorescent protein (his₆GFPuv) was prepared and used as a reference in the determinations of heavy metals. The lowest concentrations of Cd, which activated the fluorescence, were 0.5 M, 50 M, and 0.5 mM for cells carrying CdBP₄GFP, his₆GFP and native GFP, respectively.     

Antigenic properties of genetic recombinants of serologically typed E. coli]

The conjugation between the typed strains of E. coli belonging to various serological groups and conjugation between typed and untyped E. coli strains were studied. Genetic determinant controlling the synthesis of the O100 antigen proved to be closely linked with histidine locus. Among recombinants obtained in crossing the typed E. coli strains there were such belonging to the serological type different from the serological types of donor and recipient cells.     

Exogenous orthophosphate regulation of ATPase activity of E. coli cells]

The effect of exogenous orthophosphate and mutations in genes, regulating the Pi transport system, on the ATPase activity of E. coli subcellular fractions was studied. It was shown that the orthophosphate starvation resulted in the cessation of the increase in the ATPase activity of membranes and was accompanied by the increase in the analogous activity of a soluble fraction at the expense of the derepression of alkaline phosphatase possessing this activity. The disturbance, resulted from the mutation of protein components participating in the specific binding and transport of orthophosphate, changed the ATPase activity of subcellular fractions: increased the ATPase activity of soluble fraction (independently of the presence of orthophosphate in medium), did not affect significantly the activity of membrane--bound ATPase in the presence of orthophosphate and decreased this activity in the absence of orthophosphate. The data obtained point to the fact that components, binding exogenous orthophosphate and transporting it into a cell, affect the rigidity of the ATPase bound E. coli cytoplasmic membrane. Mutations resulting in the defect in these components relax this bound and lead to the detection of ATPase proper in the periplasm.     

Isolation and properties of DNA-cytosine-methyltransferase EcoRII and E. coli K12]

The methods of isolation and partial purification of two DNA-cytosine-methylases (DC-methylases) EcoRII and E. coli K12 are described. After chromatography on phosphocellulose the enzymes were purified 100-fold, the yield being 30%. Further purification of the enzymes was performed by sedimentation in a sucrose concentration gradient. Both enzymes have native molecular weights of 50,000; DC-methylase from E. coli K12 may simultaneously occur in the forms with molecular weights of 70,000, 90,000 and 110,000. Both DC-methylases modify identical nucleotide sequences of DNA, have equal numbers (90) of methylation sites in phage lambda DNA and provide in vitro a complete protection of phage lambda DNA against restriction endonuclease EcoRII. DC-methylases E. Coli K12 and EcoRII differ in their chromatographic behaviour on phosphocellulose and capacity to form compexes with the cell DNA-adenine-methylase.     

Biological properties of L-asparaginase preparations from E. coli in cell cultures]

Non-specific cytotoxicity and specific antitumor activity of 5 preparations of L-asparaginase from E. coli were studied. Two cell line, i.e. the asparagine-dependent (Berkitt lymphoma cells) and asparagin-independent (human ovary cancer cells) were used as the test-system. Incorporation of 3H-thimidine into DNA was the criterion of the preparation effect on the cells. Preparation I with the specific activity of 60-90 IU per 1 mg of protein obtained at the first stages of purification had high non-specific cytotoxicity. Preparation II obtained after further purification of preparation I, as well as preparation II without any stabilizer with the specific activity of 200 IU/mg were not inferior to the "Bayer" preparation by their biological properties. Addition of L-asparaginase to the preparation as a stabilizer of excessive glycine (preparation IV) increased its non-specific cytotoxicity and interfered with the study of its properties in the cell systems. Mannitol (preparation V) had no effect on the biological activity of L-asparaginase preparation.     

Fractionation and purification of DNA methylases and specific endonucleases from cells of Escherichia coli SK]

Fractionation and purification of DNA methylases and specific endonucleases from E. coli SK responsible for DNA specificity to host prokaryotic cells were studied. The most efficient purification was achieved by precipitation of proteins by 0.6 saturated ammonium sulfate with subsequent chromatography on KM-cellulose and concentration of fractions by dialysis against glycerol. Under these conditions the methylase activity produced 4 discrete fractions. Due to purification the specific activity of methylases increased 11--20-fold in various fractions. Methylase from the first (A) and fourth (BII) peaks catalyzed the methylation of cytosine to produce 5-methylcytosine; methylase from the third peak (BI) methylated adenine to produce 6-methylaminopurine. The chemical specificity of the second peak (B) methylase could not be established due to very high lability of the enzyme in this fraction. Specific endonuclease was found in the gradient zones eluted by 0.1--0.2 M and 0.65--0.75 M NaCl. It is assumed that those enzymes providing for DNA hydrolysis up to the formation of high--molecular discrete fragments, are restricting endonucleases of the SK system. The results obtained strongly suggest the existence of several types of methylases and restricting endonucleases in E. coli SK cells.     

Penicillin amidase from E. coli. Some physicochemical properties of the soluble enzyme]

Homogeneity of the enzyme was shown with the methods of gel filtration and disc electrophoresis. The molecular mass of penicillinamidase (PA) was determined. Sorption of PA by a carboxylic ion exchanger within a wide range of pH was studied. The values of pH in the ion exchanger phase under the conditions of the enzyme sorption were estimated. The ion exchange technique for determination of the isoelectric points of the proteins is described and the isoelectric point of PA is determined. It is proposed to use the method for estimation of close ionization constants of amphoteric an weak electrolites for interpretation of the bell-like pH dependence of kinetic and equilibrium parameters of the enzymatic reaction. The ionization constants of Michaelis complex of PA were evaluated. The activation energy of benzylpenicillin hydrolysis catalized by PA was determined.     

On heterogeneity of DNA methylases from Escherichia coli SK cells

Summary The presence ofE. coli SK cells of five different DNA-methylases differing in specificity to the methylated sequence is documented has been proven. Two enzymes methylate cytosine with the formation of 5′-methylcytosine and three enzymes methylate adenine with formation of 6′-methylaminopurine. A method for simultaneous isolation of the five individual enzymes including gel filtration on Biogel A-0.5 M is proposed. The direct evidence has been presented showing that the additional methylation test in our method modification actually can discriminate between enzymes differing in sensitive sites.     

Some properties of the alpha-hemolysin produced by a hemolytic strain of E. coli]

It was found that alpha-hemolysin of E. coli P 678 HIy+ was maximally active against human erythrocytes at pH 6.5. The hemolytic activity is characterized in time by a distinct lag-phase and a phase of the greatest velocity of the reaction immediately following it. The duration of the lag-phase and also the rate of hemolysis depends on alpha-hemolysin concentration, whose increase is accompanied by a decrease of the lag-phase and acceleration of hemolysis. There is a definite limit below which the duration of the lag-phase remains unchanged with further increase of hemolysin concentration. There was noted a linear relationship between the amount of erythrocytes taken for the test and the rate of hemoglobin release and also a temperature activation of the hemolytic reaction.