[The isolation and characteristics of Yersinia pestis membranes]. / Vydelenie i kharakteristika membran Yersinia pestis.

A method for fractionation of membrane structures of Yersinia pestis is developed. It involves the following basic stages: the cultivation of bacteria in a liquid nutrient medium, mechanical destruction from the solid state in the X-press or ultrasound treatment of the suspension, subsequent two-stage centrifugation in the step (70-15%) and linear (70-45%) gradients of the sucrose density, collection of fractions and their storage. The method makes it possible to separate rapidly and efficiently the outer and cytoplasmic membranes which preserve biochemical and morphological integrity. This is confirmed by the distribution pattern of marker enzyme activities, by the electron microscopic control as well as by other modern sediment tests. High heterogeneity of the polypeptide composition of the isolated membrane preparations has been shown by electrophoresis in PAAG in the presence of sodium dodecyl sulphate as well as definite sensitivity of certain protein subunits to variations of the temperature (28 or 37 degrees C) during cultivation of a plague agent.

[Isolation and purification of the chloramphenicol-acetyltransferase from Y. pestis EV cells with extrachromosomal resistance to the antibiotic by affinity chromatography]. / Vydelenie i ochistka khloramfenikol-atsetiltransferazy iz kletok Y. pestis EV s vnekhromosomnoi rezistentnost'iu k antibiotiku metodom affinnoi khromatografii.

Purification of chloramphenicol acetyltransferase isolated from the cells of Y. pestis EV-R with extrachromosomal resistance to chloramphenicol included ultrafiltration on the microporous membranes Diaflo-XM 300 and biospecific chromatography on columns with cepharose 4B covalently bound with the reduced chloramphenicol. Elution of chloramphenicol acetyltransferase was achieved by means of addition of native chloramphenicol to the buffer solution. Such native chloramphenicol had higher affinity to the enzyme than the reduced antibiotic. As a result the enzyme preparations with 250 times higher purity levels and 70 per cent of the initial activity were obtained. The disc electrophoresis of the sample in polyacrylamide gel produced a single protein zone capable of chloramphenicol acetylation in the presence of Ac approximately CoA.

[Pathways of enzymatic inactivation of levomycetin in El Tor vibrios with plasmid and chromosome resistance to the antibiotic]. / Nekotorye puti énzimaticheskoi inactivatsii levomitsetina u vibrionov El-Tor splasmidnoi i khromosomnoi rezistentnost'iu k antibiotiku.

Two possible mechanisms of enzymatic inactivation of levomycetin, i.e. acetylation of OH-groups and reduction of the n-nitrophenylic component by the cells and cell-free extracts of V. eltor 2044 with the plasmid or chromosome types of antibiotic resistance were studied in vitro. The vibrio containing the extrachromosome determinants were resistant to a number of antibiotics. The rate of levomycetin acetylation by them under conditions of intensive aeration and reduction of the antibiotic aromatic nitrogroup in the absence of oxygen was high. The cells with the chromosome resistance had a trace activity of levomycetin acetyltransferase. Still, they rather rapidly reduced levomycetin into its aminoderivative (during 2-hour incubation in the atmosphere of nitrogen 70-80% of the substrate are transformed into its summary arylamine). The antibiotic sensitive vibrio practically had no capacity for acetylation of levomycetin but could transform it into the reduced aminoderivative though to a less extent than the antibiotic resistant cells.

[Enzymatic inactivation of levomycetin and penicillin by cells of the plague and pseudotuberculosis microbes that contain R plasmid depending on cultivation conditions]. / Enzimaticheskaia inaktivatsiia levomitsetina i penitsillina kletkami chumnogo i psevdotuberkuleznogo mikrobov, soderzhashchimi R-plazmidu, v zavisimosti ot uslovii kul'tivirovaniia.

The activity of enzymes, inactivating levomycetin and penicillin in the cells of plague and pseudotuberculosis microbes bearing extrachromosomal determinants resistant to a number of antibiotics was studied as dependent on some cultivation parameters: population age, aeration rate and temperature. It was shown that the highest capacity for levomycetin acetylation was characteristic of the cells in the late logarithmic and early stationary growth phages. Accumulation of levomycetin O-acetothers in the incubation medium markedly increased, when the cells were grown under the conditions of intensive aeration. An increase in the cultivation temperature up to 37 degrees C was accompanied by a reliable decrease in the activity of levomycetin acetylase in the transconjugant plague and pseudotuberculosis microbes though no correlation with the resistance levels in the same strains to the above antibiotics was observed. Optimal conditions for penicillinase production were determined. The maximum levels of penicillinase were found in the cells of Y. pestis 556/106 Rn with the episotic resistance type in the early exponential developmental phase under the aeration conditions and the temperature of 28 degrees C.

[Resistance to levomycetin and activity of several enzymes in Escherichia coli and the agent of plague]. / Rezistentnost' k levomitsetinu i aktivnost' nekotorykh fementov u vozbuditaelia chumy i kishechnoi palochki

The authors compared the activity of acetyl-CoA-synthetase and of the enzymes belonging to the group of asparaginic acid in levomycetin sensitive and resistant strains of Y. pestis and E. coli. There were revealed marked differences in the activity of aspartase, fumarase, synthetase and desamidase of L-asparagin, and also of the enzyme activated by acetate in the E. coli strains with plasmide resistance. Transmission of R-factor to the pestis was accompanied by decomposition of L-asparadein, formation of AC-CoA. At the same time transformation of L-asparaginic acid catalyzed by aspartase remained on the same low level in the sensitive pestis cultures and their variants with the R-factor. When the resistance was controlled by chromosomal resistance markers, the activity of the enzymes providing formation of L-asparagic acid, its amide and L-malic acid showed no significant change. In chromosomal type of resistance in the mutants of pestis and E. coli the acetyl-CoA-synthetase reaction was as a rule somewhat increased.

[Study of the mechanisms of levomycetin inactivation by the palgue causative agent and Escherichia coli with episomal and chromosomal resistance. The enzymatic acetylation of levomycetin]. / Izuchenie mekhanizmov inaktivatsii levomitsetina vozbuditelem chumy i kishechnoi palochkoi pri épisomnoi i khromosomnoi rezistentnosti. Enzimaticheskoe atsetilirovanie levomitsetina

One of the mechanisms of levomycetin inactivation, i.e. enzymatic acetylation of the plague causative agent and Coli bacteria with chromosomic and episomic type of resistance was studied. It was shown that resistance to levomycetin in the recombinants of the plague microbe and Coli bacteria stipulated by one and the same R-factor was associated with their capacity for the antibiotic inactivation mainly with the help of levomycetinacetyltransferase. At the same time the enzyme activity in the mutants was very low and in the cells of Y. pestis and E. coli sensitive to the antibiotic it was absent. Levomycetinacetyltransferase is to somee extent soluble, still a singificant amount of it is connected with the structures of the bacterial cells and in particular with the cytoplasmic membranes. Comparison of the activity of levomycetinacetyltransferase as dependent on the incubation time, substrate concentration, pH and thermal treatment provided determination of quantitative differences in the properties of the enzyme of Y. petis and E. coli with episomic multiple drug resistance. However, under the experimental conditions transfer of R-factor from one host to the other induced no qualitative changes in the mechanism of levomycetin inactivation.