[Cloning and expression of the gene for thermostable pullulanase from Clostridium thermohydrosulfuricum in Escherichia coli]. / Klonirovanie i ékspressiia gena termostabil'noi pullulanazy Clostridium thermohydrosulfuricum v kletkakh Escherichia coli.

Using a pUC19-based genomic library of the anaerobic thermophilic bacterium C. thermohydrosulfuricum a DNA fragment that confers pullulanase activity to E. coli cells has been identified. Subcloning and restriction mapping procedures was carried out and the primary structure of the 5'-region of the pullulanase gene (pul) was determined. The pul enzyme was shown to be a protein with molecular weight of approximately 60,000. It was found that both pullulanase and glucoamylase activities resides in pullulanase. The intracellular distribution of pullulanase was studied. An E. coli strain that produces large amounts of thermostable pullulanase has been constructed.

[Isolation and characteristics of thermostable pullulanase from Clostridium thermohydrosulfuricum produced by Escherichia coli cells]. / Vydelenie i kharakteristika termostabil'noi pullulanazy Clostridium thermohydrosulfuricum, produtsiruemoi kletkami Escherichia coli.

The expressed gene (pul) for a thermostable pullulanase from Clostridium thermohydrosulfuricum was cloned into Escherichia coli. The enzyme was purified from cell extracts of E. coli by thermoinactivation, ammonium sulphate precipitation and gel exclusion. The purified enzyme was characterized as monomer with both pullulanase and glucoamylase activities. The general physico-chemical and catalytic properties of this enzyme were obtained. In particular, pullulanase and glucoamylase activities were stable and optimally active at 65 degrees C. The pH optimum for activity was 5.8. The amino acid composition and amino acid sequence of N-terminal end were estimated.

Cloning of Clostridium thermocellum endoglucanase genes in Escherichia coli.

Six independent and distinct cel genes coding endoglucanases have been selected from C. thermocellum pUC19-based gene bank in E. coli TG1. E. coli-derived Cel-proteins possessing Mr from 39,000 to 61,000 are able to cleave lichenan, as well as xylan and carboxymethyl cellulose. Cel 7- and Cel 8-endoglucanases are characterized by cellobiohydrolase type substrate specificity, being able to cleave model fluorogenic aryldisaccharide substrate MU-G2. The clone pCU110 (cel 7) produces about 10-fold more endoglucanase activity than other clones.

[Cloning the pyruvate decarboxylase gene of Zymomonas mobilis and its expression in Escherichia coli]. / Klonirovanie gena piruvatdekarboksilazy Zymomonas mobilis i ego ékspressiia v Escherichia coli.

The pyruvate decarboxylase gene of Zymomonas mobilis CP4 has been cloned in Escherichia coli strain TG1 cells on the pUC18 vector plasmid. Activity of the enzyme in the lysates of the obtained clones is about 30 units per 1 mg of protein. Neither the dependence of the pyruvate decarboxylase activity on the presence of IPTG or glucose in the cultivation medium nor the difference in activity of the enzyme for the clones harbouring the recombinant plasmids with the different orientation of the pyruvate decarboxylase gene to lac-promoter have been demonstrated in the presented work. These facts imply the transcription of pyruvate decarboxylase gene from its own promoter.

[A new type of Clostridium thermocellum endoglucanase produced by the recombinant strain of E. coli. Some properties and identification in donor cells]. / Novyi tip éndogliukanazy Clostridium thermocellum, obrazuemoi rekombinantnym shtammom E. coli. Svoistva i identifikatsiia v organizme donora.

The properties of endoglucanase produced by the recombinant strain of E. coli carrying plasmid pCU 104 with a 2.9 kb insert of chromosomal DNA of C. thermocellum encoding the multiple forms of the 35.5 kD polypeptide (pI 4.3-4.7) were studied. The enzyme has a broad pH optimum of activity (6.0-7.5). The half-inactivation time for different forms of the enzyme at 65 degrees C is similar and is equal to 25-30 minutes. The enzyme is related to endoglucanases weakly adsorbed on cellulose (Kp = 0.065 1/g). Hydrolysis of microcrystalline cellulose is completed within 7 days (7-9%) and is accompanied by the formation of cellobiose and cellotriose. The enzyme splits dyed lichenan (mixed 1,3-1,4-beta-glucane) at a higher rate than the dyed CM-cellulose. A guinea pig antiserum to enzyme isoforms with a pI of 4.46-4.54 was obtained. Using direct solid phase immunoenzymatic analysis, it was demonstrated that all the enzyme isoforms under study (pI 4.3-4.7) are immunologically related (serum titers for different enzyme isoforms vary from 1:20,000 to 1:50,000). In the original culture fluid of C. thermocellum, the antigen related to the enzyme isolated from the recombinant strain was unobserved. However, SDS-PAAG electrophoresis of SDS- and mercaptoethanol-treated culture fluids revealed among 11 protein bands at least 4 antigens interacting with antibodies (Mr = 107, 76, 67 and 37 kD), although their antibody titers were far lower and did not exceed 1:300-1:500. The cumulative data suggest that the endoglucanase under study is not identical to the earlier described enzymes encoded by the cel A- and ceI B-genes of C. thermocellum.

[Genetic analysis of Escherichia coli min81 mutation blocking the development of bacteriophage Mu]. / Geneticheskoe izuchenie mutatsii min81 Escherichia coli, blokiruiushchei razvitie bakteriofaga Mu.

Data characterizing mim81 mutation obtained by the method for direct selection of transposition mutations are presented. The development of Mu is shown to be dramatically suppressed in the mutant strain both upon infection and after induction from the lysogenic state. Frequencies of lysogenization and mini-Mu-dependent formation of cointegrates in the mutant strain are comparable with those in the wild-type strain. Mu development prohibition is removed if expression of early Mu gene is provided from the modified Pe promoter. The results obtained make us believe that the mechanism of mim81 mutation action involves reduction of early gene expression to the level that is sufficient for Mu DNA integration into the chromosome during infection and for single replicative events, but insufficient for vegetative development of bacteriophage Mu.

[Development of bacteriophage Mu in E. coli gyrBts mutant strain]. / Razvitie bakteriofaga Mu v kletkakh mutantnogo shtamma E. coli gyrBts.

The study deals with Mu growth in cells carrying a temperature-sensitive mutation in the gene of DNA gyrase B subunit. At a nonpermissive temperature the Mu growth is shown to be blocked in the host gyrB ts mutant both on infection and on prophage induction. Mu DNA does not get integrated in the host chromosome upon the infection of mutant cells, as demonstrated by DNA-DNA hybridization experiments. In the case of prophage induction in mutant cells, as opposed to the wild type cells early mRNA synthesis is practically fully inhibited while the total RNA synthesis is three times reduced after 20 min of induction. The transcription of phage DNA associated with the changed superhelicity of DNA in the cell.

[Transposition immunity in bacteriophage Mu. The effect of a mutation at the kil gene on the establishment of immunity]. / Immunnost' transpozitsii bakteriofaga Mu. Vliianie mutatsii po genu kil na ustanovlenie immunnosti.

Bacteriophage Mu is characterized by a phenomenon similar to the transposition immunity of TnA: the frequency of transposition of Mu or mini-Mu into plasmids containing certain phage sequences is reduced by two orders of magnitude. In order to lend transposition immunity to Mu, the recipient replicon must contain a sequence of phage DNA including a 5.1 kb early region from the c-end of Mu. The product of the kil (or cim) gene takes part in establishing the immunity. The transposition immunity of Mu is connected with the disturbance of cointegrate formation.

[Construction and characteristics of mini-Mu phages]. / Konstruirovanie i kharakteristika mini-Mu-fagov.

The paper reports on the principles of construction, physical characterization and results of preliminary genetic investigation of hybrid plasmids containing Mu DNA sequences or deletion derivatives of phage Mu, the so-called mini-Mu phages. The mini-Mu were obtained by joining both phage ends within one plasmid in a regular orientation. A collection obtained by in vitro manipulations included 14 recombinant plasmids containing different DNA fragments of the Mu genome. Seven plasmids have both ends of phage Mu, three plasmids containing regularly oriented ends, i.e. mini-phages of different size: the mini-Mu5 (11 kb) within pRM8 plasmid, the mini-Mu4 Ap (18 kb) within pRM6 and the mini-mini-Mu (4.4 kb) within pRM5. The collection comprises mini-Mu phages with the gene kil inactivated after treatment with hydroxylamine. Biological properties of the hybrid plasmids have been preliminary studied.

[Integration of the mini-Mu phage into multicopy plasmids]. / Integratsiia mini-Mu-faga v mul'tikopiinye plazmidy.

Preparation of mini-Mu4 phage introduced into the pRP1.2 plasmid and carrying a 18 kb deletion of the central EcoRI fragment is described. The ability of the mini-Mu4 for independent transposition is demonstrated in experiments conducted for cointegrate formation (replicon fusion). The system developed is applicable to studying the mechanism of transposition, since the frequency of cointegrate formation may be descriptive of the transposition process. Frequency changes due to some factors may point to a possible influence of these factors on the mini-Mu transcription. The described mini-Mu integration into small multicopy plasmids of Col type makes it possible to conduct a simple physical analysis of structures formed in the course of transposition.