[Thermotoga neopolitina gene cluster, participating in degradation of starch and maltodextrins: expression of aglB and aglA gene in Escherichia coli, properties of recombinant enzymes]. / Klaster genov Thermotoga neapolitana, uchastvuiushchikh v degradatsii krakhmala i mal'todekstrinov: éksptressiia genov aglB i aglA v Escherichia coli, svoistva rekombinantykh fermentov.

The aglB and aglA genes from the starch/maltodextrin utilization gene cluster of Thermotoga neapolitana were subcloned into pQE vectors for expression in Escherichia coli. The recombinant proteins AglB and AglA were purified to homogeneity and characterized. Both enzymes are hyperthermostable, the highest activity was observed at 85 degrees C. AglB is an oligomer of identical 55-kDa subunits capable of aggregation. This protein hydrolyses cyclodextrins and linear maltodextrins to glucose and maltose by liberating glucose from the reducing end of the molecules, and it is a cyclodextrinase with alpha-glucosidase activity. The pseudo-tetrasaccharide acarbose, a potent alpha-amylase and alpha-glucosidase inhibitor, does not inhibit AglB but, on the contrary, acarbose is degraded quantitatively by AglB. Recombinant AglB is activated in the presence of CaCl2, KCl, and EDTA, as well as after heating of the enzyme. AglA is a dimer of two identical 54-kDa subunits, and it hydrolyses the alpha-glycoside bonds of disaccharides and short maltooligosaccharides, acting on the substrate from the non-reducing end of the chain. It is a cofactor-dependent alpha-glucosidase with a wide action range, hydrolysing both oligoglucosides and galactosides with alpha-link. Thereby, the enzyme is not specific with respect to the configuration at the C4 position of its substrate. For the enzyme to be active, the presence of NAD+, DTT, and Mn2+ is required. Enzymes AglB and AglA supplement one another in substrate specificity and ensure complete hydrolysis to glucose for the intermediate products of starch degradation.

[Organization of the genes responsible for colicin Ib synthesis and immunity to it in plasmid ColIb-P9]. / Izuchenie organizatsii genov, otvetstvennykh za sintez kolitsina Ib i immunitet k nemu, v plazmide ColIb-P9.

To study the localization and expression of the ColIb-P9 plasmid genes responsible for colicin Ib synthesis and immunity to it, we isolated a series of Tn5 insertion mutants of recombinant plasmid pIV41 containing the colicin Ib gene in EcoRI fragment of ColIb-P9 (2.7 kb) and the deletion plasmid carrying only a part of the colicin gene. The direction of colicin Ib gene transcription was determined by the analysis of the polypeptides synthesized in minicells carrying the mutant plasmids. The pIV41 plasmid containing cells have been shown to be resistant to both colicin Ib and Ia activities. This type of resistance is usually associated with chromosomal mutations resulting in loss of cell receptors for colicin Ib adsorption. Apparently, the EcoRI fragment of ColIb-P9 studied contains no gene responsible for immunity to colicin. It has been shown that this gene is a portion of SalI fragment (22 kb) of the ColIb-P9, the fragment also carrying the gene which mediates synthesis of colicin Ib.

[Characteristics of hybrid plasmids carrying the genes of colicinogenic plasmid Collb-P9 responsible for colicin Ib synthesis and inhibition of phage T5 development]. / Kharakteristika gibridnykh plazmid, nesushchikh geny kolitsino-gennoi plazmidy Collb-P9, otvetstvenny za sintez kolitsina Ib i podavlenie razvitiia faga T5.

The EcoRI and HindII restriction endonucleases and pBR325 vector plasmid were used to obtain a set of hybrid plasmids containing ColIb-P9 fragments carrying the characters for colicin Ib synthesis and immunity and the ability to inhibit T5 phage growth. The genes responsible for colicin synthesis and immunity are closely linked and localized in the EcoRI fragment with a molecular weight of 1.85 MD (pIV41) or in the HindII fragment of 2.4 MD (pIV1). The clones containing these plasmids show an increased level of both spontaneous and mitomycin C-induced colicin synthesis and an increased level of immunity due to a larger dosage of the genes. The genes controlling T5 growth inhibition are localized in other restriction fragments of ColIb DNA: the EcoRI fragment of 1.45 MD (pIV7) and the HindII fragment of 4.3 MD (pIV5). We have demonstrated by means of hybrid plasmids that T5 growth inhibition is not connected with the colicin Ib synthesized in infected cells and is controlled by other specific product(s) of the ColIb plasmid genes. T5 phage growth was as efficient in clones containing plasmids with cloned colicin Ib genes as in a strain without plasmids. An investigation of the expression of the genes inhibiting T5 phage growth in an in vitro protein synthesis system has revealed a protein with a molecular weight of 36 000 which seems to take part in the process.

[Episome stability in Escherichia coli K-12]. / Izuchenie stabil'nosti épisom v shtammakh Escherichia coli K-12.

Arg+ and trp+ carrying episomes which have markedly different spontaneous efficiency of elimination, were obtained in experiments with AB673 and KL99 Escherichia coli donor strains. The level of episome maintenance is the result of mutation (deletion, insertion) that occurred in episomes during their formation. Differences in the efficiency of spontaneous elimination are independent of the origin of the donor strain used. The rate of loss of stably maintained episomes is not enhanced significantly by exposure of cells to AO. According to molecular characteristics, the episome demonstrating stable maintenance has a lower molecular mass than that of unstable maintenance.

[Replication characteristics of colicinogenic plasmid ColK in Escherichia coli cells]. / Osobennosti replikatsii kolitsinogennoi plazmidy ColK v kletkakh Escherichia coli.

A fraction of plasmid DNA from K and X colicins producing Escherichia coli K235 cells was studied. Cells of this strain are shown to contain four types of plasmids with molecular weights of 21.6.10(6), 38.8.10(6) daltons. Transformation of E. coli C600 by a total plasmid DNA yielded clones containing a single plasmid - colicinogenic K factor (ColK, a mol wt 4.4.10(6)). ColK DNA is present in cells in a large number of copies, replicates in the presence of chloramphenicol, requires DNA polymerase I. Two fragments with mol wts 3.4 and 1.1.10(6) are formed when ColK DNA is treated with EcoRI enzyme. After circularization using phage T4 DNA ligase, the 3.4.10(6) fragment was capable of autonomous replication and stable maintenance in E. coli cells, replicated in the presence of chloramphenicol and though unable to synthesize colicin, confered upon cells resistance to colicin K. The mode of ColK DNA replication is studied in mutants temperature-sensitive for the replication of chromosomal DNA. ColK DNA replication is shown to be virtually independent of the dnaA gene product and only slightly dependent on that of the dnaC gene. No replication occurs in the dnaB, dnaF and dnaG mutants at non-permissive temperature.

[Effect of colicinogenic plasmids on mutagenesis and the induction of colicin E1 synthesis]. / Vliianie kolitsinogennykh plazmid na mutagenez i induktsiiu sinteza kolitsina E1.

The frequency of UV-induced arg+ and his+ reversions is found to be enhanced by the presence of colicinogenic plasmids Ib-P9 and Ia-CA53 and reduced by the presence of plasmid V-K30. The effect of Ib-P9 on the UV-induced mutagenesis depended on recA and lexA genes and was independent on uvrA and recB genes. Plasmids Ib-P9 and V-K30 proved to exert no influence on the frequency of 2-aminopurine-induced mutations. The presence of Ib-P9 reduced the ability of cells carrying the colicinogenic factor E1 to induce colicin E1 synthesis under the action of DNA-damaged agents (UV, nitrosoguanidine, mitomycin C), while plasmid V-K30 enhanced the induction of colicin E1 synthesis. However this effect of plasmids on the induction of colicin E1 synthesis was not observed when the induction occurred under the action of chloramphenicol. Possible mechanisms governing the participation of plasmid gene products in processes related to the repair of DNA are discussed.

[Protective action of colicinogenic factor Ib-P9 on Escherichia coli cells defective in known repair functions after ultraviolet irradiation]. / Zashchitnoe deistvie kolitsinogennogo faktora Ib-P9 na kletki Escherichia coli, defektnye po izvestnym reparativnym funktsiiam, posle ul'trafioletovogo oblucheniia.

The presence of the plasmid colicinogenic factor Ib-P9 in Escherichia coli wild type cells is shown to increase bacterial survival after UV irradiation and the action of N-methyl-N'-nitro-N-nitrosoguanidine. The ability of the plasmid to cause the UV protection is observed in uvrA, uvrB, uvrC, polA, recB, recF E. coli strains, but the plasmid does not restore the UV resistance of the mutant cells to the wild type level. The protective effect of the plasmid CoI Ib-P9 depends on the recA+lexA+ genotype of the cells. The inhibition of protein synthesis (amino acid starvation) before and after UV irradiation does not prevent the UV protection by ColIb-P9. The nature of the plasmid-associated repair functions is discussed.

[Induction of colicin E1 synthesis in Escherichia coli cells with different concentrations of plasmid DNA]. / Induktsiia sinteza kolitsina E1 v kletkakh Escherichia coli s razlichnym soderzhaniem plazmidnoi DNK.

RRelationship between the level of induction of colicin E1 synthesis and the content of plasmid DNA in Escherichia coli K12S (E1) cells was studied. The number of Col E1 DNA copies per cell was found to decrease with growing generation time for different growth media and to increase continuously for the time of synchronous cell division cycle. No apparent relation of the level of spontaneous induction and of induction caused by N-methyl-N'-nitro-N-nitrosoguanidine to the number of Col E1 DNA copies per cell was observed. It is inferred that the content of plasmid DNA in the cells is not among the main factors determining the level of induction of colicin synthesis.