[Isolation and analysis of protease-deficient mutants of Bacillus amyloliquefaciens]. / Poluchenie i analiz defektnykh po proteazam mutantov Bacillus amyloliquefaciens.

Four types of protease negative mutants of Bacillus amyloliquefaciens A50 were selected after four stages of step-by-step UV light mutagenesis. EDTA, and PMSF were used as inhibitors of protease activity to characterize the protease negative mutants with regard to the protease type. The electrophoretic patterns of proteases from culture medium of B. amyloliquefaciens protease deficient mutants were studied. The proinsulin stability in the culture medium of different mutants was analysed. Protease deficient B. amyloliquefaciens strain A50-32 may be used as a recipient for cloning and expression of a foreign proteins genes.

[Mutations in the alpha-amylase gene of Bacillus amyloliquefaciens, leading to a decrease in the temperature of protein inactivation]. / Mutatsii v gene alpha-amilazy Bacillus amyloliquefaciens, privodiashchie k snizheniiu temperatury inaktivatsii belka.

Alpha-amylase genes of Bacillus amyloliquefaciens, coding proteins with reduced thermostability, had been obtained as a result of hydroxylamine mutagenesis. Temperature, pH and starch concentration dependences of two mutant alpha-amylases were investigated. The synthesis of the alpha-amylases by several B. subtilis strains with different levels of extracellular proteases was also studied. The mutation containing fragments were localized and the structures of the mutations were determined. It was found that the decrease of thermostability of mutant No 141 was due to Asp to Asn change at the position No 194 of the mature protein, and for mutant No 191--due to Glu to Lys change at the position No 185.

[Cloning of genetic material in Bacillus]. / Klonirovanie geneticheskogo materiala v batsillakh.

Plasmid vectors capable for propagation of Bacillus subtilis DNA fragments containing riboflavin genes were constructed. Cloning of rib operon using pUB110 derivatives was performed in recE4 strain by using sequentional rescue of plasmids containing subfragments of the operon. Also, rib operon was cloned on the vectors containing DNA repeats. It was shown that the presence of direct and inverted repeats within plasmids allows to transform B. subtilis cells by monomers of plasmid DNA. Vectors that contained repeated sequences of DNA and ensured efficient cloning of genetic material in B. subtilis recipient cells were constructed. The use of streptococcal plasmid pSM19035 allowed to obtain vectors which were suitable for cloning large DNA fragments (6 MD and even more) in B. subtilis. A model of B. subtilis transformation by various types of plasmid DNA is presented. The model is in agreement with the general conception of chromosomal DNA transformation.

[Cloning the operon genes of riboflavin biosynthesis in Bacillus subtilis on plasmid vector pBR322 in Escherichia coli]. / Klonirovanie genov operona biosinteza riboflavina Bacillus subtilis na plazmidnyi vektore pBR322 v Escherichia coli.

The operon for riboflavine biosynthesis of Bacillus subtilis wild type and its operator-constitutive derivative have been cloned in Escherichia coli cells on the plasmid pBR322 vector. The plasmids constructed were able to transform strains of E. coli and Bac. subtilis from Rib- to Rib+ phenotype. A DNA insert into the EcoRI site of pBR322 causes a decrease in tetracycline gene expression. The operator of the riboflavine operon of Bac. subtilis does not participate in regulation of the operon expression in E. coli cells.

[Ontogenetic switchover in Bacillus subtilis. II. The dynamics of the stationary phase processes during growth under conditions of catabolite repression]. / Izuchenie ontogeneticheskogo perekliucheniia u Bacillus subtilis. Soobshchenie II.Dinamika protsessov statsionarnoi fazy pri roste v usloviiakh katabolitnoi repressii.

To reveal the pecularities of the growth under the conditions of catabolite repression (medium 2) of Bacillus subtilis and the mutants obtained, the investigations of dynamics of the following processes were carried out: alteration of the pH of the culture exhaustion of glucose in the medium, appearance of the activity of both aconitase in the cells and extracellular metal- and serine proteases in the supernatant, and the appearance of the thermoresistant spores. The following features were observed during the growth under the conditions of catabolite repression: 1. Bacillus subtilis WB 746 and cgs mutants: the death of the main part of the culture after the Iogarithmic phase of growth (LPG), the presence of the secondary LPG of the survived cells which have the increasing activity of aconitase, the appearance and sharp increase in the extracellular serine protease activity 6 hours before thermoresistant spore formation. In the case of cgs mutants the activity of metal proteases appears and increases during the secondary LPG; 2. In the culture of cgl mutants the pH is lowered to 5.1 at the end of the LPG and after the glucose exhaustion the death of almost all the culture follows; 3. cgr mutants: a comparatively high activity of aconitase in the cells is found by the time of the early LPG, and at the end of the LPG the activity of both metal- and serine proteases appear in the supernatant of the culture and the secondary induction of the serine protease activity 6 hours before thermoresistant spore formation is observed. The serine protease activity found in the supernatant before and after the secondary induction of the enzyme belongs to the identical protein. During the stationary phase of the growth of cgr mutants, the high rate of 3H-uridine incorporation into the RNA molecules which have the electrophoretic mobility of mRNA was observed. The sporulation of Bac. subtilis strains under investigation, except cgl mutants, occurs when the culture has reached the definite state: the alkaline pH, the presence of the aconitase activity in the cells and the induced activity of serine protease.