[Use of catabolic repression in the selection of the glucoamylase-producer Aspergillus niger]. / Ispol'zovanie katabolitnoi rerpessii v selektsii produtsenta gliukoamilazy Aspergillus niger.

The effect of various carbon sources and cAMP on the glucoamylase synthesis in Aspergillus niger was studied to find carbon sources repressed the enzyme synthesis and conditions for the selection of catabolite stable mutants. Maltose at a concentration of 0.5% stimulated the glucoamylase synthesis, but at a concentration of 4% it repressed not only the enzyme synthesis but the growth of the parental strain on the agar medium. The more active mutant 66 was obtained as a result of treatment of Asp. niger st 6 with NG. This mutant is able to grow on the Czapek's medium containing maltose at concentrations 4 or 6%. The mutant 66 produced about 2.9 times more glucoamylase than its parent when maltose was added at 0.5% concentration to the medium. The glucoamylase synthesis in the parental strain was completely repressed under repressing conditions, while the level of the mutant strain activity was 35% from the level of enzyme activity on the medium without the repressor. The addition of cAMP (5.10(-5] resulted in a partial release of maltose (4%) repression of the glucoamylase synthesis in both strains. The results obtained indicate a possibility to select Asp niger mutants with the partially derepressed glucoamylase synthesis. Other regulation mechanisms in addition to catabolite repression may be involved in the regulation of the glucoamylase synthesis.

[Incorporation of adenine derivatives into the cells of Brevibacterium ammoniagenes ganA mutants producers of inosinic acid]. / Vkliuchenie proizvodnykh adenina v kletki mutantov Brevibacterium ammoniagenes ganA--produtsentov inozinovoi kisloty.

The ganA mutants (growth ability with nucleotides), overproducers of inosinic acid, were isolated with the aid of specially developed techniques aimed at obtaining mutants with the increased ability of assimilating adenine derivatives. The selection techniques used, as well as certain properties of ganA mutants distinguishing them from their parent strains auxotropic for adenine, led to suggesting that ganA mutation affects cell permeability for exogenous adenine derivatives. The present paper reports on a comparative study of two ganA mutants and their parent strains regarding their ability to grow on a minimal medium supplemented with various concentrations of adenine, adenosine on adenylic acid. The mutants grew better than the initial strains at concentrations of adenine or adenosine ranging from 0.005 to 0.05 mM and at a concentration of adenylic acid equal to 0.5 mM or higher. The enhanced ability of ganA mutants of assimilate exogenous adenine sources was found to correlate with an increased rate of 14C-adenine and 3H-adenosine uptake by cells. The results indicate that the ganA mutation leads to an increased permeability of adenine-dependent Brevibacterium ammoniagenes cells for adenine derivatives and suggest a participation of this gene in the formation of the cell envelope.

[Immunochemical study of subtilisins obtained from Bacillus subtilis A-50 and some of its mutants]. / Immunokhimicheskoe izuchenie subtilizinov, produtsiruemykh shtammom Bacillus subtilis A-50 i nekotorymi mutantami.

Physico-chemical parameters of subtilisins from the original Bacillus subtilis A-50 strain (proteolytic activity, electrophoretic mobility, molecular weight, reactions with specific inhibitors) were similar to those mentioned in the literature for the enzymes of other strains. Immunological experiment has shown, that Bacillus subtilis A-50 subtilisins with various electrophoretic mobility do not differ in their antigenic properties. Enzymes with high electrophoretic mobility from mutant strains were similar to I--III subtilisin fractions from Bac. subtilis A-50 in the antigenic characteristics. However, the antigenic heterogeneity was observed in I, II and III enzyme fractions of some mutant strains. Subtilisins studied appear to form the isoenzyme system.

[Effect of medium components on the metabolism of Brevibacterium ammoniagenes-producer of 5'-inosinic acid]. / Vliianie komponentov sredy na metabolizm kul'tury Brevibacterium ammoniagenes-produtsenta 5'-inozinovoi kisloty.

The effect of different carbon and nitrogen sources on the biosynthesis of 5'-inosinic acid (5'-IMP) by the culture Brevibacterium ammoniagenes 225-5 was studied. With respect to the yield of 5'-IMP, glucose was found to be the best carbon source and urea--the best nitrogen source. The proper ratio of concentrations of carbon and nitrogen sources in the nutrient medium was very important for a high 5'-IMP accumulation. The study of the effect of phosphate and magnesium concentrations on 5'-IMP biosynthesis demonstrated that their high concentrations were needed for intensive 5'-IMP salvage-synthesis from hypoxanthine and that they did not influence the de novo synthesis of hypoxanthine.

[Nature of the mutations that determine the ability of Bacillus subtilis A-50 to sporulate at high glucose concentrations in the medium]. / Priroda mutatsii, opredeliaiushchikh sposobnost' Bacillus subtilis A-50 sporulirovat' pri vysokikh kontsentratsiiakh gliukozy v srede.

The ability of Bacillus subtilis A-50 to sporulate in the medium containing high glucose concentrations is caused by at least two mutation types: pts mutations and cat (or tgl) mutations, both of them affecting differently the level of alkaline proteinase synthesis. The decrease of the level of enzyme activity in the case of pts mutation (gluR3 mutant) occurs at the expense of glucose transport disturbance. The mutation cat (tgl) (mutant gluR5) causes the increase in enzyme synthesis at the expense of catabolic resistance to glucose of genes controlling alkaline proteinase synthesis and the spore formation in Bac. subtilis A-50. cat5(gluR5) and pts3(gluR3) mutations are located on the chromosome of Bac. subtilis in the region metD and argC respectively. The over-synthesis of alkaline proteinase characteristic of Bac. subtilis A-50 is controlled by the polygenic system, as the level of alkaline proteinase synthesis in argA+ transformants makes up 25% of the level of activity of the original strain. The productivity of Bac. subtilis A-50 can be enhanced by introducing an additional cat mutation.

[Effects of mutations in Bacillus subtilis genome decreasing the protease activity on the formation of subtilisin molecular forms]. / Vliianie mutatsii Bacillus subtilis, snizhaiushchikh proteoliticheskuiu aktivnost; na obrazovanie mnozhestvennykh molekuliarnykh form subtilizina.

Multiple molecular forms of subtilisin--extracellular serine protease produced by the wild strain Bac. subtilis A-50 and its mutant strains with the protease activity decreased two-fold and more were studied. Six molecular forms of subtilisin were found on the whole when 33 mutant strains have been investigated under the experimental conditions. It is essential that both the wild and each of mutant strains under study produced not more than three out of these six forms. Three molecular forms of subtilisin from the mutant strains are similar to those found in the wild strain A-50, and have the molecular weight, of 27 000-30 000. Three other forms of subtilisin were revealed only in the mutant strains, and had the molecular weight of about 20 000. Apparently there is only one structural gene for subtilisin in Bac. subtilis genome. The appearence of multiple molecular forms of subtilisin may be due to the post-translational modifications (limited proteolysis) of the initial type of enzyme, i.e. pre-subtilisin. Probably, that certain mulations not affecting the structural gene can significantly change the expression of such gene by varying of the degree of product modifications.

[Multiple forms of Bacillus subtilis subtilisin and effects of mutations on the distribution of its molecular forms]. / Mnozhestvennost' subtilizinov Bacillus subtilis i vliianie mutatsii na raspredelenie molekuliarnykh form

Using polyacrylamide-gel electrophoresis, isoelectric focusing and gel-filtration it was demonstrated that the auxotrophic mutant strains of Bac. subtilis A-50 and their prototrophic revertant strains produce multiple molecular forms of subtilisin. Three of them are the same as the corresponding molecular forms of subtilisin from the wild strain A-50. In different mutant strains the relative amounts of the main three forms varies considerably resulting in the absence of certain forms in several strains. There is the additional minor form of subtilisin possessing high electrophoretic mobility in four prototrophic revertant strains and one Arg--auxotrophic strain of Bac. subtilis A-50. It would be reasonable to suppose that different molecular forms of subtilisin derive from the product of its single structural gene as a result of post-translational modifications (limited proteolysis). This enzyme and probably most, if not all secretory proteins may be synthesised as larger precursors and then specifically modified in the bacterial cell membranes. Thus, certain mutations, without affecting the structural gene of this secretory protein -- subtilisin -- have pronounced effects on this structural gene expression, varying the degree of its product modification and the amount of resulting secretory molecular forms of subtilisin.

[Temperature-dependent mutants of Bacillus subtilis A-50 with a diminished level of alkaline proteinase synthesis]. / Temperaturochuvstvitel'nye mutanty Bacillus subtilis A-50 so snizhennym urovnem sinteza shchelochnoi proteinazy

Temperature-sensitive proteolytically inactive mutants of Bacillus subtilis A-50 are obtained. The optimal ability to synthetise the enzyme both in the original strain and in mutants is expressed at 37 degrees C. The level of proteolytic activity of selected prot(ts) mutants depends both on the temperature of growth and on the temperature at which the activity of subtilisin is analysed. The optima of functioning ts-proteinases are shifted in mutants as compared with the original strain. The ability to form spores at 37 degrees C and 45 degrees C are considerably reduced as compared with this process at 28 degrees C. The results of disc electrophoresis in polyacrylamide gel indicate the presence of structural alteration of proteinases in the mutants obtained.

[Mutants of Bacillus subtilis--producer of alkaline proteinase, sporulating in the presence of high concentrations of glucose]. / Mutanty Bacillus subtilis--produtsenta shchelochnoi proteinazy, sporuliruiushchie v prisutstvii vysokikh kontsentratsii gliukozy

Glur mutants of Bacillus subtilis A-50 capable of sporulating in the medium containing 5% glucose are isolated. The mutants are divided into three groups. The first group comprises glur mutants whose level of proteolytic activity is lower than that of the initial strain A-50. The productivity of mutants of the 2nd group is equal to the level of alkaline proteinase synthesis characteristics of the original strain. In mutants of the third group the level of alkaline protease synthesis exceeds the productivity of the initial strain. There was no correlation between the efficiency of spore formation in the mutants obtained and the level of alkaline protease activity. The mutants of different groups differ in their ability to grow on different sugars.

[Preparation of Bacillus subtilis A-50 mutants by exposure to nitrosoguanidine and ICR-191]. / Poluchenie mutantov Bacillus subtilis A-50 pod deistviem nitrozoguanidina i JCR-191

Spontaneous and NG- and ICR-191-induced variability of Bacillus subtilis A-50 with respect to the production of alkaline proteinase (subtilisine) is studied. It is found that ICR-191 induces greater variability as compared with NG under conditions of a low survival. Six mutants synthesizing proteinases showing a changed ability to form enzymes were obtained under the action of the same mutagens.

[Regulation of alkaline protease synthesis in Bacillus subtilis A-50]. / K voprosu o reguliatsii sinteza shchelochnoi proteazy u Bacillus subtilis A-50

The effect of glucose and amino acids on the synthesis of alkaline protease of the prototroph strain and auxotrophs of Bacillus subtilis A-50 was studied. There are both general and different from one another mechanisms of regulation for sporulation and protease synthesis as one of early stages of sporulation. Most auxotroph mutations pleiotropically decrease the level of protease synthesis and the efficiency of sporulation. At the same time the differences depending on sporulation and protease formation from glucose, amino acids and caseinoacids are observed. The increase in glucose concentration in the medium from 0.5 to 5% leads to intensification of protease synthesis and decrease of the sporulation efficiency. The suppression of alkaline protease formation in B. subtilis occurs when amino acid concentration is of the order of 1.5 mg/ml in the studied combination in the presence of 1--5% glucose. The strongest inhibitory effect was discovered for 2d, 3d and 4th amino acid groups in the presence of 5% glucose, whereas amino acids of the first group stimulate the protease synthesis in the presence of 2% glucose. The stimulatory effect of amino acids of the first groups is due to histidine and arginine.