[Nitrogen assimilation enzymes in Bacillus subtilis mutants with hyperproduction of riboflavin].

Three groups of the nitrogen assimilation cycle enzymes (glutamate synthases (GTS), glutamine synthases (GS), and glutamate dehydrogenases (GD)) were studied in Bacillus subtilis strains with hyperproduction of riboflavin (vitamin B2). It was found that in all strains tested activity of GS was virtually the same, activity of GD was absent, and activity of GTS was reduced. In strains 41 and 24, riboflavin producers, activity of GTS was 30-60% the enzyme activity in the original strain (wild-type RosR). The most pronounced decrease in the activity of GTS (0-12% relative to RosR) was observed in the strain AS5, which had the highest level of biosynthetic activity relative to the other strains. According to the results of determination of the sensitivity of induction of beta-xylosidase to glucose- and fructose-induced catabolic repression, none of the strains studied was characterized by disorders in the protein CcpA, a global regulator of the catabolic repression in gram-positive bacteria, which is required for reducing amination and resulting activation of biosynthesis of glutamic acid in cell. It was suggested that mutations responsible for partial or complete inhibition of GTS biosynthesis caused an increase in the intracellular pool of glutamine. The intracellular pool of glutamine is a nitrogen source for riboflavin in cell. It follows from the results of this work that there is a trend toward an increase in the rate of biosynthesis of vitamin B2 in mutants with inhibited GTS activity. However, the complexity of the processes of regulation of nitrogen assimilation enzymes makes it difficult to find a distinct correlation between GTS activity and riboflavin biosynthesis in these strains.

[Transketolase mutation in riboflavin-synthesizing strains of Bacillus subtilis]. / Mutatsiia po transketolaze u riboflavinsinteziruiushchikh shtammov Bacillus subtilis]

Unlike its predecessors B. subtilis rosR and 41, riboflavin producing B. subtilis 24 strain does not utilize pentose and gluconate and poorly assimilates glucose. Simultaneous addition of glutamic and shikimic acid restored its capacity to grow and produce riboflavin in medium with pentose and gluconate. This strain lacks the activity of transketolase, the key enzyme of the pentose phosphate cycle, and possesses normal ribulose-5-phosphate-epimerase and glucose phosphate isomerase activities. Like enterobacteria, B. subtilis has two different transport systems for glucose and mannose. The data are discussed from the viewpoint of increasing riboflavin production by transketolase mutants. Probable consequences of cell wall and cytoplasmatic membrane damage in B. subtilis with this mutation are discussed.

[Fusion of Bacillus subtilis and Bacillus licheniformis protoplasts. The mapping of the mutations leading to the supersynthesis of riboflavin in interspecies hybrids]. / Sliianie protoplastov Bacillus subtilis i Bacillus licheniformis. Kartirovanie mutatsii, privodiashchikh k sverkhsintezu riboflavina u mezhvidovykh gibridov.

As a result of fusion of the protoplasts of B. subtilis and B. licheniformis the majority of the prototrophic hybrids, as well as the auxotrophs with the Thi-Hom-phenotype or the Thi-phenotype acquired capacity for over-production of riboflavin lacking in the initial parent strains. When grown on the minimal Spizizen medium with aeration at 37 degrees C the auxotrophic recombinants accumulated 10-60 micrograms/ml of riboflavin for 2 days, while the prototrophic recombinants accumulated up to 90 micrograms/ml of riboflavin. The respective figures for their cultivation in the sucrose fermentation medium were 100-330 and 600 micrograms/ml. For mapping 3 random variants of them with different phenotypes, i.e. SL-7 (prototroph), SL-15 Thi-and SL-52 Thi-Hom-were used. Localization of the mutation on the chromosome of B. subtilis was based on transformation experiments with three marker crossing, where hybrid strains SL-7, S1-15 and SL-52 were used as the DNA donor and strain lys-rib-of B. subtilis was used as the recipient. The analysis showed that the required mutation designated as R1 was localized on the chromosome of B. subtilis in the regulatory region rib0 of the riboflavin operon.

[Bacillus subtilis mutants resistant to roseoflavin]. / Mutanty Bacillus subtilis, ustoichivye k rozeoflavinu.

Bacillus subtilis mutants resistant to 100 mkg/ml of roseoflavin/8-dimethylamino (nor) riboflavin/have been shown to excrete 0.5 to 20 mkg/ml riboflavin and small amounts of FMN and FAD into the culture medium. The rosR mutations are localized in the operator region of riboflavin operon. The combination of rosR and ribC mutations (the latter being mutation in the regulator gene) leads to hyperproduction of riboflavin.

[Analgos of riboflavin, lumiflavin and alloxazine derivatives. II. Effect of roseoflavin on 6,7-dimethyl-8-ribityllumazine and riboflavin synthetase synthesis and growth of Bacillus subtilis]. / Analogi riboflavina, liumiflavina i proizvodnye alloksazina. Soobshchenie II. Vliianie rozeoflavina na sintez 6,7-dimetil-8-ribitillumazina, riboflavinsintetazy i rost Bacillus subtilis.

The replacement of 8-CH3 group in the riboflavin molecule results in the formation of specific antimetabolites. They are rozeoflavin, 7-desmethylrozeoflavin, 8-amino (nor) riboflavin, 8-ribitylamino (nor) riboflavin. Effect of rozeoflavin and other riboflavin analogues on the growth and regulatory characteristics of Bacillus subtilis strains with different genetic state of riboflavin operon is studied. Roseoflavin at a concentration of 0.05 mkg/ml inhibits DRL synthesis in rib-b110 strain. An analogue inhibits the growth of auxotrophic and prototrophic strains at concentrations of 0.5 mkg/ml and 50 mkg/ml respectively. Riboflavin (1 mkg/ml) recovers the growth of bacteria. The curve of rozeoflavin regulation of DRL and riboflavin synthetase synthesis is shifted in 100 times in the direction of lesser concentrations as compared with riboflavin and 8 amino (nor) riboflavin. 180 mutants resistant to 100 mkg/ml of rozeoflavin were selected. 150 mutants over-synthetize riboflavin.

[Riboflavin and lumiflavin analogs and alloxazine derivatives. I. Effect on riboflavin synthesis by and growth of Bacillus subtilis]. / Analogi riboflavina, liumiflavina i proizvodnye alloksazina. Soobshchenie I. Vliianie analogov na sintex riboflavina i rost Bacillus subtilis

Effect of riboflavin, lumiflavin and a number of alloxazine derivatives is studied on the growth of three Bacillus subtilis strains with different genetic state of riboflavin operon as well as their ability to show coenzyme and regulating properties characteristic of vitamin B2. The fission of the ribityl chain of the riboflavin molecule results in the complete loss of both coenzyme and regulating properties of vitamin B2 (analogues of lumiflavin and alloxazine derivatives). Different types of esterification of hydroxyl groups of ribityl chain or its replacement by dulcityl chain results in the decrease of regulating properties characteristic of riboflavin as an end product of biosynthetic pathway (tetraacetylriboflavin and its derivatives, tetrapropionyl- and tetrabutyrylriboflavin) or to the loss of coenzyme and regulating activities (galactoflavin and tetrabenzoylriboflavin). The length increase of hydrocarbon chain under esterification of hydroxyl groups in ribityl chain results in enhancing vitamin B2 non-specific inhibitory effect on the growth of Bacillus subtilis strains studied. The replacement of 8-CH3 group in the riboflavin molecule by amino- or D-ribitylaminogroup results in the formation of specific antimetabolites, 8-amino(nor)riboflavin and 8-D-ribitylamino(nor)riboflavin respectively. Depending on the nature of other alterations or replacements of the 8-CH3 group of riboflavin molecule and their combinations with alterations of ribityl chain the reduction of the regulating activity (8-alpha-bromtetraacetylriboflavin) or a sharp decrease of coenzyme and regulating functions (analogues N 9 to N 15) takes place. The replacement of riboflavin-5-N-oxide and 2-thioriboflavin type did not affect the studied properties of riboflavin.