Processes of spore formation and gramicidin C formation by Bacillus brevis var. G.B.]

Correlation between gramidicin C biosynthesis and sporulation in the process of Bac. brevis var. G.B. cultivation under various aeration conditions was studied. It was shown that biosynthesis of gramicidin C was characteristic of the young cells and its level was the highest during the culture active growth. The time of the sporulating forms appearance depended on the aeration rate which defined the quantitative composition of the population during the phase of the culture active growth and the stationary phase. Under the optimal aeration conditions the spore formation started during the phase of the culture active growth after some decrease in the maximum level of the cell productivity with respect to the antibiotic. When the aeration rate was increased the spore formation was shifted to later periods of the culture development, i.e. the stationary phase and the phase of the cell autolysis, the gap between the highest levels of gramicidin C buosynthesis and the beginning of sporulation being increased. Under certain aeration conditions the spore formation was not observed, while gramicidin C was synthesized. A conclusion has been made that there is no correlation between gramacidine C biosynthesis and sporualtion in Bacillus brevis var. G.B.     

Specificity of methylation of cytosine risidues in DNA of Bacillus brevis var. G-B]

On growing the cells of Bacillus brevis S methionine-auxotroph mutant in the presence of (methyl-3H)-methionine practically the total radioactivity included into DNA is found to exist in 5-methylcytosine (MC) and 6N-methyladenine (MA). The analysis of pyrimidine isopliths isolated from DNA shows that radioactivity only exists in mono- and dinucleotides and the content of MC in Pur-MC-Pur and Pur-MC-T-Pur oligonucleotides is equal. The analysis of dinucleotides isolated from DNA by means of pancreatic DNAase hydrolysis allows the nature of purine residues neighbouring with MC to be revealed and shows that MC localizes in G-MC-A and G-MC-T-Pu fragments. Bac. brevis S DNA-methylase modifying cytosine residues recognizes the GCAT GC degenerative nucleotide sequence which is a part of the following complementary structure with rotational symmetry: (5') ... N'--G--MC--T--G--C--N ... (3') (3') ... N--C--G--A--MC--G--N' ... (5') Cytosine modifying DNA-methylase activity is isolated from Bac. brevis cells; it is capable of methylating in vitro homologous and heterologous DNA. Hence, DNA in bacterial cells can be partially undermethylated. This enzyme methylates cytosine residues in native and deneaturated DNA in the same nucleotide sequences. As compared to the native DNA, the denaturated DNA is indicative of a decrease in the level of methylation of adenine, rather than cytosine residues. Specificity of methylation of cytosine residues in vitro and in vivo does not depend on the nature of substrate DNA (calf thymus, Pseudomonas aeruginosa etc.). DNA-methylases of different variants of Bac. brevis (R, S, P+, P-) methylate cytosine residues in the same nucleotide sequences. It means that specificity of methylation of DNA cytosine residues in the cells of different variants of Bac. brevis is the same.     

Lyophilization of the spores and vegetative cells of Bacillus brevis var. G-B.--producer of gramicidin S

Viability, antibiotic properties and variation of 4 variants of Bac. brevis var. G.-B. were studied after lyophilization and storage for a year in the lyophilized state. It was shown that the spores and vegetative cells of S and P- variants not synthesizing gramicidin S were somewhat more stable than the spores and cells of R and P+ variants producing the antibiotic. The latter dissociated by 10 per cent towards the cells producing and not producing gramicidin. The developmental rate of the lyophilized vegetative cells was higher than that of the lyophilized spores. Under analogous cultivation conditions they produced higher amounts of the biomass and antibiotic. The lyophilization method described may be recommended for the maintenance of viability and stability of the spores and vegetative cells of Bacillus brevis var. G.-B. producing gramicidin S.     

Sporulation in Bacillus subtilis. Antigenic changes during spore formation

1. Antisera, prepared against extracts of cells and spores of Bacillus subtilis, were used in immunoelectrophoretic studies of the changes occurring in cell extracts during the course of spore formation. 2. At least 15 antigens could be detected in vegetative-cell extracts by the antiserum prepared against cell extracts and at least seven could be demonstrated in spore extracts by the homologous antiserum. 3. Cross-absorption studies showed that two of these antigens were probably completely specific for vegetative-cell extracts and that one was probably completely specific for spore extracts. The remainder were probably present in very small quantities in the heterologous extract. 4. In extracts of cells sporulating in an ;exhaustion medium' those antigens characteristic of the spore began to appear about 1hr. after the end of exponential growth. 5. In cells sporulating in a resuspension medium, spore antigens were detected at 4hr., and by 7hr. a decrease in vegetative-cell antigens was observed. 6. In an asporogenous mutant blocked early in sporulation there was neither an increase in spore antigens nor a decrease in vegetative-cell antigens. 7. In an asporogenous mutant blocked later in sporulation, there was an increase in spore antigens similar to that which occurred in the sporogenous strain.     

Growth limitations and biosynthesis of gramicidin in Bacillus brevis var. G.-B

Gramicidin S biosynthesis was studied in Bacillus brevis var. G.-B. during its batch and continuous cultivation when the culture growth was limited with nutrient sources (glycerol, ammonium nitrogen, phosphate), oxygen deficiency and the action of a physical factor (a low temperature). The antibiotic biosynthesis was shown to be induced by a change in the growth rate caused by the action of any factor decelerating the growth. The authors propose a mathematical model for the antibiotic synthesis, biomass accumulation and the utilization of a substrate limiting the growth. The model is based on the age separation of cells. The model is analyzed in terms of optimizing the one-stage continuous cultivation process. The model allows one to calculate optimal conditions of the antibiotic synthesis in the process of one-stage continuous cultivation.     

Various characteristics of lipid metabolism of Bacillus brevis var. G.-B

Some characteristic features of the lipid metabolism of Bacillus brevis var. G.-B. natural variants and Bacillus brevis mutant 101 were studied. The authors found that upon submerged cultivation gramicidine S-producing P+-variant and B. brevis mutant 101 synthesized higher amounts of tocopherols as compared to other colonial-morphological variants. The highest tocopherol content was observed in P+-variant, whose cells contained the highest amount of total lipids as compared to other gramicidine S-producers.     

四溴甘脲对枯草杆菌黑色变种芽胞损伤作用的电镜观察

为探索四溴甘脲消毒剂杀灭细菌的机理,采用透射电镜技术对四溴甘脲消毒剂处理过的枯草杆菌黑色变种芽胞的超微结构进行了分析和比较.结果显示,以含有效溴274mg/L的四溴甘脲消毒剂作用30min,可使枯草杆菌黑色变种芽胞杀灭率达到100%.在透射电镜下观察到,经该消毒剂作用的枯草杆菌黑色变种芽胞壳质破损断裂明显,壳内结构模糊,核心溶解,有的芽胞近似空壳.结果显示,四溴甘脲消毒剂杀灭芽胞效果优于普通含氯消毒剂,对细菌芽胞超微结构破坏明显.     

Biological role of gramicidin S in spore functions. Studies on gramicidin-S-negative mutants of Bacillus brevis ATCC9999.

Gramicidin-S-negative mutants of Bacillus brevis ATCC9999 have been isolated with a remarkly higher yield after ethidium bromide or acridine orange treatment, than after N-methyl-N'-nitro-N-nitrosoguanidine treatment. Four (MIV, Smr170, R5 and EB 16) of 38 isolated mutants were characterized with respect to the lesion in gramicidin-S-synthesizing activity. The mutants sporulate to the same extent as the parental strain except mutant Smr 170 which sporulates less. However, mutant spores were more heat-sensitive and possessed a reduced level of dipicolinic acid content. No significant difference was observed in the germination time of wild-type and mutant spores. All spores germinated after 80--110 min, but the outgrowth time was different: all gramicidin-S-negative mutants grew out immediately after germination whereas wild-type spores required a lag period of 9--10 h. When the mutants were allowed to sporulate in the presence of gramicidin S, the spores were found to be heat-resistant and their outgrowth postponed to the same period as the parent spores. The addition of gramicidin also eliminated the deficiency of dipicolinic acid. A new class of gramicidin-S-negative mutant, R5, which only activates L-valine and L-leucine, is described. A possible biological function of gramicidin S in the heat-resistance and in the timing of spore outgrowth is discussed.