Role of gramicidin S in the sporulation of the R+ and R- variants of Bacillus brevis var. G.-B.]

The effect of gramicidin S added to the cultivation medium on sporulation of the gramicidin S-producing P+ variant and gramicidin S-nonproducing P- variant of Bacillus brevis var. G.-B. was studied. Gramicidin S added to the synthetic medium with glucose in an amount of 30 and 100 microgram/ml 4 and 7 hours after inoculation with the vegetative cells of R- variant had no effect on the growth of the culture but retarded its sporulation. When gramicidin S was added in an amount of 100 microgram/ml 4 hours after inoculation, the sporulation rate of R- variant strongly decreased, rohile sporulation was not suppressed as it was noted before with respect to R+ variant. Active stimulation of Bacillus brevis var. G.-B. sporulation was observed after addition of gramicidin S 13 hours after development of R+ and R- variants without the antibiotic biosynthesis. Synthesis of gramicidin S by R+ strain was suppressed by the specific inhibitor beta-phenyl-beta-alanine. The amount of gramicidin S added to the medium during the sporulation process of R+ and R- variants decreased. On addition of 30 microgram/ml of the antibiotic it was practically not detectable when the culture showed the greatest number of the spores. Therefore, gramicidin S added to the medium is probably adsorbed by the cells of Bac. brevis var. G.-B. and affects sporulation of R- and R+ variants thus accelerating or retarding this process depending on the cultivation conditions.     

Effect of glycerin on the biosynthesis of heliomycin by Streptomyces olivocinereus

Glycerol as the sole carbon source was added to the medium or biosynthesis of heliomycin by Streptomyces olivocinereus and the effect of its concentration on the culture growth and antibiotic production was studied. The culture growth and the amount of the antibiotic synthesized per 1 unit of the fermentation broth were limited by glycerol added in quantities of 0.05 to 1 per cent. Further increasing of the glycerol concentration had no significant effect on the culture growth and antibiotic biosynthesis. The amount of the antibiotic synthesized per 1 unit of the mycelial mass relatively slightly depended on the glycerol concentration. The rate of glycerol consumption by the young 24-hour culture in batch fermentations markedly exceeded that of glycerol consumption by the 48-hour culture. The younger mycelium significantly increased its rate of glycerol consumption when the initial concentration was increased whereas the rate of glycerol consumption by the more mature mycelium did not depend on the initial concentration of the carbon source (within 0.5-2 per cent). The rate of heliomycin biosynthesis practically slightly depended on the initial concentration of glycerol.     

The action of its own antibiotic on the producer of imbricin growing on an agarized medium]

The action of imbricin on its own producer Streptomyces imbricatus grown on an agarized medium was studied. Comparatively low concentrations of the antibiotic were shown to have a high lethal action on the streptomycete. The morphological and cultural features of S. imbricatus did not change under the action of imbricin while the variation with respect to the antibiotic production property markedly increased. After the strain exposure to 200 micrograms/ml of imbricin, a stable variant with the antibiotic potency 20 per cent higher than that of the initial organism was isolated.     

Effect of lincomycin on its own producer, Actinomyces roseolus, in periodic cultivation in a liquid medium

Lincomycin added to the cultivation medium induced a number of changes in the organism producing it during its ontogenesis when grown recurrently on liquid media. It was found that lincomycin inhibited the culture growth and decreased the absolute amount of the antibiotic synthesized while the specific activity of the culture increased. A number of cytomorphological rearrangements relevant to the adaptive protective reactions was found. It is suggested that an increase in the resistance of the culture to the antibiotic produced by it at the late developmental stages is the result of the above protective reactions.     

Effect of aeration and redox potential on the biosynthesis of the antibiotic imbricin]

The relationship between imbricin biosynthesis by Streptomyces imbricatus and the medium aeration and redox potential (Eh) was studied. The influence of the oxygen dissolution velocity within the ranges of 2.9 to 0.5 g O2/l.h was investigated and it was shown that the highest yield of the antibiotic was provided by the maximum velocity. At the background of the intensive aeration (2.9 g O2/l.h) decreasing of Eh by reducing agents such as ascorbic acid, L-thyrosin or K4Fe(CN)6 stimulated the biosynthesis whereas at the lower velocities the process proved to be inhibited. Under conditions of insufficient aeration the biosynthesis stimulation could be provided by increasing the medium Eh by acidifying agents such as K2Cr2O7, K3Fe(CN)6 or KMnO4. It was concluded that intensive synthesis of imbricin required not only efficient aeration but also definite levels of the medium redox potential.     

Nisin inactivation in a culture of the producer Streptococcus lactis strain MGU

The intensive biosynthesis of nizin on the glucose-yeast medium is observed during the logarithmic and early lag phases of the staphylococcal growth. The ratio of nizin in the fermentation broth (free nazin) and that bound with the cells depended on pH of the medium. When pH was maintained at 6.6-6.8, the amount of nazin in the cells during and growth logarithmic phase was equal to its amount in the fermentation broth filtrate. During the lag phase marked inactivation of nizin was noted. periodical feeding of casein prevented the nizin inactivation. The preliminary data are indicative of the enzymatic nature of the antibiotic.     

Effect of ammonium on chloramphenicol production by Streptomyces venezuelae in batch and continuous cultures

Cultures of Streptomyces venezuelae presented with a mixture of ammonium and an amino acid as nitrogen sources used both compounds together. Absence of ammonium repression of alternative nitrogen assimilation pathways was also observed when ammonium was added to cultures already growing on proline. The presence of ammonium in the medium ab initio depressed the yield of chloramphenicol. However, its addition to a culture growing on proline caused only a temporary inhibition of antibiotic synthesis, even when sufficient ammonium was added to create an excess. Continuous cultures supplied with ammonium as the growth-limiting nutrient showed no significant change in specific antibiotic production at different specific growth rates. The overall results indicate that in S. venezuelae neither nitrogen utilization pathways nor chloramphenicol biosynthesis is controlled by nitrogen repression.     

Effect of chloramphenicol and actinomycin D on extracellular alkaline RNAse biosynthesis by Bacillus intermedius

By means of chloramphenicol it was found that biosynthesis of alkaline exocellular RNAase was repressed in Bacillus intermedius by inorganic phosphate. Actinomycin D at a low concentration stimulates RNAase biosynthesis in a medium with a minimal phosphorus concentration in model experiments with washed cells and in the batch culture. As a result, the activity of RNAase rises 2-4 times. The stimulating effect of actinomycin D decreases when phosphorus concentration in the medium is increased The effect of actinomycin D is maximal if the antibiotic is added to the medium when the specific growth rate of the bacterium falls down and the rate of RNAase biosynthesis rises.     

Elevated yield of monacolin K in Monascus purpureus by fungal elicitor and mutagenesis of UV and LiCl

In China, Monascus spp., a traditional fungus used in fermentation, is used as a natural food additive. Monascus spp. can produce a secondary metabolite, monacolin K namely, which is proven to be a cholesterol-lowering and hypotensive agent. Hence, recently, many researchers have begun focusing on how to increase the production of monacolin K by Monascus purpureus. In the present study, we investigated the effect of the fungal elicitor and the mutagenesis of UV & LiCl on the amount of monacolin K produced by Monascus purpureus. The fugal elicitor, Sporobolomyces huaxiensis, was isolated from tea leaves and its filtrate was added into the culture filtrate of Monascus purpureus during growth to induct the production of monacolin K. The results showed that the highest amount of monacolin K produced by the liquid fermentation was 446.92 mg/mL, which was produced after the fungal elicitor was added to the culture filtrate of Monascus purpureus on the day 4; this amount was approximately 6 times greater than that of the control culture filtrate, whereas the highest amount of monacolin K produced by the mutated strain was 3 times greater than the control culture after the irradiation of UV light in the presence of 1.0 % LiCl in the medium.     

Effect of a Microbial Acid Protease Inhibitor (S-PI) on the Production of Fungal Acid Protease

Cladosporium sp. No. 45–2, an acid protease-producing microorganism, was cultured in medium containing a microbial acid protease inhibitor (S–PI). By the addition of S–PI, the amount of acid protease in the culture broth showed an increase of 50~80% over those of normal culture (S–PI-free). Acid protease was purified from the S–PI-added culture filtrate, and its enzymatic and physicochemical properties were compared with those of acid protease obtained from normal culture. It was determined that the acid protease obtained from S–PI-added culture was the same as that of normal culture, but that the productivity was increased by the addition of S–PI.

The increase in acid protease productivity is assumed to be due to a change in metabolism by the addition of S–PI.     

The lantibiotic mersacidin is an autoinducing peptide

The lantibiotic (lanthionine-containing antibiotic) mersacidin is an antimicrobial peptide consisting of 20 amino acids and is produced by Bacillus sp. strain HIL Y-85,54728. The structural gene (mrsA) and the genes for producer self-protection, modification enzymes, transport proteins, and regulator proteins are organized in a 12.3-kb biosynthetic gene cluster on the chromosome of the producer strain. Mersacidin is produced in stationary phase in a synthetic medium (K. Altena, A. Guder, C. Cramer, and G. Bierbaum, Appl. Environ. Microbiol. 66:2565-2571, 2000). To investigate the influence of the alternative sigma factor H on mersacidin biosynthesis, a SigH knockout was constructed. The knockout mutant was asporogenous, and a comparison to the wild-type strain indicated no significant differences concerning mersacidin production and immunity. Characterization of the mrsA promoter showed that the gene is transcribed by the housekeeping sigma factor A. The biosynthesis of some lantibiotic peptides like nisin or subtilin is regulated in a cell-density-dependent manner (M. Kleerebezem, Peptides 25:1405-1414, 2004). When mersacidin was added at a concentration of 2 mg/liter to an exponentially growing culture, an earlier production of antibacterial activity against Micrococcus luteus ATCC 4698 in comparison to that of the control culture was observed, suggesting that mersacidin itself functions as an autoinducer. In real-time PCR experiments, the expression of mrsA was remarkably increased in the induced culture compared to the control. In conclusion, mersacidin is yet another lantibiotic peptide whose biosynthesis can be regulated by an autoinducing mechanism.     

Method of determining the antibiotic sensitivity of anaerobic microorganisms using standard disks

Three variants of the procedure for determination of antibiotic sensitivity in anaerobic microorganisms with the use of standard paper discs were developed. According to the first variant the solid nutrient medium is melted at 46 degrees C and mixed with the culture of the microbe being tested. The mixture is added to the cover of a Petri dish. When the medium becomes solid, antibiotic sensitivity discs are placed onto the agar surface. After that one more layer of the medium is added. The medium is allowed to solidify and some more medium is poured near the cover edge. Immediately after that the Petri dish is placed with its flat surface onto the agar layer in its cover. According to the first and second variants the mixture of the medium and culture is added to a Petri dish and immediately a transparent gas-proof polymer film of the dish size is placed onto the agar surface. Previously antibiotic paper discs or solutions are fixed on the films. THe incubation temperature for all three variants is 37 degrees C. The procedure allows one to observe the culture growth and to obtain the results earlier than in case the culture is incubated in an aerostate. The procedure is simple and saves labor and time.     

Penicillin biosynthesis and amino acid metabolism in Penicillium chrysogenum in experiments with washed mycelium]

The study of the amino acid metabolism in Penicillium chrysogenum with the use of washed mycelium showed that the amount of the free intracellular amino acids significantly decreased during the process of penicillin production. Still, such a decrease did not cover the nitrogen requirements of the culture for the antibiotic synthesis and mobilization of the protein nitrogen took place. By the end of the process the amount of the protein nitrogen markedly decreased. At the same time alpha-amino nitrogen was absent in the fermentation broth filtrate. About 14 amino acids (including cysteine and valine) which participate in constriuction of the penicillin molecule nucleus were found in the amino acid poll. However, the amounts of cysteine and valine were not high and probably other free intracellular amino acids participated in their synthesis. It was shown that one of the limiting factors in the process of penicillin biosynthesis was synthesis of cysteine, a sulphur-containing amino acid which is one of the precursors of the antibiotic molecule nucleus.     

Study of the carbohydrate metabolic enzyme activity of Act. noursei]

Activity of aldolase and threosophosphate dehydrogenase, transketolase and phosphogluconate dehydrogenase in Act. noursei, strain 153 and its inactive mutant 149 was studied comparatively. The enzyme activity of the inactive mutant was investigated in the absence of the antibiotic production and under conditions of reduced biosynthesis of nystatin in this strain after addition of the fermentation broth filtrate of the inactive mutant 369 to the medium. The activity of the enzymes of the hexosomonophosphate metabolic pathway in the active strain 153 of Act. noursei was 2-4 times higher than that of the inactive mutant 149. The activity of the enzymes of the hexosomonophosphate metabolic pathways increased and reached the level of the enzyme of the active mutant. The high level of the enzyme activity of the hexosomonophosphate glycolysis pathway is probably one of the necessary conditions for nystatin production.     

强壮前沟藻化感物质分析

微藻化感作用是一种极其复杂的生理、生态学现象。选取强壮前沟藻指数生长初期Ⅰ和平台生长初期Ⅱ两个阶段的滤液对中肋骨条藻、海洋原甲藻、锥状斯氏藻及球等鞭金藻生长的影响进行了研究,并萃取了阶段Ⅱ的粗提物,抑藻检测表明其具有"杀藻"效应,通过GC/MS分析该粗提物中具有潜在化感作用的物质种类。研究发现强壮前沟藻两个生长阶段的滤液对中肋骨条藻均产生强烈致死效应(phaseⅠ:F=15.18475,P=0.00298<0.05;phaseⅡ:F=6.24559,P=0.03149<0.05);锥状斯氏藻在强壮前沟藻滤液中生长,实验结束时两个阶段中的细胞密度分别是对照组的79.3%和68.9%;海洋原甲藻在强壮前沟藻生长阶段Ⅱ滤液实验的最后3d,其生长受到显著抑制(F=4.84438,P=0.04925<0.05);而等鞭金藻在强壮前沟藻两个生长阶段滤液中被抑制现象不明显(P>0.05)。强壮前沟藻滤液实验表明,强壮前沟藻能够向微环境中分泌代谢产物来抑制中肋骨条藻和海洋原甲藻的生长,并且这种抑制效应具有种类特殊对应性。上述实验结果还表明,强壮前沟藻生长阶段Ⅱ的滤液具有的生长抑制作用较为明显。采用乙酸乙酯萃取强壮前沟藻生长阶段Ⅱ滤液中的代谢产物,检测发现其代谢粗提物具有溶藻效应,GC/MS分析结果表明粗提物中存在4种可能产生化感抑制作用的物质,其中二丁基羟基甲苯(Butylated Hydroxytoluene BHT)被认为具有抗滤过性病原体和抗微生物活性。     

Dependence of erythromycin biosynthesis on the active acidity of the medium]

Dependence of erythromycin biosynthesis on the medium active acidity was studied by the following methods: by changing pH of the initial medium, by changing the concentration of the medium components determining the active acidity of the culture, by using buffer mixtures by automatic control of pH. It was found that pH of the initial medium within 5.7-8.1 had no effect on the culture growth. Biosynthesis of erythromycin markedly decreased at pH 6.3 or lower. The values of pH within 6.6-7.5 (optimal values 6.7-6.9) were favourable for the antibiotic biosynthesis. At pH 6.2-6.3 the antibiotic accumulation was equal to 5-10 per cent of the control.     

The Lantibiotic Mersacidin Is an Autoinducing Peptide

The lantibiotic (lanthionine-containing antibiotic) mersacidin is an antimicrobial peptide consisting of 20 amino acids and is produced by Bacillus sp. strain HIL Y-85,54728. The structural gene (mrsA) and the genes for producer self-protection, modification enzymes, transport proteins, and regulator proteins are organized in a 12.3-kb biosynthetic gene cluster on the chromosome of the producer strain. Mersacidin is produced in stationary phase in a synthetic medium (K. Altena, A. Guder, C. Cramer, and G. Bierbaum, Appl. Environ. Microbiol. 66:2565-2571, 2000). To investigate the influence of the alternative sigma factor H on mersacidin biosynthesis, a SigH knockout was constructed. The knockout mutant was asporogenous, and a comparison to the wild-type strain indicated no significant differences concerning mersacidin production and immunity. Characterization of the mrsA promoter showed that the gene is transcribed by the housekeeping sigma factor A. The biosynthesis of some lantibiotic peptides like nisin or subtilin is regulated in a cell-density-dependent manner (M. Kleerebezem, Peptides 25:1405-1414, 2004). When mersacidin was added at a concentration of 2 mg/liter to an exponentially growing culture, an earlier production of antibacterial activity against Micrococcus luteus ATCC 4698 in comparison to that of the control culture was observed, suggesting that mersacidin itself functions as an autoinducer. In real-time PCR experiments, the expression of mrsA was remarkably increased in the induced culture compared to the control. In conclusion, mersacidin is yet another lantibiotic peptide whose biosynthesis can be regulated by an autoinducing mechanism.     

Regulation of Exocellular Proteases in Neurospora crassa: Role of Neurospora Proteases in Induction

Cells of Neurospora crassa strain 74A, grown on sucrose for 12 h and transferred to a medium containing protein as sole carbon source, would not produce exocellular protease in significant amounts. When a filtrate from a culture induced to make protease by normal growth on a medium containing protein as principal carbon source was added to an exponential-phase culture in protein medium, exocellular protease was made in amounts similar to those made during normal induction. The material in the culture filtrate that participated in the induction process was identified as protease by its heat lability, molecular weight, and the dependence of induction rate on units of proteolytic activity added to the exponential-phase culture. Induction of the formation of exocellular protease by exponential-phase cells appears to require a protein substrate, added proteolytic activity, and protein synthesis. The protease produced by induced exponential-phase cells was as efficient in promoting induction as normally induced enzyme, whereas constitutive intracellular enzyme was only 50% as efficient. The bacterial protease thermolysin was able to induce exocellular protease at 90.7% of the rate observed with added N. crassa exocellular protease.     

Some characteristics of fucidin biosynthesis]

Some features of fusidin biosynthesis by 2 strains of Fusidium coccineum were studied proceeding from the peculiar properties of the antibiotic molecule structure. It was found that an increase in the levels of the carbon sources in the medium stimulated the biosynthesis of fusidin, while excessive concentrations of nitrogen especially in its inorganic and amino acidpeptide forms stimulated the organism growth and lowered the antibiotic activity levels. The concentration of nitrogen in the medium is considered as one of the possible control mechanisms in the processes of the fungus growth and biosynthesis of fusidin.