Effects of the peptide antibiotics tyrocidine and the linear gramicidin on RNA SYNTHESIS AND SPORULATION OF Bacillus brevis.

Effects of the two peptide antibiotics tyrocidine and the linear gramicidin on exponential growing cultures of $\text{Bacillus brevis}$ (ATCC 8185) were studied. Both antibiotics are produced by this bacterial strain. Our results demonstrate that the addition of tyrocidine leads to inhibition of RNA synthesis followed by a cessation of growth. In contrast, gramicidin does not affect net RNA synthesis and the cells continue to grow. However, sporulation is inhibited by gramicidin. The addition of a mixture of tyrocidine and gramicidin prevents partially the inhibitory effect of tyrocidine on RNA synthesis. The results presented are essentially in agreement with $\text{in vitro}$ results described previously (8,9).     

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.     

Tyrocidine-induced modulation of the DNA conformation in Bacillus brevis

Using the [3H]trimethylpsoralen photobinding method [Sinden, R.R., Carlson, J.O. & Pettijohn, D.E. (1980) Cell 21, 773-783], a decrease in unrestrained torsional tension of DNA was detected in Bacillus brevis cells when they had entered the sporulation phase. This decrease in superhelicity was found in cells which synthesized the peptide antibiotic tyrocidine and which were stimulated to sporulate. Fluctuations in superhelicity probably reflect a highly complicated picture of tension-relaxing and tension-inducing activities. Addition of tyrocidine to vegetative cells reduced by one-half the torsional tension from DNA, whereas ethidium bromide relaxes DNA completely. Cross-links between DNA and tyrocidine were introduced with ultraviolet light in vitro and in vivo indicating that the modulation of the DNA conformation in the cell may in fact be due to a DNA-tyrocidine interaction. In a growing B. brevis culture exogenous [3H]tyrocidine could only be photobound to DNA after the cells had entered the sporulation phase. Our results could mean that the peptide antibiotic tyrocidine is active in B. brevis on the DNA level as one regulatory factor controlling DNA functions.     

Sporulation and spore properties of Bacillus brevis and its gramicidin S-negative mutant

The function(s) of the peptide antibiotic, gramicidin S, in its producer, Bacillus brevis Nagano, was investigated. Particular attention was paid to the possible role of gramicidin S in sporulation and spore properties. Sporulation was similar in both the gramicidin S-producing parental strain and a gramicidin S-negative mutant of this strain. Mature parental and mutant spores were equally resistant to UV irradiation, solvents (reported previously) and heat. Thus, the lack of gramicidin S synthesis impairs none of these properties. Contrary to results reported by others, we also found no difference in heat resistance between spores of B. brevis ATCC 8185 and its linear gramicidin-negative mutant, Ml.     

Enhancement of linear gramicidin expression from Bacillus brevis ATCC 8185 by casein peptide

Bacillus brevis (Brevibacillus parabrevis) ATCC 8185 synthesizes two kinds of antibiotic peptides, cyclopeptide tyrocidine and linear gramicidin. The production of linear gramicidin can be induced by the standard method (using a skim milk medium for pre-culture and beef broth for the main culture) employed for the induction of tyrocidine. In this study, we tried to determine the optimal growth medium for B. brevis ATCC 8185 for synthesizing linear gramicidin. The yield of linear gramicidin produced by the standard method was 3.11 microg/ml. When beef broth was used both as the pre-medium and the main medium, the yield of the antibiotic was only 0.59 microg/ml. To confirm the influence of skim milk, the strain was grown in a 1% skim milk medium. As a result, the amount of linear gramicidin produced reached 20.3 microg/ml. These findings show the importance of skim milk in the production of linear gramicidin. In the skim milk medium, the cells produced an extracellular protease 2 h before the linear gramicidin was expressed. The 1% skim milk medium pretreated by this protease did not allow the induction of linear gramicidin into the cells, and protease activity was not detected in the supernatant of the culture. When the cells were cultivated in a 1% egg albumin medium, protease activity from the supernatant of the culture was detected, but production of linear gramicidin was not observed. Therefore, a 1% casein medium was used for production of linear gramicidin. As a result, the yield of linear gramicidin produced in the medium reached 6.69 microg/ml. We concluded that a digested product of the extracellular protease from casein enhances linear gramicidin production.     

Structuro-functional properties of the bacteria Bacillus brevis in relation to the accumulation of gramicidin S in cells

The culture of Bacillus brevis var. G-B R-form was grown in the presence of beta-phenyl-beta-alanine, the inhibitor of gramicidin S synthesis, is characterized by enhanced endogenous respiration and the DPI-reductase activity as compared to the culture synthezising antibiotic. The increased synthesis of the antibiotic in the region of the culture transition from the logarithmic growth phase to the linear one is associated with a decrease in the number of viable cells despite the fact that the culture on the whole does not die but continues to grow. The membranes prepared from young gramicidin S-free cells and from the cells enriched with the antibiotic possess identical electron micrograph images, IR spectra and protein sets as determined by polyacrylamide gel electrophoresis in a Na-DS system. However, in young cell membranes NADH and succinate dehydrogenase are insensitive to gramicidin S and only malate dehydrogenase is inhibited by this antibiotic. In aged cell membranes the activities of all mentioned dehydrogenases are suppressed. Malate dehydrogenase from young cells is weakly inhibited by thyrotrycin obtained from Bac. brevis ATCC 10068; succinate dehydrogenase is entirely insensitive to this antibiotic, while NADH-dehydrogenase is almost completely inhibited by it. The specificity of action on the respiratory chain of peptide antibiotics synthesized by the cells of one strain of Bac. brevis is suggestive of a possible regulatory role of these peptides in the metabolism of the producent. Hence the accumulation of gramicidin S which is adsorbed on the membrane and destroys the respiratory chain function to the cause of the low rate of oxygen uptake by the culture of Bac. brevis var. G-B R-form and of the low activities of DPI-reductases.     

DNA-supercoiling is affected in vitro by the peptide antibiotics tyrocidine and gramicidin

Tyrocidine, a peptide antibiotic produced by Bacillus brevis (ATCC 8185), relaxes superhelical DNA in a biphasic manner and induces 'packaging' of the DNA at higher concentrations. This was concluded from studies using the sensitive 4,5',8-trimethylpsoralen photobinding technique [Sinden, R. R., Carlson, J. O. & Pettijohn, D.-E. (1980) Cell 21, 773-783]. Relaxed DNA is not affected by tyrocidine whereas linearized molecules become packaged. The linear gramicidin synthesized by the same strain reverses the tyrocidine-induced relaxation as well as the packaging, an observation which might be of biological relevance.     

Effect of gramicidin C on the formation and germination of Bacillus brevis var. GB (P+-variant) spores]

The effect of gramicidin C added to the medium at various periods of cultivation in concentrations of 20, 40 and 100 gamma/ml on sporulation of P+-variant of Bac. brevis var. GB was studied. The most effective increase in the sporulation rate and percentage of the cells germinating into the spores was observed on addition of the antibiotic to the medium in amounts of 20 and 40 gamma/ml in 13 hours of the culture development. The amount of gramicidin C during sporulation decreased and partially passed into the spores which did not differ after germination from those of P+-variant grown on the synthetic medium with glucose and without preliminary addition of the antibiotic. Addition of gramicidin C in an amount of 100 gamma/ml at the end of the lag phase, i.e. 4 hours after the culture inoculation suppressed sporulation and had no effect on growth of the cells of its own producing organism.     

The relation between sporulation and the induction of antibiotic synthesis and of amino acid uptake in Bacillus brevis

The induction and localization of tyrocidine-synthesizing enzymes is shown to be parallel, during growth of Bacillus brevis (ATCC 8185, American Type Culture Collection, Rockville, Md.), with the induction of uptake of constitutive amino acids and of components of pantetheine, a coenzyme of tyrocidine synthesis. Antibiotic synthesis appears at the end of logarithmic growth when the first soluble enzymes may be obtained from homogenates. During this period, binding proteins for metabolite uptake were isolated by intensive sonication which, when studied by chromatography, were identified by the appearance of low molecular weight fractions binding the radioactively marked metabolites; their induction was prevented by addition of rifampicin. The major purpose of this study was a comparison of antibiotic production and sporulation, the progress of which was followed by electron microscopy. The onset of tyrocidine synthesis and metabolite uptake coincided with the appearance of septum formation indicating that sporulation had progressed to stage II. With the progress of spore encapsulation, the tyrocidine production migrated from the soluble fraction into the forespore, terminating with the separation of forespores from the sporangium membrane. The resulting concentration of antibiotic in the forespore may indicate its function in sporulation, the nature of which, however, was not explored.     

Interrelationships between tyrocidine and gramicidin A' in their interaction with phospholipids in model membranes

(1) The interaction of tyrocidine with different lipids is studied in model membranes and the results are compared to the gramicinid-lipid interaction. (2) The tyrocidine-dielaidoylphosphatidylethanolamine interaction gives rise to a population of phospholipids with a lower gel to liquid-crystalline transition temperature and to an abolition of the bilayer to HII phase transition, resulting in a macroscopic organization with dynamic and structural properties different from those of the pure lipid. (3) Tyrocidine has a strong fluidizing effect on the acyl chains of phosphatidylcholines, manifested by a decrease in enthalpy of the main thermotropic transition. (4) No evidence of a gramicidin A'-like lipid-structure modulating activity was found. However, tyrocidine inhibits the formation by gramicidin of an HII phase in dioleoylphosphatidylcholine model membranes. Instead, a cubic type of lipid organization is observed. (5) Tyrocidine greatly perturbs the barrier properties of dioleoylphosphatidylcholine model membrane. (6) Gramicidin A' reverses the effect of tyrocidine on membrane permeability by forming a complex in the model membrane with an apparent 1:1 stoichiometry. (7) The results suggest that both peptide antibiotics, which are produced by Bacillus brevis ATC 8185 prior to sporulation, show antagonism in their effect on membrane structure similar to their effect on superhelical DNA (Bogh, A. and Ristow, H. (1986) Eur. J. Biochem. 160, 587-591. The possible underlying basic mechanism is indicated.     

Comparison of the effect of linear gramicidin analogues on bacterial sporulation, membrane permeability, and ribonucleic acid polymerase.

Various analogues of linear gramicidin were tested for their biological activity in restoring the normal spore phenotype of gramicidin-negative mutants of Bacillus brevis and for their ability to increase cation conductivity of black lipid membranes and to inhibit bacterial RNA polymerase. Whereas many biologically active gramicidin analogues had no effect on membrane permeability, all biologically active peptides were able to inhibit ribonucleic acid (RNA) polymerase. These observations make it unlikely that membranes are the site of action of gramicidin during bacterial sporulation, but they are consistent with the notion that gramicidin functions to control RNA synthesis during the transition from vegetative growth to sporulation (Sarkar & Paulus, 1972). The relationship between peptide structure and the ability to restore normal sporulation and inhibit RNA polymerase showed that the eight amino-terminal residues have little influence on the function of gramicidin, whereas the highly nonpolar repeating sequence D-leucyl-L-tryptophan is essential for biological activity and may represent the site of interaction with RNA polymerase.