Studies on gramicidin S synthetase. Purification of the heavy enzyme obtained from some mutants of Bacillus brevis.

The heavy enzyme of gramicidin S synthetase was purified to an almost homogeneous state by a combination of ammonium sulfate fractionation, ornithine-Sepharose 4B chromatography, DEAE-cellulose chromatography, and Ultrogel AcA 22 chromatography. The enzyme was proved to be essentially homogeneous by ultracentrifugation and polyacrylamide disc gel electrophoresis. The heavy enzymes of gramicidin S synthetase from various groups of mutant strains lacking the ability to form gramicidin S were also purified to a similar extent. The sedimentation rates of the purified enzymes from a wild strain and the mutant strains (BI-3, BII-3, BI-9) were studied by analytical centrifugation and sucrose density gradient centrifugation. The enzymes from the wild strain and these mutant strains were all found to have an S20,W value of 12.2 at a protein concentration of 2.5 mg per ml. These results strongly suggest that the failure of specific amino acid activation in the heavy enzyme of these gramicidin-lacking mutants might be due to some modification at the active center of the corresponding amino acid-activating enzyme rather than to a complete absence of the amino acid-activating enzyme protein in the heavy enzyme.     

Studies on gramicidin S synthetase. Purification and properties of the light enzyme obtained from some mutants of Bacillus brevis.

The phenylalanine-activating and/or-racemizing enzyme, i.e., the light enzyme, of gramicidin S synthetase was purified to a homogenous state by D-phenylalanine-Sepharose 4B chromatography from a wild and some gramicidin S-lacking mutant strains of Bacillus brevis. The light enzyme obtained from a mutant strain E-1 could activate phenylalanine but not racemize it, and had no phenylalanine-dependent ATP-[14C]AMP exchange activity, whereas the same enzyme obtained from other mutants and the wild strain had all three activities. Furthermore, the light enzyme of the mutant E-1 could form only acid-labile enzyme-bound phenylalanine, while the same fraction of the wild strain carried half of the enzyme-bound phenylalanine as acid-labile adenylate and half as a acid-stable thioester. These results suggest that the thiol site of the light enzyme of mutant E-1 might be damaged.     

Oxygen-dependent inactivation of gramicidin S synthetase in Bacillus brevis.

Incorporation of L-[14C]ornithine into gramicidin S by crude, unfractionated lysozyme extracts of Bacillus brevis ATCC 9999 was shown to represent the activity of the gramicidin synthetase complex. Frozen-thawed cells were the source of active extracts, but when cells were shaken in air at 37 degrees C, they rapidly lost activity in a first-order reaction with a half-life of 13 min. Protease inhibitors and inhibitors of energy metabolism had no effect on the inactivation process in frozen-thawed cells. Stabilization was achieved when the cells were shaken in nitrogen or helium instead of air. The addition of dithiothreitol produced a moderate degree of stabilization. The L-ornithine- and D-phenylalanine-activating activities of the gramicidin S synthetase complex were also lost during aeration of the cells. Crude cell-free extracts also lost activity when they were shaken in oxygen, but, in this case, inactivation was slower (half-life of 80 min). Nitrogen also stabilized these cell-free extracts.     

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.     

Absence of pantothenic acid in gramicidin S synthetase 2 obtained from some mutants of Bacillus brevis

The pantothenic acid content of gramicidin S synthetase 2(GS 2) was estimated microbiologically with enzymes obtained from the wild strain and gramicidin S-lacking mutant strains of Bacillus brevis. Four mutant enzymes from BI-4, C-3, E-1, and E-2 lacked pantothenic acid. Other mutant enzymes from BII-3, BI-3, BI-9, and BI-2 contained the same amount of pantothenic acid as the wild-type enzyme. Pantothenic acid-lacking GS 2 belonged to group V of mutant enzymes, which could activate all amino acids related to gramicidin S; their complementary enzyme, gramicidin S synthetase 1(GS 1), lacked racemizing activity. To ascertain whether 4'-phosphopantetheine is involved in the formation of D-phenylalanyl-L-prolyl diketopiperazine (DKP) and gramicidin S, combinations were tested of intact GS 1 from the wild strain with various mutant GS 2 either containing or lacking pantothenic acid. Only the combinations of wild-type GS 1 with mutant GS 2 containing pantothenic acid could synthesize DKP. Combinations with pantothenic acid-lacking GS 2 also failed to elongate peptide chains. Pantothenic acid-lacking GS 2 could bind the four amino acids which constitute gramicidin S as acyladenylates and thioesters, but the binding abilities were lower than those of the wild-type enzyme and other mutant enzymes containing the pantothenic group.     

Production of gramicidin S synthetases by Bacillus brevis in continuous culture.

The effects of different nutrient limitations on the production of the two enzymes of gramicidin S biosynthesis were studied during continuous culture of Bacillus brevis. Gramicidin S synthetases I and II were produced in the chemostat under carbon, nitrogen, phosphorus or sulphur limitation. The growth rate, rather than the nature of the limitation, was the major controlling factor in regulating the level of the gramicidin S synthetases. Synthetase production was low at high dilution rates (0.45 to 0.50 h-1) but increased as the dilution rate was lowered. The highest specific activities occurred at dilution rates that were different for each type of limitation: 0.40 h-1 for nitrogen, 0.32 h-1 for carbon, 0.24 h-1 for sulphur and 0.20 h-1 for phosphorus. Phosphorus limitation gave the highest specific activities. At low dilution rates (0.10 to 0.15 h-1), enzyme activities were again low. Sporulation occurred under carbon limitation, but at a lower dilution rate than that which supported optimal gramicidin S synthetase formation. The specific productivity of the synthetases in the chemostat was higher than the highest productivity obtained in batch growth.     

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.     

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.     

Membranous phosphoglyceride-linked biosynthesis of pentadecapeptide, linear gramicidin, by Bacillus brevis ATCC 8185

A peptide antibiotic, linear gramicidin A, from Bacillus brevis ATCC 8185 was biosynthesized with a cell-free preparation. An ethanolamine donor required for masking of a carboxyl terminal in this linear peptide was detected. Phosphatidylethanolamine, one of phosphoglycerides and a major structural element of membranes in bacterial cells, was verified to be the primary donor of terminal ethanolamine in the total synthesis of the peptide. This paper suggests that one of the non-ribosomal peptidyl products undergoes tight linkage to a component of cellular membranes.     

The influence of gramicidin S on hydrophobicity of germinating Bacillus brevis spores

Gramicidin S is known to prolong the outgrowth stage of spore germination in the producing culture. Bacillus brevis strain Nagano and its gramicidin S-negative mutant, BI-7, were compared with respect to cell-surface hydrophobicity and germination of their spores. Parental spores were hydrophobic as determined by adhesion to hexadecane, whereas mutant spores showed no affinity to hexadecane. Addition of gramicidin S to mutant spores resulted in a high cell surface hydrophobicity and a delay in germination outgrowth. The hydrophobicity of parental spores was retained throughout most of the germination period. Hydrophobicity was lost as outgrowing spores entered into the stage of vegetative growth. The data indicate that gramicidin S is responsible for the hydrophobicity of B. brevis spores. It is suggested that in making spores hydrophobic, the antibiotic plays a role in concentrating the spores at interfaces where there is a higher probability of finding nutrients for germination and growth.Abbreviation GS Gramicidin S