R. Barraclough, S. Laland
Aug 1, 1975
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Journal
Biochemical Society transactions
Abstract
which is synthesised by Bacillus brevis A.T.C.C. 9999 (Laland & Zimmer, 1973). It is synthesized by the enzyme gramicidin S synthetase, which activates and forms thioester bonds with each amino acid before peptide-bond formation (Gevers et al., 1969). Gramicidin S synthetase consists of two enzymes; the smaller, of mol. wt. 1OOOOO (Yamada & Kurahashi, 1969), activates and forms a thioester bond with D-phenylalanine (Itoh et al., 1968); the secondand largerenzymehasmol. wt. 280000(Kleinkaufet al., 1969), andactivates and forms a thioester bond withL-proline, L-valine,L-ornithineand L-leucine (Itoh et al., 1968). Pentapeptide units (D-Phe-L-Pro-L-Val-L-orn-L-Leu) are formed, and two pentapeptides, probably synthesized by the same enzyme molecule, dimerize in a head-to-tail manner to form the cyclic decapeptide (Stoll et al., 1970). Gramicidin S synthetase may be assayed in at least two different ways. The first is by measuring the synthesis of gramicidin S; this requires the presence of both large and small enzyme components. The second assay involves the measurement of an ATP-PP, exchange in the presence of D-phenylalanine (which is a reaction given by the light enzyme and not by the heavy enzyme), or in the presence of L-ornithine (which is a reaction given by the heavy enzyme but not by the light enzyme). Early experiments showed that cell-free extracts of Bacillus brevis could only synthesize gramicidin S during the late exponential phase of growth and that the activity of synthetase declined as the cells moved into the stationary phase (Laland & Zimmer, 1973). In order to begin a study of factors controlling the onset of gramicidin S production, experiments were undertaken to find out whether the activities of both light and heavy components of gramicidin S synthetase increased concomitantly with the ability to synthesize gramicidin S. Bacillus brevis A.T.C.C. 9999 was grown in a 10-litre New Brunswick Laboratory Fermenter at 37°C as described previously (Bredesen et al., 1968). At various times, culture samples of 500ml and lOml were withdrawn and rapidly cooled. The lOml samples were used for determination of the total gramkidins in the culture, by a modification of the method described by Eikhom et al. (1963). Cells from the 500ml samples were harvested and cell-free extracts prepared by treatment of the cells with lysozyme and deoxyribonuclease. Cell debris was removed by centrifuging for 20min at 11 OOOg,,.. The supernatants were dialysed overnight against 0.05M-potassium phosphate buffer, pH7.6, containing 0.25 m~-EDTA, 1 mM-dithiothreitol and 20 %(v/v) glycerol, and samples were assayed for gramicidin S synthetase activity as described previously (Freryshov et al., 1970). The light and heavy enzymes of gramicidin S synthetase were assayed by measuring ATP-PP, exchange reactions in the presence of D-phenylalanine or L-ornithine by a method similar to that described by Simlot & Pfaender (1973). Controls, in which amino acid was omitted, were set up. Fig. 1 shows that the gramicidin S synthetase activity of the crude extracts increased when they were prepared from samples taken during the exponential phase of growth. This increase in activity was accompanied by an increase in the activities characteristic of both the light and heavy components. This result is a direct confirmation of a result inferred by Kambe et al. (1974). The onset of gramicidin S synthesis in Bacillus brevis is dependent on the simultaneous appearance of both components. I I