Menaquinone Function in the Electron Transport System of an Antibiotic-producing Strain of Bacillus brevis

Abstract

Bacillus brevis ATCC I 0068 grown under appropriate growth conditions produces a mixture of tyrocidine and gramicidin, which are potent inhibitors of electron transport and oxidative phosphorylation processes. We have recently characterized the electron-transport system of this strain and have identified a menaquinone (MK-7) by thin-layer chromatography and by U.V. and mass spectroscopy to be the sole quinone component (Fynn, Thomas & Seddon, 1972). Pentane extraction (Szarkowska, I 966) and acetone extraction (Fynn & Redfearn, 1964) were used to remove the endogenous quinone in an attempt to confirm a functional role for MK-7 in the electron transport system. Although these methods were successful in removing 90 to 95 % of the quinone and largely abolishing the NADH oxidase activity of the preparation, the addition of MK-7 in various carrier systems failed to restore the latter activity. However, from the literature it is clear that while restoration of succinoxidase activity of acetone-extracted preparations has been successfully demonstrated, the reactivation of the NADH oxidase pathway has been more difficult to achieve. Also the pentane extraction technique suffers from the disadvantage that it can only be successfully employed to extract lyophilized material. The lyophilization process itself involves some structural damage to the electron transport particle since the NADH oxidase activity falls to 40 to 60 % of its original value. We now report on the U.V. irradiation of fresh material as a means of inactivating the endogenous menaquinone and providing the basis for a further investigation of quinone function in the organism. METHODS U.V. irradiation. Irradiation with 364 nm light was most effective in abolishing the NADH oxidase activity of the freshly prepared 105 P material : shorter wavelength (254 nm) was less effective and produced a general inactivation of the enzyme activities of the preparation. Irradiation was carried out with a 125 W Phillips 57236 F/70 mercury arc source placed at a distance of 10 cm above a Petri dish containing a 3 m~ depth of 105 P preparation in tris-HC1 buffer (0.1 M, p H 7.4). The preparation was stirred on ice by means of a magnetic stirrer and kept covered with a borosilicate glass plate to avoid evaporation losses.Quinone/phospholipid rnicelles. The appropriate phospholipid (see Results) to give a final concentration of 15 mg/ml was dissolved in a minimum volume of chloroform and the calculated volume of a I O -~ M-ethanolic solution of quinone added. The mixture was evaporated to dryness on a rotary evaporator at 20 "C and resuspended in 0.01 M, pH 7.8, tris-EDTA buffer. The suspension was sonicated for 15 min at o "C using a MSE-Mullard type 4200 60 W ultrasonic disintegrator fitted with a stainless-steel vibrator probe of 6 mm end diameter and 9: I end ratio. The sonicated material was centrifuged at Iooooog for 45 min and any insoluble residue discarded.

The Effect of Inhibitors on the Electron-transport Chain of Bacillus brevis. Evidence for Branching of the NADH Oxidase Respiratory Chain

“…The level of tyrocidine or tyrothricin inhibition with the NADH-linked oxidation must occur prior to any merging of pathways with succinate oxidation, otherwise succinate oxidation would be inhibited to the same extent as NADH oxidation (see below). Although the results are not unequivocal, the following electrontransport sequence for the NADH oxidase chain of B. brevis is proposed from the results presented here and in earlier reports (Fynn, 1973 …”

Role of Menaquinone in Nitrate Respiration in Staphylococcus aureus

“…Menaquinones or vitamins K2 are components of the respiratory chain in many bacteria (1,3,6,7,11,12,16). They play an essential role in gram-positive bacteria, where ubiquinone is absent (1,7,11). When both quinones are present (e.g., Escherichia coli and Proteus vulgaris, cf.…”