The Na+-motive terminal oxidase activity in an alkalo- and halo-tolerant Bacillus

Abstract

An alkalo- and halo-tolerant aerobic microorganism has been isolated which, according to microbiological analysis data and the ribosomal 5S RNA sequence, is a Bacillus similar, but not identical, to B. licheniformis and B. subtilis. The microorganism, called Bacillus FTU, proved to be resistant to the protonophorous uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). The fast growth of Bacillus FTU in the presence of CCCP was shown to require a high Na+ concentration in the medium. A procedure was developed to exhaust endogenous respiratory substrates in Bacillus FTU cells so that fast oxygen consumption by the cells was observed only when an exogenous respiratory substrate was added. The exhausted cells were found to oxidize ascorbate in the presence of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) in a cyanide-sensitive fashion. The ascorbate oxidation was coupled to the uphill Na+ extrusion which was stimulated by CCCP and a penetrating weak base, diethylamine, as well as by valinomycin with or without diethylamine. Operation of the Bacillus FTU terminal oxidase resulted in the generation of a delta psi which, in the Na+ medium, was slightly decreased by CCCP and strongly decreased by CCCP + diethylamine. In the K+ medium, CCCP discharged delta psi even without diethylamine. Ascorbate oxidation was competent in ATP synthesis which was resistant to CCCP in the Na+ medium and sensitive to CCCP in the K+ medium as if Na+- and H+-coupled oxidative phosphorylations were operative in the Na+ and K+ media, respectively. Inside-out subcellular vesicles of Bacillus FTU were found to be competent in the Na+ uptake supported by oxidation of ascorbate + TMPD or diaminodurene. CCCP or valinomycin + K+ increased the Na+ uptake very strongly. The process was completely inhibited by cyanide or monensin, the former, but not the latter, being inhibitory for respiration. The data obtained indicate that in Bacillus FTU there is not only H+-motive but also Na+-motive terminal oxidase activity.