Nigericin-induced Death of an Acidophilic Bacterium

Abstract

201At an external pH of 3.5, nigericin (which catalyses an electroneutral H+/K+ exchange) abolished the transmembrane proton gradient (ApH) of Bacillus acidocaldarius, causing a rapid acidification of the cytoplasm from approximately pH 6.0 to pH 3.5. A pronounced loss of viability and fine-structural changes rapidly followed treatment with nigericin. A marked decline in respiration and an even more rapid decrease in cytoplasmic ATP were observed. Activity of at least one cytoplasmic enzyme decreased more slowly. There was no generalized loss in the integrity of the cytoplasmic membrane, as assayed by permeability to inulin or Na+ or by release of ultraviolet light-absorbing compounds. The loss of viability upon treatment with carbonyl cyanide m-chlorophenylhydrazone was similar to that observed with nigericin, so proton influx alone, rather than together with K+ efflux, was probably involved in the death of the organism. Moreover, acidification of the cytoplasm rather than abolition of the ApH was the lethal event, since no loss of viability was observed when the ApH was abolished by elevation of the external pH. I N T R O D U C T I O NIn accord with Mitchell's chemiosmotic hypothesis (196 1, 1963), bacteria generate, via respiration and/or ATP hydrolysis, a protonmotive force generally consisting of a transmembrane pH gradient (ApH, exterior acid) and a transmembrane electrical potential (A@, exterior positive). The protonmotive force, in turn, energizes a variety of energyrequiring processes, as reviewed by Harold (1977). When we became interested in the protonmotive force in acidophilic bacteria, it was with the expectation that such organisms would maintain extremely large pH gradients across the membrane. Thus, bacteria which could grow at external pH values of 2 to 3 would presumably require cytoplasmic pH values much closer to neutrality in order to maintain viability. The consistently large ApH resulting from this requirement would, moreover, probably preclude the existence of any appreciable A~, exterior positive. In fact, the occurrence of a 'reversed' A+, i.e. interior positive, seemed a likely adaptation in such organisms.Our own studies with Bacillus acidocaldarius (Krulwich et al., 1978) and those of others with different acidophiles (Hsung & Haug, 1975, 1977Searcy, 1976) have indeed shown that such organisms maintain a cytoplasmic pH of greater than 5, thus generating large ApH values, and concomitantly exhibiting reversed A+ values. The assumption that organisms would need to maintain cytoplasmic pH values much closer to neutrality than a highly acidic milieu appeared validated. Thus, if the large ApH in an acidophile such as B. acidocaldarius were abolished so that rapid acidification of the cytoplasm occurred, viability of the organism would be threatened. In the study presented here, nigericin was used to abolish the ApH in B. acidocazdarius and the ensuing death of the organism was monitored and characterized. The results indicated that the constraints imposed by a lower cytoplasmic pH limit...

Regulation of cytoplasmic pH in bacteria.

“…Initial studies on a range of acidophiles suggested that uncouplers and inhibitors of respiration did not cause the complete dissipation of the pH gradient (67,73). However, transport of solutes via H+-linked systems was inhibited (39). The maintenance of ApH in the presence of uncouplers has been attributed to either limited cation permeability across the membranes of acidophiles or high cytoplasmic buffering capacity in the cells (126,127).…”

“…If these cations were to be exchanged for H+, this would result in the buffering capacity being outstripped. Indeed, there is evidence that, when K+-H+ exchange is stimulated by the addition of nigericin at acid pH, acidification of the cytoplasm occurs to an extent that causes denaturation of the proteins (39). Thus the intrinsic cation impermeability of the membrane of acidophiles may be important in protection against excessive acidification.…”

Energy conservation in acidophilic bacteria.

“…Cells treated with 2,4-dinitrophenol were used as a control to determine the extent of nonspecific binding of aspirin, and the extent of this nonspecific binding was not stated (72). It is, however, clear that the action of either uncouplers or the antibiotic nigericin (which catalyzes H+/Na+ or H+/K+ exchange) can result in a collapse of ApH of sufficient magnitude to cause disorganization of cell contents and complete loss of cell viability (45). Oshima et al (105) have also measured l\pH in B. acidocaldarius, but since they used DMO as a probe their data are open to question.…”

Acidification of intracellular pH results in neutral pH-sensitive growth of an alkalophilic Bacillus sp.

“…Acidification of the pH;n in the absence of K+ or Rb+ implies that these cations have roles in pHin regulation at neutral pH (see Table 1). It has been reported that the acidification of pHin inhibits the growth of E. coli (2) and other bacteria (5). But in obligatory alkalophilic bacteria the explanation for the failure to grow at neutral pH is not the excessive acidification of the pHin but an inadequate ability of the respiratory chain to generate a dfH at neutral pH (2,9,(17)(18)(19).…”