PROTONOPHORE-RESISTANCE AND CYTOCHROME EXPRESSION IN MUTANT STRAINS OF THE FACULTATIVE ALKALIPHILE BACILLUS-FIRMUS OF4

Two protonophore-resistant mutants, designated strains CC1 and CC2, of the facultative alkaliphile Bacillus firmus OF4 811M were isolated. The ability of carbonyl cyanide m-chlorophenylhydrazone (CCCP) to collapse the protonmotive force (DELTA-mu-BAR(H+)) was unimpaired in both mutants. Both resistant strains possessed elevated respiratory rates when grown at pH 7.5, in either the presence or absence of CCCP. Membrane cytochromes were also elevated: cytochrome o in particular in strain CC1, and cytochromes aa3, b, c and o in strain CC2. Strain CC2 also maintained a higher-DELTA-mu-BAR(H+) than the others when grown in the absence of CCCP. When grown in the presence of low concentrations of CCCP, strains CC1 and CC2 both maintained higher values of DELTA-mu-BAR(H+) than the wild-type parent and correspondingly higher capacities for ATP synthesis. In large-scale batch culture at pH 10.5, both mutant strains grew more slowly than the parent and contained significantly reduced levels of cytochrome o. Cells of strain CC1 also displayed a markedly altered membrane lipid composition when grown at pH 10.5. Unlike previously characterized protonophore-resistant strains of B. subtilis and B. megaterium, neither B. firmus mutant possessed any ability above that of the parent strain to synthesize ATP at given suboptimal values of DELTA-mu-BAR(H+). Instead, both resistant alkaliphile strains maintained a higher DELTA-mu-BAR(H+) and a correspondingly higher DELTA-G(p) than the parent strain when growing in sublethal concentrations of CCCP, apparently as a result of mutational changes affecting respiratory chain composition. Also of note in both the mutant and the wild-type strains was a marked elevation in the level of one of the multiple terminal oxidases, an aa3-type cytochrome, during growth at pH 7.5 in the presence of CCCP or during growth at pH 10.5, i.e. two conditions that reduce the bulk DELTA-mu-BAR(H+).