Isolation and characterization of uncoupler-resistant mutants of Bacillus subtilis

Abstract

Three mutant strains of Bacillus subtilis were isolated on the basis of their ability to grow in the presence of 5 ,IM carbonyl cyanide m-chlorophenylhydrazone (CCCP). The mutants (AG2A, AG1A3, and AG3A) were also resistant to 2,4-dinitrophenol, and AG2A exhibited resistance to tributyltin and neomycin. The mutants all exhibited (i) elevated levels of membrane ATPase activity relative to the wild type; (ii) slightly elevated respiratory rates, with the cytochrome contents of the membranes being the same as or slightly lower than those of the wild type; (3) a passive membrane permeability to protons that was indistinguishable from that of the wild type in the absence of CCCP and that was increased by addition of CCCP to the same extent as observed with the wild type; and (4) an enhanced sensitivity to valinomycin with respect to the ability of the ionophore to reduce the transmembrane electrical potential. Finally and importantly, starved whole cells of all the mutants synthesized more ATP than the wild type did upon energization in the presence of any one of several agents that lowered the proton motive force. Studies of revertants indicated that the phenotype resulted from a single mutation. Since a mutation in the coupling membrane might produce such pleiotropic effects, an analysis of the membrane lipids was undertaken with preparations made from cells grown in the absence of CCCP. The membrane lipids of the uncoupler-resistant strains differed from those of the wild type in having reduced amounts of monounsaturated C16 fatty acids and increased ratios of iso/anteiso branches on the C15 fatty acids. Correlations between protonophore resistance and the membrane lipid compositions of the wild type, mutants, and revertants were most consistent with the hypothesis that a reduction in the content of monounsaturated C16 fatty acids in the membrane phospholipids is related, perhaps causally, to the ability to synthesize ATP at low bulk transmembrane electrochemical gradients of protons.During the past 15 years, the chemiosmotic model of energy coupling that had been advanced by Mitchell (28) almost 10 years earlier has achieved widespread confirmation and acceptance. According to this model, the energy made available by photosynthetic light capture, oxidation of substrates by the respiratory chain, or ATP hydrolysis by the membrane-associated F,FO-ATPase is transduced via an intermediary electrochemical proton gradient (4UH+). That is, these energy-producing processes are all associated with proton translocation across a relatively proton-impermeant coupling membrane such that a transmembrane pH gradient (ApH) and transmembrane electrical potential (AJi) are produced. In a bacterial cell or mitochondrion, the orientation of the 4iiiH+ is outside positive and acid relative to the interior of the cell or organelle. Completing the energetic circuit are the energy-consuming processes that are catalyzed by integral membrane porters or enzymes. These processes, e.g., ATP synthesis and ion-coupled solute uptake, utilize a...

Mutants of Bacillus species isolated on the basis of protonophore resistance are deficient in fatty acid desaturase activity

“…Palmitoyl-CoA was preferred to stearoyl-CoA as the substrate for the fatty acid desaturase of B. subtilis. Consistently, the only monounsaturated fatty acid esterified in the membrane phospholipids is C16:1 (15). This contrasts with the observation of the stearyl-CoA desaturase of eukaryotes (25), for which stearyl-CoA is the preferred substrate.…”

“…These temperature-sensitive, protonophore-resistant strains thus showed temperature-sensitive desaturase activity that correlated inversely with their pattern of protonophore resistance. Full revertants, which had previously been shown to have restored levels of C16:1 fatty acids (15), also exhibited restored levels of desaturase activity at both temperatures and had lost the ability to grow at either temperature in the presence of the protonophore. All strains were grown at 300C to late log phase.…”

“…The mutants did not, however, inactivate or exclude the protonophore (15). Like a similar mutant strain of Bacillus megaterium described by Decker and Lang (4, 5), these strains synthesized more ATP than the wild type at submaximal levels of the proton motive force (5,15,17). All of the mutants had reduced unsaturated fatty acid content in their membrane phospholipids relative to wild-type B. subtilis and B. megaterium.…”

“…Three protonophore-resistant strains of Bacillus subtilis that were isolated in our laboratory grew on nonfermentable carbon sources in the presence of protonophore concentrations that completely inhibited growth of the wild type (15). The mutants did not, however, inactivate or exclude the protonophore (15).…”

“…The conversion of radioactive palmitoyl-coenzyme A (palmitoyl-CoA) to a monounsaturated product has been used previously for assays of the fatty acid desaturase of alkaliphilic Bacillus extracts (8) Isolation of temperature-sensitive revertants. B. subtilis AG1A3 and AG2A were grown to late log phase, and 10 ml of these cultures was subjected to ethyl methanesulfonate mutagenesis (15) in standard growth medium (24 Locant of the double bond. 13C nuclear magnetic resonance and gas chromatography-mass spectroscopy was used to determine the double-bond positions of the monounsaturated fatty acids (11).…”