title: Maintenance of DeltapH by a butanol-tolerant mutant of Clostridium beijerinckii.
abstract: The isolation of Clostridium beijerinckii mutants that are more tolerant of butanol than the wild-type offered the opportunity to investigate whether the membrane activities which are required for maintaining the transmembrane DeltapH (the difference in pH between the cellular interior and exterior) are sensitive targets of butanol toxicity. The DeltapH was measured by the accumulation of [14C]benzoate using late-exponential-phase cells which were suspended in citrate/phosphate buffer at pH 5 (to maximize the DeltapH component of the protonmotive force) and supplemented with glucose and Mg2+. The DeltapH of the butanol-tolerant tolerant mutant, strain BR54, of C. beijerinckii NCIMB 8052 was found to be significantly more tolerant of added butanol than the wild-type. Thus, in potassium citrate/phosphate buffer the mutant cells maintained a DeltapH of 1.4 when butanol was added to a concentration of 1.5 % (w/v), while the wild-type DeltapH was reduced to 0.1. The DeltapH of both strains was completely dissipated with 1.75 % butanol, an effect attributed to a chaotropic effect on the membrane phospholipids. Similar results were obtained in sodium citrate/phosphate buffer. In the absence of added Mg2+, the DeltapH of the mutant decreased in both sodium and potassium citrate/phosphate buffer, but more rapidly in the former. Interestingly, the addition of butanol at low concentrations (0.8 %) prevented this DeltapH dissipation, but only in cells suspended in sodium citrate/phosphate buffer, and not in potassium citrate/phosphate buffer. In wild-type cells the decrease in DeltapH occurred more slowly than in the mutant, and sparing of the DeltapH by 0.8 % butanol was less pronounced. The authors interpret these data to mean that the DeltapH is dissipated in the absence of Mg2+ by a Na+- or K+-linked process, possibly by a Na+/H+ or a K+/H+ antiporter, and that the former is inhibited by butanol. Apparently, butanol can selectively affect a membrane-associated function at concentrations lower than required for the complete dissipation of transmembrane ion gradients. Additionally, since the butanol-tolerant mutant BR54 is deficient in the ability to detoxify methylglyoxal (MG) and contains higher levels of MG than the wild-type, the higher Na+/H+ antiporter activity of the mutant may be due to the greater degree of protein glycation by MG in the mutant cells. The mechanism of butanol tolerance may be an indirect result of the elevated glycation of cell proteins in the mutant strain. Analysis of membrane protein fractions revealed that mutant cells contained significantly lower levels of unmodified arginine residues than those of the wild-type cells, and that unmodified arginine residues of the wild-type were decreased by exposure of the growing cells to added MG.
authors.affiliation: Department of Microbiology, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118-2526, USA.
authors.forename: Fanqiang
authors.initials: F
authors.lastname: Wang
authors.affiliation: The Forsyth Institute, 140 The Fenway, Boston, MA 02115, USA.
authors.forename: Shelby
authors.initials: S
authors.lastname: Kashket
authors.affiliation: Department of Microbiology, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118-2526, USA.
authors.forename: Eva R
authors.initials: ER
authors.lastname: Kashket
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D000440:Butanols / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects; D002463:Cell Membrane Permeability; D046970:Clostridium beijerinckii / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D003470:Culture Media; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D009154:Mutation; D011188:Potassium; D012964:Sodium
chemical_list: D001426:Bacterial Proteins; D000440:Butanols; D003470:Culture Media; D008565:Membrane Proteins; D012964:Sodium; D008274:Magnesium; D011188:Potassium