Uncoupler Of Proton Motive Force Research Articles

The Acid-Base Equilibrium of Pyrazinoic Acid Drives the pH Dependence of Pyrazinamide-Induced Mycobacterium tuberculosis Growth Inhibition.

Pyrazinamide, a first-line antibiotic used against Mycobacterium tuberculosis, has been shown to act in a pH-dependent manner in vitro. Why pyrazinamide, an antitubercle prodrug discovered more than 65 years ago, exhibits this pH-dependent activity was unclear. Upon entering mycobacterial cells, pyrazinamide is deamidated to pyrazinoate by an enzymatic process and exists in an acid-base equilibrium with pyrazinoic acid. Thus, the effects of total pyrazinoic acid (pyrazinoic acid + pyrazinoate) on M. tuberculosis growth, pH homeostasis, and proton motive force over a range of pH values found in host tissues were investigated. Although M. tuberculosis was able to maintain pH homeostasis over an external pH range of 7.0 to 5.5, total pyrazinoic acid induced growth inhibition increased as culture medium pH was decreased from 7.3 to 6.4. Consistent with growth inhibition, total pyrazinoic acid increased both acidification of the bacterial cytoplasm and dissipation of membrane potential as the environmental pH decreased when added to the bacterial suspensions. The results suggest pyrazinoic acid is the active form of the drug, which acts as an uncoupler of proton motive force, likely a protonophore, providing a mechanistic explanation for the pH dependence of the drug activity.

Identification of Gsr1 in Arabidopsis thaliana: A locus inferred to regulate gene expression in response to exogenous glutamine

An altered response to methylamine (MA) has been used to identify genes involved in nitrogen metabolism in many microbes and as an uncoupler of proton motive force in both prokaryotes and eukaryotes. The aim of this study was to attempt the use of MA to identify critical genes for response to exogenous nitrogen sources in Arabidopsis thaliana (thale cress). EMS mutagenesis and selection on MA were used to identify a series of mutants that showed increased sensitivity to MA but failed to identify any MA resistant mutants. The eight sensitive mutants were allelic as judged by segregation and map location so one mutant, gsr1-1, was selected for intensive analysis. Co-segregation of gsr1 with DNA markers showed the gene was on chromosome 5 between markers CA72 and nga151, an interval of about 0.42 Mbp. The co-segregating mutant phenotypes associated with gsr1 included; yellow color caused by a reduction in chloroplast number; reduced chlorophyll content; decreased nitrate reductase (EC 1.7.1.3) activity in the root; and increased glutamate dehydrogenase (EC 1.4.1.2) activity in root and shoot. Decreased transcripts included those encoding several nitrogen metabolism enzymes. In contrast, the mutation increased only 4 and 7 transcript abundances by more than two fold. RT-PCR showed that in the dark, regulation of transcript abundance by exogenously applied glutamine and to a lesser extent sucrose were altered. Gene action of the gsr1 mutant was recessive for chloroplast number but co-dominant for transcript abundances and co-dominant for enzyme activities. Supplementary data from the analysis of preliminary microarray data from AFGC used 10,560 targets and two reverse labeled slides to show that more than 351 ESTs were increased in transcript abundance by more than 2 fold in the green plants. In yellow plants only seventy-three ESTs were up-regulated in transcript abundance by more than 2 fold. Therefore, the gsr1 locus may regulate responses to endogenous and exogenous glutamine in an organ-specific fashion that could coordinately regulate the activity of genes enzymes and metabolic pathways that determine cellular nitrogen, carbon and bioenergetic (redox) status.

Export of the periplasmic NADP-containing glucose-fructose oxidoreductase of Zymomonas mobilis.

Glucose-fructose oxidoreductase (GFOR) of the gram-negative bacterium Zymomonas mobilis is a periplasmic enzyme with the tightly bound cofactor NADP. The preprotein carries an unusually long N-terminal signal sequence of 52 amino acid residues. A sorbitol-negative mutant strain (ACM3963) was found to be deficient in GFOR activity and was used for the expression of plasmid-borne copies of the wild-type gfo gene or of alleles encoding alterations in the signal sequence of the pre-GFOR protein. Z. mobilis cells with the wild-type gfo allele translocated pre-GFOR, at least partially, via the Sec pathway since CCCP (carboxylcyanide-m-chlorophenylhydrazone; uncoupler of proton motive force) or sodium azide (inhibitor of SecA) abolished the processing of GFOR. A gfo allele with the hydrophobic region of the signal sequence removed (residues 32-46; Delta32-46) led to a protein that was no longer processed, but showed full enzymatic activity (180 U/mg) and had the cofactor NADP firmly bound. A deletion in the n-region of the signal sequence (residues 2-20; Delta2-20) or exchange of the entire GFOR signal sequence with the signal sequence of gluconolactonase of Z. mobilis led to active and processed GFOR. Strain ACM3963 could not grow in the presence of high sugar concentrations (1 M sucrose) unless sorbitol was added. The presence of the plasmid-borne gfo wild-type allele or of the Delta2-20 deletion led to the restoration of growth on media with 1 M sucrose, whereas the presence of the Delta32-46 deletion led to a growth behavior similar to that of strain ACM3963, with no sorbitol formation from sucrose.