The F[1]F[O]-ATP synthase found in bacteria, mitochondria, and chloroplasts is responsible for synthesizing ATP from the precursors ADP and P[i]. In bacteria, the enzyme is used to synthesize ATP when the electrochemical gradient of protons ([Delta][mu]H⁺) or sodium ions ([Delta][mu]Na⁺) is high, and the phosphorylation potential is low inside the cell. Conversely, the enzyme hydrolyses ATP to generate a [Delta][mu]H⁺ or [Delta][mu]Na⁺ when these gradients are low, and the phosphorylation potential is high inside the cell. This is coupled to intracellular pH homeostasis in some fermentative bacteria. The aim of this project was to determine the role(s) of the F[1]F[O]-ATP synthase in the physiology of two anaerobic extremophiles: Clostridium paradoxum, a thermoalkaliphilic bacterium that grows over the pH range 7.5 to 10, and Thermotoga maritima, a hyperthermophilic bacterium that thrives at neutral to acidic pH. These bacteria represent good models to study membrane-bound energetic processes as a function of pH and temperature.
The energetics of C. paradoxum growth was investigated in batch culture. Growth of C. paradoxum was inhibited by the F-type ATP synthase inhibitor N,N�-dicyclohexylcarbodiimide (DCCD) and monensin, suggesting an important role for an F-type ATP synthase and a chemical gradient of sodium ([Delta]pNa⁺) in the growth of this bacterium. Protonophores had no effect on the growth of C. paradoxum. Inverted membrane vesicles contained DCCD-sensitive ATPase activity and this activity was extracted using the detergent Triton X-100. The solubilized enzyme was purified 30-fold by polyethylene glycol-6000 precipitation. The purified enzyme displayed the typical subunit pattern for an F[1]F[O]-ATP synthase, but also included the presence of a stable oligomeric c-ring that could be dissociated by trichloroacetic acid treatment into its monomeric c subunits. The c-ring was purified, crystallized in 2D, and the projection map indicated that C. paradoxum contains an undecameric c-ring. The purified ATPase was stimulated by Na⁺ ions, and sodium provided protection against inhibition by DCCD that was pH-dependent. ATP synthesis in inverted membrane vesicles was driven by an artificially imposed [Delta]pNa⁺ in the presence of a transmembrane electrical potential ([Delta][phi]) and was sensitive to monensin. Cloning and sequencing of the atp operon revealed the presence of a sodium-binding motif in the membrane-bound c subunit (viz., Q�⁸, E⁶�, and S⁶�). On the basis of these properties, the ATPase is a sodium-translocating enzyme that generates a [Delta][mu]Na⁺ that could be used to drive other membrane-bound bioenergetic processes (e.g., solute transport or flagellar rotation). In support of this proposal are the low rates of ATP synthesis catalyzed by the enzyme and the lack of the C-terminal region of the [epsilon] subunit that has been shown to be essential for coupled ATP synthesis. To facilitate future work on this enzyme at a molecular level, we developed a heterologous over-expression/production system to produce the ATPase in E. coli DK8 ([Delta]unc) in the presence of the helper plasmid pLysRARE. In future studies, this system will be used for the purification of mutant enzyme complexes.
Thermotoga maritima is an anaerobic hyperthermophilic bacterium that grows at 80�C and pH 7.0. Growth is sensitive to DCCD and monensin, but resistant to protonophores. These data suggest a [Delta]pNa⁺ is indispensable for growth and a F-type ATP synthase plays an important role under these conditions. The ATPase activity was extracted from inverted membrane vesicles using the detergent n-dodecyl β-D-maltoside. To investigate the role of the ATPase, the enzyme complex was purified 13-fold by polyethylene glycol-6000 precipitation, anionic exchange, and gel filtration chromatography. The purified enzyme was stimulated in the presence of low concentrations of Na⁺ ions, and ATP-dependent uptake of ��Na⁺ into inverted membrane vesicles was observed to be sensitive to DCCD and monensin. Furthermore, analysis of the published genome sequence revealed the presence of a sodium-binding motif in the membrane-bound c subunit (viz., E��, E⁶⁵ and T⁶⁶) and analysis of the ATPase by SDS-PAGE revealed the presence of a putative SDS-stable c-ring. On the basis of these properties, the ATPase from T. maritima appears to be a sodium-translocating enzyme. While we were unable to establish a physiological role for the ATPase, we propose that the enzyme generates a [Delta][mu]Na⁺ that could be used to drive other membrane-bound bioenergetic processes (e.g., solute transport or flagellar rotation). To improve purification and yield, the atp operon was cloned to allow the heterologous over-production of the ATPase. No expression of the ATPase (F[1]F[O]) could be achieved in E. coli BL21(DE3)-CodonPlus, C41(DE3) or E. coli DK8 ([Delta]unc). However, the overproduction of the F₁-ATPase utilizing the helper plasmid pLysRARE was succesful and the enzyme was purified 18-fold by heat precipitation, followed by anionic exchange and gel filtration chromatography, yielding a specific activity of 2.5 U/mg. This complex will be used in the future to investigate the biochemical properties of an F₁-ATPase from a hyperthermophilic anaerobe.
| Identifer | oai:union.ndltd.org:ADTP/217809 |
| Date | January 2007 |
| Creators | Ferguson, Scott A, n/a |
| Publisher | University of Otago. Department of Microbiology & Immunology |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright Scott A Ferguson |
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