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Clostridium pasteurianum F1Fo ATP synthase: operon, composition, and some properties.

ABSTRACT: The atp operon encoding F1Fo ATP synthase in the fermentative obligate anaerobic bacterium Clostridium pasteurianum was sequenced. It consisted of nine genes arranged in the order atpI(i), atpB(a), atpE(c), atpF(b), atpH(delta), atpA(alpha), atpG(gamma), atpD(beta), and atpC(epsilon), which was identical to that found in many bacteria. Reverse transcription-PCR confirmed the presence of the transcripts of all nine genes. The amount of ATPase activity in the membranes of C. pasteurianum was low compared to what has been found in many other bacteria. The F1Fo complexes solubilized from membranes of C. pasteurianum and Escherichia coli had similar masses, suggesting similar compositions for the F1Fo complexes from the two bacteria. Western blotting experiments with antibodies raised against the purified subunits of F1Fo detected the presence of eight subunits, alpha, beta, gamma, delta, epsilon, a, b, and c, in the F1Fo complex from C. pasteurianum. The F1Fo complex from C. pasteurianum was activated by thiocyanate, cyanate, or sulfhydryl compounds; inhibited by sulfite, bisulfite, or bicarbonate; and had tolerance to inhibition by dicyclohexylcarbodiimide. The target of thiol activation of the F1Fo complex from C. pasteurianum was F1. Thiocyanate and sulfite were noncompetitive with respect to substrate Mg ATP but competitive with respect to each other. The F1 and Fo parts of the F1Fo complexes from C. pasteurianum and E. coli bound to each other, but the hybrid F1Fo complexes were not functionally active.

SUBMITTER: Das A

PROVIDER: S-EPMC193768 | biostudies-literature | 2003 Sep

REPOSITORIES: biostudies-literature

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Clostridium pasteurianum F1Fo ATP synthase: operon, composition, and some properties.

Das Amaresh A Ljungdahl Lars G LG

Journal of bacteriology 20030901 18

The atp operon encoding F1Fo ATP synthase in the fermentative obligate anaerobic bacterium Clostridium pasteurianum was sequenced. It consisted of nine genes arranged in the order atpI(i), atpB(a), atpE(c), atpF(b), atpH(delta), atpA(alpha), atpG(gamma), atpD(beta), and atpC(epsilon), which was identical to that found in many bacteria. Reverse transcription-PCR confirmed the presence of the transcripts of all nine genes. The amount of ATPase activity in the membranes of C. pasteurianum was low c ...[more]

PMID: 12949105

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Project description:The F(1)F(o)-ATP synthases of alkaliphilic bacteria exhibit latent ATPase activity, and for the thermoalkaliphile Bacillus sp. strain TA2.A1, this activity is intrinsic to the F(1) moiety. To study the mechanism of ATPase inhibition, we developed a heterologous expression system in Escherichia coli to produce TA2F(1) complexes from this thermoalkaliphile. Like the native F(1)F(o)-ATP synthase, the recombinant TA2F(1) was blocked in ATP hydrolysis activity, and this activity was stimulated by the detergent lauryldimethylamine oxide. To determine if the C-terminal domain of the epsilon subunit acts as an inhibitor of ATPase activity and if an electrostatic interaction plays a role, a TA2F(1) mutant with either a truncated epsilon subunit [i.e., TA2F(1)(epsilon(DeltaC))] or substitution of basic residues in the second alpha-helix of epsilon with nonpolar alanines [i.e., TA2F(1)(epsilon(6A))] was constructed. Both mutants showed ATP hydrolysis activity at low and high concentrations of ATP. Treatment of the purified F(1)F(o)-ATP synthase and TA2F(1)(epsilon(WT)) complex with proteases revealed that the epsilon subunit was resistant to proteolytic digestion. In contrast, the epsilon subunit of TA2F(1)(epsilon(6A)) was completely degraded by trypsin, indicating that the C-terminal arm was in a conformation where it was no longer protected from proteolytic digestion. In addition, ATPase activity was not further activated by protease treatment when compared to the untreated control, supporting the observation that epsilon was responsible for inhibition of ATPase activity. To study the effect of the alanine substitutions in the epsilon subunit in the entire holoenzyme, we reconstituted recombinant TA2F(1) complexes with F(1)-stripped native membranes of strain TA2.A1. The reconstituted TA2F(o)F(1)(epsilon(WT)) was blocked in ATP hydrolysis and exhibited low levels of ATP-driven proton pumping consistent with the F(1)F(o)-ATP synthase in native membranes. Reconstituted TA2F(o)F(1)(epsilon(6A)) exhibited ATPase activity that correlated with increased ATP-driven proton pumping, confirming that the epsilon subunit also inhibits ATPase activity of TA2F(o)F(1).

Dataset Information

Clostridium pasteurianum F1Fo ATP synthase: operon, composition, and some properties.

Publications

Clostridium pasteurianum F1Fo ATP synthase: operon, composition, and some properties.

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