Replacement of Amino Acid Sequence Features of a- and c-Subunits of ATP Synthases of Alkaliphilic Bacillus with the Bacillus Consensus Sequence Results in Defective Oxidative Phosphorylation and Non-fermentative Growth at pH 10.5

Zhenxiong Wang,David B Hicks,Arthur A Guffanti,Katisha Baldwin,Terry Ann Krulwich

doi:10.1074/jbc.m401206200

Abstract

Mitchell's (Mitchell, P. (1961) Nature 191, 144-148) chemiosmotic model of energy coupling posits a bulk electrochemical proton gradient (Deltap) as the sole driving force for proton-coupled ATP synthesis via oxidative phosphorylation (OXPHOS) and for other bioenergetic work. Two properties of proton-coupled OXPHOS by alkaliphilic Bacillus species pose a challenge to this tenet: robust ATP synthesis at pH 10.5 that does not correlate with the magnitude of the Deltap and the failure of artificially imposed potentials to substitute for respiration-generated potentials in energizing ATP synthesis at high pH (Krulwich, T. (1995) Mol. Microbiol. 15, 403-410). Here we show that these properties, in alkaliphilic Bacillus pseudofirmus OF4, depend upon alkaliphile-specific features in the proton pathway through the a- and c-subunits of ATP synthase. Site-directed changes were made in six such features to the corresponding sequence in Bacillus megaterium, which reflects the consensus sequence for non-alkaliphilic Bacillus. Five of the six single mutants assembled an active ATPase/ATP synthase, and four of these mutants exhibited a specific defect in non-fermentative growth at high pH. Most of these mutants lost the ability to generate the high phosphorylation potentials at low bulk Deltap that are characteristic of alkaliphiles. The aLys(180) and aGly(212) residues that are predicted to be in the proton uptake pathway of the a-subunit were specifically implicated in pH-dependent restriction of proton flux through the ATP synthase to and from the bulk phase. The evidence included greatly enhanced ATP synthesis in response to an artificially imposed potential at high pH. The findings demonstrate that the ATP synthase of extreme alkaliphiles has special features that are required for non-fermentative growth and OXPHOS at high pH.

Highlights

Aerobic organisms maximize catabolic energy conservation by carrying out oxidative phosphorylation (OXPHOS).1 Energy stored in NADH or FADH2
Two properties of proton-coupled OXPHOS by alkaliphilic Bacillus species pose a challenge to this tenet: robust ATP synthesis at pH 10.5 that does not correlate with the magnitude of the ⌬p and the failure of artificially imposed potentials to substitute for respirationgenerated potentials in energizing ATP synthesis at high pH (Krulwich, T. (1995) Mol
The alignments used to identify these features included the two alkaliphiles that were in the earlier alignments, B. pseudofirmus OF4 and Bacillus alcalophilus, both of which are categorized as extreme alkaliphiles because they can grow at pH values of 11 and above [13, 39]; only partial sequence of the a-subunit is available for B. alcalophilus

Summary

Introduction

Aerobic organisms maximize catabolic energy conservation by carrying out OXPHOS. Energy stored in NADH or FADH2. Robust Hϩ-coupled OXPHOS by extremely alkaliphilic Bacillus strains growing on non-fermentable carbon sources at external pH values Ն10.5 poses one of the most striking challenges to the strictly bulk energization model (10 –13). At such pH values, maintenance of a cytoplasmic pH that is much lower than the external pH, i.e. a ⌬pH that is acid in, lowers the total chemiosmotic driving force, and yet OXPHOS proceeds optimally [10, 13]. ⌬Gp, phosphorylation potential; ⌬p, transmembrane electrochemical proton gradient; ⌬pH, transmembrane pH gradient; ⌬⌿, transmembrane electrical potential; DCCD, dicyclohexylcarbodiimide; F0-ATP synthase, membrane-embedded sector of the F1F0-ATP synthase; TMH, transmembrane helix; MOPS, 4-morpholinepropanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine

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