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Transcription of nhaA, the main Na(+)/H(+) antiporter of Escherichia coli, is regulated by Na(+) and growth phase.

ABSTRACT: The transcription of nhaA, encoding the main Na(+)/H(+) antiporter of Escherichia coli, is induced by Na(+), regulated by NhaR, and affected by H-NS. In this work the roles of the two nhaA promoters (P1 and P2) were studied by analysis of transcription both in vivo and in vitro and promoter mutations. We found that P1 is an NhaR-dependent, Na(+)-induced, and H-NS-affected promoter both in the exponential and stationary phases. An in vitro transcription assay demonstrated that P1 is activated by sigma(70)-RNA polymerase and both NhaR and H-NS increase the specificity of P1. Remarkably, in marked contrast to P1, P2 exhibits very low activity during the exponential phase but is induced in the stationary phase to become the major promoter. Furthermore, P2 is activated by sigma(S) and is neither induced by Na(+) nor dependent on NhaR or affected by H-NS. Hence, this work establishes that nhaA has a dual mode of regulation, each involving a different promoter, and reveals that P2 and sigma(S) together are responsible for the survival of stationary-phase cells in the presence of high Na(+), alkaline pH, and the combination of high Na(+) and alkaline pH, the most stressful condition.

SUBMITTER: Dover N

PROVIDER: S-EPMC94921 | biostudies-literature | 2001 Jan

REPOSITORIES: biostudies-literature

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Transcription of nhaA, the main Na(+)/H(+) antiporter of Escherichia coli, is regulated by Na(+) and growth phase.

Dover N N Padan E E

Journal of bacteriology 20010101 2

The transcription of nhaA, encoding the main Na(+)/H(+) antiporter of Escherichia coli, is induced by Na(+), regulated by NhaR, and affected by H-NS. In this work the roles of the two nhaA promoters (P1 and P2) were studied by analysis of transcription both in vivo and in vitro and promoter mutations. We found that P1 is an NhaR-dependent, Na(+)-induced, and H-NS-affected promoter both in the exponential and stationary phases. An in vitro transcription assay demonstrated that P1 is activated by ...[more]

PMID: 11133959

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Project description:Na+/H+ antiporters are integral membrane proteins that are present in almost every cell and in every kingdom of life. They are essential for the regulation of intracellular pH-value, Na+-concentration and cell volume. These secondary active transporters exchange sodium ions against protons via an alternating access mechanism, which is not understood in full detail. Na+/H+ antiporters show distinct species-specific transport characteristics and regulatory properties that correlate with respective physiological functions. Here we present the characterization of the Na+/H+ antiporter NhaA from Salmonella enterica serovar Thyphimurium LT2, the causing agent of food-born human gastroenteritis and typhoid like infections. The recombinant antiporter was functional in vivo and in vitro. Expression of its gene complemented the Na+-sensitive phenotype of an E. coli strain that lacks the main Na+/H+ antiporters. Purified to homogeneity, the antiporter was a dimer in solution as accurately determined by size-exclusion chromatography combined with multi-angle laser-light scattering and refractive index monitoring. The purified antiporter was fully capable of electrogenic Na+(Li+)/H+-antiport when reconstituted in proteoliposomes and assayed by solid-supported membrane-based electrophysiological measurements. Transport activity was inhibited by 2-aminoperimidine. The recorded negative currents were in agreement with a 1Na+(Li+)/2H+ stoichiometry. Transport activity was low at pH 7 and up-regulation above this pH value was accompanied by a nearly 10-fold decrease of KmNa (16 mM at pH 8.5) supporting a competitive substrate binding mechanism. K+ does not affect Na+ affinity or transport of substrate cations, indicating that selectivity of the antiport arises from the substrate binding step. In contrast to homologous E. coli NhaA, transport activity remains high at pH values above 8.5. The antiporter from S. Typhimurium is a promising candidate for combined structural and functional studies to contribute to the elucidation of the mechanism of pH-dependent Na+/H+ antiporters and to provide insights in the molecular basis of species-specific growth and survival strategies.

Dataset Information

Transcription of nhaA, the main Na(+)/H(+) antiporter of Escherichia coli, is regulated by Na(+) and growth phase.

Publications

Transcription of nhaA, the main Na(+)/H(+) antiporter of Escherichia coli, is regulated by Na(+) and growth phase.

Similar Datasets

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