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Mutational Loss of a K⁺ and NH₄⁺ Transporter Affects the Growth and Endospore Formation of Alkaliphilic Bacillus pseudofirmus OF4

Yi Wei,¹ Thomas W. Southworth,² Hilde Kloster,^1, Masahiro Ito,³ Arthur A. Guffanti,¹ Anne Moir,² and Terry A. Krulwich^1*

Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, New York 10029,¹ Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, United Kingdom,² Faculty of Life Sciences, Toyo University, Oura-gun, Gunma 374-0193, Japan³

Received 5 May 2003/ Accepted 18 June 2003

A putative transport protein (Orf9) of alkaliphilic Bacillus pseudofirmus OF4 belongs to a transporter family (CPA-2) of diverse K⁺ efflux proteins and cation antiporters. Orf9 greatly increased the concentration of K⁺ required for growth of a K⁺ uptake mutant of Escherichia coli. The cytoplasmic K⁺ content of the cells was reduced, consistent with an efflux mechanism. Orf9-dependent translocation of K⁺ in E. coli is apparently bidirectional, since ammonium-sensitive uptake of K⁺ could be shown in K⁺-depleted cells. The upstream gene product Orf8 has sequence similarity to a subdomain of KTN proteins that are associated with potassium-translocating channels and transporters; Orf8 modulated the transport capacities of Orf9. No Orf9-dependent K⁺(Na⁺)/H⁺ antiport activity was found in membrane vesicles. Nonpolar deletion mutants in the orf9 locus of the alkaliphile chromosome exhibited no K⁺-related phenotype but showed profound phenotypes in medium containing high levels of amine-nitrogen. Their patterns of growth and ammonium content suggested a physiological role for the orf9 locus in bidirectional ammonium transport. Orf9-dependent ammonium uptake was observed in right-side-out membrane vesicles of the alkaliphile wild type and the mutant with an orf8 deletion. Uptake was proton motive force dependent and was inhibited by K⁺. Orf9 is proposed to be designated AmhT (ammonium homeostasis). Ammonium homeostasis is important in high-amine-nitrogen settings and is particularly crucial at high pH since cytosolic ammonium accumulation interferes with cytoplasmic pH regulation. Endospore formation in amino-acid-rich medium was significantly defective and germination was modestly defective in the orf9 and orf7-orf10 deletion mutants.

Present address: Smerud Medical Research International, Oslo, Norway.

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