title: Mutational loss of a K+ and NH4+ transporter affects the growth and endospore formation of alkaliphilic Bacillus pseudofirmus OF4.
abstract: 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.
authors.affiliation: Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Wei
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Southworth
authors.forename: Hilde
authors.initials: H
authors.lastname: Kloster
authors.forename: Masahiro
authors.initials: M
authors.lastname: Ito
authors.forename: Arthur A
authors.initials: AA
authors.lastname: Guffanti
authors.forename: Anne
authors.initials: A
authors.lastname: Moir
authors.forename: Terry A
authors.initials: TA
authors.lastname: Krulwich
mesh_terms: D000641:Ammonia / Q000378:metabolism; D017920:Antiporters / Q000235:genetics*; D001407:Bacillus / Q000235:genetics / Q000254:growth & development* / Q000502:physiology*; D001692:Biological Transport; D027682:Cation Transport Proteins; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D006863:Hydrogen-Ion Concentration; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D011188:Potassium / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013171:Spores, Bacterial / Q000502:physiology
chemical_list: D017920:Antiporters; D027682:Cation Transport Proteins; C043130:ammonium-potassium antiporter; D000641:Ammonia; D011188:Potassium