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.

FIG. 1.

Organization of the genes in the orf9 locus. (A) Chromosomal organization of the orf9 gene locus in B. pseudofirmus OF4. The accession number for the full sequence of this region is U89914.2. A predicted promoter (P), most likely to be a sigma A type, is marked by an arrow pointing to the right, and rectangles indicate the predicted ORFs. The arrow under orf6 indicates that the direction of transcription is the same as that for orf7 to orf16 whereas that under orf17, pointing to the left, indicates that orf17 is transcribed in the opposite direction. “t” indicates a stem-loop between orf10 and orf11 (predicted ΔG = −18.1 kcal/mol). “T” between orf16 and orf17 indicates a second stem-loop structure (predicted ΔG = −19.7 kcal/mol). The arrowheads indicate the sites and directions of primers that were used in RT-PCR. (B) Primers and templates used in RT-PCR analyses. The details of the RT-PCR experiments are discussed in the text. WT, wild type.

FIG. 2.

Effect of increasing KCl concentrations on the growth of K⁺-uptake-defective E. coli TK2420. The growth of E. coli TK2420, transformed with pBK36 (vector control), pORF8, pORF9, or pORF8-9, in the presence of the indicated KCl concentrations was assessed by measuring the A₆₀₀ after 15 h. The results represent the averages of at least four independent duplicate experiments, with error bars representing the standard deviations.

FIG. 3.

Uptake of K⁺ by K⁺-depleted cells of E. coli transformants and the effect of added ammonium. The figure shows the uptake of K⁺ by K⁺-depleted cells of E. coli TK2420 transformants expressing orf8, orf9, orf8-orf9, or a vector control. Cells of the transformants of E. coli TK2420 with empty vector or expressing orf8, orf9, or orf8-orf9 were depleted of K⁺ as described under Materials and Methods. Zero time samples were taken immediately before the addition of KCl to 25 mM. Subsequent samples were taken at the indicated times and assayed for cellular K⁺ content by flame photometry. Where ammonium sulfate was also added, closed symbols are used. (Left) The K⁺ content of the transformants is shown as a function of time. (Middle) The same experiment as that shown in the left panel, except that 10 mM (NH₄)₂SO₄ was added at the same time as the 25 mM K⁺. (Right) The data shown in the left panel (i.e., the transformants to which K⁺ alone was added) and the data for the orf9 transformant to which both K⁺ and (NH₄)₂SO₄ were added (middle panel, closed squares) are shown after correction for the amount of K⁺ associated with the vector control transformant. The data are the averages of duplicate determinations from at least two independent experiments, and the error bars represent the standard deviations.

FIG. 4.

Effect of in-frame deletions in the orf9 locus on growth of B. pseudofirmus OF4 in semidefined malate-containing medium at pH 7.5 and 10.5. Cells of the wild-type (Wt) and deletion strains were grown in the semidefined medium at either pH 7.5 or pH 10.5, as described under Materials and Methods, and containing the indicated final concentrations of added K⁺. After 7 h of growth, the A₆₀₀ was recorded. The data shown, with standard deviations, are the results of at least two independent experiments conducted in duplicate.

FIG. 5.

Effect of in-frame deletions in the orf9 locus on growth of B. pseudofirmus OF4 in malate-containing QA medium at pH 7.5 and 10.5. (A) Cells of the wild-type and deletion strains were grown in the malate-containing QA medium, pH 7.5 or 10.5, described under Materials and Methods. As indicated in the figure, the effects of supplemental NaCl or KCl were also examined. After growth for 20 h, the A₆₀₀ was recorded. The values shown are from at least two independent experiments that were conducted in duplicate. Error bars represent the standard deviations of the values. (B) The growth rates of the wild-type and orf8 deletion strains were compared at pH 10.5 in modified malate-QA medium, containing glutamine and alanine (QA) at 25% of the standard QA concentration, i.e., at an amine-nitrogen concentration that is suboptimal for growth. The effect of added ammonium sulfate, at concentrations indicated in the figure, was monitored. After growth for 20 h, the A₆₀₀ was recorded. The values from at least two independent experiments, conducted in duplicate, are shown together with the standard deviations. wt, wild type.

FIG. 6.

Transmission electron microscopy showing the sporulation defect of the orf9 mutant of B. pseudofirmus. Images are shown of the wild-type strain at 24 and 48 h of incubation on sporulation medium (A and B, respectively) and of comparable data for the orf9 mutant strain (C and D).

FIG. 7.

Germination of spores from B. pseudofirmus OF4 wild-type and orf9 mutant strains. (A) Germination of B. pseudofirmus OF4 wild type in 200 mM NaCl (triangles), 200 mM NaCl plus 10 mM l-alanine (squares), and 200 mM NaCl plus 10 mM inosine (circles). (B) Germination in 200 mM NaCl and 10 mM inosine of B. pseudofirmus OF4 wild type (triangles), orf7-orf10 deletion mutant (squares), and orf9 mutant (circles). (C) Germination in 200 mM NaCl and 10 mM l-alanine of B. pseudofirmus OF4 wild type (triangles), orf7-orf10 deletion mutant (squares), and orf9 mutant (circles).