Two Ways To Convert a Low-Affinity Potassium Channel to High Affinity: Control of Bacillus subtilis KtrCD by Glutamate

Abstract

Potassium and glutamate are the major cation and anion, respectively, in every living cell. Due to the high concentrations of both ions, the cytoplasm of all cells can be regarded as a potassium glutamate solution. This implies that the concentrations of both ions need to be balanced. While the control of potassium uptake by glutamate is well established for eukaryotic cells, much less is known about the mechanisms that link potassium homeostasis to glutamate availability in bacteria. Here, we have discovered that the availability of glutamate strongly decreases the minimal external potassium concentration required for the highly abundant Bacillus subtilis potassium channel KtrCD to accumulate potassium. In contrast, the inducible KtrAB and KimA potassium uptake systems have high apparent affinities for potassium even in the absence of glutamate. Experiments with mutant strains revealed that the KtrD subunit responds to the presence of glutamate. For full activity, KtrD synergistically requires the presence of the regulatory subunit KtrC and of glutamate. The analysis of suppressor mutants of a strain that has KtrCD as the only potassium uptake system and that experiences severe potassium starvation identified a mutation in the ion selectivity filter of KtrD (Gly282 to Val) that similarly results in a strongly glutamate-independent increase of the apparent affinity for potassium. Thus, this work has identified two conditions that increase the apparent affinity of KtrCD for potassium, i.e., external glutamate and the acquisition of a single point mutation in KtrD. IMPORTANCE In each living cell, potassium is required for maintaining the intracellular pH and for the activity of essential enzymes. Like most other bacteria, Bacillus subtilis possesses multiple low- and high-affinity potassium uptake systems. Their activity is regulated by the second messenger cyclic di-AMP. Moreover, the pools of the most abundant ions potassium and glutamate must be balanced. We report two conditions under which the low-affinity potassium channel KtrCD is able to mediate potassium uptake at low external potassium concentrations: physiologically, the presence of glutamate results in a severely increased potassium uptake. Moreover, this is achieved by a mutation affecting the selectivity filter of the KtrD channel. These results highlight the integration between potassium and glutamate homeostasis in bacteria.

Essentiality of c-di-AMP inBacillus subtilis: Bypassing mutations converge in potassium and glutamate homeostasis

“…5B), however, the mechanism responsible for this control has yet to be uncovered. We have previously demonstrated that a potassium channel (KtrCD) is directly activated and thus converted from a low-to a high-affinity uptake system in the presence of glutamate [41]. Here, we show that similarly the affinity of the high affinity glutamate transporter GltT is controlled by the presence of potassium: At high potassium concentrations, the affinity of GltT for glutamate is increased fivefold.…”

“…A B. subtilis strain lacking all three diadenylate cyclases is unable to grow at potassium concentrations of 5 mM or higher or in the presence of glutamate (even at low potassium concentrations) [21,41]. In order to get more comprehensive insights into the toxicity of glutamate for the c-di-AMP-free strain GP2222 (designated Δdac from here on), we decided to isolate and study a larger set of suppressor mutants that tolerate the presence of glutamate.…”

“…1 1 Growth on different media was judged after plating on corresponding agar plates. 2 This strain was isolated in a previous study [41].…”

“…5C, 5D). Moreover, we performed this experiment in the presence of 0.1 mM and 5 mM KCl in the medium to see whether potassium has an effect on glutamate uptake as it has been observed with an activating effect of glutamate on potassium uptake by KtrCD [41]. As control, we used strain GP3071 that lacks AimA, GltT, and GltP.…”

“…GP2222 (Δdac), GP2814 (Δdac yfkC*), GP2223 (Δdac nhaK*), GP2259 (Δdac ΔgltT), and GP3054 (Δdac ΔaimA) were cultivated in MSSM medium with glutamate and 0.1 mM KCl and suppressors were isolated and analyzed by whole genome sequencing. Note that for GP2222 (Δdac), 14 mutants were isolated in this study and one mutant was isolated previously [41] but studied in depth here, thus resulting in a total number of 15 mutants. Fig.…”