High-affinity potassium uptake system in Bacillus acidocaldarius showing immunological cross-reactivity with the Kdp system from Escherichia coli.

During growth with low levels of K+, Bacillus acidocaldarius expressed a high-affinity K+ uptake system. The following observations indicate that this system strongly resembles the Kdp-ATPase of Escherichia coli: (i) its high affinity for K+ (Km of 20 microM or below); (ii) its poor transport of Rb+; (iii) the enhanced ATPase activity of membranes derived from cells grown with low levels of K+ (this activity was stimulated by K+ and inhibited by vanadate); (iv) the expression of an extra protein with a molecular weight of 70,000 in cells grown with low levels of K+; and (v) the immunological cross-reactivity of this 70,000-molecular-weight protein with antibodies against the catalytic subunit B of the E. coli Kdp system. Antibodies against the complete E. coli Kdp system, which immunoprecipitated the whole E. coli KdpABC complex, almost exclusively precipitated the 70,000-molecular-weight protein from detergent-solubilized B. acidocaldarius membranes. The possibility that the B. acidocaldarius Kdp system consists of a single, KdpB-type subunit is discussed.     

A K+ transport ATPase in Escherichia coli.

A K+ -stimulated ATPase in membranes of Escherichia coli has been identified as an activity of the Kdp system, and ATP-driven K+ transport system. Three characteristics support association of the ATPase with the Kdp system: (i) ATPase and Kdp transport are both repressed by growth in media containing high concentrations of K+; (ii) the ATPase and Kdp system accept only K+ as substrate, neither requires Na+ nor accepts Rb+ as a substrate; (iii) the affinity of the ATPase and that of th Kdp system for K+ is similar and is altered by mutations in the structural genes of the Kdp system. Discovery of an ATPase associated with a bacterial transport system suggests functional similarities with the ATP-driven transport systems of animal cells.     

Discrimination between Rb+ and K+ by Escherichia coli.

1. The K+ requirment of Escherichia coli is only partially fulfilled by Rb+. The molar growth yield on Rb+ was about 5% of that on K+ and the growth rate in Rb+-supplemented media is lower thatn in K+ influx by any of the four K+ transport systems of E. coli. The high-affinity Kdp system (Km = 2 micron) is poorly traced by 86Rb+. It discriminates against a 86Rb+ tracer at least 1000-fold. The two moderate affinity systems, the high-rate TrkA system (Km = 1.5 mM) and the moderate rate TrkD system (Km = 0.5 mM), discriminate against a 86Rb+ tracer by approximately 10-fold and 25-fold, respectively. 86Rb+ is preferred by the low-rate TrkF system and overestimates its K+ influx by 40%.     

Kup is the major K+ uptake system in Escherichia coli upon hyper-osmotic stress at a low pH

The K+ uptake was observed in washed cells of Escherichia coli, wild-type, upon hyper-osmotic stress at pH 5.5 when glucose was supplemented. This uptake had apparent a Km of 0.58 mM and Vmax of 0.10 micromol K+/min/mg protein. Such a K+ uptake was investigated using a mutant defective in Kdp and TrkA but with a functional Kup and a mutant defective in Kdp and Kup but having an active TrkA. The K+ uptake to reach the steady state level as well as the initial K+ influx rate in the first mutant were at least 3.5-fold greater than these values with the second mutant and similar to those of the wild-type. Such differences in the K+ uptake activity were correlated with K+ requirements for growth of these mutants. Moreover, the K+ uptake in the wild-type was blocked by a protonophore (carbonyl cyanide m-chlorophenylhydrazone). Valinomycin, arsenate and N,N'-dicyclohexylcarbodiimide were not effective in changing the K+ uptake. It is suggested that Kup is the major K+ uptake system in E. coli upon hyper-osmotic stress at a low pH.     

K+ influx by Kup in Escherichia coli is accompanied by a decrease in H+ efflux

Escherichia coli accumulates K+ by means of multiple uptake systems of which Kup is the major transport system at acidic pH. In cells grown under fermentative conditions at pH 5.5, K+ influx by a wild-type strain upon hyper-osmotic stress at pH 5.5 was accompanied by a marked decrease in H+ efflux, with a 1:1 ratio of K+ to H+ fluxes. This was observed with cells treated with N,N'-dicyclohexylcarbodiimide. Similar results with a mutant defective in Kdp and TrkA but with a functional Kup system but not in a mutant defective in Kdp and Kup but having an active TrkA system suggest that Kup operates as a H+ -K+ -symporter.     

Thallous ion is accumulated by potassium transport systems in Escherichia coli.

The accumulation of 204T1+ by Escherichia coli occurs primarily via either of two K+ transport systems called Kdp and TrkA. T1+ influx is inhibited and T1+ efflux is stimulated by the addition of K+ to the assay medium. Mutants defective in both the Kdp and TrkA systems accumulate little T1+. Uptake of triphenylmethylphosphonium, a lipid-soluble cation whose distribution is widely used to estimate the membrane electrical potential in bacteria, occurs to about the same extent in mutants that accumulate little T1+ as in strains that accumulate T1+ to high levels. These findings indicate that T1+ may be useful as a probe of bacterial K+ transport systems but is not a reliable indicator of the membrane electrical potential in E. coli.     

Cation transport in Escherichia coli. VIII. Potassium transport mutants

Analysis of K transport mutants indicates the existence of four separate K uptake systems in Escherichia coli K-12. A high affinity system called Kdp has a Km of 2 muM, and Vmax at 37 degrees C of 150 mumol/g min. This system is repressed by growth in high concentrations of K. Two constitutive systems, TrkA and TrkD, have Km's of 1.5 and 0.5 mM and Vmax's of 550 and 40 at 37 and 30 degrees C, respectively. Mutants lacking all three of these saturable systems take up K slowly by a process, called TrkF, whose rate of transport is linearly dependent on K concentration up to 105 mM. On the whole, each of these systems appears to function as an independent path for K uptake since the kinetics of uptake when two are present is the sum of each operating alone. This is not true for strains having both the TrkD and Kdp systems, where presence of the latter results in K uptake which saturates at a K concentration well below 0.1 mM. This result indicates some interaction between these systems so that uptake now has the affinity characteristic of the Kdp system. All transport systems are able to extrude Na during K uptake. The measurements of cell Na suggest that growing cells of E. coli have very low concentrations of Na, considerably lower than indicated by earlier studies.     

The K+-translocating Kdp-ATPase from Escherichia coli. Purification, enzymatic properties and production of complex- and subunit-specific antisera

The Kdp system from Escherichia coli is a derepressible high-affinity K+-uptake ATPase. Its membrane-bound ATPase activity was approximately 50 mumol g-1 min-1. The Kdp-ATPase complex was purified from everted vesicles by solubilization with the nonionic detergent Aminoxid WS 35 followed by DEAE-Sepharose CL-6B chromatography at pH 7.5 and pH 6.4 and gel filtration on Fractogel TSK HW-65. The overall yield of activity was 6.5% and the purity at least 90%. The isolated KdpABC complex had a high affinity for its substrates K+ (Km app. = 10 microM) and Mg2+-ATP (Km = 80 microM) and a narrow substrate specificity. The ATPase activity was inhibited by vanadate (Ki = 1.5 microM), fluorescein isothiocyanate (Ki = 3.5 microM), N,N'-dicyclohexylcarbodiimide (Ki = 60 microM) and N-ethylmaleimide (Ki = 0.1 mM). The purification protocol was likewise applicable to the isolation of a KdpA mutant ATPase which in contrast to the wild-type enzyme exhibited an increased Km value for K+ of 6 mM and a 10-fold lowered sensitivity for vanadate. Starting from the purified Kdp complex the single subunits were obtained by gel filtration on Bio-Gel P-100 in the presence of SDS. Both the native Kdp-ATPase and the SDS-denatured polypeptides were used to raise polyclonal antibodies. The specificity of the antisera was established by immunoblot analysis. In functional inhibition studies the anti-KdpABC and anti-KdpB sera impaired ATPase activity in the membrane-bound as well as in the purified state of the enzyme. In contrast, the anti-KdpC serum did not inhibit enzyme activity.     

Interconversion of components of the bacterial proton motive force by electrogenic potassium transport.

The influence of K+ ions on the components of the transmembrane proton motive force (delta mu H+) in intact bacteria was investigated. In K+-depleted cells of the glycolytic bacterium STreptococcus faecalis the addition of K+ ions caused a depolarization of the membrane by about 60 mV. However, since the depolarization was compensated for by an increase in the transmembrane pH gradient (delta pH), the total proton motive force remained almost constant at about 120 mV. Half-maximal changes in the potential were observed at K+ concentrations at which the cells accumulated K+ ions extensively. In EDTA-treated, K+-depleted cells of Escherichia coli K-12, the addition of K+ ions to the medium caused similar, although smaller changes in the components of delta mu H+. Experiments with various E. coli K-12 K+ transport mutants showed that for the observed potential changes the cells required either a functional TrkA or Kdp K+ transport system. These data are interpreted to mean that the inward movement of K+ ions via each of these bacterial transport systems is electrogenic. Consequently, it leads to a depolarization of the membrane, which in its turn allows the cell to pump more protons into the medium.     

Energy coupling to net K+ transport in Escherichia coli K-12.

Energy coupling for three K+ transport systems of Escherichia coli K-12 was studied by examining effects of selected energy sources and inhibitors in strains with either a wild type or a defective (Ca2+, Mg2+)-stimulated ATPase. This approach allows discrimination between transport systems coupled to the proton motive force from those coupled to the hydrolysis of a high energy phosphate compound (ATP-driven). The three K+ transport systems here studied are: (a) the Kdp system, a repressible high affinity (Km=2 muM) system probably coded for by four linked Kdp genes; (b) the Trka system, a constitutive system with high rate and modest affinity (Km=1.5 mM) defined by mutations in the single trkA gene; and (c) the TrkF system, a nonsaturable system with a low rate of uptake (Rhoads, D.B., Waters, F.B., and Epstein, W. (1976) J. Gen. Physiol. 67, 325-341). Each of these systems has a different mode of energy coupling: (a) the Kdp system is ATP-driven and has a periplasmic protein component; (b) the TrkF system is proton motive force-driven; and (c) the TrkA system is unique among bacterial transport systems described to date in requiring both the proton motive force and ATP for activity. We suggest that this dual requirement represents energy fueling by ATP and regulation by the proton motive force. Absence of ATP-driven systems in membrane vesicles is usually attributed to the requirement of such systems for a periplasmic protein. This cannot explain the failure to demonstrate the TrkA system in vesicles, since this system does not require a periplasmic protein. Our findings indicate that membrane vesicles cannot couple energy to ATP-driven transport systems. Since vesicles can generate a proton motive force, the inability of vesicles to generate ATP or couple ATP to transport (or both) must be invoked to explain the absence of TrkA in vesicles. The TrkF system should function in vesicles, but its very low rate may make it difficult to identify.     

Cation transport in Escherichia coli. IX. Regulation of K transport

Kinetics of K exchange in the steady state and of net K uptake after osmotic upshock are reported for the four K transport systems of Escherichia coli: Kdp, TrkA, TrkD, and TrkF. Energy requirements for K exchange are reported for the Kdp and TrkA systems. For each system, kinetics of these two modes of K transport differ from those for net K uptake by K-depleted cells (Rhoads, D. B. F.B. Walters, and W. Epstein. 1976. J. Gen. Physiol. 67:325-341). The TrkA and TrkD systems are inhibited by high intracellular K, the TrkF system is stimulated by intracellular K, whereas the Kdp system is inhibited by external K when intracellular K is high. All four systems mediate net K uptake in response to osmotic upshock. Exchange by the Kdp and TrkA systems requires ATP but is not dependent on the protonmotive force. Energy requirements for the Kdp system are thus identical whether measured as net K uptake or K exchange, whereas the TrkA system differs in that it is dependent on the protonmotive force only for net K uptake. We suggest that in both the Kpd and TrkA systems formation of a phosphorylated intermediate is necessary for all K transport, although exchange transport may not consume energy. The protonmotive-force dependence of the TrkA system is interpreted as a regulatory influence, limiting this system to exchange except when the protonmotive force is high.     

Improvement in K+-limited growth rate associated with expression of the N-terminal fragment of one subunit (KdpA) of the multisubunit Kdp transporter in Escherichia coli

Mutations in any one of three genes, kdpA, -B, or -C, in Escherichia coli abolish the activity of Kdp, a multisubunit K+-ATPase that belongs to the P-type ATPase family of cation transporters. We found in this study that expression in vivo of a 135-amino-acid-long N-terminal fragment (KdpA'), less than one-quarter the length of native KdpA, was able to mediate an improvement in K+-limited growth rates in two different contexts, even in the absence of both KdpC and the ATPase subunit KdpB. The first context was when KdpA' was overexpressed in cells from a heterologous inducible promoter, and the second was when KdpA' was provided with a C-terminally altered extension (following a spontaneous genetic rearrangement). Our results suggest that KdpA' provides an incipient pathway for K+ translocation which can serve to transport K+ into the cells in response to the cytoplasmic membrane potential.     

The sensor kinase KdpD of Escherichia coli senses external K+

The Kdp system of Escherichia coli is composed of the high‐affinity K⁺ transporter KdpFABC and the two regulatory proteins KdpD (sensor kinase) and KdpE (response regulator), which constitute a typical two‐component system. The kdpFABC operon is induced under K⁺‐limiting conditions and, to a lesser extent, under high osmolality in the medium. In search for the stimulus sensed by KdpD, we studied the inhibitory effect of extracellular K⁺ on the Kdp system at pH 6.0, which is masked by unspecific K⁺ transport at higher pH values. Based on KdpD derivatives carrying single aspartate replacements in the periplasmic loops which are part of the input domain, we concluded that the inhibition of the Kdp system at extracellular K⁺ concentrations above 5 mM is mediated via KdpD/KdpE and not due to inhibition of the K⁺‐transporting KdpFABC complex. Furthermore, time‐course analyses of kdpFABC expression revealed that a decline in the extracellular K⁺ concentration efficiently stimulates KdpD/KdpE‐mediated signal transduction. In this report we provide evidence that the extracellular K⁺ concentration serves as one of the stimuli sensed by KdpD.     

The Kdp-ATPase of Escherichia coli mediates an ATP-dependent, K+-independent electrogenic partial reaction

Charge transport by the K+ transporting Kdp-ATPase from Escherichia coli was investigated using planar lipid membranes to which liposomes reconstituted with the enzyme were adsorbed. To study reactions in the absence of K+, given some contamination of solutions with K+, we used a mutant of Kdp whose affinity for K+ was 6 mM instead of the wild-type whose affinity is 2 microM. Upon rapid release of ATP from caged ATP, a transient current occurred in the absence of K+. In the presence of K+, a stationary current was seen. On the basis of their structural similarity, we propose a kinetic model for the Kdp-ATPase analogous to that of the Na+K+-ATPase. In this model, the first, K+-independent step is electrogenic and corresponds to the outward transport of a negative charge. The second, K+-translocating step is probably also electrogenic and corresponds to transport of positive charge to the intracellular side of the protein.     

Role of Na+ and Li+ in thiomethylgalactoside transport by the melibiose transport system of Escherichia coli.

Thiomethyl-beta-galactoside (TMG) accumulation via the melibiose transport system was studied in lactose transport-negative strains of Escherichia coli. TMG uptake by either intact cells or membrane vesicles was markedly stimulated by Na+ or Li+ between pH 5.5 and 8. The Km for uptake of TMG was approximately 0.2 mM at an external Na+ concentration of 5 mM (pH 7). The alpha-galactosides, melibiose, methyl-alpha-galactoside, and o-nitrophenyl-alpha-galactoside had a high affinity for this system whereas lactose, maltose and glucose had none. Evidence is presented for Li+-TMG or Na+-TMG cotransport.     

Potassium extrusion by the moderately halophilic and alkaliphilic methanogen methanolobus taylorii GS-16 and homeostasis of cytosolic pH.

Methanolobus taylorii GS-16, a moderately halophilic and alkaliphilic methanogen, grows over a wide pH range, from 6.8 to 9.0. Cells suspended in medium with a pH above 8.2 reversed their transmembrane pH gradient (delta pH), making their cytosol more acidic than the medium. The decreased energy in the proton motive force due to the reversed delta pH was partly compensated by an increased electric membrane potential (delta psi). The cytosolic acidification by M. taylorii at alkaline pH values was accompanied by K+ extrusion. The cytosolic K+ concentration was 110 mM in cells suspended at pH 8.7, but it was 320 mM in cells suspended at neutral pH values. High external K+ concentrations (210 mM or higher) inhibited the growth of M. taylorii at alkaline pH values, perhaps by preventing K+ extrusion. Cells suspended at pH 8.5 and 300 mM external K+ failed to acidify their cytosol. The key observation indicative of the involvement of K+ transport in cytosolic acidification was that valinomycin (0.8 microM), a K+ uniporter, inhibited the growth of M. taylorii only at alkaline pH values. Experiments with resting cells indicated that at alkaline pH values valinomycin uncoupled catabolic reactions from ATP synthesis. Thus, K+/H+ antiport activity was proposed to account for the K+ extrusion and the uncoupling effect of valinomycin at alkaline pH values. Such antiport activity was demonstrated by the sharp drop in pH of the bulk medium of the cell suspension upon the addition of 0.1 M KCl. The antiporter appeared to be active only at alkaline pH values, which was in accordance with a possible role in pH homeostasis by M. taylorii growing at alkaline pH values.     

The KdpF subunit is part of the K(+)-translocating Kdp complex of Escherichia coli and is responsible for stabilization of the complex in vitro

The kdpABC operon codes for the high affinity K(+)-translocating Kdp complex (P-type ATPase) of Escherichia coli. Upon expression of this operon in minicells, a so far unrecognized small hydrophobic polypeptide, KdpF, could be identified on high resolution SDS-polyacrylamide gels in addition to the subunits KdpA, KdpB, and KdpC. Furthermore, it could be demonstrated that KdpF remains associated with the purified complex. As determined by mass spectrometry, this peptide is present in its formylated form and has a molecular mass of 3100 Da. KdpF is not essential for growth on low K(+) (0.1 mM) medium, as shown by deletion analysis of kdpF, but proved to be indispensable for a functional enzyme complex in vitro. In the absence of KdpF, the ATPase activity of the membrane-bound Kdp complex was almost indistinguishable from that of the wild type. In contrast, the purified detergent-solubilized enzyme complex showed a dramatic decrease in enzymatic activity. However, addition of purified KdpF to the KdpABC complex restored the activity up to wild type level. It is interesting to note that the addition of high amounts of E. coli lipids had a similar effect. Although KdpF is not essential for the function of the Kdp complex in vivo, it is part of the complex and functions as a stabilizing element in vitro. The corresponding operon should now be referred to as kdpFABC.     

Transport of N-acetyl-D-galactosamine in Escherichia coli K92: effect on acetyl-amino sugar metabolism and polysialic acid production

The N-acetyl-D-galactosamine (GalNAc) transport system of Escherichia coli K92 was studied when the bacterium was grown in a chemically defined medium containing GalNAc as a carbon source. Kinetic measurements were carried out in vivo at 37 degrees C in 25 mM phosphate buffer, pH 7.0. Under these conditions, the uptake rate was linear for at least 3 min and the calculated Km for GalNAc was 3 microM. The transport system was strongly inhibited by sodium arsenate (70%), potassium cyanide (62%) and 2,4-dinitrophenol (75%). Analysis of bacterial GalNAc phosphotransferase activity revealed in vitro GalNAc phosphorylation activity only when phosphoenolpyruvate was present. These results strongly support the notion that GalNAc uptake depends on a specific phosphotransferase system. Study of activity regulation showed that N-acetylglucosamine and mannosamine specifically inhibit the transport of GalNAc in this bacterium. Analysis of expression revealed that the GalNAc transport system is specifically induced by GalNAc but not by N-acetylglucosamine (GlcNAc) or N-acetylmannosamine (ManNAc), two intimately related sugars. Moreover, full induction of GalNAc transport required the presence of both cAMP and GalNAc. Comparative studies revealed that E. coli K92 has developed a regulation mechanism that specifically induces the appropriate permease based on the presence of each respective phospho-amino sugar (GlcNAc, ManNAc and GalNAc). In this regulation system, GlcNAc is the preferred amino sugar as the carbon source. Finally, when E. coli K92 was grown using GalNAc, capsular polysialic acid production was strongly affected. The presence of intracellular phosphoderivative acetylamino sugars, generated by the action of the phosphotransferase transport system, can be responsible for this effect.     

The high-affinity K+-translocating ATPase complex from Bacillus acidocaldarius consists of three subunits

Cells of the thermoacidophilic bacterium Bacillus acidocaldarius express a high-affinity K+-uptake system when grown at low external K+. A vanadate-sensitive, K+- and Mg2+-stimulated ATPase was partially purified from membranes of these cells by solubilization with a non-ionic detergent followed by ion-exchange chromatography of the extract. Combinations of non-denaturing and denaturing electrophoretic separation methods revealed that the ATPase complex consisted of three subunits with molecular weights almost identical to those of the KdpA, B and C proteins, which together form the Kdp high-affinity, K+-translocating ATPase complex of Escherichia coli. The affinity of the partially purified ATPase from B. acidocaldarius for its substrates K+ (Km 2-3 microM) and ATP (Km 80 microM), its stimulation by various divalent cations, and its inhibition by vanadate (Ki 1-2 microM), bafilomycin A1 (Ki 20 microM), DCCD (Ki 200 microM) or Ca2+ were also similar to those of the E. coli enzyme, indicating that the two K+-translocating ATPases have almost identical properties.     

Effect of Cd2+ on phosphate uptake by cadmium-resistant and cadmium-sensitive Staphylococcus aureus.

The effect of Cd2+ on phosphate (Pi) uptake was investigated in the growing cells of Cd(2+)-resistant Staphylococcus aureus 1781OR and Cd(2+)-sensitive S. aureus 17810S. Inhibitor and ionophore studies showed that 32Pi uptake in the two strains occurred via the Pi porter down pH gradient (delta pH) generated by the respiratory chain. Cd2+ inhibited 32Pi uptake in the cadmium-sensitive strain 1781OS at all concentrations used (10 microM-1 mM). In strain 1781OR, possessing the plasmid-coded Cd2+ efflux system, 10-100 microM Cd2+ did not inhibit 32Pi uptake. Even at 1 mM Cd2+, inhibition of 32Pi uptake in strain 1781OR was reversed when the external Cd2+ was chelated with cysteine and activity of Cd2+ efflux system was restored. Cd2+ efflux induced by cysteine was energized either by membrane potential (delta psi) or by delta pH, which indicated that electrochemical gradient of protons (delta mu H+) was required for this efflux.