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.     

Negatively charged phospholipids influence the activity of the sensor kinase KdpD of Escherichia coli

Synthesis of the high-affinity K⁺-translocating Kdp-ATPase of Escherichia coli, encoded by the kdpFABC operon, is regulated by the membrane-bound sensor kinase KdpD and the soluble response regulator KdpE. K⁺ limitation or a sudden increase in osmolarity induces the expression of kdpFABC. Due to the importance of K⁺ to maintain turgor, it has been proposed that KdpD is a turgor sensor. Although the primary stimulus that KdpD senses is unknown, alterations in membrane strain or the interaction between KdpD and membrane components might be good candidates. Here, we report a study of the influence of the membrane phospholipid composition on the function of KdpD in vivo and in vitro using various E. coli mutants defective in phospholipid biosynthesis. Surprisingly, neither the lack of the major E. coli phospholipid phosphatidylethanolamine nor the drastic reduction of the phosphatidylglycerol/cardiolipin content influenced induction of kdpFABC expression significantly. However, in vitro reconstitution experiments with synthetic phospholipids clearly demonstrated that KdpD kinase activity is dependent on negatively charged phospholipids, whereas the structure of the phospholipids plays a minor role. These results indicate that electrostatic interactions are important for the activity of KdpD. Received: 29 March 1999 / Accepted: 26 July 1999     

The Universal Stress Protein UspC Scaffolds the KdpD/KdpE Signaling Cascade of Escherichia coli under Salt Stress

The sensor kinase KdpD and the response regulator KdpE control induction of the kdpFABC operon encoding the high-affinity K⁺-transport system KdpFABC in response to K⁺ limitation or salt stress. Under K⁺ limiting conditions the Kdp system restores the intracellular K⁺ concentration, while in response to salt stress K⁺ is accumulated far above the normal content. The kinase activity of KdpD is inhibited at high concentrations of K⁺, so it has been puzzling how the sensor can be activated in response to salt stress. Here, we demonstrate that the universal stress protein UspC acts as a scaffolding protein of the KdpD/KdpE signaling cascade by interacting with a Usp domain in KdpD of the UspA subfamily under salt stress. Escherichia coli encodes three single domain proteins of this subfamily, UspA, UspC, and UspD, whose expression is up-regulated under various stress conditions. Among these proteins only UspC stimulated the in vitro reconstructed signaling cascade (KdpD→KdpE→DNA) resulting in phosphorylation of KdpE at a K⁺ concentration that would otherwise almost prevent phosphorylation. In agreement, in a ΔuspC mutant KdpFABC production was down-regulated significantly when cells were exposed to salt stress, but unchanged under K⁺ limitation. Biochemical studies revealed that UspC interacts specifically with the Usp domain in the stimulus perceiving N-terminal domain of KdpD. Furthermore, UspC stabilized the KdpD/KdpE∼P/DNA complex and is therefore believed to act as a scaffolding protein. This study describes the stimulation of a bacterial two-component system under distinct stress conditions by a scaffolding protein, and highlights a new role of the universal stress proteins.     

The KdpD/KdpE Two-Component System: Integrating K+ Homeostasis and Virulence

The two-component system (TCS) KdpD/KdpE, extensively studied for its regulatory role in potassium (K⁺) transport, has more recently been identified as an adaptive regulator involved in the virulence and intracellular survival of pathogenic bacteria, including Staphylococcus aureus, entero-haemorrhagic Escherichia coli, Salmonella typhimurium, Yersinia pestis, Francisella species, Photorhabdus asymbiotica, and mycobacteria. Key homeostasis requirements monitored by KdpD/KdpE and other TCSs such as PhoP/PhoQ are critical to survival in the stressful conditions encountered by pathogens during host interactions. It follows these TCSs may therefore acquire adaptive roles in response to selective pressures associated with adopting a pathogenic lifestyle. Given the central role of K⁺ in virulence, we propose that KdpD/KdpE, as a regulator of a high-affinity K⁺ pump, has evolved virulence-related regulatory functions. In support of this hypothesis, we review the role of KdpD/KdpE in bacterial infection and summarize evidence that (i) KdpD/KdpE production is correlated with enhanced virulence and survival, (ii) KdpE regulates a range of virulence loci through direct promoter binding, and (iii) KdpD/KdpE regulation responds to virulence-related conditions including phagocytosis, exposure to microbicides, quorum sensing signals, and host hormones. Furthermore, antimicrobial stress, osmotic stress, and oxidative stress are associated with KdpD/KdpE activity, and the system''s accessory components (which allow TCS fine-tuning or crosstalk) provide links to stress response pathways. KdpD/KdpE therefore appears to be an important adaptive TCS employed during host infection, promoting bacterial virulence and survival through mechanisms both related to and distinct from its conserved role in K⁺ regulation.     

Signal transduction between the two regulatory components involved in the regulation of the kdpABC operon in Escherichia coli: Phosphorylation-dependent functioning of the positive regulator, KdpE

The proteins KdpD and KdpE are crucial to the osmotic regulation of the kdpABC operon that is responsible for the high-affinity K⁺ ion transport system in Escherichia coli. We demonstrated previously that the response regulator, KdpE, is capable of undergoing Phosphorylation mediated by the sensory protein kinase, KdpD. In this study, we obtained biochemical evidence supporting the view that when KdpE is phosphorylated, it takes on an active form that exhibits relatively high affinity for the kdpABC promoter, which in turn results in activation of the kdpABC operon. It was also suggested that the central hydrophobic domain of KdpD, which is conceivably responsible for membrane anchoring of this protein, plays a role in the signalling mechanism underlying KdpE Phosphorylation in response to hyperosmotic stress.     

On the osmoregulation in Atriplex hymenelytra (Torr.) Wats. (Chenopodiaceae)

Summary Aspects of osmoregulation were studied in leaves of irrigated and nonirrigated plants of Atriplex hymenelytra (Torr.) Wats. (Chenopodiaceae) from their natural habitat in Death Valley, California. Using a set of several data concentrations of inorganic electrolytes (Na⁺, K⁺, Cl^-) and of oxalate in the mesophyll of this salt secreting species were calculated. The osmotic potential resulting from these solutes (under consideration of an empirically estimated osmotic coefficient) is in good agreement with field measurements of the overall osmotic potential in the leaf mesophyll as determined by pressure-volume curves. This indicates that these 4 electrolytes are the main osmotically active solutes. Oxalate is present in comparably high concentrations and is used to achieve ion balance.Organic solutes analyzed include soluble carbohydrates (mono-, di- and oligosaccharides), amino- and organic acids as well as glycinebetaine. Of these, organic- and amino acids (including proline) contribute only little to osmoregulation. Soluble carbohydrates and especially glycinebetaine exhibit concentrations high enough for generating considerable osmotic potentials, at least if these compounds are regarded to be restricted to the cytoplasm acting as compatible solutes.     

Depolarization-induced changes in neurite elongation and intracellular Ca2+ in isolated Helisoma neurons

This study focuses on the effects of K⁺ depolarization on neurite elongation of identified Helisoma neurons isolated into culture. Application of K⁺ to the external medium caused a dose-dependent suppression of neurite elongation. Lower concentrations of K⁺ were associated with a slowing in the rate of neurite elongation, whereas higher concentrations produced neurite retraction. Surprisingly, the effects of K⁺ depolarization were transient, and neurite elongation rates recovered towards control levels within 90 min even though the neurons remained in high-K⁺ solution. Identified neurons differed in the magnitude of their response to K⁺ depolarization; neurite elongation of buccal neuron B4 was inhibited at 5 mM K⁺, but elongation in B5 and B19 was not affected until concentrations of 25 mM. Electrophysiologically, K⁺ application evoked a brief period (5–10 s) of action potential activity that was followed by a steady-state membrane depolarization lasting 2 h or more. The changes in neurite elongation induced by K⁺ depolarization occurred in isolated growth cones severed from their neurites and were blocked by application of calcium antagonists. Intracellular free Ca²⁺ levels in growth cones of B4 and B19 increased and then decreased during the 90-min depolarization, corresponding to the changes in elongation. B4 and B19 showed differences in the magnitude, time course, and spatial distribution of the Ca²⁺ change during depolarization, reflecting their different sensitivities to depolarization.     

Effects of NaCl on ion relations and carbohydrate status of roots and on osmotic regulation of roots and shoots of Atriplex amnicola

Abstract Atriplex amnicola was grown at 25, 200 or 400 mol m³ NaCl. Root tissues at different stages of development were investigated for concentrations of K⁺, Na⁺ and Mg²⁺, and in some cases for Cl^?. Sugar and starch concentrations were measured for plants grown at 25 or 400 mol m³ NaCl. In the ‘slightly vaeuolated’ root tips, Na⁺ was only 40 mol m^?3 at an external concentration of 400 mol m^?3 NaCl. The concentrations of K⁺ were not affected substantially by external NaCl between 25 mol m^?3 and 400 mol m^?3. The ‘highly vacuolated’ root tissues had substantially higher concentrations of K⁺, Na⁺ and Cl^? in plants grown at 200 and 400 mol m ³ NaCl than in plants grown at 25 mol m^?3 NaCl. Concentrations of Cr and of the sum of the cations in recently expanded tissue were similar to those in the bulk of the roots, consisting mainly of old cells. However, the K⁺: Na⁺ decreased with age; at 400 mol m^?3 external NaCl with a K⁺: Na⁺ of 0.012, the K⁺: Na⁺ in recently expanded 12 mm root tips was as high as 1.6, compared with 0.7 for the bulk of the roots. These ion data were used to estimate cytoplasmic and vacuolar concentrations of K⁺ and Na ⁺. Such calculations indicated that between 25 mol m³ and 400 mol m^?3 external NaCl the concentration of the sum of (Na⁺+K⁺) in the cytoplasm was maintained at about 180–200 mol m^?3 (cell water basis). In contrast, the (Na⁺+ K⁺) concentration in the vacuole was 170 mol m^?3 for plants grown at 25 mol m^?3 NaCl and 420 mol 400 mol m^?3 NaCl. The expanding root (issues exhibited greatly decreased soluble sugars and starch between dusk and dawn. Ai both times, sugar and starch concentrations in these tissues were 2.5–4.0 times greater in plants grown at 400 mol m^?3 NaCl compared with plants grown at 25 mol m^?3 NaCl. In contrast, carbohydrate concentrations in expanded root tissues were very similar at 25 and 400 mol m^?3 and showed little diurnal fluctuation. This paper considers the causes for the slower growth of A. amnicola at 400 than at 25 mol m”³ NaCl, using the data for the roots described here, and those for the shoots presented in the preceding paper (Aslam et al., 1986). There is no support for possible adverse effects by high internal ion concentrations. Instead, there may be deficiencies in supply of organic solutes for osmotic regulation; during part of the night a limited supply of such solutes may well restrict the rate of expansion of cells in plants growing at 400 mol m^?3 NaCl. There is insufficient evidence to decide whether this limitation in the expanding tissues is particularly prominent for the roots or for the shoots.     

Domain swapping reveals that the N-terminal domain of the sensor kinase KdpD in Escherichia coli is important for signaling

Background  

The KdpD/KdpE two-component system of Escherichia coli regulates expression of the kdpFABC operon encoding the high affinity K⁺ transport system KdpFABC. The input domain of KdpD comprises a domain that belongs to the family of universal stress proteins (Usp). It has been previously demonstrated that UspC binds to this domain, resulting in KdpD/KdpE scaffolding under salt stress. However the mechanistic significance of this domain for signaling remains unclear. Here, we employed a "domain swapping" approach to replace the KdpD-Usp domain with four homologous domains or with the six soluble Usp proteins of E. coli.     

Ion regulation in potassium-sensitive mutants of paramecium tetraurelia

Two recessive mutations of Paramecium tetraurelia confer sensitivity to potassium: While wild-type cells survive when up to 30 mM KCI is added to their growth medium, mutants cease to grow and die when levels of added KCl reach 20–25 mM. Similar sensitivities are seen to Rb⁺ and Cs⁺, but not to Na⁺. Swimming behavior of mutants is indistinguishable from wild type when place in stimulating solutions containing Na⁺, K⁺, or Ba²⁺. Behavioral adaptation to low levels of K⁺ also is indistiguishable from wild type. Flame photometry reveals that one mutant is unable to keep out K⁺ when that ion is at high levels in the medium, while the other mutant readily leaks K⁺ and Na⁺ when those ions are at low levels in the medium. Both mutants have markedly lower internal Na⁺ than does wild type. Problem with K⁺ permeability account for the sensitivity of the one mutant to elevated external K⁺, but the basis of sensitivity in the other mutant is unclear. These mutants expand the range of ion regulation mutants in Paramecium and demonstrate that lesions in cellular ion regulation in this organism need not result in changes in swimming behavior.     

第二信使分子c-di-AMP调控细菌中钾离子转运的机制

钾离子(K~+)是维持生命体存活的必需元素。原核生物进化出一系列K~+转运系统,如Kdp系统﹑Ktr系统和Trk系统等,来维持胞内相对恒定的K~+浓度。环二腺苷酸单磷酸(cyclic diadenosine monophosphate,c-di-AMP)是新发现的第二信使分子,可以与K~+转运系统中的KdpD、KtrA和TrkA结合。当胞内c-di-AMP浓度高时,c-di-AMP会与K~+转运蛋白结合,降低其转运活性。c-di-AMP的靶标除蛋白质外,还有RNA元件,即c-di-AMP的核糖开关。高浓度的c-di-AMP与其核糖开关结合后,可抑制下游K~+转运蛋白编码基因,如kdp、ktr和trk操纵子以及kup基因的转录,从而调控K~+的转运。总之,胞内高浓度的c-di-AMP抑制细菌对K~+的吸收。c-di-AMP调控K~+转运机制的研究,不仅丰富了K~+转运的调控方式,而且也扩大了c-di-AMP的调控范围,为细菌的利用与防治提供了新思路。     

Potassium channel AKT1 is involved in the auxin‐mediated root growth inhibition in Arabidopsis response to low K+ stress

The changes in external K⁺ concentration affect plant root growth. However, the molecular mechanism for perceiving a K⁺ signal to modulate root growth remains unknown. It is hypothesized that the K⁺ channel AKT1 is involved in low K⁺ sensing in the Arabidopsis root and subsequent regulation of root growth. Along with the decline of external K⁺ concentration, the primary root growth of wild‐type plants was gradually inhibited. However, the primary root of the akt1 mutant could still grow under low K⁺ (LK) conditions. Application of NAA inhibited akt1 root growth, but promoted wild‐type root growth under LK conditions. By using the ProDR5:GFP and ProPIN1:PIN1‐GFP lines, we found that LK treatment reduced auxin accumulation in wild‐type root tips by degrading PIN1 proteins, which did not occur in the akt1 mutant. The LK‐induced PIN1 degradation may be due to the inhibition of vesicle trafficking of PIN1 proteins. In conclusion, our findings indicate that AKT1 is required for an Arabidopsis response to changes in external K⁺, and subsequent regulation of K⁺‐dependent root growth by modulating PIN1 degradation and auxin redistribution in the root.     

Susceptibility of the guard-cell K+-uptake channel KST1 to Zn2+ requires histidine residues in the S3-S4 linker and in the channel pore

 Potassium channels are inhibited by several mono- and divalent cations. To identify sites involved in the interaction between K⁺ channels and cationic effectors, we expressed the potato (Solanum tuberosum L.) guard-cell K⁺-uptake channel KST1 in Xenopus oocytes. This channel was reversibly blocked by extracellular Zn²⁺ in the micromolar range. In the presence of this heavy metal, steady-state currents were reduced in a pH-dependent but voltage-independent manner. Since Zn²⁺-inhibition was less effective at elevated external proton concentrations, we generated alanine mutants with respect to both extracellular histidines in KST1. Whereas substitution of the pore histidine H271 resulted in a reduced blockade by Zn²⁺, the channel mutant KST1-H160A in the S3-S4 linker lost most of its Zn²⁺ sensitivity. Since both histidines alter the susceptibility of KST1 to Zn²⁺, the block may predominantly result from these two sites. We thus conclude that the S3-S4 linker is involved in the formation of the outer pore. Received: 3 May 1999 / Accepted: 8 July 1999     

Effects of Cationic Substitutions on Delayed Rectifier Current in Type I Vestibular Hair Cells

The resting potassium current (I _KI ) in gerbil dissociated type I vestibular hair cells has been characterized under various ionic conditions in whole cell voltage-clamp. When all K⁺ in the patch electrode solution was replaced with Na⁺, (Na⁺) _in or Cs⁺, (Cs⁺) _in , large inward currents were evoked in response to voltage steps between −90 and −50 mV. Activation of these currents could be described by a Hodgkin-Huxley-type kinetic scheme, the order of best fit increasing with depolarization. Above ∼−40 mV currents became outward and inactivated with a monoexponential time course. Membrane resistance was inversely correlated with external K⁺ concentration. With (Na⁺) _in , currents were eliminated when K⁺ was removed from the external solution or following extracellular perfusion of 4-aminopyridine, indicating that currents flowed through I _KI channels. Also, reduction of K⁺ entry through manipulation of membrane potential reduced the magnitude of the outward current. Under symmetrical Cs⁺, 0 K⁺ conditions I _KI is highly permeable to Cs⁺. However, inward currents were reduced when small amounts of external K⁺ were added. Higher concentrations of K⁺ resulted in larger currents indicating an anomalous mole fraction effect in mixtures of external Cs⁺ and K⁺. Received: 23 June 1999/Revised: 27 September 1999     

Cation flux in the ehrlich ascites tumor cell evidence for Na-for-Na and K-for-K exchange diffusion

In a previous study, evidence was presented for an external Na⁺-dependent, ouabain-insensitive component of Na⁺ efflux and an external K⁺-dependent component of K⁺ efflux in the Ehrlich ascites tumor cell. Evidence is now presented that these components are inhibited by the diuretic furosemide and that under conditions of normal extracellular Na⁺ and K⁺ they represent Na⁺-for-Na⁺ and K-⁺for-K⁺ exchange mechanisms. Using ⁸⁶Rb to monitor K⁺ movements, furosemide is shown to inhibit an ouabain-insensitive component of Rb⁺ influx and a component of Rb⁺ efflux, both representing approx. 30% of the total fux. Inhibition of Rb⁺ efflux is greatly reduced by removal of extracellular K⁺. Furosemide does not alter steady-state levels of intracellular K⁺ and it does not prevent cells depleted of K⁺ by incubation in the cold from regaining K⁺ upon warming. Using ²²Na to monitor Na⁺ movements, furosemide is shown to inhibit an ouabain-insensitive component of unidirectional Na⁺ efflux which represents approx. 22% of total Na⁺ efflux. Furosemide does not alter steady-state levels of intracellular Na⁺ and does not prevent removal of intracellular Na⁺ upon warming from cells loaded with Na⁺ by preincubation in the cold. The ability of furosemide to affect unidirectional Na⁺ and K⁺ fluxes but not net fluxes is consistent with the conclusion that these components of cation movement across the cell membrane represent one-for-one exchange mechanisms. Data are also presented which demonstrate that the uptake of α-aminoisobutyrate is not affected by furosemide. This indicates that these components of cation flux are not directly involved in the Na⁺-dependent amino acid transport system A.     

The influence of potassium content on the kinetics of potassium influx into excised ryegrass and barley roots

The influx of K⁺ into excised roots of barley (Hordeum vulgare L.) and ryegrass (Lolium multiflorum L.) previously grown with or without K⁺ was measured in K⁺ solutions ranging in concentration from 0.01 to 50 mM. In both species the K⁺ influx was lower in the roots with high K⁺ content. The extent of reduction by high internal [K⁺] decreased with external concentration above 1 mM. These results support the contention that at high external concentrations passive diffusion makes significant contributions to observed fluxes.     

Investigations on the growth and metabolism of cultured explants of Daucus carota

Summary Earlier papers of this series relate to different growth-promoting substances and systems which, singly and in combination, have interacted with trace elements (Mn and Mo) and Fe to induce growth and to affect the metabolism of aseptic cultures of carrot. The solutes of cultured carrot cells (K⁺, Na⁺, Cl^–, total solutes) are also affected. Two clones were grown in 9 combinations of growth factors and under 4 trace-element regimes (a complete complement including Fe, and this complement lacking either Mn or Mo, or both Mn and Mo), a total of 36 treatments under otherwise standardized experimental conditions. Under the treatments applied the number of cells varied over a 35fold range and their average size over a 7fold range; the concomitant effects on their solutes are expressed in terms of concentrations and of total content per cell. Both growth and the solutes accumulated were variously affected by carrot growth-promoting system I (mediated by inositol), by system II (mediated by IAA), and by coconut milk in the presence of Fe, with and without Mn, Mo, or Mn and Mo.The greatest concentrations of total solutes occurred in tissue cultured in nutrient solutions which lacked the stimuli to rapid cell multiplication and were also limited by the trace elements Mn and Mo. Moreover, specific regulatory effects of the trace elements on solute content, not solely attributable to their effects on cell growth, have been noted. An imbalanced growth-factor regime (zeatin acting alone, i.e. without IAA) shifted the normal preference for K⁺ over Na⁺ strongly toward Na⁺, a trend which could also be induced by certain trace elements and more balanced growth-factor regimes, e.g. in a basal coconut milk medium lacking only Mn.The data are interpreted in the context of views on the de-novo uptake of salts and solutes in cultured cells as they grow. These cells respond to a network, or matrix, of interacting factors by distinctive effects that are attributable to the component parts of the culture medium acting singly and in various combinations. These interactions (involving trace elements and exogenous growth factors) control growth (fresh weight, number and size of cells) and regulate the solutes (organic and inorganic; K⁺ vs. Na⁺; organic anions vs. Cl^–) which the cells acquire as they grow and develop. The intensity of the response of the cultures to balanced, or imbalanced, growth factors creates the internal spaces accessible to solutes; and the metabolism, as it is also affected by growth factors and trace elements, determines how these spaces are to be filled at a given osmotic value. The evidence shows the range of factors that affect the accumulation of solutes in cells as they grow and is to be contrasted with conventional observations on mature cells held in steady states under conditions that preclude all growth and when only a single ionic species is followed over a very short interval of time.