Arabidopsis thaliana vacuolar TPK channels form functional K+ uptake pathways in Escherichia coli

Very few vacuolar two pore potassium channels (TPKs) have been functionally characterized. In this paper we have used complementation of K⁺ uptake deficient Escherichia coli mutant LB2003 to analyze the functional properties of Arabidopsis thaliana TPK family members. The four isoforms of AtTPKs were cloned and expressed in LB2003 E. coli background.The expression of channels in bacteria was analyzed by RT-PCR. Our results show that AtTPK1, AtTPK2 and AtTPK5 are restoring the LB2003 growth on low K⁺ media. The analysis of potassium uptake exhibited elevated level of K⁺ uptake in the same three types of AtTPKs transformants.     

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.     

Induction of <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> Pyruvate Decarboxylase Genes

A thalium chloride-resistant (TlCl^r) mutant strain and a sodium chloride-resistant (NaCl^r) mutant strain of the diazotrophic cyanobacterium Anabaena variabilis have been isolated by spontaneous and chemical mutagenesis by using TlCl, a potassium (K⁺) analog, and nitrosoguanidine (NTG), respectively. The TlCl^r mutant strain was found to be defective in K⁺ transport and showed resistance against 10 μM TlCl. However, it also showed sensitivity against NaCl (LD₅₀, 50 mM). In contrast, neither wild-type A. variabilis nor its NaCl^r mutant strain could survive in the presence of 10 μM TlCl and died even at 1 μM TlCl. The TlCl^r mutant strain exhibited almost negligible K⁺ uptake, indicating the lack of a K⁺ uptake system. High K⁺ uptake was, however, observed in the NaCl^r mutant strain, reflecting the presence of an active K⁺ uptake system in this strain. DCMU, an inhibitor of PS II, inhibited the K⁺ uptake in wild-type A. variabilis and its TlCl^r and NaCl^r mutant strains, suggesting that K⁺ uptake in these strains is an energy-dependent process and that energy is derived from photophosphorylation. This contention is further supported by the inhibition of K⁺ uptake under dark conditions. Furthermore, the inhibition of K⁺ uptake by KCN, DNP, and NaN₃ also suggests the involvement of oxidative phosphorylation in the regulation of an active K⁺ uptake system. The whole-cell protein profile of wild-type A. variabilis and its TlCl^r and NaCl^r mutant strains growing in the presence of 50 mM KCl was made in the presence and absence of NaCl. Lack of transporter proteins in TlCl^r mutant strain suggests that these proteins are essentially required for the active transport and accumulation of K⁺ and make this strain NaCl sensitive. In contrast, strong expression of the transporter proteins in NaCl^r mutant strain and its weak expression in wild-type A. variabilis is responsible for their resistance and sensitivity to NaCl, respectively. Therefore, it appears that the increased salt tolerance of the NaCl^r mutant strain was owing to increased K⁺ uptake and accumulation, whereas the salt sensitivity of the TlCl^r mutant strain was owing to the lack of K⁺ uptake and accumulation. Received: 7 March 2002 / Accepted: 8 April 2002     

Molecular cloning and expression analysis of a gene encoding KUP/HAK/KT-type potassium uptake transporter from Cryptomeria japonica

Key message

The molecular mechanism of potassium ion transport across membranes in conifers is poorly known. We isolated and analyzed a gene encoding a potassium transporter from the conifer Cryptomeria japonica.

Abstract

Potassium ion (K⁺) is an essential and the most abundant intracellular cation in plants. The roles of K⁺ in various aspects of plant life are closely linked to its transport across biological membranes such as the plasma membrane and the tonoplast, which is mediated by membrane-bound transport proteins known as transporters and channels. Information on the molecular basis of K⁺ membrane transport in trees, especially in conifers, is currently limited. In this study, we isolated one complementary DNA, CjKUP1, which is homologous to known plant K⁺ transporters, from Cryptomeria japonica. Complementation tests using an Escherichia coli mutant, which is deficient in K⁺ uptake activity, was conducted to examine the K⁺ uptake function of the protein encoded by CjKUP1. Transformation of the K⁺-uptake-deficient mutant with CjKUP1 complemented the deficiency of this mutant. This result indicates that CjKUP1 has a function of K⁺ uptake. The expression levels of CjKUP1 in male strobili were markedly higher from late September to early October than in other periods. The expression levels in male and female strobili were higher than those in other organs such as needles, inner bark, differentiating xylem, and roots. These results indicate that CjKUP1 is mainly involved in K⁺ membrane transport in the cells of reproductive organs of C. japonica trees, especially in male strobili during pollen differentiation.     

Alternating Hemiplegia of Childhood-Related Neural and Behavioural Phenotypes in Na+,K+-ATPase α3 Missense Mutant Mice

Missense mutations in ATP1A3 encoding Na⁺,K⁺-ATPase α3 have been identified as the primary cause of alternating hemiplegia of childhood (AHC), a motor disorder with onset typically before the age of 6 months. Affected children tend to be of short stature and can also have epilepsy, ataxia and learning disability. The Na⁺,K⁺-ATPase has a well-known role in maintaining electrochemical gradients across cell membranes, but our understanding of how the mutations cause AHC is limited. Myshkin mutant mice carry an amino acid change (I810N) that affects the same position in Na⁺,K⁺-ATPase α3 as I810S found in AHC. Using molecular modelling, we show that the Myshkin and AHC mutations display similarly severe structural impacts on Na⁺,K⁺-ATPase α3, including upon the K⁺ pore and predicted K⁺ binding sites. Behavioural analysis of Myshkin mice revealed phenotypic abnormalities similar to symptoms of AHC, including motor dysfunction and cognitive impairment. 2-DG imaging of Myshkin mice identified compromised thalamocortical functioning that includes a deficit in frontal cortex functioning (hypofrontality), directly mirroring that reported in AHC, along with reduced thalamocortical functional connectivity. Our results thus provide validation for missense mutations in Na⁺,K⁺-ATPase α3 as a cause of AHC, and highlight Myshkin mice as a starting point for the exploration of disease mechanisms and novel treatments in AHC.     

Relation of potassium uptake to proton transport and activity of hydrogenases in Escherichia coli grown at low pH

Escherichia coli grown under anaerobic conditions in acidic medium (pH 5.5) upon hyperosmotic stress accumulates potassium ions mainly through the Kup system, the functioning of which is associated with proton efflux decrease. It was shown that H⁺ secretion but not glucose-induced K⁺ uptake was inhibited by N,N′-dicyclohexylcarbodiimide (DCC). The inhibitory effect of DCC on the H⁺ efflux was stronger in the trkA mutant with defective potassium transport. The K⁺ and H⁺ fluxes depended on the extent of hyperosmotic stress in the absence or presence of DCC. The decrease in external oxidation/reduction potential and H₂ liberation insensitive to DCC were recorded. It was found that the atpD mutant with nonfunctional F₀F₁-ATPase produced a substantial amount of H₂, while in the hyc mutant (but not the hyf mutant defective in hydrogenases 3 (Hyd-3) and 4 (Hyd-4)) the H₂ production was significantly suppressed. At the same time, the rate of K⁺ uptake was markedly lower in hyfR and hyfB-R but not in hycE or hyfA-B mutants; H⁺ transport was lowered and sensitive to DCC in hyf but not in hyc mutants. The results point to the relationship of K⁺ uptake with the Hyd-4 activity. Novel options of the expression of some hyf genes in E. coli grown at pH 5.5 are proposed. It is possible that the hyfB-R genes expressed under acidic conditions or their gene products interact with the gene coding for the Kup protein or directly with the Kup system.     

Alternating Hemiplegia of Childhood mutations have a differential effect on Na+,K+-ATPase activity and ouabain binding

De novo mutations in ATP1A3, the gene encoding the α3-subunit of Na⁺,K⁺-ATPase, are associated with the neurodevelopmental disorder Alternating Hemiplegia of Childhood (AHC). The aim of this study was to determine the functional consequences of six ATP1A3 mutations (S137Y, D220N, I274N, D801N, E815K, and G947R) associated with AHC. Wild type and mutant Na⁺,K⁺-ATPases were expressed in Sf9 insect cells using the baculovirus expression system. Ouabain binding, ATPase activity, and phosphorylation were absent in mutants I274N, E815K and G947R. Mutants S137Y and D801N were able to bind ouabain, although these mutants lacked ATPase activity, phosphorylation, and the K⁺/ouabain antagonism indicative of modifications in the cation binding site. Mutant D220N showed similar ouabain binding, ATPase activity, and phosphorylation to wild type Na⁺,K⁺-ATPase. Functional impairment of Na⁺,K⁺-ATPase in mutants S137Y, I274N, D801N, E815K, and G947R might explain why patients having these mutations suffer from AHC. Moreover, mutant D801N is able to bind ouabain, whereas mutant E815K shows a complete loss of function, possibly explaining the different phenotypes for these mutations.     

A study of the K+-site mutant of ascorbate peroxidase: mutations of protein residues on the proximal side of the heme cause changes in iron ligation on the distal side

A series of ferric and ferrous derivatives of wild-type ascorbate peroxidase (APX) and of an engineered K⁺-site mutant of APX that has had its potassium cation binding site removed have been examined by electronic absorption and magnetic circular dichroism (MCD) spectroscopy at 4??°C. Wild-type ferric APX has spectroscopic properties that are very similar to those of ferric cytochrome c peroxidase (CCP) and likely exists primarily as a five-coordinate high-spin heme ligated on the proximal side by a histidine at pH 7. There is also evidence for minority contributions from six-coordinate high- and low-spin species (histidine-water, histidine-hydroxide, and bis-histidine). The K⁺-site mutant of APX varies considerably in the electronic absorption and MCD spectra in both the ferric and ferrous states when compared with spectra of the wild-type APX. The electronic absorption and MCD spectra of the engineered K⁺-site APX mutant are essentially identical to those of cytochrome b ₅, a known bis-imidazole (histidine) ligated heme system. It therefore appears that the K⁺-site mutant of APX has undergone a conformational change to yield a bis-histidine coordination structure in both the ferric and ferrous oxidation states at neutral pH. This conformational change is the result of mutagenesis of the protein to remove the K⁺-binding site which is located ～8?Å from the peroxide binding pocket. Thus, mutations of protein residues on the proximal side of the heme cause changes in iron ligation on the distal side.     

Vacuolar Cation/H+ Antiporters of Saccharomyces cerevisiae

We previously demonstrated that Saccharomyces cerevisiae vnx1Δ mutant strains displayed an almost total loss of Na⁺ and K⁺/H⁺ antiporter activity in a vacuole-enriched fraction. However, using different in vitro transport conditions, we were able to reveal additional K⁺/H⁺ antiporter activity. By disrupting genes encoding transporters potentially involved in the vnx1 mutant strain, we determined that Vcx1p is responsible for this activity. This result was further confirmed by complementation of the vnx1Δvcx1Δ nhx1Δ triple mutant with Vcx1p and its inactivated mutant Vcx1p-H303A. Like the Ca²⁺/H⁺ antiporter activity catalyzed by Vcx1p, the K⁺/H⁺ antiporter activity was strongly inhibited by Cd²⁺ and to a lesser extend by Zn²⁺. Unlike as previously observed for NHX1 or VNX1, VCX1 overexpression only marginally improved the growth of yeast strain AXT3 in the presence of high concentrations of K⁺ and had no effect on hygromycin sensitivity. Subcellular localization showed that Vcx1p and Vnx1p are targeted to the vacuolar membrane, whereas Nhx1p is targeted to prevacuoles. The relative importance of Nhx1p, Vnx1p, and Vcx1p in the vacuolar accumulation of monovalent cations will be discussed.     

Bioelectric Signaling Regulates Size in Zebrafish Fins

The scaling relationship between the size of an appendage or organ and that of the body as a whole is tightly regulated during animal development. If a structure grows at a different rate than the rest of the body, this process is termed allometric growth. The zebrafish another longfin (alf) mutant shows allometric growth resulting in proportionally enlarged fins and barbels. We took advantage of this mutant to study the regulation of size in vertebrates. Here, we show that alf mutants carry gain-of-function mutations in kcnk5b, a gene encoding a two-pore domain potassium (K⁺) channel. Electrophysiological analysis in Xenopus oocytes reveals that these mutations cause an increase in K⁺ conductance of the channel and lead to hyperpolarization of the cell. Further, somatic transgenesis experiments indicate that kcnk5b acts locally within the mesenchyme of fins and barbels to specify appendage size. Finally, we show that the channel requires the ability to conduct K⁺ ions to increase the size of these structures. Our results provide evidence for a role of bioelectric signaling through K⁺ channels in the regulation of allometric scaling and coordination of growth in the zebrafish.     

Intracellular Na+ and K+ contents of Zygosaccharomyces rouxii mutants defective in salt tolerance

Two of five Zygosaccharomyces rouxii mutants defective in salt tolerance, 152S (sat1) and 1717S (SAT3), were inviable in a nutrient medium (YPD) containing more than 1% NaCl. These two mutant cells contained significantly higher amounts of Na⁺ (298 μmol and 285 μmol per g cells of 152S and 1717S, respectively) but lower amounts of K⁺ (242 μmol and 176 μmol per g cells of 152S and 1717S, respectively) than three other mutants, 41S (sat2-1 [98 μmol Na⁺ and 326 μmol K⁺/g cells]), 197S (sat2-2 [103μmol Na⁺ and 336 μmol K⁺/g cells]), 1611S (SAT4 [139 μmol Na⁺ and 294 μmol K⁺/g cells]), as well as a wild-type strain, AN39 (61 μmol Na⁺ and 349 μmol K⁺/g cells), when cultured in YPD medium containing 0.8% NaCl. A KCl supplement, optimally 0.6 M, added to the medium somewhat restored the NaCl-hypersensitivity of 152S and 1717S with a concomitant decrease of intracellular Na⁺. This finding suggests that the NaCl-hypersensitive mutations are due to a defect in the Na⁺-regulating mechanism. The other three mutants showed weak responses to KCl in high NaCl-YPD. These five salt sensitive mutants and the wild-type strain retained the same levels of intracellular glycerol and arabitol when transferred into NaCl (5%)-YPD from YDP medium. This suggests that polyol accumulation is not the only mechanism of salt tolerance in Z. rouxii.     

Comparative Analysis of kdp and ktr Mutants Reveals Distinct Roles of the Potassium Transporters in the Model Cyanobacterium Synechocystis sp. Strain PCC 6803

Photoautotrophic bacteria have developed mechanisms to maintain K⁺ homeostasis under conditions of changing ionic concentrations in the environment. Synechocystis sp. strain PCC 6803 contains genes encoding a well-characterized Ktr-type K⁺ uptake transporter (Ktr) and a putative ATP-dependent transporter specific for K⁺ (Kdp). The contributions of each of these K⁺ transport systems to cellular K⁺ homeostasis have not yet been defined conclusively. To verify the functionality of Kdp, kdp genes were expressed in Escherichia coli, where Kdp conferred K⁺ uptake, albeit with lower rates than were conferred by Ktr. An on-chip microfluidic device enabled monitoring of the biphasic initial volume recovery of single Synechocystis cells after hyperosmotic shock. Here, Ktr functioned as the primary K⁺ uptake system during the first recovery phase, whereas Kdp did not contribute significantly. The expression of the kdp operon in Synechocystis was induced by extracellular K⁺ depletion. Correspondingly, Kdp-mediated K⁺ uptake supported Synechocystis cell growth with trace amounts of external potassium. This induction of kdp expression depended on two adjacent genes, hik20 and rre19, encoding a putative two-component system. The circadian expression of kdp and ktr peaked at subjective dawn, which may support the acquisition of K⁺ required for the regular diurnal photosynthetic metabolism. These results indicate that Kdp contributes to the maintenance of a basal intracellular K⁺ concentration under conditions of limited K⁺ in natural environments, whereas Ktr mediates fast potassium movements in the presence of greater K⁺ availability. Through their distinct activities, both Ktr and Kdp coordinate the responses of Synechocystis to changes in K⁺ levels under fluctuating environmental conditions.     

Cloning of two SOS1 transporters from the seagrass <Emphasis Type="Italic">Cymodocea nodosa</Emphasis>. SOS1 transporters from <Emphasis Type="Italic">Cymodocea</Emphasis> and <Emphasis Type="Italic">Arabidopsis</Emphasis> mediate potassium uptake in bacteria

Two cDNAs isolated from Cymodocea nodosa, CnSOS1A, and CnSOS1B encode proteins with high-sequence similarities to SOS1 plant transporters. CnSOS1A expressed in a yeast Na⁺-efflux mutant under the control of a constitutive expression promoter mimicked AtSOS1 from Arabidopsis; the wild type cDNA did not improve the growth of the recipient strain in the presence of Na⁺, but a cDNA mutant that expresses a truncated protein suppressed the defect of the yeast mutant. In similar experiments, CnSOS1B was not effective. Conditional expression, under the control of an arabinose responsive promoter, of the CnSOS1A and CnSOS1B cDNAs in an Escherichia coli mutant defective in Na⁺ efflux was toxic, and functional analyses were inconclusive. The same constructs transformed into an E. coli K⁺-uptake mutant revealed that CnSOS1A was also toxic, but that it slightly suppressed defective growth at low K⁺. Truncation in the C-terminal hydrophilic tail of CnSOS1A relieved the toxicity and proved that CnSOS1A was an excellent low-affinity K⁺ and Rb⁺ transporter. CnSOS1B mediated a transient, extremely rapid K⁺ or Rb⁺ influx. Similar tests with AtSOS1 revealed that it was not toxic and that the whole protein exhibited excellent K⁺ and Rb⁺ uptake characteristics in bacteria.     

Molecular cloning and functional expression in bacteria of the potassium transporters CnHAK1 and CnHAK2 of the seagrass Cymodocea nodosa

The cDNAs CnHAK1 and CnHAK2, encoding K⁺ transporters, were amplified from the leaves of the seagrass Cymodocea nodosa. None of these transporters suppressed the K⁺ deficiency of a Saccharomyces cerevisiae mutant, but both suppressed the equivalent defect of an Escherichia coli mutant. Overexpression of the transporter CnHAK1, but not CnHAK2, mediated very rapid K⁺ or Rb⁺ influxes in the E. coli mutant. The concentration dependence of these influxes demonstrated that CnHAK1 is a low-affinity K⁺ transporter, which does not discriminate between K⁺ and Rb⁺. CnHAK1 expressed in E. coli worked in reverse when the external K⁺ concentrations were low, and we established the condition of a simple functional test of K⁺ loss for transporters of the Kup-HAK family. In comparison with its homologue barley transporter HvHAK2, CnHAK1 was insensitive to Na⁺.     

Relationship between the F0F1-ATPase and the K+-transport system within the membrane of anaerobically grownEscherichia coli. N,N′-dicyclohexylcarbodiimide-sensitive ATPase activity in mutants with defects in K+-transport

A considerable (2-fold) stimulation of the DCCD-sensitive ATPase activity by K⁺ or Rb⁺, but not by Na⁺, over the range of zero to 100mM was shown in the isolated membranes ofE. coli grown anaerobically in the presence of glucose. This effect was observed only in parent and in thetrkG, but not in thetrkA, trkE, ortrkH mutants. ThetrkG or thetrkH mutant with anunc deletion had a residual ATPase activity not sensitive to DCCD. A stimulation of the DCCD-sensitive ATPase activity by K⁺ was absent in the membranes from bacteria grown anaerobically in the presence of sodium nitrate. Growth of thetrkG, but not of othertrk mutants, in the medium with moderate K⁺ activity did not depend on K⁺ concentration. Under upshock, K⁺ accumulation was essentially higher in thetrkG mutant than in the othertrk mutant. The K⁺-stimulated DCCD-sensitive ATPase activity in the membranes isolated from anaerobically grownE. coli has been shown to depend absolutely on both the F₀F₁ and theTrk system and can be explained by a direct interaction between these transport systems within the membrane of anaerobically grown bacteria with the formation of a single supercomplex functioning as a H⁺-K⁺ pump. ThetrkG gene is most probably not functional in anaerobically grown bacteria.This study was performed at the Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois 60637.     

Two major potassium uptake systems, KtrI and KtrII, in Enterococcus hirae

We isolated a mutant, JEMK1, which did not grow at pH 6.0 in medium containing 0.5 mM KCl, derived from a Enterococcus hirae mutant deficient in the KtrII K⁺ uptake system. This mutant showed an impairment in the proton potential-dependent K⁺ uptake, the activity of the KtrI K⁺ uptake system, and did not grow well in K⁺-poor media in a wide pH range from 6 to 10, suggesting that KtrI and KtrII are the main systems for potassium accumulation of this bacterium.     

OsPRX2 contributes to stomatal closure and improves potassium deficiency tolerance in rice

Peroxiredoxins (Prxs) which are thiol-based peroxidases have been implicated in the toxic reduction and intracellular concentration regulation of hydrogen peroxide. In Arabidopsis thaliana At2-CysPrxB (At5g06290) has been demonstrated to be essential in maintaining the water-water cycle for proper H₂O₂ scavenging. Although the mechanisms of 2-Cys Prxs have been extensively studied in Arabidopsis thaliana, the function of 2-Cys Prxs in rice is unclear. In this study, a rice homologue gene of At2-CysPrxB, OsPRX2 was investigated aiming to characterize the effect of 2-Cys Prxs on the K⁺-deficiency tolerance in rice. We found that OsPRX2 was localized in the chloroplast. Overexpressed OsPRX2 causes the stomatal closing and K⁺-deficiency tolerance increasing, while knockout of OsPRX2 lead to serious defects in leaves phenotype and the stomatal opening under the K⁺-deficiency tolerance. Detection of K⁺ accumulation, antioxidant activity of transgenic plants under the starvation of potassium, further confirmed that OsPRX2 is a potential target for engineering plants with improved potassium deficiency tolerance.     

Reversible Repression of Early Enzyme Synthesis in Bacteriophage T4-infected Escherichia coli

A mutant of Escherichia coli B, defective in its accumulation of K⁺, was found to synthesize protein at a rate proportional to the level of this cation in the growth medium. When bacteriophage T4-infected cells were incubated in growth medium containing 1 mm K⁺, phage deoxyribonucleic acid (DNA) was synthesized at a rate 25% that of normal, and phage protein was synthesized at a rate of 50% of normal. Deoxycytidine pyrophosphatase, a phage-directed early enzyme, shut off at a level of 55% that of normal when infected cells were incubated in medium containing 1 mm K⁺. However, deoxycytidine pyrophosphatase synthesis resumed in these cells when they were shifted to medium containing the normal K⁺ concentration (33 mm). DNA synthesis also attained the rate characteristic of this K⁺ concentration. These results suggest that phage DNA synthesis is not sufficient to repress early protein formation and also indicate that the inhibitor of early protein formation is an early function whose synthesis is sensitive to the same repression as that of the early proteins.     

Early changes in membrane potential of Saccharomyces cerevisiae induced by varying extracellular K+, Na+ or H+ concentrations

Recently we introduced a fluorescent probe technique that makes possible to convert changes of equilibrium fluorescence spectra of 3,3’-dipropylthiadicarbocyanine, diS-C₃(3), measured in yeast cell suspensions under defined conditions into underlying membrane potential differences, scaled in millivolts (Plasek et al. in J Bioenerg Biomembr 44: 559–569, 2012). The results presented in this paper disclose measurements of real early changes of plasma membrane potential induced by the increase of extracellular K⁺, Na⁺ and H⁺ concentration in S. cerevisiae with and without added glucose as energy source. Whereas the wild type and the ?tok1 mutant cells exhibited similar depolarization curves, mutant cells lacking the two Trk1,2 potassium transporters revealed a significantly decreased membrane depolarization by K⁺, particularly at lower extracellular potassium concentration [K⁺]_out. In the absence of external energy source plasma membrane depolarization by K⁺ was almost linear. In the presence of glucose the depolarization curves exhibited an exponential character with increasing [K⁺]_out. The plasma membrane depolarization by Na⁺ was independent from the presence of Trk1,2 transporters. Contrary to K⁺, Na⁺ depolarized the plasma membrane stronger in the presence of glucose than in its absence. The pH induced depolarization exhibited a fairly linear relationship between the membrane potential and the pH_o of cell suspensions, both in the wild type and the Δtrk1,2 mutant strains, when cells were energized by glucose. In the absence of glucose the depolarization curves showed a biphasic character with enhanced depolarization at lower pH_o values.     

Growth of Bradyrhizobium sp. SEMIA 6144 in Response to Methylglyoxal: Role of Glutathione

We previously showed the important role of glutathione (GSH) in the protection mechanism against different stresses, such as acid pH, saline, and oxidative stress, using a GSH-deficient mutant of Bradyrhizobium sp. (peanut microsymbiont). In this work, we studied the role of GSH in the protection mechanism against methylglyoxal (MG) toxicity. MG is a naturally occurring toxic electrophilic compound, and it has been shown that GSH is involved in the detoxification of MG in Escherichia coli. One recognized component of this detoxification process is the formation of a GSH adduct, which in turn transports potassium (K⁺) out of bacterial cells. Our results showed that growth of wild-type strain Bradyrhizobium sp. SEMIA 6144 was not affected at a MG concentration of 0.5 mM in the yeast extract–mannitol culture medium. However, a reduction of growth, at concentrations of 1.5 and 2.5 mM MG and reaching complete growth inhibition at 3.0 mM MG, was observed. In wild-type strain, intracellular GSH content decreased, and intracellular K⁺ content was unchanged, whereas GSH-deficient mutant SEMIA 6144-S7Z was unable to grow at 1.5 mM MG. The addition of external GSH to the incubation medium did not restore the growth rate either in wild-type or mutant strains. Our findings showed that GSH has not proven to be protective against the cell-growth inhibiting activity of MG. Therefore, the response of Bradyrhizobium sp. growth to MG is different from that reported in E. coli and other Gram-negative bacteria.