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     

Functional expression and characterization of the wild-type mammalian renal cortex sodium/phosphate cotransporter and an 215R mutant in Saccharomyces cerevisiae.

The wild-type and an R215E mutant of the rat renal cortex sodium/phosphate cotransporter type 2 (NaPi-2) were functionally expressed in the yeast Saccharomyces cerevisiae strain MB192, a cell line lacking the high-affinity endogenous H+/P(i) cotransporter. The expression of the mRNA molecules and corresponding proteins was confirmed by Northern and Western blot analysis, respectively. As detected by indirect immunofluorescence and antibody capture assay, both wild-type and mutant NaPi-2 proteins are expressed in the yeast plasma membrane in comparable amounts. In the presence of 5 microM phosphate, Na+ promotes phosphate uptake into yeast cells expressing the wild-type NaPi-2 with a K(0.5) of 5.6 +/- 1.1 mM. The maximum uptake of phosphate (649 +/- 30 pmol/10 min) is approximately 8-fold higher than the uptake obtained with nontransformed cells (76.8 +/- 8 pmol/10 min). Yeast cells expressing the R215E mutant of NaPi-2 accumulate 213 +/- 9 pmol of phosphate/10 min under the same conditions. The K(0.5) for the stimulation of phosphate uptake by Na+ is 4.2 +/- 0.8 mM for the R215E mutant and thus not significantly different from the value obtained with cells expressing the wild-type cotransporter. The reduced level of accumulation of phosphate in yeast cells expressing the R215E mutant is probably due to a reduction of the first-order rate constant k for phosphate uptake: while cells expressing wild-type NaPi-2 accumulate phosphate with a k of 0.06 min(-1), the rate for phosphate uptake into cells expressing the R215E mutant (k) is 0.016 min(-1) and therefore about 4-fold lower. In comparison, the rate for phosphate uptake into nontransformed cells (k) is 0.0075 min(-1). Phosphate uptake into yeast cells that express the wild-type NaPi-2 in the presence of 150 mM NaCl is promoted by extracellular phosphate with a K(0.5) of 45 +/- 4 microM. A phosphate-dependent phosphate accumulation is also observed with cells expressing the R215E mutant, but the K(0.5) is twice as high (86 +/- 5 microM) as that obtained with the wild-type cotransporter. We conclude that the yeast expression system is a useful tool for the investigation of structure-function relationships of the renal sodium/phosphate cotransporter and that (215)R, although not involved in Na+ recognition, is a part of the structure involved in phosphate recognition and considerably influences the rate of phosphate uptake by the NaPi-2 cotransporter.     

Role of glutathione in detoxification of metal(loid)s by Saccharomyces cerevisiae

Cellular glutathione (GSH) was implicated in tolerance to potentially toxic metal(loid)s using two strains of Saccharomyces cerevisiae, a wild-type (sigma 1278b) and a GSH-deficient mutant strain (gshA-2). Both yeast strains exhibited no significant difference in tolerance to tellurite, zinc, cobalt, copper, manganese, nickel and chromate. There was no marked influence of glutathione on the accumulation of Te, Co, Cu, and Mn, although the absence of cellular glutathione significantly increased the cellular content of Zn and Ni, but greatly decreased Cr content without significant alteration of tolerance. These results indicated the independence of cellular glutathione activity from tolerance to Te, Zn, Co, Cu, Mn, Ni, and Cr. However, involvement of glutathione in Zn, Ni and Cr uptake is possible. The glutathione-deficient strain displayed a high sensitivity to selenite and cadmium in comparison to the wild-type strain of S. cerevisiae. The minimum inhibitory concentrations of Se and Cd for the glutathione-deficient strain were 980 +/- 13 and 32 +/- 4 microM, respectively, whereas the wild strain tolerated up to 4080 +/- 198 microM Se and 148 +/- 5 microM Cd. A relationship between tolerance and reduced cellular content of both Se and Cd was also shown: the mutant strain accumulated approximately three-fold more Se and two-fold more Cd than that accumulated by the wild-type strain. This suggests an influence of GSH on cellular uptake of Se and Cd, and also directly confirms the protective action of such a cellular thiol compound against Se and Cd toxicity.     

Influx and accumulation of Cs(+) by the akt1 mutant of Arabidopsis thaliana (L.) Heynh. lacking a dominant K(+) transport system.

An extensive literature reports that Cs(+), an environmental contaminant, enters plant cells through K(+) transport systems. Several recently identified plant K(+) transport systems are permeable to Cs(+). Permeation models indicate that most Cs(+) uptake into plant roots under typical soil ionic conditions will be mediated by voltage-insensitive cation (VIC) channels in the plasma membrane and not by the inward rectifying K(+) (KIR) channels implicated in plant K nutrition. Cation fluxes through KIR channels are blocked by Cs(+). This paper tests directly the hypothesis that the dominant KIR channel in plant roots (AKT1) does not contribute significantly to Cs(+) uptake by comparing Cs(+) uptake into wild-type and the akt1 knockout mutant of Arabidopsis thaliana (L.) Heynh. Wild-type and akt1 plants were grown to comparable size and K(+) content on agar containing 10 mM K(+). Both Cs(+) influx to roots of intact plants and Cs(+) accumulation in roots and shoots were identical in wild-type and akt1 plants. These data indicate that AKT1 is unlikely to contribute significantly to Cs(+) uptake by wild-type Arabidopsis from 'single-salt' solutions. The influx of Cs(+) to roots of intact wild-type and akt1 plants was inhibited by 1 mM Ba(2+), Ca(2+) and La(3+), but not by 10 microM Br-cAMP. This pharmacology resembles that of VIC channels and is consistent with the hypothesis that VIC channels mediate most Cs(+) influx under 'single-salt' conditions.     

Two types of Bacillus subtilis tetA(L) deletion strains reveal the physiological importance of TetA(L) in K(+) acquisition as well as in Na(+), alkali, and tetracycline resistance

The chromosomally encoded TetA(L) protein of Bacillus subtilis is a multifunctional tetracycline-metal/H(+) antiporter that also exhibits monovalent cation/H(+) antiport activity and a net K(+) uptake mode. In this study, B. subtilis mutant strains JC112 and JC112C were found to be representative of two phenotypic types of tetA(L) deletion strains that are generated in the same selection. Both strains exhibited increased sensitivity to low tetracycline concentrations as expected. The mutants also had significantly reduced ability to grow in media containing low concentrations of K(+), indicating that the net K(+) uptake mode is of physiological consequence; the deficit in JC112 was greater than in JC112C. JC112 also exhibited (i) greater impairment of Na(+)- or K(+)-dependent growth at pH 8.3 than JC112C and (ii) a greater degree of Co(+2) as well as Na(+) sensitivity. Studies were initiated to explore the possibility of two different patterns of compensatory changes in other ion-translocating transporters in these mutants. Increased expression of two loci has thus far been shown. Increased expression of czcD-trkA, a locus with a proposed involvement in K(+) uptake, occurred in both mutants. The increase was highest in the presence of Co(2+) and was higher in JC112 than in JC112C. Deletion of czcD-trkA resulted in diminished growth of the wild-type and both mutant strains at low [K(+)], supporting a significant role for this locus in K(+) uptake. Expression of yheL, which is a homologue of the Na(+)/H(+) antiporter-encoding nhaC gene from Bacillus firmus OF4, was also increased in both tetA(L) deletion strains, again with higher up-regulation in JC112. The phenotypes resulting from deletion of yheL were consistent with a modest role for YheL in Na(+)-dependent pH homeostasis in the wild type. No major role for YheL was indicated in the mutants in spite of the overexpression. The studies underscore the multiple physiological functions of TetA(L), including tetracycline, Na(+), and alkali resistance and K(+) acquisition. The studies also reveal and begin to detail the complexity of the response to mutational loss of these functions.     

Protection of plasma membrane K+ transport by the salt overly sensitive1 Na+-H+ antiporter during salinity stress

Physicochemical similarities between K(+) and Na(+) result in interactions between their homeostatic mechanisms. The physiological interactions between these two ions was investigated by examining aspects of K(+) nutrition in the Arabidopsis salt overly sensitive (sos) mutants, and salt sensitivity in the K(+) transport mutants akt1 (Arabidopsis K(+) transporter) and skor (shaker-like K(+) outward-rectifying channel). The K(+)-uptake ability (membrane permeability) of the sos mutant root cells measured electrophysiologically was normal in control conditions. Also, growth rates of these mutants in Na(+)-free media displayed wild-type K(+) dependence. However, mild salt stress (50 mm NaCl) strongly inhibited root-cell K(+) permeability and growth rate in K(+)-limiting conditions of sos1 but not wild-type plants. Increasing K(+) availability partially rescued the sos1 growth phenotype. Therefore, it appears that in the presence of Na(+), the SOS1 Na(+)-H(+) antiporter is necessary for protecting the K(+) permeability on which growth depends. The hypothesis that the elevated cytoplasmic Na(+) levels predicted to result from loss of SOS1 function impaired the K(+) permeability was tested by introducing 10 mm NaCl into the cytoplasm of a patch-clamped wild-type root cell. Complete loss of AKT1 K(+) channel activity ensued. AKT1 is apparently a target of salt stress in sos1 plants, resulting in poor growth due to impaired K(+) uptake. Complementary studies showed that akt1 seedlings were salt sensitive during early seedling development, but skor seedlings were normal. Thus, the effect of Na(+) on K(+) transport is probably more important at the uptake stage than at the xylem loading stage.     

Mutational Engineering of the cyanobacterium Nostoc muscorum for resistance to growth-inhibitory action of LiCl and NaCl

The effect of NaCl on two vital processes of cyanobacterial metabolism, viz. N(2) fixation and oxygenic photosynthesis, was studied in the cyanobacterium Nostoc muscorum grown diazotrophically. An increase in NaCl concentration suppressed the formation of heterocyst and adversely affected the nitrogenase activity in the parent, whereas in Li(+)-R and Na(+)-R mutants NaCl stress did not cause any adverse effect. The rate of photosynthetic O(2)-evolution was also adversely affected by the NaCl stress, but the magnitude was less than that of nitrogenase activity. L-Proline, the well-known osmoprotectant, provided protection to the cyanobacterium against NaCl stress. The parent strain utilized L-proline as a nitrogen source and suppressed heterocyst formation and nitrogenase activity, while mutants showed normal heterocyst frequency and nitrogenase activity. Therefore, it may be that the proline metabolism is altered as a result of mutation. The intracellular levels of proline in the parent were enhanced about threefold in the medium containing 1 mol x m(-3) proline, while in mutants there was no significant increase in the intracellular level of proline. In the medium containing both NaCl and proline, the intracellular level of proline was enhanced in the parent as well as in both mutant strains. This suggests that the parent strain possessed both normal proline uptake and salt-induced proline uptake systems, whereas the mutant strains were defective in normal proline uptake and had only salt-induced proline uptake. The over-accumulation of proline in the presence of NaCl stress is due either to the loss of proline oxidase activity or to the accumulation of exogenous proline.     

Genes from Debaryomyces hansenii increase salt tolerance in Saccharomyces cerevisiae W303

The yeast Debaryomyces hansenii has been chosen as a model for molecular studies of tolerance to NaCl. A gene library was built and transformants of Saccharomyces cerevisiae W303 containing genes from D. hansenii were selected for their ability to grow in the presence of high concentrations of NaCl and/or low concentrations of KCl. In three of these transformants 500 mM NaCl improved growth at pH 7.6 like in D. hansenii but not in S. cerevisiae. One of the plasmids restored growth at 50 microM KCl and K(+) uptake in a mutant of S. cerevisiae lacking genes that encode K(+) transporters.     

Mutant Phe788 --> Leu of the Na+,K+-ATPase is inhibited by micromolar concentrations of potassium and exhibits high Na+-ATPase activity at low sodium concentrations.

Mutant Phe788 --> Leu of the rat kidney Na+,K(+)-ATPase was expressed in COS cells to active-site concentrations between 40 and 60 pmol/mg of membrane protein. Analysis of the functional properties showed that the discrimination between Na+ and K+ on the two sides of the system is severely impaired in the mutant. Micromolar concentrations of K+ inhibited ATP hydrolysis (K(0.5) for inhibition 107 microM for the mutant versus 76 mM for the wild-type at 20 mM Na+), and at 20 mM K+, the molecular turnover number for Na+,K(+)-ATPase activity was reduced to 11% that of the wild-type. This inhibition was counteracted by Na+ in high concentrations, and in the total absence of K+, the mutant catalyzed Na(+)-activated ATP hydrolysis ("Na(+)-ATPase activity") at an extraordinary high rate corresponding to 86% of the maximal Na+,K(+)-ATPase activity. The high Na(+)-ATPase activity was accounted for by an increased rate of K(+)-independent dephosphorylation. Already at 2 mM Na+, the dephosphorylation rate of the mutant was 8-fold higher than that of the wild-type, and the maximal rate of Na(+)-induced dephosphorylation amounted to 61% of the rate of K(+)-induced dephosphorylation. The cause of the inhibitory effect of K+ on ATP hydrolysis in the mutant was an unusual stability of the K(+)-occluded E2(K2) form. Hence, when E2(K2) was formed by K+ binding to unphosphorylated enzyme, the K(0.5) for K+ occlusion was close to 1 microM in the mutant versus 100 microM in the wild-type. In the presence of 100 mM Na+ to compete with K+ binding, the K(0.5) for K+ occlusion was still 100-fold lower in the mutant than in the wild-type. Moreover, relative to the wild-type, the mutant exhibited a 6-7-fold reduced rate of release of occluded K+, a 3-4-fold increased apparent K+ affinity in activation of the pNPPase reaction, a 10-11-fold lower apparent ATP affinity in the Na+,K(+)-ATPase assay with 250 microM K+ present (increased K(+)-ATP antagonism), and an 8-fold reduced apparent ouabain affinity (increased K(+)-ouabain antagonism).     

Rhizobium japonicum mutants that are hypersensitive to repression of H2 uptake by oxygen.

The synthesis of an H2 oxidation system in free-living Rhizobium japonicum wild-type strain SR is repressed by oxygen. Maximal H2 uptake rates were obtained in strain SR after derepression in 11 microM or less dissolved oxygen. Oxygen levels above 45 microM completely repressed H2 uptake in strain SR. Five R. japonicum mutant strains that are hypersensitive to repression or H2 oxidation by oxygen were derived from strain SR. The mutants were obtained by screening H2 uptake-negative mutants that retained the ability to oxidize H2 as bacteroids from soybean nodules. As bacteroids, the five mutant strains were capable of H2 oxidation rates comparable to that of the wild type. The mutants did not take up H2 when derepressed in 22 microM dissolved oxygen, whereas strain SR had substantial activity at this oxygen concentration. The O2 repression of H2 uptake in both the wild-type and two mutant strains, SR174 and SR200, was rapid and was similar to the effect of inhibiting synthesis of H2 uptake system components with rifampin. None of the mutant strains was able to oxidize H2 when the artificial electron acceptors methylene blue or phenazine methosulfate were provided. The mutant strains were not sensitive to killing by oxygen, they took up O2 at rates similar to strain SR, and they did not produce an H2 uptake system that was oxygen labile. Cyclic AMP levels were comparable in strain SR and the five mutant strains after subjection of the cultures to the derepression conditions.     

Absence of light-induced proton extrusion in a cotA-less mutant of Synechocystis sp. strain PCC6803.

cotA of Synechocystis sp. strain PCC6803 was isolated as a gene that complemented a mutant defective in CO2 transport and is homologous to cemA that encodes a chloroplast envelope membrane protein (A. Katoh, K.S. Lee, H. Fukuzawa, K. Ohyama, and T. Ogawa, Proc. Natl. Acad. Sci. USA 93:4006-4010, 1996). A mutant (M29) constructed by replacing cotA in the wild-type (WT) Synechocystis strain with the omega fragment was unable to grow in BG11 medium (approximately 17 mM Na+) at pH 6.4 or at any pH in a low-sodium medium (100 microM Na+) under aeration with 3% (vol/vol) CO2 in air. The WT cells grew well in the pH range between 6.4 and 8.5 in BG11 medium but only at alkaline pH in the low-sodium medium. Illumination of the WT cells resulted in an extrusion followed by an uptake of protons. In contrast, only proton uptake was observed for the M29 mutant in the light without proton extrusion. There was no difference in sodium uptake activity between the WT and mutant. The mutant still possessed 51% of the WT CO2 transport activity in the presence of 15 mM NaCl. On the basis of these results we concluded that cotA has a role in light-induced proton extrusion and that the inhibition of CO2 transport in the M29 mutant is a secondary effect of the inhibition of proton extrusion.     

Cadmium-resistant mutant of Bacillus subtilis 168 with reduced cadmium transport.

Cd2+ and Mn2+ accumulation was studied with wild-type Bacillus subtilis 168 and a Cd2+-resistant mutant. After 5 min of incubation in the presence of 0.1 microM 109Cd2+ or 54Mn2+, both strains accumulated comparable amounts of 54Mn2+, while the sensitive cells accumulated three times more 109Cd2+ than the Cd2+-resistant cells did. Both 54Mn2+ and 109Cd2+ uptake, which apparently occur by the same transport system, demonstrated cation specificity; 20 microM Mn2+ or Cd2+ (but not Zn2+) inhibited the uptake of 0.1 microM 109Cd2+ or 54Mn2+. 54Mn2+ and 109Cd2+ uptake was energy dependent and temperature sensitive, but 109Cd2+ uptake in the Cd2+-resistant strain was only partially inhibited by an uncoupler or by a decrease in temperature. 109Cd2+ uptake in the sensitive strain followed Michaelis-Menten kinetics with a Km of 1.8 microM Cd2+ and a Vmax of 1.5 mumol/min X g (dry weight); 109Cd2+ uptake in the Cd2+-resistant strain was not saturable. The apparent Km value for the saturable component of 109Cd2+ uptake by the Cd2+-resistant strain was very similar to that of the sensitive strain, but the Vmax was 25 times lower than the Vmax for the sensitive strain. The Km and Vmax for 54Mn2+ uptake by both strains were very similar. Cd2+ inhibition of 54Mn2+ uptake had an apparent Ki of 3.4 and 21.5 microM Cd2+ for the sensitive and Cd2+-resistant strains, respectively. Mn2+ had an apparent Ki of 1.2 microM Mn2+ for inhibition of 109Cd2+ uptake by the sensitive strain, but the Cd2+-resistant strain had no defined Ki value for inhibition of Cd2+ uptake by Mn2+.     

The Wheat cDNA LCT1 Generates Hypersensitivity to Sodium in a Salt-Sensitive Yeast Strain

Salinity affects large areas of agricultural land, and all major crop species are intolerant to high levels of sodium ions. The principal route for Na(+) uptake into plant cells remains to be identified. Non-selective ion channels and high-affinity potassium transporters have emerged as potential pathways for Na(+) entry. A third candidate for Na(+) transport into plant cells is a low-affinity cation transporter represented by the wheat protein LCT1, which is known to be permeable for a wide range of cations when expressed in yeast (Saccharomyces cerevisiae). To investigate the role of LCT1 in salt tolerance we have used the yeast strain G19, which is disrupted in the genes encoding Na(+) export pumps and as a result displays salt sensitivity comparable with wheat. After transformation with LCT1, G19 cells became hypersensitive to NaCl. We show that LCT1 expression results in a strong decrease of intracellular K(+)/Na(+) ratio in G19 cells due to the combined effect of enhanced Na(+) accumulation and loss of intracellular K(+). Na(+) uptake through LCT1 was inhibited by K(+) and Ca(2+) at high concentrations and the addition of these ions rescued growth of LCT1-transformed G19 on saline medium. LCT1 was also shown to mediate the uptake of Li(+) and Cs(+). Expression of two mutant LCT1 cDNAs with N-terminal truncations resulted in decreased Ca(2+) uptake and increased Na(+) tolerance compared with expression of the full-length LCT1. Our findings strongly suggest that LCT1 represents a molecular link between Ca(2+) and Na(+) uptake into plant cells.     

Block of sodium channels by divalent mercury: role of specific cysteinyl residues in the P-loop region

Divalent mercury (Hg(2+)) blocked human skeletal Na(+) channels (hSkM1) in a stable dose-dependent manner (K(d) = 0.96 microM) in the absence of reducing agent. Dithiothreitol (DTT) significantly prevented Hg(2+) block of hSkM1, and Hg(2+) block was also readily reversed by DTT. Both thimerosal and 2,2'-dithiodipyridine had little effect on hSkM1; however, pretreatment with thimerosal attenuated Hg(2+) block of hSkM1. Y401C+E758C rat skeletal muscle Na(+) channels (mu1) that form a disulfide bond spontaneously between two cysteines at the 401 and 758 positions showed a significantly lower sensitivity to Hg(2+) (K(d) = 18 microM). However, Y401C+E758C mu1 after reduction with DTT had a significantly higher sensitivity to Hg(2+) (K(d) = 0.36 microM) than wild-type hSkM1. Mutants C753Amu1 (K(d) = 8.47 microM) or C1521A mu1 (K(d) = 8.63 microM) exhibited significantly lower sensitivity to Hg(2+) than did wild-type hSkM1, suggesting that these two conserved cysteinyl residues of the P-loop region may play an important role in the Hg(2+) block of the hSkM1 isoform. The heart Na(+) channel (hH1) was significantly more sensitive to low-dose Hg(2+) (K(d) = 0.43 microM) than was hSkM1. The C373Y hH1 mutant exhibited higher resistance (K(d) = 1.12 microM) to Hg(2+) than did wild-type hH1. In summary, Hg(2+) probably inhibits the muscle Na(+) channels at more than one cysteinyl residue in the Na(+) channel P-loop region. Hg(2+) exhibits a lower K(d) value (<1. 23 microM) for inhibition by forming a sulfur-Hg-sulfur bridge, as compared to reaction at a single cysteinyl residue with a higher K(d) value (>8.47 microM) by forming sulfur-Hg(+) covalently. The heart Na(+) channel isoform with more than two cysteinyl residues in the P-loop region exhibits an extremely high sensitivity (K(d) < 0. 43 microM) to Hg(+), accounting for heart-specific high sensitivity to the divalent mercury.     

Functional expression of Sinorhizobium meliloti BetS, a high-affinity betaine transporter, in Bradyrhizobium japonicum USDA110

Among the Rhizobiaceae, Bradyrhizobium japonicum strain USDA110 appears to be extremely salt sensitive, and the presence of glycine betaine cannot restore its growth in medium with an increased osmolarity (E. Boncompagni, M. Osteras, M. C. Poggi, and D. Le Rudulier, Appl. Environ. Microbiol. 65:2072-2077, 1999). In order to improve the salt tolerance of B. japonicum, cells were transformed with the betS gene of Sinorhizobium meliloti. This gene encodes a major glycine betaine/proline betaine transporter from the betaine choline carnitine transporter family and is required for early osmotic adjustment. Whereas betaine transport was absent in the USDA110 strain, such transformation induced glycine betaine and proline betaine uptake in an osmotically dependent manner. Salt-treated transformed cells accumulated large amounts of glycine betaine, which was not catabolized. However, the accumulation was reversed through rapid efflux during osmotic downshock. An increased tolerance of transformant cells to a moderate NaCl concentration (80 mM) was also observed in the presence of glycine betaine or proline betaine, whereas the growth of the wild-type strain was totally abolished at 80 mM NaCl. Surprisingly, the deleterious effect due to a higher salt concentration (100 mM) could not be overcome by glycine betaine, despite a significant accumulation of this compound. Cell viability was not significantly affected in the presence of 100 mM NaCl, whereas 75% cell death occurred at 150 mM NaCl. The absence of a potential gene encoding Na(+)/H(+) antiporters in B. japonicum could explain its very high Na(+) sensitivity.     

GerN, an endospore germination protein of Bacillus cereus, is an Na(+)/H(+)-K(+) antiporter

GerN, a Bacillus cereus spore germination protein, exhibits homology to a widely distributed group of putative cation transporters or channel proteins. GerN complemented the Na(+)-sensitive phenotype of an Escherichia coli mutant that is deficient in Na(+)/H(+) antiport activity (strain KNabc). GerN also reduced the concentration of K(+) required to support growth of an E. coli mutant deficient in K(+) uptake (strain TK2420). In a fluorescence-based assay of everted E. coli KNabc membrane vesicles, GerN exhibited robust Na(+)/H(+) antiport activity, with a K(m) for Na(+) estimated at 1.5 mM at pH 8.0 and 25 mM at pH 7.0. Li(+), but not K(+), served as a substrate. GerN-mediated Na(+)/H(+) antiport was further demonstrated in everted vesicles as energy-dependent accumulation of (22)Na(+). GerN also used K(+) as a coupling ion without completely replacing H(+), as indicated by partial inhibition by K(+) of H(+) uptake into right-side-out vesicles loaded with Na(+). K(+) translocation as part of the antiport was supported by the stimulatory effect of intravesicular K(+) on (22)Na(+) uptake by everted vesicles and the dependence of GerN-mediated (86)Rb(+) efflux on the presence of Na(+) in trans. The inhibitory patterns of protonophore and thiocyanate were most consistent with an electrogenic Na(+)/H(+)-K(+) antiport. GerN-mediated Na(+)/H(+)-K(+) antiport was much more rapid than GerN-mediated Na(+)/H(+) antiport.     

Biochemical analysis of neomycin-resistance in the methanoarchaeon Methanothermobacter thermautotrophicus and some implications for energetic processes in this strain

Methanogenesis-driven ATP synthesis in a neomycin-resistant mutant of Methanothermobacter thermautotrophicus (formerly Methanobacterium thermoautotrophicum strain DeltaH) was strongly inhibited at both pH 6.8 and pH 8.5 by the uncoupler 3,3',4',5 -tetrachlorosalicylanilide (TCS) in the presence of either 1 or 10 mM NaCl. The generation of a membrane potential in the mutant cells at pH 6.8 was also strongly inhibited by TCS in the presence of 1 or 10 mM NaCl. On the other hand, at pH 8.5 in the presence of 10mM NaCl, a protonophore-resistant membrane potential of approximately 150 mV was found. These results indicate that in the mutant cells the process of energy transduction between methanogenesis and membrane potential generation is not impaired. In contrast to the wild-type strain, ATP synthesis in the mutant cells was driven by an electrochemical gradient of H(+) under alkaline conditions. Unlike wild-type cells, the mutant lacks the capacity to transduce an uncoupler-resistant membrane potential energy at pH 8.5 into ATP synthesis. Na(+)/H(+) exchange was comparable in the wild type and the mutant cells. Western blots of sub-cellular fractions with polyclonal antiserum reactive to the B-subunit of the halobacterial A-type H(+)-translocating ATPase confirmed the presence of A-type ATP synthase in the mutant cells. Furthermore, in the mutant cells a protein band of molecular mass about 45 kDa is absent but there was an abundant protein band at about 67 kDa. Based on the observed bioenergetic features of the mutant cells, neither the A(1)A(o) ATP synthase alone nor together with the Na(+)/H(+) antiporter seems to be responsible for ATP synthesis driven by sodium motive force. Rather, some other links between neomycin-resistance and failure of sodium motive force-dependent ATP synthesis in the neomycin resistant mutant are discussed.     

Arabidopsis thaliana cyclic nucleotide gated channel 3 forms a non-selective ion transporter involved in germination and cation transport

The Arabidopsis thaliana genome contains 20 cyclic nucleotide gated channel (CNGC) genes encoding putative non-selective ion channels. Classical and reverse genetic approaches have revealed that two members of this family (CNGC2 and CNGC4) play a role in plant defence responses whereas CNGC1 and CNGC10 may participate in heavy metal and cation transport. Yet, it remains to be resolved how the ion transport attributes of CNGCs are integrated into their physiological function. In this study, CNGC3 is characterized through heterologous expression, GUS- and GFP-reporter gene fusions, and by adopting a reverse genetics approach. A CNGC3-GFP fusion protein shows that it is mainly targeted to the plasma membrane. Promoter GUS studies demonstrate CNGC3 expression predominantly in the cortical and epidermal root cells, but also a ubiquitous presence in shoot tissues. Expression of CNGC3 in yeast indicates it can function as a Na(+) uptake and a K(+) uptake mechanism. cngc3 null mutations decreased seed germination in the presence of NaCl but not KCl. Relative to the wild type, mutant seedling growth is more resistant to the presence of toxic concentrations of NaCl and KCl. The ionic composition and ion uptake characteristics of wild-type and mutant seedlings suggests that the growth advantage in these conditions may be due to restricted ion influx in mutant plants, and that CNGC3 functions in the non-selective uptake of monovalent cations in Arabidopsis root tissue.     

Hexavalent chromium uptake by sensitive and tolerant mutants of Schizosaccharomyces pombe

Lysine and leucine auxotrophic, heterothallic (h+, h-) strains of Schizosaccharomyces pombe were used to obtain chromium (VI)-sensitive and -tolerant mutants by ultraviolet radiation-induced and nitrosoguanidine-induced mutagenesis. The minimal inhibitory concentrations of K2Cr2O7 on YEA media were 225 microM for the wild-type strain CW-6, 125 microM for the sensitive mutant CS-6.51 and 275 microM for the tolerant mutant CT-6.66. The mutants exhibited cross-sensitivity of various patterns to Cd2+, Cu2+, Ni2+, Zn2+ and VO3-(4). Cr(VI) was added to the actively growing cultures and the total chromium (TOCr) content of the cells was determined. The sensitive mutant exhibited a high bioaccumulation ability, with a dry biomass of 810 micrograms g-1 after 30 min, while the tolerant mutant had a significantly lower ability than the wild-type strain. In PIPES buffer, washed, lysine-starved biomasses were treated with 75 microM Cr(VI) and after 2 h, the TOCr and the organically bound chromium (OBCr) were determined. Under these conditions, the sensitive and tolerant mutants had the same TOCr content, 50% of which was OBCr. The wild-type strain exhibited a lower TOCr content than that of its mutants and only 35% of this was OBCr. The Cr(VI)-sensitivity was due to a significantly increased uptake of Cr(VI).     

Degradation of pyrene by indigenous fungi from a former gasworks site

Salt tolerance in Saccharomyces cerevisiae is a complex trait, involving regulation of membrane polarization, Na(+) efflux and sequestration of Na(+) in the vacuole. Since transmembrane transport energized by H(+)-adenosine triphosphatases (ATPases) is common to all of these tolerance mechanisms, the objective of this study was to characterize the responses of the plasma membrane H(+)-ATPase, vacuolar H(+)-ATPase and mitochondrial F(1)F(0)-ATPase to NaCl stress. We hypothesized that since the vacuolar ATPase is responsible for generating the proton motive force required for import of cations (such as Na(+)) into the vacuole, strains lacking this activity should be hypersensitive to NaCl. We found that strains lacking vacuolar ATPase activity were in fact hypersensitive to NaCl, while strains lacking ATP synthase were not. This effect was specific to the ionic component of NaCl stress, since the mutant strains were indistinguishable from wild-type and complemented strains in the presence of sorbitol.