Succinate transport in Bacillus subtilis. Dependence on inorganic anions

Cations were generally ineffective in stimulating succinate transport in a succinate dehydrogenase mutant of Bacillus subtilis unless accompanied by polyvalent anions; phosphate and sulfate being particularly active. The K_m values for the phosphate or sulfate requirement were approx. 3 mM.Biphasic kinetics were characteristic of both the succinate (K_m values 0.1 and 1 mM), and inorganic phosphate (K_m values 0.1 and 3 mM) transport system(s). The phosphate transport system(s) was repressed by high inorganic phosphate and a coordinate increase in the transport of phosphate, arsenate, and phosphate-stimulated succinate transport accompanied growth in low phosphate media.A class of arsenate resistant mutants were simultaneously defective in the transport of arsenate, phosphate and succinate when cells were repressed for phosphate transport, however, the transport of these ions was regained in these mutants when grown in low phosphate media. Organic phosphate esters did not stimulate succinate transport in arsenate resistant mutants but were effective after growth in low phosphate media. Growth under phosphate limitation permitted the simultaneous regain of both phosphate and sulfate dependent succinate transport activities whereas sulfate limitation alone was ineffective.Succinate was not transported by an anion exchange diffusion mechanism since phosphate efflux was low or absent during succinate transport.The transport of C₄-dicarboxylates in B. subtilis is strongly stimulated by intracellular polyvalent anions. The absence of an anion permeability mechanism precludes succinate transport but partial escape from this restriction is mediated by the derepression of a phosphate transport system.     

Effect of arsenate on inorganic phosphate transport in Escherichia coli.

The effect of arsenate on strains dependent on the two major inorganic phosphate (Pi) transport systems in Escherichia coli was examined in cells grown in 1 mM phosphate medium. The development of arsenate-resistant Pi uptake in a strain dependent upon the Pst (phosphate specific transport) system was examined. The growth rate of Pst-dependent cells in arsenate-containing medium was a function of the arsenate-to-Pi ratio. Growth in arsenate-containing medium was not due to detoxification of the arsenate. Kinetic studies revealed that cells grown with a 10-fold excess of arsenate to Pi have almost a twofold increase in capacity (Vmax) for Pi, but maintained the same affinity (Km). Pi accumulation in the Pst-dependent strain was still sensitive to changes in the arsenate-to-Pi ratio, and a Ki (arsenate) for Pi transport of 39 microM arsenate was determined. The Pst-dependent strain did not accumulate radioactive arsenate, and showed only a transient decrease in intracellular adenosine triphosphate levels after arsenate was added to the medium. The Pi transport-dependent strain ceased growth in arsenate-containing media. This strain accumulated 74As-arsenate, and intracellular adenosine triphosphate pools were almost completely depleted after the addition of arsenate to the medium. Arsenate accumulation required a metabolizable energy source and was inhibited by N-ethylmaleimide. Previously accumulated arsenate could exchange with arsenate or Pi in the medium.     

Arsenate substitutes for phosphate in the human red cell sodium pump and anion exchanger

The sodium pump of human red blood cells mediates a Rb:Rb exchange that is dependent for maximal rates upon the simultaneous presence of intracellular ATP (or ADP) and phosphate. We have measured ouabain-sensitive 86Rb uptake into resealed ghosts of human red cells containing ADP and show that arsenate will substitute for phosphate in supporting the Rb:Rb exchange transport mode. The concentration dependence of arsenate-supported Rb:Rb exchange in ghosts containing 2 mM ADP shows both activating and inhibiting phases; the dependence upon phosphate shows similar characteristics. Elevation of the external [Rb] lowers the apparent affinity for arsenate since there is a shift to higher concentrations of arsenate in the activating and inhibiting phases of the arsenate concentration dependence curve. Similarly, elevation of [ADP] substantially reduces the inhibition of Rb:Rb exchange observed at higher [arsenate]. These effects are also observed in phosphate-supported Rb:Rb exchange. The phosphate requirement for Rb:Rb exchange involves phosphorylation of the sodium pump protein; the close agreement between the effects of arsenate and phosphate in supporting Rb:Rb exchange makes it likely that arsenylation of the sodium pump occurs during Rb:Rb exchange. Arsenate efflux from red blood cell ghosts into arsenate-free chloride medium is partially inhibited (77-80%) by DNDS (4,4'-dinitro-2,2'-stilbenedisulfonic acid), this compares with 82-87% inhibition by DNDS of phosphate efflux under the same conditions. It appears that Band III, the red cell anion transport system, accepts arsenate in a similar fashion to phosphate and that a fraction of the flux of both anions may occur through pathways other than Band III. Thus, in human red blood cells, both the sodium pump and the anion exchange transport system will accept arsenate as a phosphate congener and the protein-arsenate interactions are very similar to those with phosphate.     

Interactions of Arsenate with the Phosphate-Transporting System of Yeast

Arsenate competes with phosphate for transport into the yeast cell. The affinity of the two substances for the transport system is about equal, but in mixtures the phosphate is taken up about twice as fast as arsenate, because the maximal transport rate for phosphate is about twice as high. In addition to the competitive effect, arsenate causes a continuous and irreversible inactivation of the transport system that can be characterized by first order kinetics. The rate of arsenate inactivation is slower in the presence of phosphate and the amount of arsenate taken up before complete block is established is also decreased. The inactivation of the transport system cannot be relieved by washing or by treatment with glucose and phosphate. The inactivation is not the result of an inhibition of metabolism.     

Continuous culture of Rhodotorula rubra: kinetics of phosphate-arsenate uptake, inhibition, and phosphate-limited growth

The pink yeast Rhodotorula rubra of marine origin was found to be capable of extended growth at very low phosphate concentrations (K(0.5) = 10.8 nm). Average intracellular phosphate concentrations, based on isotope exchange techniques, were 15 to 200 nm, giving concentration gradients across the cell envelope of about 10(6). Sensitivity to metabolic inhibitors occurred at micromolar concentrations. Inability of the phosphate transport system, K(s) = 0.5 to 2.8 mum, V(max) = 55 mumoles per g of cells per min, to discriminate against arsenate transport led to arsenate toxicity at 1 to 10 nm, whereas environmental arsenate levels are reportedly much higher. Phosphate competitively prevented arsenate toxicity. The K(i) for phosphate inhibition of arsenate uptake was 0.7 to 1.2 mum. Phosphate uptake experiments showed that maximal growth rates could be achieved with approximately 4% of the total phosphate-arsenate transport system. Organisms adapted to a range both of concentration of NaCl and of pH. Maximal affinity for phosphate occurred at pH 4 and at low concentrations of NaCl; however, V(max) for phosphate transport was little affected. Maximal specific growth rates on minimal medium were consistent in batch culture but gradually increased to the much higher rates found with yeast extract media when the population was subjected to long-term continuous culture with gradually increasing dilution rates. Phosphate initial uptake rates that were in agreement with the steady-state flux in continuous culture were obtained by using organisms and medium directly from continuous culture. This procedure resulted in rates about 500 times greater than one in which harvested batch-grown cells were used. Discrepancies between values found and those reported in the literature for other organisms were even larger. Growth could not be sustained below a threshold phosphate concentration of 3.4 nm. Such thresholds are explained in terms of a system where growth rate is set by intracellular nutrient concentrations. Threshold concentrations occur in response to nutrient sinks not related to growth, such as efflux and endogenous metabolism. Equations are presented for evaluation of growth rate-limiting substrate concentrations in the presence of background substrate and for evaluating low inhibitor concentration inhibition mechanisms by substrate prevention of inhibitor flux.     

Methionine transport in Yersinia pestis.

Yersinia pestis TJW, an avirulent wild-type strain, requires phenylalanine and methionine for growth. It was of interest to examine and define the methionine transport system because of this requirement. The methionine system showed saturation kinetics with a Km for transport of approximately 9 times 10(-7) M. After 8 s of methionine transport, essentially all of the methionine label appeared in S-adenosyl-L-methionine (SAM) as detected in ethanol extracts. Small amounts of free methionine was detected intracellularly after 1 min of transport. Addition of glucose increased significantly the amount of intracellular methionine at 1 min. A series of SAM metabolic products was detected after 90 s to 5 min of transport including: 5'-thiomethyladenosine, homoserine lactone, S-adenosyl homoserine, and a fluorescent methyl receptor compound. Results from assays for SAM synthetase in spheroplast fractions showed a small (16%) but significant portion of synthetase associated with the membrane. However, most of the enzyme activity was associated with the cytoplasmic fraction. Methionine transport was characterized by a high degree of stereospecificity. No competition occurred from structurally unrelated amino acids. Although uptake was inhibited by uncoupling and sulfhydryl reagents, no efflux was observed. Results using energy inhibitors on unstarved and starved cells showed that respiratory inhibitors such as potassium cyanide (KCN) and amytal were most effective, and that arsenate was least effective. KCN plus arsenate completely blocked utilization of energy derived from glucose, and KCN completely blocked utilization of energy deived from D-lactate. The data indicate that methionine transport in Y. pestis is linked to the trapping of methionine in SAM. The results further suggest that this transport system can be classified as a permease-bound system where transport is coupled to an energized membrane state and to respiration.     

Phosphate and glucose accumulation by Pseudomonas cultures in relation to their arsenic resistance

The effect of arsenite and arsenate on 14C-glucose and 32-P-phosphate transport was studied in the cells of Pseudomonas aeruginosa 561 sensitive to arsenite and in the cells of Pseudomonas putida 18 oxidizing arsenite and resistant to arsenic. Transport and accumulation of phosphate and glucose were inhibited in the presence of arsenite in the cells of P. aeruginosa 561 whereas arsenate inhibited only phosphate accumulation. Arsenite and arsenate had hardly any effect at the initial transport rate and on the overall accumulation of phosphate and glucose in the cells of P. putida 18. The resistance to arsenite is supposed to be caused by selective impermeability of the cellular membranes to arsenite and arsenate.     

Relationship between phospholipid biosynthesis and the efficiency of the arsenate transport system in yeasts

In studying the possibility that phosphoinositides which formed complexes with arsenic were involved in the arsenate transport system of yeasts, a comparative study of the phospholipid composition and metabolism was carried out both in Saccharomyces carslbergensis and in its arsenate-adapted variant, which showed a deficient inflow of arsenate. It was found that the lipid composition of the two organisms was quite similar, the main classes of phospholipids being phosphatidylcholine, phosphatidylethanolamine, and phosphoinositides. The only difference was a 1.5- to 2-fold increase in the proportion of inositides in the arsenate-adapted cells. When the transport of arsenate became inactivated in the nonadapted yeasts after a 30- to 60-min exposure to 10(-2)m arsenate, an increment of inositides of 29 to 50% over the original level was also detected. A study of the incorporation of radioactivity from uniformly labeled (14)C-maltose and from (32)P-orthophosphate ((32)P(i)) demonstrated a decreased rate of lipid biosynthesis in the arsenate-adapted cells as compared to the normal nonadapted ones. The turnover of the phosphate in phospholipids demonstrated no turnover in phosphatidylcholine and phosphatidylethanolamine, and a slow turnover in phosphoinositides. It could be inferred that a normal rate of phospholipid (phosphoinositides) biosynthesis is necessary to have a normal arsenate uptake and that inositide accumulation impairs both the mechanism responsible for the uptake and accumulation of arsenate and the rate of lipid biosynthesis. No differences were found in the deoxyribonucleic acid or protein content of the two types of cells. Also, the arsenate-adapted cells, once freed of external arsenate, showed an increased uptake of (32)P(i) from low external concentrations of phosphate (10(-6) to 10(-8)m, 10-fold over that observed in AsS cells). These results are indicative of independent behavior in phosphate and arsenate transport systems.     

Interaction of arsenate with phosphate-transport systems in wild- type and mutant Streptococcus faecalis

Harold, F. M. (National Jewish Hospital, Denver, Colo.), and J. R. Baarda. Interaction of arsenate with phosphate-transport systems in wild-type and mutant Streptococcus faecalis. J. Bacteriol. 91:2257-2262. 1966.-Arsenate competitively inhibits the growth of Streptococcus faecalis, primarily by competition with phosphate for a common transport system. Arsenate is itself accumulated by the cells; the uptake requires metabolic energy, and the intracellular arsenate level may reach 0.01 m. Cells loaded with arsenate have lost the capacity to take up radioactive glutamate, rubidium, phosphate, or arsenate itself, apparently by the uncoupling of adenosine triphosphate generation. The pH dependence of arsenate uptake is complex. At low concentrations of extracellular arsenate, uptake by the wild-type strain 9790 exhibits a single maximum about pH 8; mutant PT-1, previously shown to be defective in phosphate uptake, takes up essentially no arsenate. At high concentrations of arsenate, uptake by the wild type is bimodal with maxima at pH 5.5 and 9; the uptake curve for mutant PT-1 corresponds to the shoulder in the curve for the wild type. The apparent dissociation constant for arsenate uptake by the wild type is approximately 10(-5)m from pH 5 to 9, whereas that for mutant PT-1 is about 5 x 10(-5) M at pH 5 and rises rapidly with increasing pH. The results confirm the earlier conclusion that the lesion in mutant PT-1 resides in the transport of phosphate and arsenate. It is proposed that the wild type has two distinct transport systems, whereas the mutant has lost the one with alkaline pH optimum.     

Arsenic-lipid complex formatinon during the active transport of arsenate in yeast

In studying formation of an arsenic-lipid complex during the active transport of (74)As-arsenate in yeast, it was found that adaptation of yeast to arsenate resulted in cell populations which showed a deficient inflow of arsenate as compared to the nonadapted yeast. Experiments with both types of cells showed a direct correlation between the arsenate taken up and the amount of As-lipid complex formed. (74)As-arsenate was bound exclusively to the phosphoinositide fraction of the cellular lipids. When arsenate transport was inhibited by dinitrophenol and sodium azide, the formation of the As-lipid complex was also inhibited. Phosphate did not interfere with the arsenate transport at a non-inhibitory concentration of external arsenate (10(-9)m). The As-adapted cells but not the unadapted cells were able to take up phosphate when growing in the presence of 10(-2)m arsenate.     

Evidence for a functional change in the plasma membrane of yeasts associated with the mechanism of arsenate resistance.

In previous reports experimental evidence has been presented indicating a possible relationship between the formation of arseno-phosphoinositides and the active transport of arsenate-phosphate in yeast cells. There is an increment in the amount of inositides in yeast cells adapted to grow in the presence of toxic concentrations of arsenate. These cells exhibit a highly reduced arsenate uptake but maintain their capacity to transport phosphate. Since, in normal (nonadapted) yeast cells, both arsenate and phosphate anions share the same transport system, a study was conducted to obtain further information about the plausible role played by the phosphoinositides in the active transport system of arsenate and their inhibition that allows the cells to grow in the presence of the toxic. Studies on [³²P]orthophosphate and [⁷⁴As]arsenate incorporation into phospholipids in normal and arsenate-adapted yeast show that: The ³²P incorporation into phospholipids is two times larger in normal yeast as compared to arsenateadapted ones. The ³²P labeling was maximum for phosphatidylinositol in normal yeasts while in the arsenate-adapted cells it was maximum for phosphatidylcholine. This incorporation was largely inhibited by arsenate in normal yeasts and minimal in the arsenate-adapted ones. Cell fractionation shows that the maximum incorporation of [³²P]orthophosphate resides in the microsomal fraction, while the incorporation of [⁷⁴As]arsenate resides mainly in the cell envelope fraction which incorporates 86% of the ⁷⁴As label. Phosphate is capable of inhibiting the ⁷⁴As-inositide complex formation and destroying the previously formed one. Yeast cells prelabeled with [2C-³H]myoinositol showed a reduced turnover rate of phosphoinositides even when transporting nontoxic amounts of arsenate. The involvement of the inositides as a regulatory mechanism in the phosphate-arsenate active transport system in yeast cells is discussed.     

Arsenate transport and reduction in the hyper-tolerant fungus Aspergillus sp. P37

Aspergillus sp. P37 is able to grow at arsenate concentrations of 0.2 M--more than 20-fold higher than that withstood by reference microorganisms such Escherichia coli, Saccharomyces cerevisiae and Aspergillus nidulans. This paper examines the transport of arsenate and phosphate and the reduction of arsenate in Aspergillus sp. P37. These properties were compared with the corresponding properties of the archetype strain Aspergillus nidulans TS1. Both uptake and efflux of arsenate were inhibited by carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, suggesting that the transport system(s) is(are) membrane-potential dependent. As uptake of arsenate and phosphate are higher in Aspergillus sp. P37 than in A. nidulans, the increase in arsenate resistance cannot be accounted for by a change in uptake. Cells of both strains loaded with arsenic slowly released the oxyanion. Speciation of the arsenic in the medium showed an enhanced level of arsenate reduction in Aspergillus sp. P37. These data suggest that increased arsenate reduction is at least in part responsible for the hyper-tolerant phenotype of this fungus.     

Phosphate transport in arsenate-resistant mutants of Micrococcus lysodeikticus.

Two types of arsenate-resistant mutants of Micrococcus lysodeikticus were found: (i) mutants that grow in the presence of 10 mM but not 1 mM phosphate (Pi) with low uptake rate for Pi and arsenate, and (ii) mutants able to grow in the presence of 10 mM and 1 mM Pi, with a near-normal uptake rate for Pi but a low one for arsenate. The Km values for Pi transport and the Ki values for its competitive inhibition by arsenate were similar for the mutants and the wild type. Similar to the wild type, the mutants also accumulated Pi to high concentrations. In all strains, the transport of Pi was subject to repression by Pi. Mutant types showed lower Vmax but unaltered Km values for arsenate as compared to the wild type, and they accumulated arsenate to markedly lower levels. The results suggest a two-component transport system common to Pi and arsenate.     

Inorganic phosphate transport inAcinetobacter lwoffi

The transport of inorganic phosphate has been studied inAcinetobacter lwoffi JW11. During growth on excess phosphate, only one transport system was present, with an apparent K_m of 1.4 M. When cells were starved for phosphate, a second uptake system with an apparent K_m of 110 nM was also synthesized. The two transport systems could be distinguished by differing sensitivities to the phosphate analogs arsenate and 2-aminoethylphosphonate. Both systems were inhibited by carbonylcyanidem-chlorophenylhydrazone, and to a lesser extent by Na azide. The high-affinity transport system was inactivated by osmotic shock treatment and by spheroplast formation. Preliminary evidence for a phosphate-binding protein in the osmotic shock fluid is presented. The isolation of a mutant constitutive for the high-affinity transport system is described.     

Accumulation of arsenate, phosphate, and aspartate by Sreptococcus faecalis.

Uptake of arsenate and phosphate by Streptococcus faecalis 9790 is strictly dependent on concurrent energy metabolism and essentially unidirectional. targinine supports uptake only in presence of glycerol or related substances; glycerol is not directly involved in transport but depletes the cellular orthophosphate pool and thus relieves feedback inhibition of transport. Uptake of phosphate and arsenate is stimulated by K+ and by other permeant cations. The results suggest that electroneutrality is preserved by compensatory movement of either H+ or OH minus. Ionophores and N,N'-dicyclohexylcarbodiimide, which prevent establishment of a proton motive force, block the accumulation of thiomethylgalactoside and of threonine but not that of arsenate or phosphate. We conclude that arsenate accumulation requires adenosine 5'-triphosphate but is not driven by the proton-motive force. However, conditions and reagents that lower the cytoplasmic pH do inhibit accumulation of arsenate and phosphate, suggesting that uptake depends on the capacity of the cells to maintain a neutral or alkaline cytoplasm. We therefore propose that phosphate accumulation is an electroneutral exchange for OH driven by adenosine 5'-triphosphate or by a metabolite thereof. Accumulation of aspartate and glutamate also requires adenosine 5'-triphosphate but not the proton-motive force and may involve a similar mechanism.     

Uptake kinetics of arsenic species in rice plants

Arsenic (As) finds its way into soils used for rice (Oryza sativa) cultivation through polluted irrigation water, and through historic contamination with As-based pesticides. As is known to be present as a number of chemical species in such soils, so we wished to investigate how these species were accumulated by rice. As species found in soil solution from a greenhouse experiment where rice was irrigated with arsenate contaminated water were arsenite, arsenate, dimethylarsinic acid, and monomethylarsonic acid. The short-term uptake kinetics for these four As species were determined in 7-d-old excised rice roots. High-affinity uptake (0-0.0532 mM) for arsenite and arsenate with eight rice varieties, covering two growing seasons, rice var. Boro (dry season) and rice var. Aman (wet season), showed that uptake of both arsenite and arsenate by Boro varieties was less than that of Aman varieties. Arsenite uptake was active, and was taken up at approximately the same rate as arsenate. Greater uptake of arsenite, compared with arsenate, was found at higher substrate concentration (low-affinity uptake system). Competitive inhibition of uptake with phosphate showed that arsenite and arsenate were taken up by different uptake systems because arsenate uptake was strongly suppressed in the presence of phosphate, whereas arsenite transport was not affected by phosphate. At a slow rate, there was a hyperbolic uptake of monomethylarsonic acid, and limited uptake of dimethylarsinic acid.     

Serum stimulation of phosphate uptake into 3T3 cells.

The stimulation by calf serum of phosphate uptake into 3T3 cells results from a change in maximum velocity of the transport process with no change in the Michaelis constant. Only arsenate among a series of inorganic structural analogs of phosphate inhibited phosphate uptake indicating a high specificity for the process. The arsenate inhibition was competitive in nature. Papaverine, theophylline, and protaglandin E1, drugs known to maintain high intracellular levels of cAMP, had little effect on serum stimulated phosphate uptake. The phosphate uptake stimulating factor(s) in serum could be distinguised from the 3T3 cell survival and migration factors by stability characteristics, but this factor(s) could not be completely separated from a uridine uptake stimulation activity or growth promoting activity using a variety of serum fractionation procedures. Only partial stimulation of the uptake process was achieved with any one serum fraction indicating a multiplicity of serum components is probably involved in this process. Because of the rapidity of serum activation of phosphate uptake and its apparent independence of intracellular cyclic nucleotide levels, it is suggested that serum factors may stimulate phosphate uptake by inducing structural changes in the phosphate carrier system.     

Suppression of the High Affinity Phosphate Uptake System: A Mechanism of Arsenate Tolerance in Holcus lanatus L.

In Holcus lanatus L. phosphate and arsenate are taken up bythe same transport system. Short-term uptake kinetics of thehigh affinity arsenate transport system were determined in excisedroots of arsenate-tolerant and non-tolerant genotypes. In tolerantplants the V_max of ion uptake in plants grown in phosphate-freemedia was decreased compared to non-tolerant plants, and theaffinity of the uptake system was lower than in the non-tolerantplants. Both the reduction in V_max and the increase in K_m ledto reduced arsenate influx into tolerant roots. When the twogenotypes were grown in nutrient solution containing high levelsof phosphate, there was little change in the uptake kineticsin tolerant plants. In non-tolerant plants, however, there wasa marked decrease in the V_max to the level of the tolerant plantsbut with little change in the K_m. This suggests that the lowrate of arsenate uptake over a wide range of differing rootphosphate status is due to loss of induction of the synthesisof the arsenate (phosphate) carrier. Key words: Arsenate, Holcus lanatus L., phosphate uptake, tolerance mechanisms, uptake mechanisms