Inducible plasmid-determined resistance to arsenate, arsenite, and antimony (III) in escherichia coli and Staphylococcus aureus.

Plasmids in both Escherichia coli and Staphylococcus aureus contain an "operon" that confers resistance to arsenate, arsenite, and antimony(III) salts. The systems were always inducible. All three salts, arsenate, arsenite, and antimony(III), were inducers. Mutants and a cloned deoxyribonucleic acid fragment from plasmid pI258 in S. aureus have lost arsenate resistance but retained resistances to arsenite and antimony, demonstrating that separate genes are involved. Arsenate-resistant arsenite-sensitive S. aureus plasmid mutants were also isolated. In E. coli, plasmid-determined arsenate resistance and reduced uptake were additive to that found with chromosomal arsenate resistance mutants. Arsenate resistance was due to reduced uptake of arsenate by the induced plasmid-containing cells. Under conditions of high arsenate, when some uptake could be demonstrated with the induced resistant cells, the arsenate was rapidly lost by the cells in the absence of extracellular phosphate. Sensitive cells retained arsenate under these conditions. When phosphate was added, phosphate-arsenate exchange occurred. High phosphate in the growth medium protected cells from arsenate, but not from arsenite or antimony(III) toxicity. We do not know the mechanisms of arsenite or antimony resistance. However, arsenite was not oxidized to less toxic arsenate. Since cell-free medium "conditioned" by prior growth to induced resistant cells with toxic levels of arsenite or antimony(III) retained the ability to inhibit the growth of sensitive cells, the mechanism of arsenite and antimony resistance does not involve conversion of AsO2- or SbO+ to less toxic forms or binding by soluble thiols excreted by resistant cells.     

Inorganic phosphate transport in Escherichia coli: involvement of two genes which play a role in alkaline phosphatase regulation

Two classes of alkaline phosphatase constitutive mutations which comprise the original phoS locus (genes phoS and phoT) on the Escherichia coli genome have been implicated in the regulation of alkaline phosphatase synthesis. When these mutations were introduced into a strain dependent on a single system, the pst system, for inorganic phosphate (P(i)) transport, profound changes in P(i) transport were observed. The phoT mutations led to a complete P(i) (-) phenotype in this background, and no activity of the pst system could be detected. The introduction of the phoS mutations changed the specificity of the pst system so that arsenate became growth inhibitory. Changes in the phosphate source led to changes in the levels of constitutive alkaline phosphatase synthesis found in phoS and phoT mutants. When glucose-6-phosphate or l-alpha-glycerophosphate was supplied as the sole source of phosphate, phoT mutants showed a 3- to 15- fold reduction in constitutive alkaline phosphatase synthesis when compared to the maximal levels found in limiting P(i) media. However, these levels were still 100 times greater than the basal level of alkaline phosphatase synthesized in wild-type strains under these conditions. The phoS mutants showed only a two- to threefold reduction when grown with organic phosphate sources. The properties of the phoT mutants selected on the basis of constitutive alkaline phosphatase synthesis were similar in many respects to those of pst mutants selected for resistance to growth inhibition caused by arsenate. It is suggested that the phoS and phoT genes are primarily involved in P(i) transport and, as a result of this function, play a role in the regulation of alkaline phosphatase synthesis.     

Arsenate arrests flagellar rotation in cytoplasm-free envelopes of bacteria.

The effect of arsenate on flagellar rotation in cytoplasm-free flagellated envelopes of Escherichia coli and Salmonella typhimurium was investigated. Flagellar rotation ceased as soon as the envelopes were exposed to arsenate. Inclusion of phosphate intracellularly (but not extracellular) prevented the inhibition by arsenate. In a parallel experiment, the rotation was not affected by inclusion of an ATP trap (hexokinase and glucose) within the envelopes. It is concluded that arsenate affects the motor in a way other than reversible deenergization. This may be an irreversible damage to the cell or direct inhibition of the motor by arsenate. The latter possibility suggests that a process of phosphorylation or phosphate binding is involved in the motor function.     

A novel aspect of the inhibition by arsenicals of binding-protein-dependent galactose transport in gram-negative bacteria.

The inhibitory effects of arsenate and arsenite on binding-protein-dependent transport systems are reconsidered. It is shown that arsenate inhibits binding-protein-dependent galactose transport in proteoliposomes energized either by dihydrolipoamide and NAD+ or by a membrane potential (under conditions where ATP metabolism is not implicated); this result is in contradiction with the current interpretation of arsenate inhibition of binding-protein-dependent transport systems (which is based on ATP depletion) and can be explained by reference to the recently discovered ATP inhibition of the binding-protein-dependent galactose transport. In whole cells, the greater inhibition by arsenate of lipoamide-dependent transport than of protonmotive-force-dependent transport may be explained by a modification by arsenate of the pools of several compounds metabolized by 2-oxo-acid dehydrogenases (which have been implicated in binding-protein-dependent transport). The inhibition of binding-protein-dependent galactose transport by arsenite is probably linked to the inhibition by arsenite of the galactose-stimulated lipoamide dehydrogenase activity implicated in this transport and is reminiscent of the known arsenite inhibition of lipoamide dehydrogenases.     

ars1, an Arabidopsis mutant exhibiting increased tolerance to arsenate and increased phosphate uptake

Arsenic is one of the most toxic pollutants at contaminated sites, yet little is known about the mechanisms by which certain plants survive exposure to high arsenic levels. To gain insight into the mechanisms of arsenic tolerance in plants, we developed a genetic screen to isolate Arabidopsis thaliana mutants with altered tolerance to arsenic. We report here on the isolation of a mutant arsenic resisant 1 (ars1) with increased tolerance to arsenate. ars1 germinates and develops under conditions that completely inhibit growth of wild-type plants and shows a semi-dominant arsenic resistance phenotype. ars1 accumulates levels of arsenic similar to that accumulated by wild-type plants, suggesting that ars1 plants have an increased ability to detoxify arsenate. However, ars1 plants produce phytochelatin levels similar to levels produced by the wild type, and the enhanced resistance of ars1 is not abolished by the gamma-glutamylcysteine synthetase inhibitor l-buthionine sulfoxime (BSO). Furthermore, ars1 plants do not show resistance to arsenite or other toxic metals such as cadmium and chromium. However, ars1 plants do show a higher rate of phosphate uptake than that shown by wild-type plants, and wild-type plants grown with an excess of phosphate show increased tolerance to arsenate. Traditional models of arsenate tolerance in plants are based on the suppression of phosphate uptake pathways and consequently on the reduced uptake of arsenate. Our data suggest that arsenate tolerance in ars1 could be due to a new mechanism mediated by increased phosphate uptake in ars1. Models discussing how increased phosphate uptake could contribute to arsenate tolerance are discussed.     

High intracellular phosphorus contents exhibit a correlation with arsenate resistance in chlamydomonas mutants

Pi in the medium relieved the toxicity of arsenate against cellular growth of Chlamydomonas reinhardtii. To investigate the relationship between intracellular P contents and arsenate resistance, we determined the intracellular P contents of arsenate-sensitive and arsenate-resistant mutants, which had been generated by random insertional mutagenesis. All 13 arsenate-resistant mutants showed higher P contents than the parent strain, while arsenate-sensitive mutants with high P contents were not found. In one of the arsenate-resistant mutants, AR3, the intracellular P content was about twice that in the wild type during growth in the absence of arsenate. Arsenate incorporation in AR3 was suppressed within 10 min after the addition of 1 mM arsenate, while Pi incorporation continued even after arsenate uptake ceased. Whereas the P content of the wild type decreased to half in the presence of 0.5 mM arsenate, almost the same degree (about 50%) of decrease was observed in AR3 cells grown in the presence of as much as 3 mM arsenate. AR3, in which PTB1, a homolog of a Pi transporter gene, had been disrupted, exhibited a higher activity of a high-affinity Pi transporter, suggesting that it may be due to a compensatory transport activity. These data suggest that the intracellular level of P is one of the important factors of arsenate resistance.     

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.     

Relationship between mutant amidases of Pseudomonas aeruginosa and hydroxyurea as an inhibitor.

Summary Hydroxyurea inhibited growth of Pseudomonas aeruginosa strain AI 3 on media containing either acetanilide (N-phenyl acetamide) or acetamide as sole carbon sources. Mutants resistant to hydroxyurea inhibition of growth on acetanilide (OUCH strains) and acetamide (AmOUCH strains) displayed altered growth properties on various amide media compared with the parent strain AI 3. AI 3 amidase, which catalyses the initial step in the metabolism of acetanilide and acetamide, was inhibited by hydroxyurea in a time-dependent reaction that was slowly reversible at pH 7.2 Compared with AI 3 amidase, amidases from the OUCH mutants were much less sensitive to inhibition by hydroxyurea and showed altered substrate specificities and pH/activity profiles; amidases from the AmOUCH mutants were more sensitive to hydroxyurea inhibition but showed increased activity towards acetamide. Association of resistance to hydroxyurea inhibition with a mutation in the amidase structural gene of strain OUCH 4 was confirmed by transduction.     

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.     

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 Pseudomonas aeruginosa.

A high-affinity (Km = 2.7 x 10(-7) M) energy-requiring methionine-transport system has been characterized in RM 46 and RM 48, two different PAO methionine auxotrophs of Pseudomonas aeruginosa. After 8 s of transport 40--60% of the methionine label in the alcohol extract appears in S-adenosyl-L-methionine (SAM) with the remaining activity in free methionine. Methionine transport required a high degree of structural specificity for transport. Stimulation of transport occurred by addition of glucose or organic acids. The ability of a given substrate to stimulate transport was related to the type of carbon source used for growth. Transport was sensitive to sulfhydryl reagents and required oxidative phosphorylation, as indicated by the inhibitory effects of anaerobiosis, cyanide, and arsenate. The degree of inhibition by arsenate correlated with the level of ATP in the cell. Rapid transport in a SAM-deficient mutant (TM 1) and inhibition by arsenate of transport in this mutant suggested that SAM formation was not directly linked to transport and that ATP supplied energy for transport. Inhibition by arsenate was more severe in glucose- compared to citrate-stimulated cells. This result was also observed with proline transport indicating that this was not a peculiarity of the methionine-transport system. These data emphasize the close link between glucose metabolism, ATP levels, and transport. This ATP level is not so critical for transport in cells metabolizing citrate.     

Phenotype microarray profiling of Staphylococcus aureus menD and hemB mutants with the small-colony-variant phenotype

Standard biochemical tests have revealed that hemin and menadione auxotrophic Staphylococcus aureus small-colony variants (SCVs) exhibit multiple phenotypic changes. To provide a more complete analysis of the SCV phenotype, two genetically defined mutants with a stable SCV phenotype were comprehensively tested. These mutants, generated via mutations in menD or hemB that yielded menadione and hemin auxotrophs, were subjected to phenotype microarray (PM) analysis of over 1,500 phenotypes (including utilization of different carbon, nitrogen, phosphate, and sulfur sources; growth stimulation or inhibition by amino acids and other nutrients, osmolytes, and metabolic inhibitors; and susceptibility to antibiotics). Compared to parent strain COL, the hemB mutant was defective in utilization of a variety of carbon sources, including Krebs cycle intermediates and compounds that ultimately generate ATP via electron transport. The phenotype of the menD mutant was similar to that of the hemB mutant, but the defects in carbon metabolism were more pronounced than those seen with the hemB mutant. In both mutant strains, hexose phosphates and other carbohydrates that provide ATP in the absence of electron transport stimulated growth. Other phenotypes of SCV mutants, such as hypersensitivity to sodium selenite, sodium tellurite, and sodium nitrite, were also uncovered by the PM analysis. Key results of the PM analysis were confirmed in independent growth studies and by using Etest strips for susceptibility testing. PM technology is a new and efficient technology for assessing cellular phenotypes in S. aureus.     

An arsenate-activated glutaredoxin from the arsenic hyperaccumulator fern Pteris vittata L. regulates intracellular arsenite

To elucidate the mechanisms of arsenic resistance in the arsenic hyperaccumulator fern Pteris vittata L., a cDNA for a glutaredoxin (Grx) Pv5-6 was isolated from a frond expression cDNA library based on the ability of the cDNA to increase arsenic resistance in Escherichia coli. The deduced amino acid sequence of Pv5-6 showed high homology with an Arabidopsis chloroplastic Grx and contained two CXXS putative catalytic motifs. Purified recombinant Pv5-6 exhibited glutaredoxin activity that was increased 1.6-fold by 10 mm arsenate. Site-specific mutation of Cys(67) to Ala(67) resulted in the loss of both GRX activity and arsenic resistance. PvGrx5 was expressed in E. coli mutants in which the arsenic resistance genes of the ars operon were deleted (strain AW3110), a deletion of the gene for the ArsC arsenate reductase (strain WC3110), and a strain in which the ars operon was deleted and the gene for the GlpF aquaglyceroporin was disrupted (strain OSBR1). Expression of PvGrx5 increased arsenic tolerance in strains AW3110 and WC3110, but not in OSBR1, suggesting that PvGrx5 had a role in cellular arsenic resistance independent of the ars operon genes but dependent on GlpF. AW3110 cells expressing PvGrx5 had significantly lower levels of arsenite when compared with vector controls when cultured in medium containing 2.5 mm arsenate. Our results are consistent with PvGrx5 having a role in regulating intracellular arsenite levels, by either directly or indirectly modulating the aquaglyceroporin. To our knowledge, PvGrx5 is the first plant Grx implicated in arsenic metabolism.     

Isolation and characterization of arsenate-sensitive and resistant mutants of Chlamydomonas reinhardtii

Arsenate-sensitive and resistant mutants of Chlamydomonas reinhardtii were obtained by screening mutants generated by random insertional mutagenesis for growth in the presence of various concentrations of arsenate. The intracellular concentrations of arsenic in the mutants kept in the arsenate-containing medium were determined with an atomic absorption spectrophotometer. The intracellular levels of arsenic in the arsenate-resistant mutants were all lower than that of the parent strain CC425. Some of the arsenate-sensitive mutants, AS1 and AS3, showed obviously higher levels of arsenic than that of CC425, while other sensitive mutant, AS2, did not accumulate arsenic so much. Analysis of the chemical species of arsenic suggested that inorganic arsenic was converted to dimethylarsinic acid (DMAA) in CC425. However, DMAA was hardly detected in AS2. The mechanisms of the resistance to arsenate are discussed on its uptake and detoxification.     

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.     

Single point mutations in either gene encoding the subunits of the heterooctameric yeast phosphofructokinase abolish allosteric inhibition by ATP

Yeast phosphofructokinase is a heterooctameric enzyme subject to a complex allosteric regulation. A mutation in the PFK1 gene, encoding the larger -subunits, rendering the enzyme insensitive to allosteric inhibition by ATP was found to be caused by an exchange of proline 728 for a leucine residue. By in vitro mutagenesis, we introduced this mutation in either PFK1 or PFK2 and found that the exchange in either subunit drastically reduced the sensitivity of the holoenzyme to ATP inhibition. This was accompanied by a lack of allosteric activation by AMP, fructose 2,6-bisphosphate, or ammonium and an increased resistance to heat inactivation. Yeast cells carrying either one mutation or both in conjunction did not display a strong phenotype when grown on fermentable carbon sources and did not show any significant changes in intermediary metabolites. Growth on non-fermentable carbon sources was clearly impaired. The strain carrying both mutant alleles was more sensitive to Congo Red than the wild-type strain or the single mutants indicating differences in cell wall composition. In addition, we found single pfk null mutants to be less viable than wild type at different storage temperatures and a pfk2 null mutant to be temperature-sensitive for growth at 37 degrees C. The latter mutant was shown to be respiration-dependent for growth on glucose.     

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.     

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.     

Characterisation of phosphate binding to mitochondrial and bacterial membrane-bound ATP synthase by studies of inhibition with 4-chloro-7-nitrobenzofurazan

The effect of phosphate on the inhibition by 4-chloro-7-nitrobenzofurazan of the ATPase activity of the proton-translocating ATP synthase in heart submitochondrial particles was investigated. Binding of phosphate protected strongly against the inhibition. A dissociation constant of 0.2 mM was determined for the enzyme X Pi complex and shown to be independent of pH in the range 7.0-8.0. The protective effect of phosphate was mimicked by arsenate but not by sulphate or malonate. Similar results were obtained for the enzyme from Paracoccus denitrificans. 2,4-Dinitrophenol enhanced phosphate binding to the mitochondrial enzyme since the protective effect of phosphate was increased. The data are compatible with protection arising from binding of phosphate to a catalytic site.