Assimilatory sulfur metabolism in marine microorganisms: a novel sulfate transport system in Alteromonas luteo-violaceus.

The sulfate transport mechanism of a marine bacterium, Alteromonas luteo-violaceus, was unique among microorganisms in its extremely low affinity for the sulfate analog thiosulfate. Distinguishing characteristics included weak inhibition of sulfate transport by thiosulfate, inability to transport thiosulfate effectively, poor growth using thiosulfate as the sole source of sulfur, and a mild effect of the sulfhydryl reagent para-hydroxymercuribenzoate. In contrast, sulfate transport by a marine pseudomonad, Pseudomonas halodurans, was strongly inhibited by thiosulfate, and para-hydroxymercuribenzoate reversibly but completely blocked sulfate transport.     

Isolation and characterization of Burkholderia cenocepacia mutants deficient in pyochelin production: pyochelin biosynthesis is sensitive to sulfur availability

The opportunistic pathogen Burkholderia cenocepacia produces the yellow-green fluorescent siderophore, pyochelin. To isolate mutants which do not produce this siderophore, we mutagenized B. cenocepacia with the transposon mini-Tn5Tp. Two nonfluorescent mutants were identified which were unable to produce pyochelin. In both mutants, the transposon had integrated into a gene encoding an orthologue of CysW, a component of the sulfate/thiosulfate transporter. The cysW gene was located within a putative operon encoding other components of the transporter and a polypeptide exhibiting high homology to the LysR-type regulators CysB and Cbl. Sulfate uptake assays confirmed that both mutants were defective in sulfate transport. Growth in the presence of cysteine, but not methionine, restored the ability of the mutants to produce pyochelin, suggesting that the failure to produce the siderophore was the result of a depleted intracellular pool of cysteine, a biosynthetic precursor of pyochelin. Consistent with this, the wild-type strain did not produce pyochelin when grown in the presence of lower concentrations of sulfate that still supported efficient growth. We also showed that whereas methionine and certain organosulfonates can serve as sole sulfur sources for this bacterium, they do not facilitate pyochelin biosynthesis. These observations suggest that, under conditions of sulfur depletion, cysteine cannot be spared for production of pyochelin even under iron starvation conditions.     

A sulfate,sulfite and thiosulfate incorporating system inCandida utilis

Sulfate, sulfite and thiosulfate incorporation in the yeastCandida utilis is inhibited by extracellular sulfate, sulfite and thiosulfate and by sulfate analogues selenate, chromate and molybdate. The three processes are blocked if sulfate, sulfite, thiosulfate, cysteine and homocysteine are allowed to accumulate endogenously. Incorporation of the three inorganic sulfur oxy anions is inactivated by heat at the same rate. Mutants previously shown to be defective in sulfate incorporation are also affected in sulfite and thiosulfate uptake. Revertants of these mutants selected by plating in ethionine-supplemented minimal medium recovered the capacity to incorporate sulfate, sulfite and thiosulfate. These results taken together with previous evidence demonstrate the existence of a common sulfate, sulfite and thiosulfate incorporating system in this yeast.     

Cysteine and Methionine Regulation of Sulfate Uptake in Potato Tuber Discs (Solanum tuberosum)

Sulfate uptake in potato tuber discs is inhibited by cysteine and methionine with an 8 h lag period. Cysteine, but not methionine, inhibition can be reversed by washing the treated discs. During the experimental period cysteine is rapidly metabolized, while methionine persists as a free amino acid. Amino acid inhibition of sulfate uptake is overcome by increasing sulfate concentration. The kinetic parameters change suggesting a loss of flexibility of the sulfate uptake system caused by sulfur amino acids.     

Two transsulfurylation pathways in Klebsiella pneumoniae

In most bacteria, inorganic sulfur is assimilated into cysteine, which provides sulfur for methionine biosynthesis via transsulfurylation. Here, cysteine is transferred to the terminal carbon of homoserine via its sulfhydryl group to form cystathionine, which is cleaved to yield homocysteine. In the enteric bacteria Escherichia coli and Salmonella enterica, these reactions are catalyzed by irreversible cystathionine-gamma-synthase and cystathionine-beta-lyase enzymes. Alternatively, yeast and some bacteria assimilate sulfur into homocysteine, which serves as a sulfhydryl group donor in the synthesis of cysteine by reverse transsulfurylation with a cystathionine-beta-synthase and cystathionine-gamma-lyase. Herein we report that the related enteric bacterium Klebsiella pneumoniae encodes genes for both transsulfurylation pathways; genetic and biochemical analyses show that they are coordinately regulated to prevent futile cycling. Klebsiella uses reverse transsulfurylation to recycle methionine to cysteine during periods of sulfate starvation. This methionine-to-cysteine (mtc) transsulfurylation pathway is activated by cysteine starvation via the CysB protein, by adenosyl-phosphosulfate starvation via the Cbl protein, and by methionine excess via the MetJ protein. While mtc mutants cannot use methionine as a sulfur source on solid medium, they will utilize methionine in liquid medium via a sulfide intermediate, suggesting that an additional nontranssulfurylation methionine-to-cysteine recycling pathway(s) operates under these conditions.     

Inhibitory effects of sulfamic acid on three thiosulfate-oxidizing chemolithotrophs

Sulfamate is an analogue of thiosulfate, and the sodium and potassium salts of sulfamic acid inhibited the chemolithoautotrophic growth on thiosulfate of Acidithiobacillus ferrooxidans and Halothiobacillus neapolitanus. The chemo-organotrophic growth of Paracoccus versutus on sucrose was similarly inhibited by sulfamate. Thiosulfate oxidation by suspensions of H. neapolitanus was, however, unaffected by sulfamate, showing that sulfamate did not directly affect thiosulfate uptake, activation or oxidation. Inhibition of P. versutus was not relieved by cysteine and methionine, indicating that sulfate uptake and sulfur amino acid biosynthesis were not directly affected by sulfamate. Sulfamate was not degraded by any of the bacteria, and so could not serve as an alternative to thiosulfate as an energy-yielding substrate. Sulfamate is also an analogue of ammonia and might act like hydrazine by inhibiting ammonium uptake or an essential enzyme activity.     

Sulfur regulation of the sulfate transporter genes sutA and sutB in Penicillium chrysogenum

Penicillium chrysogenum uses sulfate as a source of sulfur for the biosynthesis of penicillin. Sulfate uptake and the mRNA levels of the sulfate transporter-encoding sutB and sutA genes are all reduced by high sulfate concentrations and are elevated by sulfate starvation. In a high-penicillin-yielding strain, sutB is effectively transcribed even in the presence of excess sulfate. This deregulation may facilitate the efficient incorporation of sulfur into cysteine and penicillin.     

Characteristics of assimilatory sulfate transport in Rhodobacter sulfidophilus

Abstract Sulfate uptake was investigated with four species of phototrophic sulfur bacteria. Rhodobacter sulfidophilus and Chromatium vinosum took up ³⁵S-labeled sulfate added in micromolar concentrations. Sulfate uptake by C. vinosum was expressed only under sulfate starvation. R. sulfidophilus took up 10 μM sulfate almost completely and accumulated it up to 5300-fold, also when grown with excess sulfate. Sulfite (1 mM) as an intermediate of sulfate assimilation inhibited sulfate uptake completely within 1 min. Moderate inhibition was observed with cysteine (1 mM) and none with sulfide (1 mM). Transport was not dependent on the cations K⁺, Na⁺, Li⁺ or protons, but was sensitive to uncouplers and to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD). The accumulation of sulfate correlated with the ATP concentration in the cells, indicating an ATP-dependent uptake mechanism.     

Uptake and efflux of sulfate in Neurospora crassa

Sulfate efflux from an intracellular pool was observed with both wild-type and cys-11 cells of Neurospora and apparently occurs by way of the sulfate transport system. Efflux requires the presence of external sulfate or the related ions, chromate, selenate, or thiosulfate, and probably occurs by an exchange reaction. The sulfur amino acids, cysteine or methionine, do not promote sulfate efflux. The K_m for efflux is much greater than the K_m for sulfate uptake, which permits the accumulation of a considerable intracellular pool before efflux becomes significant. Substantial transmembrane movement of sulfate both influx and exit, was found to occur in azidetreated cells, although the net uptake of sulfate was abolished by this inhibitor. Both sulfate uptake and efflux are inhibited by p-chloromercuribenzoate which suggests that the sulfate permease possesses an essential sulfhydryl group.     

Metabolism of Sulfur Compounds in Bacillus subtilis during Sporulation

Metabolism of various sulfur compounds in Bacillus subtilis during growth and sporulation was investigated by use of tracer techniques, in an attempt to clarify the mechanism involved in the formation of cystine rich protein of the spore coat.

Methionine, homocysteine, cystathionine, cysteine and some inorganic sulfur compounds (sulfate, sulfite and thiosulfate) were utilized by this organism as sulfur sources for its growth and sporulation. Biosynthesis of methionine from sulfate during growth was more or less inhibited by the addition of cysteine, homocysteine or cystathionine to the culture.

It is suggested from these results that in Bacillus subtilis methionine is synthesized from sulfate through cysteine, cystathionine and homocysteine as is the case in Salmonella or Neurospora. The results also suggest that the metabolism of sulfur-containing amino acids in Bacillus subtilis is strongly regulated by methionine and homocysteine.     

Sulfur Regulation of the Sulfate Transporter Genes sutA and sutB in Penicillium chrysogenum

Penicillium chrysogenum uses sulfate as a source of sulfur for the biosynthesis of penicillin. Sulfate uptake and the mRNA levels of the sulfate transporter-encoding sutB and sutA genes are all reduced by high sulfate concentrations and are elevated by sulfate starvation. In a high-penicillin-yielding strain, sutB is effectively transcribed even in the presence of excess sulfate. This deregulation may facilitate the efficient incorporation of sulfur into cysteine and penicillin.     

Sulfur uptake in the ectomycorrhizal fungus Laccaria bicolor S238N

The importance of the ectomycorrhiza symbiosis for plant acquisition of phosphorus and nitrogen is well established whereas its contribution to sulfur nutrition is only marginally understood. In a first step to investigate the role of ectomycorrhiza in plant sulfur nutrition, we characterized sulfate and glutathione uptake in Laccaria bicolor. By studying the regulation of sulfate uptake in this ectomycorrhizal fungus, we found that in contrast to bacteria, yeast, and plants, sulfate uptake in L. bicolor was not feedback-inhibited by glutathione. On the other hand, sulfate uptake was increased by sulfur starvation as in other organisms. The activity of 3′-phosphoadenosine 5′-phosphosulfate reductase, the key enzyme of the assimilatory sulfate reduction pathway in fungi, was increased by sulfur starvation and decreased after treatment with glutathione revealing an uncoupling of sulfate uptake and reduction in the presence of reduced sulfur compounds. These results support the hypothesis that L. bicolor increases sulfate supply to the plant by extended sulfate uptake and the plant provides the ectomycorrhizal fungus with reduced sulfur.     

Methionine-to-cysteine recycling in Klebsiella aerogenes

In the enteric bacteria Escherichia coli and Salmonella enterica, sulfate is reduced to sulfide and assimilated into the amino acid cysteine; in turn, cysteine provides the sulfur atom for other sulfur-bearing molecules in the cell, including methionine. These organisms cannot use methionine as a sole source of sulfur. Here we report that this constraint is not shared by many other enteric bacteria, which can use either cysteine or methionine as the sole source of sulfur. The enteric bacterium Klebsiella aerogenes appears to use at least two pathways to allow the reduced sulfur of methionine to be recycled into cysteine. In addition, the ability to recycle methionine on solid media, where cys mutants cannot use methionine as a sulfur source, appears to be different from that in liquid media, where they can. One pathway likely uses a cystathionine intermediate to convert homocysteine to cysteine and is induced under conditions of sulfur starvation, which is likely sensed by low levels of the sulfate reduction intermediate adenosine-5'-phosphosulfate. The CysB regulatory proteins appear to control activation of this pathway. A second pathway may use a methanesulfonate intermediate to convert methionine-derived methanethiol to sulfite. While the transsulfurylation pathway may be directed to recovery of methionine, the methanethiol pathway likely represents a general salvage mechanism for recovery of alkane sulfide and alkane sulfonates. Therefore, the relatively distinct biosyntheses of cysteine and methionine in E. coli and Salmonella appear to be more intertwined in Klebsiella.     

Sulfate assimilation in Rhodopseudomonas globiformis

Rhodopseudomonas globiformis is able to grow on sulfate as sole source of sulfur, but only at concentrations below 1 mM. Good growth was observed with thiosulfate, cysteine or methionine as sulfur sources. Tetrathionate supported slow growth. Sulfide and sulfite were growth inhibitory. Growth inhibition by higher sulfate concentrations was overcome by the addition of O-acetylserine, which is known as derepressor of sulfate-assimilating enzymes, and by reduced glutathione. All enzymes of the sulfate assimilation pathway. ATP-sulfurylase, adenylylphosphate-sulfotransferase, thiosulfonate reductase and O-acetylserine sulfhydrylase are present in R. globiformis. Sulfate was taken up by the cells and the sulfur incorporated into the amino acids cysteine, methionine and homocysteine. It is concluded, that the failure of R. globiformis to grow on higher concentrations of sulfate is caused by disregulation of the sulfate assimilation pathway. Some preliminary evidence for this view is given in comparing the activities of some of the involved enzymes after growth on different sulfur sources and by examining the effect of O-acetylserine on these activities.Abbreviations DTE dl-dithioerythritol - APS adenosine 5-phosphosulfate, adenylyl sulfate - PAPS 3-phosphoadenosine 5-phosphosulfate, 3-phosphoadenylylsulfate     

Microbial Recovery of Sulfur from Thiosulfate-Bearing Wastewater with Phototrophic and Sulfur-Reducing Bacteria

The spent caustic wastewater from the oxidation of sulfide present in offshore natural gas production mainly comprises thiosulfate and sulfate. A biocatalytic process, employing phototrophic green sulfur bacteria in symbiosis with sulfate-reducing bacteria, is described in this paper for the production of sulfur from the spent caustic wastewater, with synthetic wastewater as the model system. The process entails the conversion of thiosulfate to sulfur and sulfate by photosynthetic green sulfur bacteria Chlorobium vibrioforme f. thiosulfatophilum. Sulfate formed in turn is removed by Desulfovibrio desulfuricans to sulfide, which is further converted to sulfur by Chlorobium limicola through photooxidation. Sulfide is also oxidized to sulfur and sulfate via thiosulfate as an intermediate by Chlorobium vibrioforme f. thiosulfatophilum.     

Studies of Sulfate Utilization by Algae. 7. In vivo Metabolism of Thiosulfate by Chlorella

Chlorella pyrenoidosa Chick (Emerson strain 3) utilizes thiosulfate for growth as effectively as sulfate, and more effectively than a variety of organic sulfur compounds containing sulfur in various oxidation states. Thiosulfates, differentially labeled with ³⁵S in either the SH— or SO₃ — sulfur moieties, were used to follow the incorporation of thiosulfate-sulfur into constituents of the insoluble fraction and of the soluble pools. Labeled sulfate was also used for purposes of comparison. Label from both sulfur atoms of thiosulfate and from sulfate is incorporated into the cysteine, homocysteine, and glutathione of the soluble pools, and into the methionine and cystine of protein in the insoluble fraction. Label from SO₃-sulfur of thiosulfate is incorporated more slowly into protein methionine and cystine than label from the SH-sulfur. Moreover, the SO₃-sulfur of thiosulfate is recovered largely as sulfate in both the soluble pools and the insoluble fraction, while only a trace of SH-sulfur is recovered as sulfate in either case. Consistent with this, the metabolism of the SO₃-sulfur of thiosulfate more closely resembles the metabolism of sulfate. Thus it would appear that exogenous thiosulfate undergoes early dismutation in which the SO₃-sulfur is preferentially oxidized, and the SH-sulfur is preferentially incorporated in a reduced state. These results are discussed in relation to the conversion of sulfate to thiosulfate by cell-free extracts of Chlorella previously described.     

The Uptake and Metabolism of Cysteine by Giardia lamblia Trophozoites

ABSTRACT. The cysteine, cystine, methionine and sulfate uptake and cysteine metabolism of Giardia lamblia was studied. Initial experiments indicated that bathocuproine sulphonate (20 μM) added to Keister's modified TYI-S-33 medium supported the growth of G. lamblia at low L-cysteine concentration. This allowed the use of high specific activity radiolabeled L-cysteine for further studies. The analyses of L-cysteine uptake by G. lamblia indicate the presence of at least two different transport systems. The total cysteine uptake was non saturable, with a capacity of 3.7 pmoles per 10⁶ cells per min per μM of cysteine, and probably represent passive diffusion. However, cysteine transport was partially inhibited by L-methionine, D-cysteine and DL-homocysteine. indicating that another system specific for SH-containing amino acids is also present. Cysteine uptake was markedly decreased in medium without serum. In contrast to cysteine, the uptake of L-methionine and sulfate were carried out by saiurable systems with apparent K_m, of 71 and 72 μM, respectively, but the Vmax of the uptake of sulfate was six orders of magnitude lower than the Vmax of methionine uptake. Cystine was not incorporated into trophozoites. [³⁵S]-labeled L-cysteine and L-methionine, but not [³⁵S]sulfate, were incorporated into Giardia proteins, indicating that the parasite lacks the capacity to synthesize cysteine or methionine from sulfate. Neither cystathionine γ lyase nor crystathionine γ synthase activities was detected in homogenates of Giardia lamblia , suggesting that the transsulfuration pathway is not active and there is no conversion of methionine to cysteine. Our data indicate that cysteine is essential for Giardia because the parasite: a) cannot take up cystine, and b) cannot synthesize cysteine de novo.     

Role of source-to-sink transport of methionine in establishing seed protein quantity and quality in legumes

Grain legumes such as pea (Pisum sativum L.) are highly valued as a staple source of protein for human and animal nutrition. However, their seeds often contain limited amounts of high-quality, sulfur (S) rich proteins, caused by a shortage of the S-amino acids cysteine and methionine. It was hypothesized that legume seed quality is directly linked to the amount of organic S transported from leaves to seeds, and imported into the growing embryo. We expressed a high-affinity yeast (Saccharomyces cerevisiae) methionine/cysteine transporter (Methionine UPtake 1) in both the pea leaf phloem and seed cotyledons and found source-to-sink transport of methionine but not cysteine increased. Changes in methionine phloem loading triggered improvements in S uptake and assimilation and long-distance transport of the S compounds, S-methylmethionine and glutathione. In addition, nitrogen and carbon assimilation and source-to-sink allocation were upregulated, together resulting in increased plant biomass and seed yield. Further, methionine and amino acid delivery to individual seeds and uptake by the cotyledons improved, leading to increased accumulation of storage proteins by up to 23%, due to both higher levels of S-poor and, most importantly, S-rich proteins. Sulfate delivery to the embryo and S assimilation in the cotyledons were also upregulated, further contributing to the improved S-rich storage protein pools and seed quality. Overall, this work demonstrates that methionine transporter function in source and sink tissues presents a bottleneck in S allocation to seeds and that its targeted manipulation is essential for overcoming limitations in the accumulation of high-quality seed storage proteins.

Methionine transporter function in pea phloem and embryo affects sulfur, nitrogen, and carbon acquisition, metabolism, and partitioning, resulting in increased seed yield, protein levels, and quality.     

Utilization of sulfonic acids as the only sulfur source for growth of photosynthetic organisms

Growth on ethanesulfonic acid as the only sulfur source was found to occur in ten of the 14 green algae tested and in three of the ten cyanobacteria analyzed. Similar growth could not be demonstrated in the higher plant Lemna minor, or in tissue cultures of anise, sunflower and tobacco. Organisms growing on sulfonic acids as the only sulfur source developed an uptake system for ethanesulfonate found neither in algae growing on sulfate nor in algae unable to utilize sulfonic acids for growth. The development of sulfonate transport was not caused by substrate induction, but by conditions of sulfate starvation. The presence of this uptake system was always correlated with an increased sulfate-uptake capacity. Enhanced sulfate uptake was found in all S-deficient and sulfonate-grown cultures tested, indicating sulfate limitation as the regulatory signal. A lag period of 2–2.5 h after transfer to sulfate deprivation was needed for expression of both enhanced sulfate uptake and ethanesulfonate uptake in case of the green alga Chlorella fusca. It is speculated that the availability of sulfate (pool size) or a metabolic product in equilibrium with oxidized sulfur compounds (sulfate ester? sulfolipids?) controls sulfate and sulfonate uptake systems. The principle of (coordinated) derepression by starvation is discussed as a general strategy in photosynthetic organisms.     

Chromate toxicity and the role of sulfur

The molecular mode(s)-of-action of the toxic metal chromium has yet to be fully resolved. This Mini review focuses on interactions between chromate and sulfur in biological systems. Cr binds sulfur ligands, with cysteine and glutathione having the capacity to aggravate or ameliorate Cr toxicity. Competition between chromate and sulfate for uptake and in metabolism provokes sulfur starvation, which can be growth limiting. Recent data indicate that sulfur deficiency determines protein damage-related Cr toxicity, due to mRNA mistranslation caused by Cr-induced S limitation. Sulfur deprivation could contribute to additional aspects of Cr toxicity, including oxidative DNA damage and Cr related disease.