Sulphate Uptake in Two Mutants of Chlorella vulgaris with High and Low Sulphur Amino Acid Content

Two mutants of Chlorella vulgaris characterized by higher and lower content of sulphur amino acids compared with the wild strain were assayed for the efficiency of the sulphate uptake mechanism. In both mutants uptake exhibited positive cooperation kinetics and was strongly stimulated by sulphate starvation. Stimulation was depressed by cysteine and to a higher extent by methionine. Mutations affected the uptake efficiency concordantly with the level of sulphur amino acids. Addition to the starved strains of sulphate or chromate reduced the induced transport to the lower values of the non-starved strains. Addition of cycloheximide during the induction period prevented a further enhancement of transport without depressing the attained rate in the low sulphur mutant; it was followed by a rapid fall to the non-induced rate in the high sulphur mutant.     

A yeast with unusual sulphur amino acid metabolism

A yeast strain highly resistant to propargylglycine (an inhibitor of cystathionine gamma-lyase) was isolated from air. It was partially characterized, but it has not been identified with any known yeast species. Its sulphur amino acid metabolism differed from that of other fungi by the lack of the reverse transsulphuration pathway from methionine to cysteine, as no activity of cystathionine beta-synthase or cystathionine gamma-lyase was found. The functional lack of this pathway was confirmed by growth tests and by experiments with [35S]methionine. In contrast to Saccharomyces cerevisiae neither homocysteine synthase nor the sulphate assimilation pathway were repressible by methionine in the new strain; on the contrary, a regulatory effect of cysteine was observed.     

Biosynthesis of thiazole from thiamine in Escherichia coli]

The incorporation into the thiazole moiety of thiamine of several labeled compounds has been studied on short time incubations of washed-cells suspensions. No incorporation of radioactivity from [G-14C] methionine was found in a mutant auxotrophic for methionine. No radioactivity was incorporated from [U-14C] aspartate or from [U-14C] serine. The incorporation of 35S from sulphate was lowered by cysteine or glutathione but was unaffected by methionine or homocystine. Although the synthesis of thiazole is dependent on methionine, neither the sulphur atom nor the carbon chain of thiazole originate from methonine in E. coli. No carbon originates from cysteine which is the likely direct donor of sulphur.     

Sulphate metabolism of selenate-resistant Schizosaccharomyces pombe mutants

Selenate-resistant mutants were obtained from several strains of Schizosaccharomyces pombe. The obtained mutants all belonged to the same genetic complementation group. They were low in sulphate uptake activity and in ATP sulphurylase activity. They grew on medium containing sulphite, thiosulphate, cysteine or glutathione but not methionine as the sole source of sulphur. From these results, the mutants were concluded to carry mutations in the ATP sulphurylase gene. Inability of the mutants to utilize methionine as a sulphur source is rationalized by the absence of the reverse transsulphurylation pathway in this organism; wild type strains must utilize methionine as a sulphur source after it is degraded to give rise to sulphate.     

The uptake of labelled sulphur by the proteins of human liver slices incubated with L-(35S)-methionine or (35S)-sulphate

Transfer of labelled sulphur from methionine into protein-bound cysteine was observed in human foetal liver $\text{in}$$\text{vitro}$. Inorganic sulphate could not be utilized as sulphur donor for cystein synthesis in human foetal liver.     

Effect of regulatory mutations of sulphur metabolism on the levels of cysteine- and homocysteine-synthesizing enzymes in Neurospora crassa

1. Regulation of four enzymes involved in cysteine and homocysteine synthesis, i.e. cysteine synthase (EC 4.2.99.8), homocysteine synthase (EC 4.1.99.10), cystathionine beta-synthase (EC 2.1.22) and gamma-cystathionase (EC 4.4.1.1) was studied in the wild type and sulphur regulatory mutants of Neurospora crassa. 2. Homocysteine synthase and cystathionine beta-synthase were found to be regulatory enzymes but only the former is under control of the cys-3 - scon system regulating several enzymes of sulphur metabolism, including gamma-cystathionase. 3. The results obtained with the mutants strongly suggest that homocysteine synthase plays a physiological role as an enzyme of the alternative pathway of methionine synthesis. Cysteine synthase activity was similar in all strains examined irrespective of growth conditions. 4. The sconc strain with derepressed enzymes of sulphur metabolism showed an increased pool of sulphur amino acids, except for methionine. Particularly characteristic for this pool is a high content of hypotaurine, a product of cysteine catabolism.     

Sulphate utilization in an aphid symbiosis

When two clones of Myzus persicae were maintained on a defined diet with inorganic sulphate as sole sulphur source, their growth and survival were inferior to that on diets containing the sulphur amino acid, methionine. This discrepancy is due, at least in part, to the phagostimulatory properties of methionine, which stimulated aphid feeding rate by 50–150%. Myzus persicae incorporated radioactivity from dietary [³⁵S]sulphate into protein and low molecular weight compounds, including cysteine and methionine. Two lines of evidence indicate that the mycetocyte-symbionts are responsible for the reductive assimilation of sulphate. (1) [³⁵S]sulphate incorporation is abolished by treatment of the aphids with the antibiotic chlortetracycline, which disrupts the symbionts; and (2) [³⁵S]sulphate is utilized by isolated embryos (which contain mycetocyte-symbionts but no gut flora) but not by isolated guts. Tracer studies suggest that 20% of dietary radiosulphur is translocated to the aphid tissues, and it is hypothesized that methionine may be the principal product released by the symbionts.     

Genetics of sulphate assimilation in Schizosaccharomyces pombe (a short review)

Sulphur plays an important role in yeasts, especially in the biosynthesis of methionine and cysteine. The inorganic sulphur source, sulphate, is taken up by the cells via the sulphate-permease(s). After its transport, it is activated and subsequently reduced to sulphide or serves as a donor for sulphurylation reactions. Selenate anion (SeO4(2-)), which has the same metabolic pathway as sulphate, is toxic for the cells of Schizosaccharomyces pombe. We isolated selenate resistant mutants which cannot utilize sulphate, therefore they need organic sulphur source for growth. One of the selenate resistant mutants was successively transformed with S. pombe genomic libraries and the gene complementing the selenate resistance was identified as that of coding for the ATP-sulphurylase enzyme.     

Regulation of sulphur amino acids metabolic enzymes in Cephalosporium acremonium strains differing in antibiotic production

Three strains of Cephalosporium acremonium with different potential of cephalosporin C production show differences in the levels of sulphur amino acid metabolic enzymes. The regulation of these enzymes is also different in the strains. In the superior producing strain two enzymes directly involved in cysteine synthesis are the least susceptible to repression by methionine. In this strain cystathionine metabolizing enzymes seem to favour cysteine synthesis. Antibiotic producing strains, in contrast to the non-producing strain, are highly sensitive to chromate.     

Isolation of a sulphate reductase-less mutant of the blue-green alga Nostoc muscorum.

The wild type Nostoc muscorum (UW strain) has yielded various physiological mutants altered in utilization of sulphate, following mutagenic treatments with N-methyl, N'-nitro N-nitrosoguanidine (NTG). One of the mutant strains designated as Sat-20 failed to grow in a medium containing sulphate (MgSO4.7 H2O). However, the mutant strain could grow when supplemented with thiosulphate (Na2S2O3.5 H2O), while methionine could fulfil the sulphur requirement only partially. On comparative reasons, the wild type as well as the mutant showed preference for thiosulphate over other sulphur sources employed.     

Nutritional factors affecting nisin production by Lactococcus lactis subsp. actis NIZO 22186 in a synthetic medium

L. DE VUYST. 1995 A minimal synthetic medium (SM8) for nisin-producing Lactococcus lactis subsp. lactis strains has been designed; it consists of eight growth-stimulating amino acids (glutamic acid, methionine, valine, leucine, threonine, arginine, isoleucine and histidine), five vitamins (biotin, calcium pantothenate, nicotinic acid, pyridoxine and riboflavin) and the mineral salts dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, magnesium sulphate and trisodium citrate. Nisin biosynthesis is strongly dependent on the presence of a sulphur source, either an inorganic salt (magnesium sulphate or sodium thiosulphate) or the amino acids methionine, cysteine or cystathionine. The amino acids serine, threonine and cysteine highly stimulate nisin production without affecting the final cell yield, indicating their precursor role during nisin biosynthesis.     

Isolation of sulphate transport defective mutants ofCandida utilis: Further evidence for a common transport system for sulphate,sulphite and thiosulphate

Selenate-resistant mutants ofCandida utilis were isolated. They did not take up sulphate while incorporation of an organic sulphur source, such asl-methionine, was similar to the wild-type strain. They grew poorly on sulphate, sulphite and thiosulphate and, as expected, grew well on methionine. Sulphite reductase activities of the mutants were similar to the wild type strain. The properties of these mutants support the view of a common transport system for sulphate, sulphite and thiosulphate.     

Plant adenosine 5'-phosphosulphate reductase: the past, the present, and the future

The sulphate assimilation pathway provides reduced sulphur for the synthesis of the amino acids cysteine and methionine. These are the essential building blocks of proteins and further sources of reduced sulphur for the synthesis of coenzymes and various secondary compounds. Several recent reports identified the adenosine 5'-phosphosulphate reductase (APR) as the enzyme with the greatest control over the pathway. In this review, a short historical excursion into the investigations of sulphate assimilation is given with emphasis on the proposed alternative pathways to APR, via 'bound sulphite' or via PAPS reductase. The evolutionary past of APR is reviewed, based on phylogenetic analysis of APR and PAPS reductase sequences. Furthermore, recent biochemical analyses of APR that identified an iron-sulphur centre as a cofactor, proposed functions for different protein domains, and addressed the enzyme mechanism are summarized. Finally, questions that have to be addressed in order to improve understanding of the molecular mechanism and regulation of APR have been identified.     

Physiological analysis of mutants of Saccharomyces cerevisiae impaired in sulphate assimilation.

The assimilation of sulphate in Saccharomyces cerevisiae, comprising the reduction of sulphate to sulphide and the incorporation of the sulphur atom into a four-carbon chain, requires the integrity of 13 different genes. To date, the functions of nine of these genes are still not clearly established. A set of strains, each bearing a mutation in one MET gene, was studied. Phenotypic studies and enzyme determinations showed that the products of at least five genes are needed for the synthesis of an enzymically active sulphite reductase. These genes are MET1, MET5, MET8, MET10 and MET20. Wild-type strains of S. cerevisiae can use organic metabolites such as homocysteine, cysteine, methionine and S-adenosylmethionine as sulphur sources. They are also able to use inorganic sulphur sources such as sulphate, sulphite, sulphide or thiosulphate. Here we show that both of the two sulphur atoms of thiosulphate are used by S. cerevisiae. Thiosulphate is cleaved into sulphite and sulphide prior to utilization by the sulphate assimilation pathway, as the metabolism of one sulphur atom from thiosulphate requires the presence of an active sulphite reductase.     

Catalytic and regulatory properties of sulphur metabolizing enzymes in cyanobacterium Synechococcus elongatus PCC 7942

Synechococcus elongatus PCC 7942 was able to grow with several S sources. The sulphur metabolizing enzymes viz. ATP sulphurylase, cysteine synthase, thiosulphate reductase and L- and D-cysteine desulphydrases were regulated by sulphur sources, particularly by sulphur amino acids and organic sulphate esters. Sulphur starvation reduced ATP sulphurylase and cysteine synthase whereas reduced glutathione appreciated Cys degradation activity. With partially purified enzymes apparent Km values for sulphate, ATP, D- and L-Cys, thiosulphate, sulphide and O-acetyl serine were in a range of 12-50 microM. p-Nitrophenyl sulphate inhibited ATP sulphurylase competitively. Met was a feedback inhibitor of several key enzymes.     

Interrelated regulation of sulphur-containing amino-acid biosynthetic enzymes and folate-metabolizing enzymes in Aspergillus nidulans

In Aspergillus nidulans homocysteine can be metabolized both to cysteine and methionine. Mutants impaired in the main pathway of cysteine synthesis or in the sulphate assimilation pathway show a low pool of glutathione and elevated levels of homocysteine synthase and of the homocysteine-to-cysteine pathway enzymes. On the other hand, the level of methionine synthase and other enzymes of folate metabolism is depressed in these mutants. This anticoordinated regulation provides a mechanism controlling the partition of homocysteine between the two diverging pathways. Homocysteine synthase was found derepressed, along with folate enzymes, in a strain carrying a mutation which suppresses mutations in metA, metB and metG genes. These results indicate that homocysteine synthase can be regarded as the enzyme of an alternative pathway of methionine synthesis and strongly suggest that the regulatory mechanisms governing sulphur-containing amino acid and folate metabolisms are interrelated.     

Sulphur acquisition by Neisseria meningitidis

Group B Neisseria meningitidis (SD1C) was grown on defined medium supplemented with each of a variety of sulphur compounds as the sole source of sulphur. The organism grew on sulphate, sulphite, bisulphite, thiosulphate, dithionite, hydrosulphide, thiocyanate, L-cysteine, L-cystine, reduced glutathione, methionine, mercaptosuccinate, and lanthionine, but not on dithionate unless previously sulphur starved. Good growth was seen on concentrations of sulphate or thiosulphate as low as 10 microM. When pregrown on and subsequently starved for sulphate, the meningococcus showed enhanced transport capacity for this ion. Optimal conditions for assessing sulphur transport by active sulphur-limited cells were determined. The maximal sulphate uptake velocity was 9.3 nmol sulphate X mg protein-1 X min-1, and the apparent Km was 1.4 microM, far below human nasopharyngeal or serum sulphate levels.     

Cysteine and methionine-precursors of methyl sulphone metabolites of 2,4',5-trichlorobiphenyl in mice

In order to account for the origin of the sulphur atom in methyl sulphones derived from polychlorinated biphenyls (PCB), groups of mice were treated with 2,4'-5-trichlorobiphenyl and [35S]cysteine or [35S]methionine, respectively. Control animals received the labelled amino acids only. The radioactive substances extracted from lung tissue were characterized by partition between hexane and sulphuric acid, thin-layer radiochromatography (TLRC), gas chromatography (GC) and mass fragmentography (MF). In lung extracts of both experimental groups sulphuric acid soluble metabolites were present: Their Rf-values on TLRC were identifical with that of 4-methylsulphonyl-2,4'-5-trichlorobiphenyl and their identity was confirmed by GC and GC-MF, which also indicated the presence of a minor sulphone isomer. The study shows that both cysteine and methionine could function as donors of the sulphur atom in the methyl sulphone metabolites derived from PCB.     

Nutritive value of lupin-seed protein and leaf-protein concentrates supplemented with various sulphur sources

The effect of DL methionine, methionine hydroxyanalogue (MHA) and sodium sulphate on the nutritive value of lupin-seed protein and leaf-protein concentrate (LPC) from lucerne for rats and chicks was evaluated. Methionine and MHA markedly improved the biological value of both lupin- and LPC-proteins, but the effect of sodium sulphate was not significant.The supplement of organic sulphur-compounds improved the performance of chicks fed on lupin-seed meal, but the effect of sodium sulphate was negligible. There was no significant response to any of the organic and inorganic sulphur compounds in chicks fed on LPC diets. These results suggest that some factor other than methionine defiency is limiting the nutritive value of LPC for these animals.