Analysis of an engineered sulfate reduction pathway and cadmium precipitation on the cell surface

We previously have genetically engineered an aerobic sulfate reduction pathway in Escherichia coli for the generation of hydrogen sulfide and demonstrated the pathway's utility in the precipitation of cadmium. To engineer the pathway, the assimilatory sulfate reduction pathway was modified so that cysteine was overproduced. Excess cysteine was then converted by cysteine desulfhydrase to an abundance of hydrogen sulfide, which then reacted with aqueous cadmium to form cadmium sulfide. In this study, observations of various E. coli clones were combined with an analysis of kinetic and transport phenomena. This analysis revealed that cysteine production is the rate-limiting step in the engineered pathway and provided an explanation for the phenomenon of cell surface precipitation. An analytical model showed that cadmium sulfide must form at the cell surface because the rate of cadmium sulfide formation is extremely fast and the rate of sulfide transport is relatively slow.     

Metabolic engineering of an aerobic sulfate reduction pathway and its application to precipitation of cadmium on the cell surface

The conversion of sulfate to an excess of free sulfide requires stringent reductive conditions. Dissimilatory sulfate reduction is used in nature by sulfate-reducing bacteria for respiration and results in the conversion of sulfate to sulfide. However, this dissimilatory sulfate reduction pathway is inhibited by oxygen and is thus limited to anaerobic environments. As an alternative, we have metabolically engineered a novel aerobic sulfate reduction pathway for the secretion of sulfides. The assimilatory sulfate reduction pathway was redirected to overproduce cysteine, and excess cysteine was converted to sulfide by cysteine desulfhydrase. As a potential application for this pathway, a bacterium was engineered with this pathway and was used to aerobically precipitate cadmium as cadmium sulfide, which was deposited on the cell surface. To maximize sulfide production and cadmium precipitation, the production of cysteine desulfhydrase was modulated to achieve an optimal balance between the production and degradation of cysteine.     

Metabolic Engineering of an Aerobic Sulfate Reduction Pathway and Its Application to Precipitation of Cadmium on the Cell Surface

The conversion of sulfate to an excess of free sulfide requires stringent reductive conditions. Dissimilatory sulfate reduction is used in nature by sulfate-reducing bacteria for respiration and results in the conversion of sulfate to sulfide. However, this dissimilatory sulfate reduction pathway is inhibited by oxygen and is thus limited to anaerobic environments. As an alternative, we have metabolically engineered a novel aerobic sulfate reduction pathway for the secretion of sulfides. The assimilatory sulfate reduction pathway was redirected to overproduce cysteine, and excess cysteine was converted to sulfide by cysteine desulfhydrase. As a potential application for this pathway, a bacterium was engineered with this pathway and was used to aerobically precipitate cadmium as cadmium sulfide, which was deposited on the cell surface. To maximize sulfide production and cadmium precipitation, the production of cysteine desulfhydrase was modulated to achieve an optimal balance between the production and degradation of cysteine.     

Bioremediation of cadmium by growing Rhodobacter sphaeroides: kinetic characteristic and mechanism studies

The removal kinetic characteristic and mechanism of cadmium by growing Rhodobacter sphaeroides were investigated. The removal data were fitted to the second-order equation, with a correlation coefficient, R2=0.9790-0.9916. Furthermore, it was found that the removal mechanism of cadmium was predominantly governed by bioprecipitation as cadmium sulfide with biosorption contributing to a minor extent. Also, the results revealed that the activities of cysteine desulfhydrase in strains grown in the presence of 10 and 20 mg/l of cadmium were higher than in the control, while the activities in the presence of 30 and 40 mg/l of cadmium were lower than in the control. Content analysis of subcellular fractionation showed that cadmium was mostly removed and transformed by precipitation on the cell wall.     

Engineering hydrogen sulfide production and cadmium removal by expression of the thiosulfate reductase gene (phsABC) from Salmonella enterica serovar typhimurium in Escherichia coli

The thiosulfate reductase gene (phsABC) from Salmonella enterica serovar Typhimurium was expressed in Escherichia coli to overproduce hydrogen sulfide from thiosulfate for heavy metal removal (or precipitation). A 5.1-kb DNA fragment containing phsABC was inserted into the pMB1-based, high-copy, isopropyl-beta-D-thiogalactopyranoside-inducible expression vector pTrc99A and the RK2-based, medium-copy, m-toluate-inducible expression vector pJB866, resulting in plasmids pSB74 and pSB77. A 3. 7-kb DNA fragment, excluding putative promoter and regulatory regions, was inserted into the same vectors, making plasmids pSB103 and pSB107. E. coli DH5alpha strains harboring the phsABC constructs showed higher thiosulfate reductase activity and produced significantly more sulfide than the control strains under both aerobic and anaerobic conditions. Among the four phsABC constructs, E. coli DH5alpha (pSB74) produced thiosulfate reductase at the highest level and removed the most cadmium from solution under anaerobic conditions: 98% of all concentrations up to 150 microM and 91% of 200 microM. In contrast, a negative control did not produce any measurable sulfide and removed very little cadmium from solution. Energy-dispersive X-ray spectroscopy revealed that the metal removed from solution precipitated as a complex of cadmium and sulfur, most likely cadmium sulfide.     

Precipitation of cadmium by Clostridium thermoaceticum.

Cadmium at an initial concentration of 1 mM was completely precipitated by cultures of Clostridium thermoaceticum in complex medium. The precipitation was energy dependent and required cysteine, although cysteine alone did not act as a growth substrate. Electron microscopic analysis revealed localized areas of precipitation at the surfaces of nonstarved cells as well as precipitate in the surrounding medium. The addition of cadmium had no apparent effect on growth or acetogenesis. However, nickel and cadmium were synergistically toxic at a concentration (1 mM) at which neither alone was toxic. The amount of protein extracted from cadmium-treated cultures was twofold higher than that in control extracts, and the amount of total sulfide was fourfold higher in cultures containing cadmium than in control cultures. Comparable levels of cysteine desulfhydrase activity were observed in extracts of both cadmium-treated and control cultures, but the enzyme activity was expressed maximally about 24 h earlier in the cadmium-treated cultures than in the untreated controls.     

Co-expression of Arabidopsis thaliana phytochelatin synthase and Treponema denticola cysteine desulfhydrase for enhanced arsenic accumulation

Arsenic is one of the most hazardous pollutants found in aqueous environments and has been shown to be a carcinogen. Phytochelatins (PCs), which are cysteine-rich and thio-reactive peptides, have high binding affinities for various metals including arsenic. Previously, we demonstrated that genetically engineered Saccharomyces cerevisiae strains expressing phytochelatin synthase (AtPCS) produced PCs and accumulated arsenic. In an effort to further improve the overall accumulation of arsenic, cysteine desulfhydrase, an aminotransferase that converts cysteine into hydrogen sulfide under aerobic condition, was co-expressed in order to promote the formation of larger AsS complexes. Yeast cells producing both AtPCS and cysteine desulfhydrase showed a higher level of arsenic accumulation than a simple cumulative effect of expressing both enzymes, confirming the coordinated action of hydrogen sulfide and PCs in the overall bioaccumulation of arsenic.     

Thiol Accumulation and Cysteine Desulfhydrase Activity in H2S-Fumigated Leaves and Leaf Homogenates of Cucurbit Plants

Fumigation of both, cucurbit plants and cucurbit leaf homogenateswith hydrogen sulfide (H₂S) resulted in an increase in solublethiol, mainly glutathione and cysteine. In leaf homogenatesthis increase was counteracted or prevented by the additionat 1 mM of inhibitors of pyridoxalphosphate dependent enzymesor of products of the cysteine desulfhydrase reaction. Thesecompounds inhibited cysteine desulfhydrase activity, but didnot severely affect O-acetylserine sulfhydrylase activity atthis concentration. These results provide circumstantial evidencethat cysteine desulfhydrase in its reverse reaction, but notO-acetylserine sulfhydrylase participates in the assimilationof atmospheric H₂S. (Received February 4, 1991; Accepted April 30, 1991)     

Hydrogen sulfide is a novel potential virulence factor of Mycoplasma pneumoniae: characterization of the unusual cysteine desulfurase/desulfhydrase HapE

Mycoplasma pneumoniae is a human pathogen causing atypical pneumonia with a minimalized and highly streamlined genome. So far, hydrogen peroxide production, cytadherence, and the ADP‐ribosylating CARDS toxin have been identified as pathogenicity determinants. We have studied haemolysis caused by M. pneumoniae, and discovered that hydrogen peroxide is responsible for the oxidation of heme, but not for lysis of erythrocytes. This feature could be attributed to hydrogen sulfide, a compound that has previously not been identified as virulence factor in lung pathogens. Indeed, we observed hydrogen sulfide production by M. pneumoniae. The search for a hydrogen sulfide‐producing enzyme identified HapE, a protein with similarity to cysteine desulfurases. In contrast to typical cysteine desulfurases, HapE is a bifunctional enzyme: it has both the cysteine desulfurase activity to produce alanine and the cysteine desulfhydrase activity to produce pyruvate and hydrogen sulfide. Experiments with purified HapE showed that the enzymatic activity of the protein is responsible for haemolysis, demonstrating that HapE is a novel potential virulence factor of M. pneumoniae.     

Cysteine desulfhydrase activity in cucurbit plants: Stimulation by preincubation with l- or d-cysteine

Cysteine desulfhydrase activity in leaf discs of cucurbit plants is enhanced 2–4-fold by preincubation with l or d-cysteine. Preincubation with structural analogs of cysteine also stimulated the activity of the enzyme, but to a smaller extent. Maximal increase in cysteine desulfhydrase activity was observed by preincubation with 5 mM or higher concentrations of cysteine. Although not caused by activation, stimulation of the enzyme activity was half-maximal within less than 15 min. Whereas the increase in cysteine desulfhydrase activity by preincubation of leaf discs with cysteine was light independent, pretreatment of the entire plant with light or dark determined the leaf discs' potential for stimulation of the enzyme. Exposure to darkness for 4 hr reduced this potential by 60%. It is concluded that the potential for stimulation of cysteine desulfhydrase activity by preincubation with cysteine is regulated by a compound not synthesized, but metabolized, in the leaf tissue. This regulatory compound may be supplied to the leaves by long-distance transport.     

Distribution of Cysteine Desulfhydrase in Microorganisms

The distribution of cysteine desulfhydrase activity in microorganisms was studied with intact cells. The enzyme activity was found mainly in strains belonging to Enterobacteriaceae, especially to genus Aerobacter (Enterobacter). Aerobacter cloacae IFO 12009 showed markedly high activity.

l-Cysteine was essential as an inducer of the enzyme formation, of which 0.2% in the medium is appropriate.

Intact cells of bacteria containing high cysteine desulfhydrase activity, prepared from broth cultured for 19hr, catalyzed the synthesis of l-cysteine from pyruvate, ammonia and hydrogen sulfide.     

Effect of cysteine desulfhydrase gene disruption on L-cysteine overproduction in Escherichia coli

In Escherichia coli, the enzyme called cysteine desulfhydrase (CD), which is responsible for L-cysteine degradation, was investigated by native-PAGE and CD activity staining of crude cell extracts. Analyses with gene-disrupted mutants showed that CD activity resulted from two enzymes: tryptophanase (TNase) encoded by tnaA and cystathionine beta-lyase (CBL) encoded by metC. It was also found that TNase synthesis was induced by the presence of L-cysteine. The tnaA and metC mutants transformed with the plasmid containing the gene for feedback-insensitive serine acetyltransferase exhibited higher L-cysteine productivity than the wild-type strain carrying the same plasmid. These results indicated that TNase and CBL did act on L-cysteine degradation in E. coli cells.     

Role of D-cysteine desulfhydrase in the adaptation of Escherichia coli to D-cysteine

D-cysteine, a powerful inhibitor of Escherichia coli growth, is decomposed in vitro into pyruvate, H2S, and NH3 by D-cysteine desulfhydrase. To assess the role of this reaction in the adaptation of the bacterium to growth on D-cysteine, the gene of the desulfhydrase was cloned. It corresponds to the open reading frame yedO at 43.03 min on the genetic map of E. coli. The amino acid sequence deduced from this gene is homologous to those of several 1-aminocyclopropane-carboxylate deaminases. However, the E. coli desulfhydrase does not use 1-aminocyclopropane-1-carboxylate as substrate. Various mutants in which the yedO gene was inactivated or overexpressed were constructed. They exhibited hypersensitivity or resistance, respectively, to the presence of d-cysteine in the culture medium. Growth protection against D-cysteine in minimal medium was conferred by the simultaneous addition of isoleucine, leucine, and valine. In agreement with this behavior, D-cysteine inhibited the activity of threonine deaminase, a key enzyme of the isoleucine, leucine, and valine pathway. Finally, in the presence of the intact yedO gene, E. coli growth was improved by addition of D-cysteine as the sole sulfur source. In agreement with a role of the desulfhydrase in sulfur metabolism, yedO expression was induced under conditions of sulfate limitation.     

Complete assimilation of cysteine by a newly isolated non-sulfur purple bacterium resembling Rhodovulum sulfidophilum (Rhodobacter sulfidophilus)

A rod-shaped, motile, phototrophic bacterium, strain SiCys, was enriched and isolated from a marine microbial mat, with cysteine as sole substrate. During phototrophic anaerobic growth with cysteine, sulfide was produced as an intermediate, which was subsequently oxidized to sulfate. The molar growth yield with cysteine was 103 g mol^–1, in accordance with complete assimilation of electrons from the carbon and the sulfur moiety into cell material. Growth yields with alanine and serine were proportionally lower. Thiosulfate, sulfide, hydrogen, and several organic compounds were used as electron donors in the light, whereas cystine, sulfite, or elemental sulfur did not support phototrophic anaerobic growth. Aerobic growth in the dark was possible with fructose as substrate. Cultures of strain SiCys were yellowish-brown in color and contained bacteriochlorophyll a, spheroidene, spheroidenone, and OH-spheroidene as major photosynthetic pigments. Taking the morphology, photosynthetic pigments, aerobic growth in the dark, and utilization of sulfide for phototrophic growth into account, strain SiCys was assigned to the genus Rhodovulum (formerly Rhodobacter) and tentatively classified as a strain of R. sulfidophilum. In cell-free extracts in the presence of pyridoxal phosphate, cysteine was converted to pyruvate and sulfide, which is characteristic for cysteine desulfhydrase activity (l-cystathionine γ-lyase, EC 4.4.1.1). Received: 15 December 1995 / Accepted: 1 April 1996     

Effect of Sulfur-Containing Compounds on Growth of Methanosarcina barkeri in Defined Medium

Methanosarcina barkeri Fusaro (DSM 804) could grow on methanol in a mineral medium containing cysteine or thiosulfate as the sole sulfur source. Optimum growth occurred at cysteine concentrations of 1 to 2.8 mM and at thiosulfate concentrations of 2.5 to 5 mM. No inhibition of growth was observed even when these concentrations were doubled in the culture medium. Under the optimum cysteine and thiosulfate concentrations, the generation times of the organism were about 8 to 10 and 10 to 12 h, respectively, giving a cell yield of about 0.14 to 0.17 and 0.08 to 0.11 g (dry weight)/g of methanol consumed. The organism metabolized cysteine and thiosulfate during growth, giving rise to sulfide in the culture medium. H₂S evolution from cysteine and thiosulfate was catalyzed by two enzymes, namely cysteine desulfhydrase and thiosulfate reductase, respectively, as revealed by enzyme assay in the crude cell-free extract of the organism.     

Cysteine metabolism in cultured tobacco cells

Transported l-[(35)S]cysteine was rapidly metabolized by cultured tobacco cells when supplied to the cells at 0.02 millimolar or 0.5 millimolar. The internal cysteine pool was expandable to approximately 2400 nmoles per gram fresh weight.The (35)S label derived from cysteine was found in several metabolites. The amount of label in glutathione and sulfate was directly proportional to the internal l-[(35)S]cysteine, while the levels of labeled methionine and protein were apparently independent of internal labeled cysteine. Cysteine was more rapidly metabolized when the external cysteine concentration was low (0.02 millimolar) with up to 90% of the (35)S label present as compounds other than cysteine.The initial step in cysteine degradation yielded pyruvate, sulfide, and presumably NH(4) (+). Stoichiometry studies using extracts prepared from acetone powders of tobacco cells indicated that pyruvate and sulfide were produced in a 1:1 ratio. The catabolic reaction was linear with respect to time and amount of protein and had a pH optimum of 8 in crude extracts. Preliminary kinetic data indicated the K(m) to be approximately 0.2 millimolar. The extractable degradative activity was enhanced 15- to 20-fold by preincubating the cells for 24 hours in 0.5 millimolar cysteine. The extractable specific enzyme activity roughly reflected the growth curve of the cells in culture. Maximal cysteine degradation was observed in extracts prepared from late log phase cultures that were preincubated in cysteine, while little activity was found in similar extracts from stationary phase cultures. These results are consistent with an inducible catabolic enzyme similar to the cysteine desulfhydrase from bacteria.     

Role of a cysteine synthase in Staphylococcus aureus

The gram-positive human pathogen Staphylococcus aureus is often isolated with media containing potassium tellurite, to which it has a higher level of resistance than Escherichia coli. The S. aureus cysM gene was isolated in a screen for genes that would increase the level of tellurite resistance of E. coli DH5alpha. The protein encoded by S. aureus cysM is sequentially and functionally homologous to the O-acetylserine (thiol)-lyase B family of cysteine synthase proteins. An S. aureus cysM knockout mutant grows poorly in cysteine-limiting conditions, and analysis of the thiol content in cell extracts showed that the cysM mutant produced significantly less cysteine than wild-type S. aureus SH1000. S. aureus SH1000 cannot use sulfate, sulfite, or sulfonates as the source of sulfur in cysteine biosynthesis, which is explained by the absence of genes required for the uptake and reduction of these compounds in the S. aureus genome. S. aureus SH1000, however, can utilize thiosulfate, sulfide, or glutathione as the sole source of sulfur. Mutation of cysM caused increased sensitivity of S. aureus to tellurite, hydrogen peroxide, acid, and diamide and also significantly reduced the ability of S. aureus to recover from starvation in amino acid- or phosphate-limiting conditions, indicating a role for cysteine in the S. aureus stress response and survival mechanisms.     

Expression of the mouse metallothionein-I gene in Escherichia coli: increased tolerance to heavy metals

The cDNA of mouse metallothionein, a small metal-binding protein rich in cysteine, has been cloned downstream from a bacterial inducible promoter and expressed in Escherichia coli. Upon induction, E. coli harboring this cDNA clone contained a protein species readily labelled by [35S]cysteine in vivo and incorporated 10-times as much 109Cd from the medium than would otherwise be the case. We show that expression of metallothionein endows resistance in E. coli to heavy metal ions such as mercury, silver, copper, cadmium and zinc by sequestering rather than exclusion or conversion, common mechanisms of metal resistance in bacteria.     

Aerobic transformation of zinc into metal sulfide by photosynthetic microorganisms

Industrial activity over the last two centuries has increased heavy metal contamination worldwide, leading to greater human exposure. Zinc is particularly common in industrial effluents and although an essential nutrient, it is highly toxic at elevated concentrations. Photoautotrophic microbes hold promise for heavy metal bioremediation applications because of their ease of culture and their ability to produce sulfide through metabolic processes that in turn are known to complex with the metal ion, Hg(II). The green alga Chlamydomonas reinhardtii, the red alga Cyanidioschyzon merolae, and the cyanobacterium Synechococcus leopoliensis were all able to synthesize sulfide and form zinc sulfide when exposed to Zn(II). Supplementation of their respective media with sulfite and cysteine had deleterious effects on growth, although ZnS still formed in Cyanidioschyzon cells to the same extent as in unsupplemented cells. The simultaneous addition of sulfate and Zn(II) had similar effects to that of Zn(II) alone in all three species, whereas supplying sulfate prior to exposure to Zn(II) enhanced metal sulfide production. The coupled activities of serine acetyltransferase and O-acetylserine(thiol)lyase (SAT/OASTL) did not increase significantly in response to conditions in which enhanced ZnS formation occurred; sulfate added prior to and simultaneously with Zn(II). However, even low activity could provide sufficient sulfate assimilation over this relatively long-term study. Because the extractable activity of cysteine desulfhydrase was elevated in cells that produced higher amounts of zinc sulfide, cysteine is the probable source of the sulfide in this aerobic process.