Assimilatory Sulfate Reduction Pathway Research Articles

A bioaugmentation strategy to recover methane production under sulfate-stressed conditions: Highlights on targeted sulfate-reducing bacteria and DIET-related species

Bioaugmentation has been recognized as a key strategy to improve the anaerobic digestion efficiency in organic waste treatment. Methanosarcina barkeri possesses direct interspecies electron transfer capability, a characteristic that allows it to outcompete other unwanted species such as sulfate-reducing bacteria. This study investigated the effects of bioaugmentation with Methanosarcina barkeri DSM800 on two continuous-stirred tank reactors fed with a sulfate-rich feedstock. One of the two reactors was supplemented with magnetite to facilitate direct interspecies electron transfer. Time series quantitative polymerase chain reactions were performed to evaluate the absolute abundance of crucial species, including the augmented Methanosarcina. Results showed increased and stabilized methane production of 22% and 21% in the reactor amended with magnetite and in the control reactor, respectively. Moreover, volatile fatty acids were almost completely consumed in the magnetite-supplemented reactor. The quantitative polymerase chain reaction was used to analyze the abundance of targeted species in response to bioaugmentation. Specifically, Methanosarcina barkeri was not retained in either reactor after one hydraulic retention time. Direct interspecies electron transfer-associated microorganisms showed opposite trends in the two reactors, highlighting the different interactions with Methanosarcina barkeri in the presence and absence of magnetite. Sulfate-reducing bacteria following the dissimilatory sulfate reduction pathway exhibited an opposite behavior in the reactor amended with magnetite, in contrast to those employing the assimilatory sulfate reduction pathway. Overall, the study demonstrated that bioaugmentation with exogenous archaea can considerably alter the microbial community, but the introduced species is not able to establish itself in a stable microbiome. In addition, the strategy could be further tested to control H2S production in real-world waste treatment scenarios. Quantitative polymerase chain reaction proved to be a useful tool for monitoring changes in the absolute abundance of microorganisms in bioreactors, implementing effective monitoring and control strategies to improve overall system performance.

Melatonin Mitigates Water Deficit Stress in Cenchrus alopecuroides (L.) Thunb through Up-Regulating Gene Expression Related to the Photosynthetic Rate, Flavonoid Synthesis, and the Assimilatory Sulfate Reduction Pathway.

Melatonin can improve plant adaptability to water deficit stress by regulating the biosynthesis of flavonoids and improving the reactive oxygen species-scavenging enzyme system. However, it remains unclear whether melatonin mitigates the effects and causes of water deficit stress in Cenchrus alopecuroides. We conducted a PEG-simulated water stress pot experiment to determine whether and how exogenous melatonin alleviates water deficit in C. alopecuroides. The experiment was divided into four treatments: (1) normal watering (Control), (2) 40% PEG-6000 treatment (D), (3) 100 μmol·L-1 melatonin treatment (MT), and (4) both melatonin and PEG-6000 treatment (DMT). The results showed that melatonin can alleviate water deficit in C. alopecuroides by effectively inhibiting plant chlorophyll degradation and MDA accumulation while increasing antioxidant enzyme activities and photosynthetic rates under water deficit stress. The transcriptome results indicated that melatonin regulates the expression of genes with the biosynthesis pathway of flavonoids (by increasing the expression of PAL, 4CL, HCT, and CHS), photosynthesis-antenna proteins (by increasing the expression of LHC), and sulfur metabolism (the expression of PAPSS and CysC is up-regulated in the assimilatory sulfate reduction pathway), while up-regulating the transcription factors (AP2/ERF-ERF-, C2H2-, WRKY-, Tify-, bHLH-, NAC-, and MYB-related). These findings revealed the possible causes by which melatonin mitigates water deficit stress in C. alopecuroides, which provided novel insights into the role of melatonin in water deficit stress.

The link between increased Desulfovibrio and disease severity in Parkinson’s disease

Parkinson's disease (PD), a progressive and incurable neurodegenerative disease, has taken a huge economic toll and medical burden on our society. Increasing evidence has shown a strong link between PD and the gut microbiome, but studies on the relationship between the gut microbiome and the severity of PD are limited. In this study, 90 fecal samples were collected from newly diagnosed and untreated patients with PD (n = 47) and matched healthy control subjects (n = 43). The 16S rRNA amplicon and shotgun metagenomic sequencing was performed, aiming to uncover the connection between the gut microbiome and disease severity in PD. The results showed that Desulfovibrio was significantly increased in PD compared to healthy controls and positively correlated with disease severity. The increase in Desulfovibrio was mainly driven by enhanced homogeneous selection and weakened drift. Moreover, through metagenome-assembled genomes (MAGs) analysis, a Desulfovibrio MAG (MAG58) was obtained which was also positively correlated with disease severity. MAG58 possesses a complete assimilatory sulfate reduction pathway and a near-complete dissimilatory sulfate reduction pathway to produce hydrogen sulfide which may influence the development of PD. Based on these results, a potential pathogenic mechanism was presented to illustrate how the increased Desulfovibrio accelerates the development of PD by producing excessive hydrogen sulfide. The present study highlighted the vital role of Desulfovibrio in the development of PD, which may provide a new target for the diagnosis and treatment of PD. KEY POINTS: • The evidence for the link between increased Desulfovibrio and disease severity in PD • A Desulfovibrio MAG was obtained which was correlated with PD • A model was presented to illustrate how increased Desulfovibrio causes PD.

Insight of the bio-cathode biofilm construction in microbial electrolysis cell dealing with sulfate-containing wastewater

Signaling molecules are useful in biofilm formation, but the mechanism for biofilm construction still needs to be explored. In this study, a signaling molecule, N-butyryl-l-Homoserine lactone (C4-HSL), was supplied to enhance the construction of the sulfate-reducing bacteria (SRB) bio-cathode biofilm in microbial electrolysis cell (MEC). The sulfate reduction efficiency was more than 90% in less time under the system with C4-HSL addition. The analysis of SRB bio-cathode biofilms indicated that the activity, distribution, microbial population, and secretion of extracellular polymers prompted by C4-HSL, which accelerate the sulfate reduction, in particular for the assimilatory sulfate reduction pathway. Specifically, the relative abundance of acidogenic fermentation bacteria increased, and Desulfovibrio was co-metabolized with acidogenic fermentation bacteria. This knowledge will help to reveal the potential of signaling molecules to enhance the SRB bio-cathode biofilm MEC construction and improve the performance of treating sulfate-containing wastewater.

An assimilatory sulfite reductase, CysI, negatively regulates the dormancy of Microbulbifer aggregans CCB-MM1T.

Sulfur is one of the common and essential elements of all life. Sulfate, which is a major source of sulfur, plays an important role in synthesizing sulfur-containing amino acids, such as cysteine and methionine, organic compounds essential to all living organisms. Some investigations reported that the assimilatory sulfate reduction pathway (ASRP) involved in cysteine synthesis is crucial to entering bacterial dormancy in pathogens. Our previous investigation reported that the halophilic marine bacterium, Microbulbifer aggregans CCB-MM1T , possesses an ASRP and the dissimilatory sulfate reduction pathway (DSRP). The bacterium might use DSRP to generate energy required for entering its dormant. However, the role of the ASRP in the dormancy of M. aggregans CCB-MM1T was so far unknown. In this study, we found that genes involved in ASRP were downregulated in the dormancy. The disruption of the gene encoding an assimilatory sulfite reductase, cysI, suppressed a completely dormant state under low nutrient conditions. In addition, the cysI mutant showed cell aggregation at the middle-exponential phase under high nutrient conditions, indicating that the mutation might be stimulated to enter the dormancy. The wild-type phenotype of the bacterium was recovered by the addition of cysteine. These results suggested that cysteine concentration may play an important role in inducing the dormancy of M. aggregans.

Substrate competition and microbial function in sulfate-reducing internal circulation anaerobic reactor in the presence of nitrate

Nitrate and sulfate often coexist in organic wastewater. In this study, an internal circulation anaerobic reactor was conducted to investigate the impact of nitrate on sulfate reduction. The results showed that sulfate reduction rate dropped from 78.4% to 41.4% at NO3- /SO42- ratios ranging from 0 to 1.03, largely attributed to the inactivity of acetate-utilizing sulfate-reducing bacteria (SRB) and preferential usage of nitrate of propionate-utilizing SRB. Meanwhile, high nitrate removal efficiency was maintained and COD removal efficiency increased with nitrate addition. Enhancement of propionate and butyrate degradation based on Modified Gompertz model and Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) analysis. Moreover, nitrate triggered the shift of microbial community and function. Twelve genera affiliated to Firmicutes, Bacteroidetes and Proteobacteria were identified as keystone genera via network analysis, which kept functional stability of the bacterial community responding to nitrate stress. Increased nitrate inhibited Desulfovibrio, but promoted the growth of Desulforhabdus. Both the predicted functional genes associated with assimilatory sulfate reduction pathway (cysC and cysNC) and dissimilatory sulfate reduction pathway (aprA, aprB, dsrA and dsrB) exhibited negative relationship with nitrate addition.

Hydrogen sulfide: An emerging signaling molecule regulating drought stress response in plants.

Hydrogen sulfide (H2 S) is a small, reactive signaling molecule that is produced within chloroplasts of plant cells as an intermediate in the assimilatory sulfate reduction pathway by the enzyme sulfite reductase. In addition, H2 S is also produced in cytosol and mitochondria by desulfhydration of l-cysteine catalyzed by l-cysteine desulfhydrase (DES1) in the cytosol and from β-cyanoalanine in mitochondria, in a reaction catalyzed by β-cyano-Ala synthase C1 (CAS-C1). H2 S exerts its numerous biological functions by post-translational modification involving oxidation of cysteine residues (RSH) to persulfides (RSSH). At lower concentrations (10-1000 μmol L-1 ), H2 S shows huge agricultural potential as it increases the germination rate, the size, fresh weight, and ultimately the crop yield. It is also involved in abiotic stress response against drought, salinity, high temperature, and heavy metals. H2 S donor, for example, sodium hydrosulfide (NaHS), has been exogenously applied on plants by various researchers to provide drought stress tolerance. Exogenous application results in the accumulation of polyamines, sugars, glycine betaine, and enhancement of the antioxidant enzyme activities in response to drought-induced osmotic and oxidative stress, thus, providing stress adaptation to plants. At the biochemical level, administration of H2 S donors reduces malondialdehyde content and lipoxygenase activity to maintain the cell integrity, causes abscisic acid-mediated stomatal closure to prevent water loss through transpiration, and accelerates the photosystem II repair cycle. Here, we review the crosstalk of H2 S with secondary messengers and phytohormones towards the regulation of drought stress response and emphasize various approaches that can be addressed to strengthen research in this area.

Metagenomic insights into aniline effects on microbial community and biological sulfate reduction pathways during anaerobic treatment of high-sulfate wastewater

For comprehensive insights into the change of sulfate reduction pathway responding to the toxic stress and the shift of microbial community and performance of sulfate reduction, we built a laboratory-scale expanded granular sludge bed reactor (EGSB) treating high-sulfate wastewater with elevated aniline concentrations from 0 to 480 mg/L. High-throughput sequencing and metagenomic approaches were applied to decipher the molecular mechanisms of sulfate reduction under aniline stress through taxonomic and functional profiles. The increasing aniline in the anaerobic system induced the accumulation of volatile fatty acids (VFA), further turned the bioreactor into acidification, which was the principal reason for the deterioration of system performance and finally resulted in the accumulation of toxic free sulfide. Moreover, aniline triggered the change of bacterial community and genes relating to sulfate reduction pathways. The increase of aniline from 0 to 320 mg/L enriched total sulfate-reducing bacteria (SRB), and the most abundant genus was Desulfomicrobium, accounting for 66.85–91.25% of total SRB. The assimilatory sulfate reduction pathway was obviously inhibited when aniline was over 160 mg/L, while genes associated with dissimilatory sulfate reduction pathways all exhibited an upward tendency with the increasing aniline content. The enrichment of aniline-resistant SRB (e.g. Desulfomicrobium) carrying genes associated with the dissimilatory sulfate reduction pathway also confirmed the underlying mechanism that sulfate reduction turned into dissimilation under high aniline condition. Taken together, these results comprehensively provided solid evidence for the effects of aniline on the biological sulfate reduction processes treating high-sulfate wastewater and the underlying molecular mechanisms which may highlight the important roles of SRB and related sulfate reduction genes during treatment.

Sulfite Reductase Co-suppression in Tobacco Reveals Detoxification Mechanisms and Downstream Responses Comparable to Sulfate Starvation.

Sulfite reductase (SIR) is a key enzyme in higher plants in the assimilatory sulfate reduction pathway. SIR, being exclusively localized in plastids, catalyzes the reduction of sulfite (SO32−) to sulfide (S2−) and is essential for plant life. We characterized transgenic plants leading to co-suppression of the SIR gene in tobacco (Nicotiana tabacum cv. Samsun NN). Co-suppression resulted in reduced but not completely extinguished expression of SIR and in a reduction of SIR activity to about 20–50% of the activity in control plants. The reduction of SIR activity caused chlorotic and necrotic phenotypes in tobacco leaves, but with varying phenotype strength even among clones and increasing from young to old leaves. In transgenic plants compared to control plants, metabolite levels upstream of SIR accumulated, such as sulfite, sulfate and thiosulfate. The levels of downstream metabolites were reduced, such as cysteine, glutathione (GSH) and methionine. This metabolic signature resembles a sulfate deprivation phenotype as corroborated by the fact that O-acetylserine (OAS) accumulated. Further, chlorophyll contents, photosynthetic electron transport, and the contents of carbohydrates such as starch, sucrose, fructose, and glucose were reduced. Amino acid compositions were altered in a complex manner due to the reduction of contents of cysteine, and to some extent methionine. Interestingly, sulfide levels remained constant indicating that sulfide homeostasis is crucial for plant performance and survival. Additionally, this allows concluding that sulfide does not act as a signal in this context to control sulfate uptake and assimilation. The accumulation of upstream compounds hints at detoxification mechanisms and, additionally, a control exerted by the downstream metabolites on the sulfate uptake and assimilation system. Co-suppression lines showed increased sensitivity to additionally imposed stresses probably due to the accumulation of reactive compounds because of insufficient detoxification in combination with reduced GSH levels.

Drought stress in maize causes differential acclimation responses of glutathione and sulfur metabolism in leaves and roots.

BackgroundDrought is the most important environmental stress that limits crop yield in a global warming world. Despite the compelling evidence of an important role of oxidized and reduced sulfur-containing compounds during the response of plants to drought stress (e.g. sulfate for stomata closure or glutathione for scavenging of reactive oxygen species), the assimilatory sulfate reduction pathway is almost not investigated at the molecular or at the whole plant level during drought.ResultsIn the present study, we elucidated the role of assimilatory sulfate reduction in roots and leaves of the staple crop maize after application of drought stress. The time-resolved dynamics of the adaption processes to the stress was analyzed in a physiological relevant situation –when prolonged drought caused significant oxidation stress but root growth should be maintained. The allocation of sulfate was significantly shifted to the roots upon drought and allowed for significant increase of thiols derived from sulfate assimilation in roots. This enabled roots to produce biomass, while leaf growth was stopped. Accumulation of harmful reactive oxygen species caused oxidation of the glutathione pool and decreased glutathione levels in leaves. Surprisingly, flux analysis using [35S]-sulfate demonstrated a significant down-regulation of sulfate assimilation and cysteine synthesis in leaves due to the substantial decrease of serine acetyltransferase activity. The insufficient cysteine supply caused depletion of glutathione pool in spite of significant transcriptional induction of glutathione synthesis limiting GSH1. Furthermore, drought impinges on transcription of membrane-localized sulfate transport systems in leaves and roots, which provides a potential molecular mechanism for the reallocation of sulfur upon prolonged water withdrawal.ConclusionsThe study demonstrated a significant and organ-specific impact of drought upon sulfate assimilation. The sulfur metabolism related alterations at the transcriptional, metabolic and enzyme activity level are consistent with a promotion of root growth to search for water at the expense of leaf growth. The results provide evidence for the importance of antagonistic regulation of sulfur metabolism in leaves and roots to enable successful drought stress response at the whole plant level.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0940-z) contains supplementary material, which is available to authorized users.

Genes of Bacillus subtilis 168 that Support Growth of the Cyanobacterium, Synechococcus leopoliensis CCAP1405/1 on Agar Media.

Synechococcus leopoliensis CCAP1405/1 cannot grow on common solid media; however, the strain can grow when co-cultured with Bacillus subtilis 168. Gene-disruptant strains of B. subtilis 168 by pMUTINs at the following sites lost the ability to support the growth of S. leopoliensis CCAP1405/1 on agar media: yxeO and yufO (transporter), yxdK (histidine kinase), sdhC (succinate dehydrogenase), yvgQR (sulfite reductase), acoB (acetoin dehydrogenase), yusE (thioredoxin), yrdA (function unknown). Involvement of the assimilatory sulfate reduction pathway was the suggested reason for loss of the function.

Characterization of the serine acetyltransferase gene family of Vitis vinifera uncovers differences in regulation of OAS synthesis in woody plants.

In higher plants cysteine biosynthesis is catalyzed by O-acetylserine(thiol)lyase (OASTL) and represents the last step of the assimilatory sulfate reduction pathway. It is mainly regulated by provision of O-acetylserine (OAS), the nitrogen/carbon containing backbone for fixation of reduced sulfur. OAS is synthesized by Serine acetyltransferase (SERAT), which reversibly interacts with OASTL in the cysteine synthase complex (CSC). In this study we identify and characterize the SERAT gene family of the crop plant Vitis vinifera. The identified four members of the VvSERAT protein family are assigned to three distinct groups upon their sequence similarities to Arabidopsis SERATs. Expression of fluorescently labeled VvSERAT proteins uncover that the sub-cellular localization of VvSERAT1;1 and VvSERAT3;1 is the cytosol and that VvSERAT2;1 and VvSERAT2;2 localize in addition in plastids and mitochondria, respectively. The purified VvSERATs of group 1 and 2 have higher enzymatic activity than VvSERAT3;1, which display a characteristic C-terminal extension also present in AtSERAT3;1. VvSERAT1;1 and VvSERAT2;2 are evidenced to form the CSC. CSC formation activates VvSERAT2;2, by releasing CSC-associated VvSERAT2;2 from cysteine inhibition. Thus, subcellular distribution of SERAT isoforms and CSC formation in cytosol and mitochondria is conserved between Arabidopsis and grapevine. Surprisingly, VvSERAT2;1 lack the canonical C-terminal tail of plant SERATs, does not form the CSC and is almost insensitive to cysteine inhibition (IC50 = 1.9 mM cysteine). Upon sulfate depletion VvSERAT2;1 is strongly induced at the transcriptional level, while transcription of other VvSERATs is almost unaffected in sulfate deprived grapevine cell suspension cultures. Application of abiotic stresses to soil grown grapevine plants revealed isoform-specific induction of VvSERAT2;1 in leaves upon drought, whereas high light- or temperature- stress hardly trigger VvSERAT2;1 transcription.

Hydrogen sulfide formation as well as ethanol production in different media by cysND- and/or cysIJ-inactivated mutant strains of Zymomonas mobilis ZM4

Many bacteria reduce inorganic sulfate to sulfide to satisfy their need for sulfur, one of the most important elements for biological life. But little is known about the metabolic pathways involving hydrogen sulfide (H₂S) in mesophilic bacteria. By genomic sequence analysis, a complete set of genes for the assimilatory sulfate reduction pathway has been identified in the ethanologen Zymomonas mobilis. In this study, the first ATP sulfurylase- and final sulfite reductase-encoding genes cysND and cysIJ, respectively, in the putative pathway from sulfate to sulfite in Z. mobilis ZM4 was singly or doubly inactivated by homologous recombination and a site-specific FLP-FRT recombination. The resultant mutants, ∆cysND, ∆cysIJ and ∆cysND-cat∆cysIJ, were unable to produce detectable H₂S in glucose or sucrose-containing rich medium and sweet sorghum juice, in which the wild-type ZM4 produced detectable H₂S. While adding sulfite (SO₃²⁻) into media impaired the growth of the mutants and ZM4 to varying degrees, the sulfite restored the H₂S formation in the ∆cysND in the above media, but not in the ∆cysIJ and ∆cysND-cat∆cysIJ mutants. Although it seemed that the inactivation of cysND and cysIJ did not exert a significant negative effect on the cell growth at least in glucose or sucrose medium, the ethanol production of all mutants was inferior to that of ZM4 in sucrose medium and sweet sorghum juice. In addition, adding L-cysteine to glucose-containing rich media restored H₂S formation of all mutants, indicating the existence of another pathway for producing H₂S in Z. mobilis. All these results would help to further elucidate the metabolic pathways involving H₂S in Z. mobilis and exploit the biotechnological applications of this industrially important bacterium.

Sulfite Reductase Defines a Newly Discovered Bottleneck for Assimilatory Sulfate Reduction and Is Essential for Growth and Development in Arabidopsis thaliana

The role of sulfite reductase (SiR) in assimilatory reduction of inorganic sulfate to sulfide has long been regarded as insignificant for control of flux in this pathway. Two independent Arabidopsis thaliana T-DNA insertion lines (sir1-1 and sir1-2), each with an insertion in the promoter region of SiR, were isolated. sir1-2 seedlings had 14% SiR transcript levels compared with the wild type and were early seedling lethal. sir1-1 seedlings had 44% SiR transcript levels and were viable but strongly retarded in growth. In mature leaves of sir1-1 plants, the levels of SiR transcript, protein, and enzymatic activity ranged between 17 and 28% compared with the wild type. The 28-fold decrease of incorporation of (35)S label into Cys, glutathione, and protein in sir1-1 showed that the decreased activity of SiR generated a severe bottleneck in the assimilatory sulfate reduction pathway. Root sulfate uptake was strongly enhanced, and steady state levels of most of the sulfur-related metabolites, as well as the expression of many primary metabolism genes, were changed in leaves of sir1-1. Hexose and starch contents were decreased, while free amino acids increased. Inorganic carbon, nitrogen, and sulfur composition was also severely altered, demonstrating strong perturbations in metabolism that differed markedly from known sulfate deficiency responses. The results support that SiR is the only gene with this function in the Arabidopsis genome, that optimal activity of SiR is essential for normal growth, and that its downregulation causes severe adaptive reactions of primary and secondary metabolism.

Natural variation for sulfate content in Arabidopsis thaliana is highly controlled by APR2

Most agronomic traits of importance, whether physiological (such as nutrient use efficiency) or developmental (such as flowering time), are controlled simultaneously by multiple genes and their interactions with the environment. Here, we show that variation in sulfate content between wild Arabidopsis thaliana accessions Bay-0 and Shahdara is controlled by a major quantitative trait locus that results in a strong interaction with nitrogen availability in the soil. Combining genetic and biochemical results and using a candidate gene approach, we have cloned the underlying gene, showing how a single-amino acid substitution in a key enzyme of the assimilatory sulfate reduction pathway, adenosine 5'-phosphosulfate reductase, is responsible for a decrease in enzyme activity, leading to sulfate accumulation in the plant. This work illustrates the potential of natural variation as a source of new alleles of known genes, which can aid in the study of gene function and metabolic pathway regulation. Our new insights on sulfate assimilation may have an impact on sulfur fertilizer use and stress defense improvement.

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.

Cadmium sequestration in Chlamydomonas reinhardtii

We demonstrated that phytochelatin (PC) is the major intracellular cadmium (Cd) chelator in the microalga, Chlamydomonas reinhardtii. From Cd challenged algal cells, two types of PC–Cd complexes were purified and characterized; the high molecular weight (HMW) complex and the lower molecular weight (LMW) complex. These complexes differed in accumulation kinetics, PC profile, acid labile sulfide content, in vivo turnover rate, UV absorption spectra, and acid sensitivity in vitro. Although the HMW complex appeared first during Cd treatment, our detailed analysis suggested the quick conversion of LMW complex to HMW complex inside the algal cells and HMW complex contributed to stable Cd sequestration. The accumulation of LMW complex appeared to be an early sign of metal stress. In several Cd resistant mutants generated in this lab, the level of PC production was proportional to the capacity of Cd accumulation. Cd-resistant mutant could grow in elevated Cd concentration through different mechanisms. One type of mutant produced high levels of HMW complex with a distinct PC profile to facilitate Cd accumulation. In this mutant, we detected higher content of reduced glutathione and cystein than the wild type. This study suggested the involvement of assimilatory sulfate reduction pathway(s) in Cd sequestration.

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.

Functional analysis of the Bacillus subtilis cysK and cysJI genes

The function of the Bacillus subtilis cysK and cysJI (previously designated yvgQR) genes, expected to be involved in the assimilatory sulfate reduction pathway, was investigated. A B. subtilis mutant with a deletion in the cysJI genes was unable to use sulfate or sulfite as sulfur source, which confirmed that these genes encode sulfite reductase. A mutant with a transposon insertion in the cysK gene, whose deduced protein sequence showed similarity to cysteine synthases, grew poorly on sulfate and butanesulfonate. A strain in which cysK and yrhA, a cysK paralog, were inactivated was unable to grow with sulfate. Whereas expression of the cysJI genes was induced by sulfate, expression of cysK was repressed both by sulfate and by cysteine.

Functional analysis of the Bacillus subtilis cysK and cysJI genes.

The function of the Bacillus subtilis cysK and cysJI (previously designated yvgQR) genes, expected to be involved in the assimilatory sulfate reduction pathway, was investigated. A B. subtilis mutant with a deletion in the cysJI genes was unable to use sulfate or sulfite as sulfur source, which confirmed that these genes encode sulfite reductase. A mutant with a transposon insertion in the cysK gene, whose deduced protein sequence showed similarity to cysteine synthases, grew poorly on sulfate and butanesulfonate. A strain in which cysK and yrhA, a cysK paralog, were inactivated was unable to grow with sulfate. Whereas expression of the cysJI genes was induced by sulfate, expression of cysK was repressed both by sulfate and by cysteine.