Cytochromes and iron sulfur proteins in sulfur metabolism of phototrophic bacteria

Dissimilatory sulfur metabolism in phototrophic sulfur bacteria provides the bacteria with electrons for photosynthetic electron transport chain and, with energy. Assimilatory sulfate reduction is necessary for the biosynthesis of sulfur-containing cell components. Sulfide, thiosulfate, and elemental sulfur are the sulfur compounds most commonly used by phototrophic bacteria as electron donors for anoxygenic photosynthesis. Cytochromes or other electron transfer proteins, like high-potential-iron-sulfur protein (HIPIP) function as electron acceptors or donors for most enzymatic steps during the oxidation pathways of sulfide or thiosulfate. Yet, heme- or siroheme-containing proteins themselves undergo enzymatic activities in sulfur metabolism. Sirohemes comprise a porphyrin-like prosthetic group of sulfate reductase. eenzymatic reactions involve electron transfer. Electron donors or acceptors are necessary for each reaction. Cytochromes and iron sulfur problems, are able to transfer electrons

β-n-oxalyl-l-α, β -diaminopropionic acid (β -odap) content in lathyrus sativus: The integration of nitrogen and sulfur metabolism through β -cyanoalanine synthase

Grass pea (Lathyrus sativus L.) is an important legume crop grown mainly in South Asia and Sub-Saharan Africa. This underutilized legume can withstand harsh environmental conditions including drought and flooding. During drought-induced famines, this protein-rich legume serves as a food source for poor farmers when other crops fail under harsh environmental conditions; however, its use is limited because of the presence of an endogenous neurotoxic nonprotein amino acid β-N-oxalyl-l-α,β-diaminopropionic acid (β-ODAP). Long-term consumption of Lathyrus and β-ODAP is linked to lathyrism, which is a degenerative motor neuron syndrome. Pharmacological studies indicate that nutritional deficiencies in methionine and cysteine may aggravate the neurotoxicity of β-ODAP. The biosynthetic pathway leading to the production of β-ODAP is poorly understood, but is linked to sulfur metabolism. To date, only a limited number of studies have been conducted in grass pea on the sulfur assimilatory enzymes and how these enzymes regulate the biosynthesis of β-ODAP. Here, we review the current knowledge on the role of sulfur metabolism in grass pea and its contribution to β-ODAP biosynthesis. Unraveling the fundamental steps and regulation of β-ODAP biosynthesis in grass pea will be vital for the development of improved varieties of this underutilized legume

Effects of inhibiting antioxidant pathways on cellular hydrogen sulfide and polysulfide metabolism

Elaborate antioxidant pathways have evolved to minimize the threat of excessive reactive oxygen species (ROS) and to regulate ROS as signaling entities. ROS are chemically and functionally similar to reactive sulfur species (RSS) and both ROS and RSS have been shown to be metabolized by the antioxidant enzymes, superoxide dismutase and catalase. Here we use fluorophores to examine the effects of a variety of inhibitors of antioxidant pathways on metabolism of two important RSS, hydrogen sulfide (H2S with AzMC) and polysulfides (H2Sn, where n = 2–7, with SSP4) in HEK293 cells. Cells were exposed to inhibitors for up to 5 days in normoxia (21% O2) and hypoxia (5% O2), conditions also known to affect ROS production. Decreasing intracellular glutathione (GSH) with l-buthionine-sulfoximine (BSO) or diethyl maleate (DEM) decreased H2S production for 5 days but did not affect H2Sn. The glutathione reductase inhibitor, auranofin, initially decreased H2S and H2Sn but after two days H2Sn increased over controls. Inhibition of peroxiredoxins with conoidin A decreased H2S and increased H2Sn, whereas the glutathione peroxidase inhibitor, tiopronin, increased H2S. Aminoadipic acid, an inhibitor of cystine uptake did not affect either H2S or H2Sn. In buffer, the glutathione reductase and thioredoxin reductase inhibitor, 2-AAPA, the glutathione peroxidase mimetic, ebselen, and tiopronin variously reacted directly with AzMC and SSP4, reacted with H2S and H2S2, or optically interfered with AzMC or SSP4 fluorescence. Collectively these results show that antioxidant inhibitors, generally known for their ability to increase cellular ROS, have various effects on cellular RSS. These findings suggest that the inhibitors may affect cellular sulfur metabolism pathways that are not related to ROS production and in some instances they may directly affect RSS or the methods used to measure them. They also illustrate the importance of carefully evaluating RSS metabolism when biologically or pharmacologically attempting to manipulate ROS

Secondary sulfur metabolism in cellular signalling and oxidative stress responses

The sulfur metabolism pathway in plants produces a variety of compounds that are central to the acclimation response to oxidative stresses such as drought and high light. Primary sulfur assimilation provides the amino acid cysteine, which is utilized in protein synthesis and as a precursor for the cellular redox buffer glutathione. In contrast, the secondary sulfur metabolism pathway produces sulfated compounds such as glucosinolates and sulfated peptides, as well as a corresponding by-product 3'-phosphoadenosine 5'-phosphate (PAP). Emerging evidence over the past decade has shown that secondary sulfur metabolism also has a crucial engagement during oxidative stress. This occurs across various cellular, tissue and organismal levels including chloroplast-to-nucleus retrograde signalling events mediated by PAP, modulation of hormonal signalling by sulfated compounds and PAP, control of physiological responses such as stomatal closure, and potential regulation of plant growth. In this review, we examine the contribution of the different components of plant secondary metabolism to oxidative stress homeostasis, and how this pathway is metabolically regulated. We further outline the key outstanding questions in the field that are necessary to understand how and why this 'specialized' metabolic pathway plays significant roles in plant oxidative stress tolerance

The methionine salvage pathway in Bacillus subtilis

BACKGROUND: Polyamine synthesis produces methylthioadenosine, which has to be disposed of. The cell recycles it into methionine through methylthioribose (MTR). Very little was known about MTR recycling for methionine salvage in Bacillus subtilis. RESULTS: Using in silico genome analysis and transposon mutagenesis in B. subtilis we have experimentally uncovered the major steps of the dioxygen-dependent methionine salvage pathway, which, although similar to that found in Klebsiella pneumoniae, recruited for its implementation some entirely different proteins. The promoters of the genes have been identified by primer extension, and gene expression was analyzed by Northern blotting and lacZ reporter gene expression. Among the most remarkable discoveries in this pathway is the role of an analog of ribulose diphosphate carboxylase (Rubisco, the plant enzyme used in the Calvin cycle which recovers carbon dioxide from the atmosphere) as a major step in MTR recycling. CONCLUSIONS: A complete methionine salvage pathway exists in B. subtilis. This pathway is chemically similar to that in K. pneumoniae, but recruited different proteins to this purpose. In particular, a paralogue or Rubisco, MtnW, is used at one of the steps in the pathway. A major observation is that in the absence of MtnW, MTR becomes extremely toxic to the cell, opening an unexpected target for new antimicrobial drugs. In addition to methionine salvage, this pathway protects B. subtilis against dioxygen produced by its natural biotope, the surface of leaves (phylloplane)

Global regulation of gene expression in response to cysteine availability in Clostridium perfringens

<p>Abstract</p> <p>Background</p> <p>Cysteine has a crucial role in cellular physiology and its synthesis is tightly controlled due to its reactivity. However, little is known about the sulfur metabolism and its regulation in clostridia compared with other firmicutes. In <it>Clostridium perfringens</it>, the two-component system, VirR/VirS, controls the expression of the <it>ubiG </it>operon involved in methionine to cysteine conversion in addition to the expression of several toxin genes. The existence of links between the <it>C. perfringens </it>virulence regulon and sulfur metabolism prompted us to analyze this metabolism in more detail.</p> <p>Results</p> <p>We first performed a tentative reconstruction of sulfur metabolism in <it>C. perfringens </it>and correlated these data with the growth of strain 13 in the presence of various sulfur sources. Surprisingly, <it>C. perfringens </it>can convert cysteine to methionine by an atypical still uncharacterized pathway. We further compared the expression profiles of strain 13 after growth in the presence of cystine or homocysteine that corresponds to conditions of cysteine depletion. Among the 177 genes differentially expressed, we found genes involved in sulfur metabolism and controlled by premature termination of transcription via a cysteine specific T-box system (<it>cysK</it>-<it>cysE</it>, <it>cysP1 </it>and <it>cysP2</it>) or an S-box riboswitch (<it>metK </it>and <it>metT</it>). We also showed that the <it>ubiG </it>operon was submitted to a triple regulation by cysteine availability via a T-box system, by the VirR/VirS system via the VR-RNA and by the VirX regulatory RNA.</p> <p>In addition, we found that expression of <it>pfoA </it>(theta-toxin), <it>nagL </it>(one of the five genes encoding hyaluronidases) and genes involved in the maintenance of cell redox status was differentially expressed in response to cysteine availability. Finally, we showed that the expression of genes involved in [Fe-S] clusters biogenesis and of the <it>ldh </it>gene encoding the lactate dehydrogenase was induced during cysteine limitation.</p> <p>Conclusion</p> <p>Several key functions for the cellular physiology of this anaerobic bacterium were controlled in response to cysteine availability. While most of the genes involved in sulfur metabolism are regulated by premature termination of transcription, other still uncharacterized mechanisms of regulation participated in the induction of gene expression during cysteine starvation.</p

Isolated sulfite oxidase deficiency: a founder mutation.

Isolated sulfite oxidase deficiency is a rare autosomal recessive inborn error of sulfur metabolism. Clinical features generally include devastating neurologic dysfunction, ectopia lentis, and increased urinary excretion of sulfite, thiosulfate, an

Assimilatory sulfur metabolism in marine microorganisms

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution December 1980The reductive assimilation of sulfate into cellular organic sulfur compounds was studied in aerobic marine bacteria, with emphasis on the relationship between sulfur metabolism and protein synthesis. The goal of the study was to develop and apply a method for the quantitative assay of total bacterial protein synthesis in aerobic ocean waters. The study consisted of four parts: (1) The sulfate transport systems of two nutritionally different marine bacteria, Pseudomonas halodurans and Alteromonas luteo-violaceus, were characterized to provide information on environmental regulation of sulfate transport capacity. In common with terrestrial bacteria, the transport systems of both marine bacteria exhibit (a) size-selective competitive inhibition of sulfate uptake by sulfate analogs, (b) requirements for energy coupling,and (c) derepression of transport capacity as a result of sulfur starvation. Features which are unique to the marine bacteria include (a) a ten-fold lower affinity for sulfate (half-saturation constant ~200 μm), (b) derepression of transport capacity when grown with methionine as the sole source of sulfur, and (c) an inability to accumulate inorganic sulfate in excess of growth requirements. The different characteristics of the sulfate transport systems of the marine bacteria relative to terrestrial microorganisms are consistent with the saturating concentration of sulfate that is always present in their environment. Substantial differences also exist between the two marine bacteria, notably in the effect of thiosulfate on sulfate uptake. P. halodurans transports thiosulfate with a ten-fold higher affinity than sulfate. Sulfate and thiosulfate are mutually competitive inhibitors of transport, and the half-saturating concentration of thiosulfate for uptake also produces half-maximal inhibition of sulfate transport. Sulfate and thiosulfate transport systems both respond similarly to all inibitors. These facts implicate a common carrier for the two compounds. In contrast, sulfate transport in A. luteo-violaceus is relatively insensitive to thiosulfate. The effect of the suIfhydryl reagent pHMB is similarly much less pronounced than in P. halodurans. These and other differences indicate that the sulfate transport system of A. luteo-violaceus is unique among microorganisms. (2) Growth experiments with P. halodurans and A. luteo-violaceus were carried out over a range of nutritional regimes. Biomass parameters (cell counts, bulk protein, particulate carbon and nitrogen), total uptake of radioactive sulfate, and the distribution of sulfur in major biochemical components (low molecular weight [L.M.W.], alcohol soluble protein, lipid, hot TCA soluble material, and residue protein) were monitored to determine the variability in cellular composition as a function of the environment. Special emphasis was placed on the quantitative relationship between incorporation of sulfur into protein and bulk protein synthesis and conditions which might alter the sulfur content of protein. It was found that sulfur metabolism is restricted predominantly to the production and utilization of protein precursors. The protein synthesis inhibitor chloramphenicol caused an immediate halt to both bulk protein synthesis and sulfur incorporation into protein, accompanied by a rapid swelling of L.M.W. organic sulfur pools, in both bacteria. Incorporation of exogenous sulfur into protein was rapid due to the very small size of the L.M.W. pool. No significant deviation from the ratio of protein-S:bulk protein determined for unperturbed exponential growth was observed as a function of carbon limitation, nitrogen limitation, treatment with chloramphenicol, or during lag and stationary phases. However, the concentration of sulfate in the growth medium exerted a strong influence on the sulfur content of both whole cells and isolated protein. At concentrations less than 500 μM (P. halodurans) or 100 μM (A. luteo-violaceus) the weight % S in protein was proportional to the silate concentration in the medium. Since the sulfate concentration is invariably high in seawater (25mM), data from sulfur-limited growth were not included in the analysis of compositional variability. Under all the conditions examined, the incorporation of sulfur into protein provided the best measurement of protein synthesis and cell growth, with a very low coefficient of variation for the protein-S:bulk protein ratio (less than 16%). The mean true weight % S in protein, 1.07 (P. halodurans) and 0.92 (A. luteoviolaceus) agrees well with the 1.1% predicted from analyses of sequenced proteins. (3) The method used for the analysis of sulfur incorporation into protein was tested with mixed natural populations of marine bacteria in enrichment culture and 13 isolates from the Sargasso Sea to establish the variability of the protein-S:bulk protein ratio among marine bacteria. The mean true weight % S in protein, 1.09, and the operational weight % S in protein, 0.93, have coefficients of variation of 13.1 and 15.1%, respectively. The values are similar to those obtained with the two marine bacteria studied in detail and to that predicted from protein composition studies. Therefore sulfur incorporation into protein measures protein synthesis in marine bacteria within a small degree of error. (4) The method was applied to unenriched natural populations of marine bacteria in waters of the continental shelf, slope, and Sargasso Sea. Time-course incorporation measurements revealed along lag period at the shelf and slope stations, whereas incorporation of sulfur into protein began immediately in the Sargasso Sea. However, long term incubations confirmed that the potential for bacterial protein synthesis decreases in an off-shore transect. These observations were confirmed by simultaneous incorporation studies using labeled ammonia, phosphate, and organic carbon compounds. The potential protein synthesis measured in the unenriched samples provides evidence suggesting that bacterial biomass may be an important contributor to marine food webs.Gracious financial support provided by the National Science Foundation (grants OCE77-12172, OCE79-19178, and OCE79-19264) and the Education Department of W.H.O.I. are appreciate

Diversity of sulfur metabolism in microalgae

openLo zolfo è un macronutriente fondamentale, ma spesso sottovalutato sostanzialmente meno informazioni su di esso in letteratura rispetto ad altri macronutrienti. Tra gli organismi fotosintetici, il metabolismo dello zolfo è generalmente si presume di essere conservato e la maggior parte delle informazioni disponibili è limitata alle pianti vascolari. L'indagine sulle alghe è limitata a pochissimi cladi, nonostante il importanza del solfato - la forma più disponibile di zolfo in natura - nell'acqua di mare. Infatti, ci sono prove che suggeriscono che la concentrazione di solfato nell'acqua di mare sia una delle maggiori importanti fattori determinanti della composizione del fitoplancton e potrebbero essere i responsabili di oggi predominanza delle microalghe clorofilla a + c nel fitoplancton - la facilitazione del solfato Ipotesi (SFH). Di fronte a questo scenario, questa tesi intende aumentare la comprensione di il ruolo dello zolfo nel fitoplancton implementando un approccio multidisciplinare coinvolgendo vari gruppi di microalghe. In primo luogo, un'ampia analisi in silico del cellulare localizzazione e relazioni filogenetiche dei sei enzimi fondamentali dell'assimilazione dei solfati mostra un'origine piuttosto complessa del percorso. I dati suggeriscono anche un ruolo rilevante di redox regolazione, che sembra funzionare in modo diverso tra i diversi gruppi di fototrofi. Quindi, la funzione, la localizzazione e la regolazione redox in vivo di uno di questi enzimi - con una filogenesi particolarmente complessa, denominata ATPS - è stata ulteriormente indagata nel modello di diatomee Phaeodactylum tricornutum utilizzando nuove tecniche di biologia molecolare. L'ATPS sembra essere cruciale per la vita ed è presente in P. tricornutum come due non ridondanti isoforme che si trovano in diversi compartimenti e possono funzionare nel partizionamento dello zolfo. Il recupero di mutanti knockout ATPS mediante complementazione con mezzo di crescita Vengono inizialmente studiate fonti alternative di zolfo. Infine, utilizzando un fisiologico e approccio biochimico, la crescita di microalghe marine appartenenti a vari gruppi è stata valutato in funzione della concentrazione di solfato nel mezzo di crescita. La concentrazione di il solfato che si traduce in limitazione della crescita è sorprendentemente diverso tra le microalghe. Infatti, le microalghe clorofilla a + c possono essere limitate in concentrazioni di solfato fino a 10000 volte superiore alle altre microalghe, che è altamente congruente con l'SFH. È interessante notare che le loro risposte alla limitazione dei solfati erano anche diverse all'interno delle alghe con plastide rosse, soprattutto nella quota di ferro nelle cellule.Sulfur is a fundamental, yet frequently underestimated, macronutrient resulting on substantially less information about it in literature when compared to other macronutrients. Among photosynthetic organisms, sulfur metabolism is generally assumed to be conserved and most of the available information is limited to vascular plants. The investigation among algae is constrained to very few clades, despite the importance of sulfate – the most available form of sulfur in nature – in seawater. In fact, there is evidence to suggest that sulfate concentration in seawater is one of the most important drivers of phytoplankton composition and may be the responsible for today’s dominance of chlorophyll a + c microalgae in the phytoplankton – the Sulfate Facilitation Hypothesis (SFH). Facing this scenario, this thesis intends to raise the understanding of the role of sulfur in phytoplankton by implementing a multidisciplinary approach involving various groups of microalgae. First, a broad in silico analysis of the cellular localization and phylogenetic relationships of the six core enzymes of sulfate assimilation shows a rather complex origin of the pathway. Data also suggests a relevant role of redox regulation, that appears to function differently among different groups of phototrophs. Then, the function, localization and the in vivo redox regulation of one of these enzymes – with a particularly complex phylogeny, named ATPS – was further investigated in the model diatom Phaeodactylum tricornutum using novel molecular biology techniques. ATPS seems to be crucial for life and is present in P. tricornutum as two non-redundant isoforms that are in different compartments and may function in sulfur partitioning. The recovery of ATPS-knockout mutants by growth medium complementation with alternative sources of sulfur is initially investigated. Finally, using a physiological and biochemical approach, the growth of marine microalgae belonging to various groups was assessed as a function of sulfate concentration in growth medium. The concentration of sulfate that results in growth limitation is strikingly different among microalgae. In fact, chlorophyll a + c microalgae can be limited in sulfate concentrations up to 10000-fold higher than the other microalgae, which is highly congruent with the SFH. Interestingly, their responses to sulfate limitation were also different within the red-plastid algae, especially in the iron quota in cells.BIOLOGIA ED ECOLOGIA MARINAopenPOUSA KURPAN NOGUEIRA, Danie