Allochromatium vinosum DsrC: solution-state NMR structure, redox properties, and interaction with DsrEFH, a protein essential for purple sulfur bacterial sulfur oxidation

Sequenced genomes of dissimilatory sulfur-oxidizing and sulfate-reducing bacteria containing genes coding for DsrAB, the enzyme dissimilatory sulfite reductase, inevitably also contain the gene coding for the 12-kDa DsrC protein. DsrC is thought to have a yet unidentified role associated with the activity of DsrAB. Here we report the solution structure of DsrC from the sulfur-oxidizing purple sulfur bacterium Allochromatium vinosum determined with NMR spectroscopy in reducing conditions, and we describe the redox behavior of two conserved cysteine residues upon transfer to an oxidizing environment. In reducing conditions, the DsrC structure is disordered in the highly conserved carboxy-terminus. We present multiple lines of evidence that, in oxidizing conditions, a strictly conserved cysteine (Cys111) at the penultimate position in the sequence forms an intramolecular disulfide bond with Cys100, which is conserved in DsrC in all organisms with DsrAB. While an intermolecular Cys111-Cys111 disulfide-bonded dimer is rapidly formed under oxidizing conditions, the intramolecularly disulfide-bonded species (Cys100-Cys111) is the thermodynamically stable form of the protein under these conditions. Treatment of the disulfidic forms with reducing agent regenerates the monomeric species that was structurally characterized. Using a band-shift technique under nondenaturing conditions, we obtained evidence for the interaction of DsrC with heterohexameric DsrEFH, a protein encoded in the same operon. Mutation of Cys100 to serine prevented formation of the DsrC species assigned as an intramolecular disulfide in oxidizing conditions, while still allowing formation of the intermolecular Cys111-Cys111 dimer. In the reduced form, this mutant protein still interacted with DsrEFH. This was not the case for the Cys111Ser and Cys100Ser/Cys111Ser mutants, both of which also did not form protein dimers. Our observations highlight the central importance of the carboxy-terminal DsrC cysteine residues and are consistent with a role as a sulfur-substrate binding/transferring protein, as well as with an electron-transfer function via thiol-disulfide interchanges.     

DsrC is involved in fermentative growth and interacts directly with the FlxABCD–HdrABC complex in Desulfovibrio vulgaris Hildenborough

DsrC is a key protein in dissimilatory sulfur metabolism, where it works as co-substrate of the dissimilatory sulfite reductase DsrAB. DsrC has two conserved cysteines in a C-terminal arm that are converted to a trisulfide upon reduction of sulfite. In sulfate-reducing bacteria, DsrC is essential and previous works suggested additional functions beyond sulfite reduction. Here, we studied whether DsrC also plays a role during fermentative growth of Desulfovibrio vulgaris Hildenborough, by studying two strains where the functionality of DsrC is impaired by a lower level of expression (IPFG07) and additionally by the absence of one conserved Cys (IPFG09). Growth studies coupled with metabolite and proteomic analyses reveal that fermentation leads to lower levels of DsrC, but impairment of its function results in reduced growth by fermentation and a shift towards more fermentative metabolism during sulfate respiration. In both respiratory and fermentative conditions, there is increased abundance of the FlxABCD–HdrABC complex and Adh alcohol dehydrogenase in IPFG09 versus the wild type, which is reflected in higher production of ethanol. Pull-down experiments confirmed a direct interaction between DsrC and the FlxABCD–HdrABC complex, through the HdrB subunit. Dissimilatory sulfur metabolism, where sulfur compounds are used for energy generation, is a key process in the ecology of anoxic environments, and is more widespread among bacteria than previously believed. Two central proteins for this type of metabolism are DsrAB dissimilatory sulfite reductase and its co-substrate DsrC. Using physiological, proteomic and biochemical studies of Desulfovibrio vulgaris Hildenborough and mutants affected in DsrC functionality, we show that DsrC is also relevant for fermentative growth of this model organism and that it interacts directly with the soluble FlxABCD-HdrABC complex that links the NAD(H) pool with dissimilatory sulfite reduction.     

The crystal structure of Desulfovibrio vulgaris dissimilatory sulfite reductase bound to DsrC provides novel insights into the mechanism of sulfate respiration

Sulfate reduction is one of the earliest types of energy metabolism used by ancestral organisms to sustain life. Despite extensive studies, many questions remain about the way respiratory sulfate reduction is associated with energy conservation. A crucial enzyme in this process is the dissimilatory sulfite reductase (dSiR), which contains a unique siroheme-[4Fe4S] coupled cofactor. Here, we report the structure of desulfoviridin from Desulfovibrio vulgaris, in which the dSiR DsrAB (sulfite reductase) subunits are bound to the DsrC protein. The alpha(2)beta(2)gamma(2) assembly contains two siroheme-[4Fe4S] cofactors bound by DsrB, two sirohydrochlorins and two [4Fe4S] centers bound by DsrA, and another four [4Fe4S] centers in the ferredoxin domains. A sulfite molecule, coordinating the siroheme, is found at the active site. The DsrC protein is bound in a cleft between DsrA and DsrB with its conserved C-terminal cysteine reaching the distal side of the siroheme. We propose a novel mechanism for the process of sulfite reduction involving DsrAB, DsrC, and the DsrMKJOP membrane complex (a membrane complex with putative disulfide/thiol reductase activity), in which two of the six electrons for reduction of sulfite derive from the membrane quinone pool. These results show that DsrC is involved in sulfite reduction, which changes the mechanism of sulfate respiration. This has important implications for models used to date ancient sulfur metabolism based on sulfur isotope fractionations.     

Cytoplasmic sulfurtransferases in the purple sulfur bacterium Allochromatium vinosum: evidence for sulfur transfer from DsrEFH to DsrC

While the importance of sulfur transfer reactions is well established for a number of biosynthetic pathways, evidence has only started to emerge that sulfurtransferases may also be major players in sulfur-based microbial energy metabolism. Among the first organisms studied in this regard is the phototrophic purple sulfur bacterium Allochromatium vinosum. During the oxidation of reduced sulfur species to sulfate this Gammaproteobacterium accumulates sulfur globules. Low molecular weight organic persulfides have been proposed as carrier molecules transferring sulfur from the periplasmic sulfur globules into the cytoplasm where it is further oxidized via the "Dsr" (dissimilatory sulfite reductase) proteins. We have suggested earlier that the heterohexameric protein DsrEFH is the direct or indirect acceptor for persulfidic sulfur imported into the cytoplasm. This proposal originated from the structural similarity of DsrEFH with the established sulfurtransferase TusBCD from E. coli. As part of a system for tRNA modification TusBCD transfers sulfur to TusE, a homolog of another crucial component of the A. vinosum Dsr system, namely DsrC. Here we show that neither DsrEFH nor DsrC have the ability to mobilize sulfane sulfur directly from low molecular weight thiols like thiosulfate or glutathione persulfide. However, we demonstrate that DsrEFH binds sulfur specifically to the conserved cysteine residue DsrE-Cys78 in vitro. Sulfur atoms bound to cysteines in DsrH and DsrF were not detected. DsrC was exclusively persulfurated at DsrC-Cys111 in the penultimate position of the protein. Most importantly, we show that persulfurated DsrEFH indeed serves as an effective sulfur donor for DsrC in vitro. The active site cysteines Cys78 of DsrE and Cys20 of DsrH furthermore proved to be essential for sulfur oxidation in vivo supporting the notion that DsrEFH and DsrC are part of a sulfur relay system that transfers sulfur from a persulfurated carrier molecule to the dissimilatory sulfite reductase DsrAB.     

Purification, crystallization and preliminary crystallographic analysis of a dissimilatory DsrAB sulfite reductase in complex with DsrC

Dissimilatory sulfite reductase (dSiR, DsrAB) is a key protein in dissimilatory sulfur metabolism, one of the earliest types of energy metabolism to be traced on earth. dSirs are large oligomeric proteins around 200kDa forming an alpha(2)beta(2) arrangement and including a unique siroheme-[4Fe-4S] coupled cofactor. Here, we report the purification, crystallization and preliminary X-ray diffraction analysis of dSir isolated from Desulfovibrio vulgaris Hildenborough, also known as desulfoviridin. In this enzyme the DsrAB protein is associated with DsrC, a protein of unknown function that is believed to play an important role in the sulfite reduction. Crystals belong to the monoclinic space group P2(1) with unit-cell parameters a=122.7, b=119.4 and c=146.7A and beta =110.0 degrees , and diffract X-rays to 2.8A on a synchrotron source.     

Structural and molecular genetic insight into a widespread sulfur oxidation pathway

Many environmentally important photo- and chemolithoautotrophic bacteria accumulate globules of polymeric, water-insoluble sulfur as a transient product during oxidation of reduced sulfur compounds. Oxidation of this sulfur requires the concerted action of Dsr proteins. However, individual functions and interplay of these proteins are largely unclear. We proved with a ΔdsrE mutant experiment that the cytoplasmic α₂β₂γ₂-structured protein DsrEFH is absolutely essential for the oxidation of sulfur stored in the intracellular sulfur globules of the purple sulfur bacterial model organism Allochromatium vinosum. The ability to degrade stored sulfur was fully regained upon complementation with dsrEFH in trans. The crystal structure of DsrEFH was determined at 2.5 Å resolution to assist functional assignment in detail. In conjunction with phylogenetic analyses, two different types of putative active sites were identified in DsrE and DsrH and shown to be characteristic for sulfur-oxidizing bacteria. Conserved Cys78 of A. vinosum DsrE corresponds to the active cysteines of Escherichia coli YchN and TusD. TusBCD and the protein TusE are parts of sulfur relay system involved in thiouridine biosynthesis. DsrEFH interacts with DsrC, a TusE homologue encoded in the same operon. The conserved penultimate cysteine residue in the carboxy-terminus of DsrC is essential for the interaction. Here, we show that Cys78 of DsrE is strictly required for interaction with DsrC while Cys20 in the putative active site of DsrH is dispensable for that reaction. In summary, our findings point at the occurrence of sulfur transfer reactions during sulfur oxidation via the Dsr proteins.     

Insight into the molecular mechanism of the sulfur oxidation process by reverse sulfite reductase (rSiR) from sulfur oxidizer <Emphasis Type="Italic">Allochromatium vinosum</Emphasis>

Sulfur metabolism is one of the oldest known biochemical processes. Chemotrophic or phototrophic proteobacteria, through the dissimilatory pathway, use sulfate, sulfide, sulfite, thiosulfate or elementary sulfur by either reductive or oxidative mechanisms. During anoxygenic photosynthesis, anaerobic sulfur oxidizer Allochromatium vinosum forms sulfur globules that are further oxidized by dsr operon. One of the key redox enzymes in reductive or oxidative sulfur metabolic pathways is the DsrAB protein complex. However, there are practically no reports to elucidate the molecular mechanism of the sulfur oxidation process by the DsrAB protein complex from sulfur oxidizer Allochromatium vinosum. In the present context, we tried to analyze the structural details of the DsrAB protein complex from sulfur oxidizer Allochromatium vinosum by molecular dynamics simulations. The molecular dynamics simulation results revealed the various types of molecular interactions between DsrA and DsrB proteins during the formation of DsrAB protein complex. We, for the first time, predicted the mode of binding interactions between the co-factor and DsrAB protein complex from Allochromatium vinosum. We also compared the binding interfaces of DsrAB from sulfur oxidizer Allochromatium vinosum and sulfate reducer Desulfovibrio vulgaris. This study is the first to provide a comparative aspect of binding modes of sulfur oxidizer Allochromatium vinosum and sulfate reducer Desulfovibrio vulgaris.     

Phylogenetic and environmental diversity of DsrAB-type dissimilatory (bi)sulfite reductases

The energy metabolism of essential microbial guilds in the biogeochemical sulfur cycle is based on a DsrAB-type dissimilatory (bi)sulfite reductase that either catalyzes the reduction of sulfite to sulfide during anaerobic respiration of sulfate, sulfite and organosulfonates, or acts in reverse during sulfur oxidation. Common use of dsrAB as a functional marker showed that dsrAB richness in many environments is dominated by novel sequence variants and collectively represents an extensive, largely uncharted sequence assemblage. Here, we established a comprehensive, manually curated dsrAB/DsrAB database and used it to categorize the known dsrAB diversity, reanalyze the evolutionary history of dsrAB and evaluate the coverage of published dsrAB-targeted primers. Based on a DsrAB consensus phylogeny, we introduce an operational classification system for environmental dsrAB sequences that integrates established taxonomic groups with operational taxonomic units (OTUs) at multiple phylogenetic levels, ranging from DsrAB enzyme families that reflect reductive or oxidative DsrAB types of bacterial or archaeal origin, superclusters, uncultured family-level lineages to species-level OTUs. Environmental dsrAB sequences constituted at least 13 stable family-level lineages without any cultivated representatives, suggesting that major taxa of sulfite/sulfate-reducing microorganisms have not yet been identified. Three of these uncultured lineages occur mainly in marine environments, while specific habitat preferences are not evident for members of the other 10 uncultured lineages. In summary, our publically available dsrAB/DsrAB database, the phylogenetic framework, the multilevel classification system and a set of recommended primers provide a necessary foundation for large-scale dsrAB ecology studies with next-generation sequencing methods.     

X-ray structure of the gamma-subunit of a dissimilatory sulfite reductase: fixed and flexible C-terminal arms

The X-ray structure of the gamma-subunit of the dissimilatory sulfite reductase (DsrC) from Archaeoglobus fulgidus was determined at 1.12 and 2.1A resolution, in the two crystal forms named DsrC(nat) and DsrC(ox) the latter being cocrystallized with the oxidizing agent tert-butyl hydroperoxide. The fold corresponds to that of the homologous protein from Pyrobaculum aerophilum but is significantly more compact. The most interesting, highly conserved C-terminal arm adopts a well-defined conformation in A. fulgidus DsrC in contrast to the completely disordered conformation in P. aerophilum DsrC. The functional relevance of both conformations and of a potentially redox-active disulfide bond between the strictly invariant Cys103 and Cys114 are discussed.     

DsrL mediates electron transfer between NADH and rDsrAB in Allochromatium vinosum

Dissimilatory sulphite reductase DsrAB occurs in sulphate/sulphite-reducing prokaryotes, in sulphur disproportionators and also in sulphur oxidizers, where it functions in reverse. Predictions of physiological traits in metagenomic studies relying on the presence of dsrAB, other dsr genes or combinations thereof suffer from the lack of information on crucial Dsr proteins. The iron–sulphur flavoprotein DsrL is an example of this group. It has a documented essential function during sulphur oxidation and was recently also found in some metagenomes of probable sulphate and sulphite reducers. Here, we show that DsrL and reverse acting rDsrAB can form a complex and are copurified from the phototrophic sulphur oxidizer Allochromatium vinosum. Recombinant DsrL exhibits NAD(P)H:acceptor oxidoreductase activity with a strong preference for NADH over NADPH. In vitro, the rDsrABL complex effectively catalyses NADH-dependent sulphite reduction, which is strongly enhanced by the sulphur-binding protein DsrC. Our work reveals NAD⁺ as suitable in vivo electron acceptor for sulphur oxidation in organisms operating the rDsr pathway and points to reduced nicotinamide adenine dinucleotides as electron donors for sulphite reduction in sulphate/sulphite-reducing prokaryotes that contain DsrL. In addition, dsrL cannot be used as a marker distinguishing sulphate/sulphite reducers and sulphur oxidizers in metagenomic studies without further analysis.     

Redox and nitric oxide homeostasis are affected in tomato (Solanum lycopersicum) roots under salinity-induced oxidative stress

The nicotinamide adenine dinucleotide phosphate (NADPH) and reduced glutathione (GSH) molecules play important roles in the redox homeostasis of plant cells. Using tomato (Solanum lycopersicum) plants grown with 120 mM NaCl, we studied the redox state of NADPH and GSH as well as ascorbate, nitric oxide (NO) and S-nitrosoglutathione (GSNO) content and the activity of the principal enzymes involved in the metabolism of these molecules in roots. Salinity caused a significant reduction in growth parameters and an increase in oxidative parameters such as lipid peroxidation and protein oxidation. Salinity also led to an overall decrease in the content of these redox molecules and in the enzymatic activities of the main NADPH-generating dehydrogenases, S-nitrosoglutathione reductase and catalase. However, NO content as well as gluthahione reductase and glutathione peroxidase activity increased under salinity stress. These findings indicate that salinity drastically affects redox and NO homeostasis in tomato roots. In our view, these molecules, which show the interaction between ROS and RNS metabolisms, could be excellent parameters for evaluating the physiological conditions of plants under adverse stress conditions.     

Assessing the regulation of leaf redox status under water stress conditions in Arabidopsis thaliana: Col-0 ecotype (wild-type and vtc-2), expressing mitochondrial and cytosolic roGFP1

Using Arabidopsis plants Col-0 and vtc2 transformed with a redox sensitive green fluorescent protein, (c-roGFP) and (m-roGFP), we investigated the effects of a progressive water stress and re-watering on the redox status of the cytosol and the mitochondria. Our results establish that water stress affects redox status differently in these two compartments, depending on phenotype and leaf age, furthermore we conclude that ascorbate plays a pivotal role in mediating redox status homeostasis and that Col-0 Arabidopsis subjected to water stress increase the synthesis of ascorbate suggesting that ascorbate may play a role in buffering changes in redox status in the mitochondria and the cytosol, with the presumed buffering capacity of ascorbate being more noticeable in young compared with mature leaves. Re-watering of water-stressed plants was paralleled by a return of both the redox status and ascorbate to the levels of well-watered plants. In contrast to the effects of water stress on ascorbate levels, there were no significant changes in the levels of glutathione, thereby suggesting that the regeneration and increase in ascorbate in water-stressed plants may occur by other processes in addition to the regeneration of ascorbate via the glutathione. Under water stress in vtc2 lines it was observed stronger differences in redox status in relation to leaf age, than due to water stress conditions compared with Col-0 plants. In the vtc2 an increase in DHA was observed in water-stressed plants. Furthermore, this work confirms the accuracy and sensitivity of the roGFP1 biosensor as a reporter for variations in water stress-associated changes in redox potentials.     

Comparative proteome analysis of propionate degradation by Syntrophobacter fumaroxidans in pure culture and in coculture with methanogens

Syntrophobacter fumaroxidans is a sulfate‐reducing bacterium able to grow on propionate axenically or in syntrophic interaction with methanogens or other sulfate‐reducing bacteria. We performed a proteome analysis of S. fumaroxidans growing with propionate axenically with sulfate or fumarate, and in syntrophy with Methanospirillum hungatei, Methanobacterium formicicum or Desulfovibrio desulfuricans. Special attention was put on the role of hydrogen and formate in interspecies electron transfer (IET) and energy conservation. Formate dehydrogenase Fdh1 and hydrogenase Hox were the main confurcating enzymes used for energy conservation. In the periplasm, Fdh2 and hydrogenase Hyn play an important role in reverse electron transport associated with succinate oxidation. Periplasmic Fdh3 and Fdh5 were involved in IET. The sulfate reduction pathway was poorly regulated and many enzymes associated with sulfate reduction (Sat, HppA, AprAB, DsrAB and DsrC) were abundant even at conditions where sulfate was not present. Proteins similar to heterodisulfide reductases (Hdr) were abundant. Hdr/Flox was detected in all conditions while HdrABC/HdrL was exclusively detected when sulfate was available; these complexes most likely confurcate electrons. Our results suggest that S. fumaroxidans mainly used formate for electron release and that different confurcating mechanisms were used in its sulfidogenic metabolism.     

Inactivation of nitric oxide by cytochrome c oxidase under steady-state oxygen conditions

We have developed a respiration chamber that allows intact cells to be studied under controlled oxygen (O₂) conditions. The system measures the concentrations of O₂ and nitric oxide (NO) in the cell suspension, while the redox state of cytochrome c oxidase is continuously monitored optically. Using human embryonic kidney cells transfected with a tetracycline-inducible NO synthase we show that the inactivation of NO by cytochrome c oxidase is dependent on both O₂ concentration and electron turnover of the enzyme. At a high O₂ concentration (70 μM), and while the enzyme is in turnover, NO generated by the NO synthase upon addition of a given concentration of l-arginine is partially inactivated by cytochrome c oxidase and does not affect the redox state of the enzyme or consumption of O₂. At low O₂ (15 μM), when the cytochrome c oxidase is more reduced, inactivation of NO is decreased. In addition, the NO that is not inactivated inhibits the cytochrome c oxidase, further reducing the enzyme and lowering O₂ consumption. At both high and low O₂ concentrations the inactivation of NO is decreased when sodium azide is used to inhibit cytochrome c oxidase and decrease electron turnover.     

Redox tuning of the catalytic activity of soluble fumarate reductases from Shewanella

Many enzymes involved in bioenergetic processes contain chains of redox centers that link the protein surface, where interaction with electron donors or acceptors occurs, to a secluded catalytic site. In numerous cases these redox centers can transfer only single electrons even when they are associated to catalytic sites that perform two-electron chemistry. These chains provide no obvious contribution to enhance chemiosmotic energy conservation, and often have more redox centers than those necessary to hold sufficient electrons to sustain one catalytic turnover of the enzyme. To investigate the role of such a redox chain we analyzed the transient kinetics of fumarate reduction by two flavocytochromes c₃ of Shewanella species while these enzymes were being reduced by sodium dithionite. These soluble monomeric proteins contain a chain of four hemes that interact with a flavin adenine dinucleotide (FAD) catalytic center that performs the obligatory two electron–two proton reduction of fumarate to succinate. Our results enabled us to parse the kinetic contribution of each heme towards electron uptake and conduction to the catalytic center, and to determine that the rate of fumarate reduction is modulated by the redox stage of the enzyme, which is defined by the number of reduced centers. In both enzymes the catalytically most competent redox stages are those least prevalent in a quasi-stationary condition of turnover. Furthermore, the electron distribution among the redox centers during turnover suggested how these enzymes can play a role in the switch between respiration of solid and soluble terminal electron acceptors in the anaerobic bioenergetic metabolism of Shewanella.     

Monitoring cellular redox dynamics using newly developed BRET-based redox sensor proteins

Reactive oxygen species are key factors that strongly affect the cellular redox state and regulate various physiological and cellular phenomena. To monitor changes in the redox state, we previously developed fluorescent redox sensors named Re-Q, the emissions of which are quenched under reduced conditions. However, such fluorescent probes are unsuitable for use in the cells of photosynthetic organisms because they require photoexcitation that may change intracellular conditions and induce autofluorescence, primarily in chlorophylls. In addition, the presence of various chromophore pigments may interfere with fluorescence-based measurements because of their strong absorbance. To overcome these problems, we adopted the bioluminescence resonance energy transfer (BRET) mechanism for the sensor and developed two BRET-based redox sensors by fusing cyan fluorescent protein–based or yellow fluorescent protein–based Re-Q with the luminescent protein Nluc. We named the resulting redox-sensitive BRET-based indicator probes “ROBINc” and “ROBINy.” ROBINc is pH insensitive, which is especially vital for observation in photosynthetic organisms. By using these sensors, we successfully observed dynamic redox changes caused by an anticancer agent in HeLa cells and light/dark-dependent redox changes in the cells of photosynthetic cyanobacterium Synechocystis sp. PCC 6803. Since the newly developed sensors do not require excitation light, they should be especially useful for visualizing intracellular phenomena caused by redox changes in cells containing colored pigments.     

Copper binding traps the folded state of the SCO protein from Bacillus subtilis

The SCO protein from the aerobic bacterium Bacillus subtilis (BsSCO) is involved in the assembly of the cytochrome c oxidase complex, and specifically with the Cu_A center. BsSCO has been proposed to play various roles in Cu_A assembly including, the direct delivery of copper ions to the Cu_A site, and/or maintaining the appropriate redox state of the cysteine ligands during formation of Cu_A. BsSCO binds copper in both Cu(II) and Cu(I) redox states, but has a million-fold higher affinity for Cu(II). As a prerequisite to kinetic studies, we measured equilibrium stability of oxidized, reduced and Cu(II)-bound BsSCO by chemical and thermal induced denaturation. Oxidized and reduced apo-BsSCO exhibit two-state behavior in both chemical- and thermal-induced unfolding. However, the Cu(II) complex of BsSCO is stable in up to nine molar urea. Thermal or guanidinium-induced unfolding of BsSCO-Cu(II) ensues only as the Cu(II) species is lost. The effect of copper (II) on the folding of BsSCO is complicated by a rapid redox reaction between copper and reduced, denatured BsSCO. When denatured apo-BsSCO is refolded in the presence of copper (II) some of the population is recovered as the BsSCO-Cu(II) complex and some is oxidized indicating that refolding and oxidation are competing processes. The proposed functional roles for BsSCO in vivo require that its cysteine residues are reduced and the presence of copper during folding may be detrimental to BsSCO attaining its functional state.     

Oxidative state in idiophase links reactive oxygen species (ROS) and lovastatin biosynthesis: Differences and similarities in submerged- and solid-state fermentations

The present work was focused on finding a relationship between reactive oxygen species (ROS) and lovastatin biosynthesis (secondary metabolism) in Aspergillus terreus. In addition, an effort was made to find differences in accumulation and control of ROS in submerged (SmF) and solid-state fermentation (SSF), which could help explain higher metabolite production in the latter. sod1 expression, ROS content, and redox balance kinetics were measured during SmF and SSF. Results showed that A. terreus sod1 gene (oxidative stress defence enzyme) was intensely expressed during rapid growth phase (trophophase) of lovastatin fermentations. This high expression decreased abruptly, just before the onset of production (idiophase). However, ROS measurements detected high concentrations only in idiophase, suggesting a link between ROS and lovastatin biosynthesis. Apparently sod1 down regulation promotes the rise of ROS during idiophase. This oxidative state in idiophase was further supported by a high redox balance observed in trophophase that changed to a low value in idiophase (around six-fold lower). The patterns of ROS accumulation, sod1 expression, and redox balance behaviour were similar in SmF and SSF. However, sod1 expression and ROS concentration (ten-fold), were higher in SmF. Our results indicate a link between ROS and lovastatin biosynthesis. Also, showed differences of physiology in SSF that yield lower but more steady ROS concentrations, which could be associated to higher lovastatin production.     

N-Propionylmannosamine-induced over-expression and secretion of thioredoxin leads to neurite outgrowth of PC12 cells

The function of the central nervous system largely depends on growth and differentiation (neurite outgrowth) of neural cells and it is well established that growth factors, especially nerve growth factor NGF stimulate neurite outgrowth. However, additional factors are implicated in this process notably the redox state of the cells. For the first time we could demonstrate that the application of recombinant thioredoxin stimulates neurite outgrowth of PC12 cells to the same extend as NGF. Thioredoxin, a small redox protein is a major player in the cellular protein reduction system. An increased expression and secretion of thioredoxin is achieved by the application of the novel sialic acid precursor N-propionylmannosamine (ManNProp). From earlier studies it is known that this N-acylmannosamine analog stimulates significantly the neurite outgrowth in cell cultures. This finding would give new insights into the mechanism of the nerve-stimulatory action of ManNProp and demonstrates the novel role of thioredoxin during the regulation of nerve growth, encouraging further studies.