Reduction in Phosphoribulokinase Amount and Re-Routing Metabolism in Chlamydomonas reinhardtii CP12 Mutants.

The chloroplast protein CP12 is involved in the dark/light regulation of the Calvin-Benson-Bassham cycle, in particular, in the dark inhibition of two enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), but other functions related to stress have been proposed. We knocked out the unique CP12 gene to prevent its expression in Chlamydomonas reinhardtii (ΔCP12). The growth rates of both wild-type and ΔCP12 cells were nearly identical, as was the GAPDH protein abundance and activity in both cell lines. On the contrary, the abundance of PRK and its specific activity were significantly reduced in ΔCP12, as revealed by relative quantitative proteomics. Isolated PRK lost irreversibly its activity over-time in vitro, which was prevented in the presence of recombinant CP12 in a redox-independent manner. We have identified amino acid residues in the CP12 protein that are required for this new function preserving PRK activity. Numerous proteins involved in redox homeostasis and stress responses were more abundant and the expressions of various metabolic pathways were also increased or decreased in the absence of CP12. These results highlight CP12 as a moonlighting protein with additional functions beyond its well-known regulatory role in carbon metabolism.

ppGpp influences protein protection, growth and photosynthesis in Phaeodactylum tricornutum.

Chloroplasts retain elements of a bacterial stress response pathway that is mediated by the signalling nucleotides guanosine penta- and tetraphosphate ((p)ppGpp). In the model flowering plant Arabidopsis, ppGpp acts as a potent regulator of plastid gene expression and influences photosynthesis, plant growth and development. However, little is known about ppGpp metabolism or its evolution in other photosynthetic eukaryotes. Here, we studied the function of ppGpp in the diatom Phaeodactylum tricornutum using transgenic lines containing an inducible system for ppGpp accumulation. We used these lines to investigate the effects of ppGpp on growth, photosynthesis, lipid metabolism and protein expression. We demonstrate that ppGpp accumulation reduces photosynthetic capacity and promotes a quiescent-like state with reduced proliferation and ageing. Strikingly, using nontargeted proteomics, we discovered that ppGpp accumulation also leads to the coordinated upregulation of a protein protection response in multiple cellular compartments. Our findings highlight the importance of ppGpp as a fundamental regulator of chloroplast function across different domains of life, and lead to new questions about the molecular mechanisms and roles of (p)ppGpp signalling in photosynthetic eukaryotes.

A new type of flexible CP12 protein in the marine diatom Thalassiosira pseudonana.

BACKGROUND: CP12 is a small chloroplast protein that is widespread in various photosynthetic organisms and is an actor of the redox signaling pathway involved in the regulation of the Calvin Benson Bassham (CBB) cycle. The gene encoding this protein is conserved in many diatoms, but the protein has been overlooked in these organisms, despite their ecological importance and their complex and still enigmatic evolutionary background. METHODS: A combination of biochemical, bioinformatics and biophysical methods including electrospray ionization-mass spectrometry, circular dichroism, nuclear magnetic resonance spectroscopy and small X ray scattering, was used to characterize a diatom CP12. RESULTS: Here, we demonstrate that CP12 is expressed in the marine diatom Thalassiosira pseudonana constitutively in dark-treated and in continuous light-treated cells as well as in all growth phases. This CP12 similarly to its homologues in other species has some features of intrinsically disorder protein family: it behaves abnormally under gel electrophoresis and size exclusion chromatography, has a high net charge and a bias amino acid composition. By contrast, unlike other known CP12 proteins that are monomers, this protein is a dimer as suggested by native electrospray ionization-mass spectrometry and small angle X-ray scattering. In addition, small angle X-ray scattering revealed that this CP12 is an elongated cylinder with kinks. Circular dichroism spectra indicated that CP12 has a high content of α-helices, and nuclear magnetic resonance spectroscopy suggested that these helices are unstable and dynamic within a millisecond timescale. Together with in silico predictions, these results suggest that T. pseudonana CP12 has both coiled coil and disordered regions. CONCLUSIONS: These findings bring new insights into the large family of dynamic proteins containing disordered regions, thus increasing the diversity of known CP12 proteins. As it is a protein that is more abundant in many stresses, it is not devoted to one metabolism and in particular, it is not specific to carbon metabolism. This raises questions about the role of this protein in addition to the well-established regulation of the CBB cycle. Choregraphy of metabolism by CP12 proteins in Viridiplantae and Heterokonta. While the monomeric CP12 in Viridiplantae is involved in carbon assimilation, regulating phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) through the formation of a ternary complex, in Heterokonta studied so far, the dimeric CP12 is associated with Ferredoxin-NADP reductase (FNR) and GAPDH. The Viridiplantae CP12 can bind metal ions and can be a chaperone, the Heterokonta CP12 is more abundant in all stresses (C, N, Si, P limited conditions) and is not specific to a metabolism. Video Abstract.

Overproduction of the Flv3B flavodiiron, enhances the photobiological hydrogen production by the nitrogen-fixing cyanobacterium Nostoc PCC 7120.

BACKGROUND: The ability of some photosynthetic microorganisms, particularly cyanobacteria and microalgae, to produce hydrogen (H2) is a promising alternative for renewable, clean-energy production. However, the most recent, related studies point out that much improvement is needed for sustainable cyanobacterial-based H2 production to become economically viable. In this study, we investigated the impact of induced O2-consumption on H2 photoproduction yields in the heterocyte-forming, N2-fixing cyanobacterium Nostoc PCC7120. RESULTS: The flv3B gene, encoding a flavodiiron protein naturally expressed in Nostoc heterocytes, was overexpressed. Under aerobic and phototrophic growth conditions, the recombinant strain displayed a significantly higher H2 production than the wild type. Nitrogenase activity assays indicated that flv3B overexpression did not enhance the nitrogen fixation rates. Interestingly, the transcription of the hox genes, encoding the NiFe Hox hydrogenase, was significantly elevated, as shown by the quantitative RT-PCR analyses. CONCLUSION: We conclude that the overproduced Flv3B protein might have enhanced O2-consumption, thus creating conditions inducing hox genes and facilitating H2 production. The present study clearly demonstrates the potential to use metabolic engineered cyanobacteria for photosynthesis driven H2 production.

Clostridial whole cell and enzyme systems for hydrogen production: current state and perspectives.

Strictly anaerobic bacteria of the Clostridium genus have attracted great interest as potential cell factories for molecular hydrogen production purposes. In addition to being a useful approach to this process, dark fermentation has the advantage of using the degradation of cheap agricultural residues and industrial wastes for molecular hydrogen production. However, many improvements are still required before large-scale hydrogen production from clostridial metabolism is possible. Here we review the literature on the basic biological processes involved in clostridial hydrogen production, and present the main advances obtained so far in order to enhance the hydrogen productivity, as well as suggesting some possible future prospects.

Phototrophic hydrogen production from a clostridial [FeFe] hydrogenase expressed in the heterocysts of the cyanobacterium Nostoc PCC 7120.

The conversion of solar energy into hydrogen represents a highly attractive strategy for the production of renewable energies. Photosynthetic microorganisms have the ability to produce H2 from sunlight but several obstacles must be overcome before obtaining a sustainable and efficient H2 production system. Cyanobacteria harbor [NiFe] hydrogenases required for the consumption of H2. As a result, their H2 production rates are low, which makes them not suitable for a high yield production. On the other hand, [FeFe] enzymes originating from anaerobic organisms such as Clostridium exhibit much higher H2 production activities, but their sensitivity to O2 inhibition impairs their use in photosynthetic organisms. To reach such a goal, it is therefore important to protect the hydrogenase from O2. The diazotrophic filamentous cyanobacteria protect their nitrogenases from O2 by differentiating micro-oxic cells called heterocysts. Producing [FeFe] hydrogenase in the heterocyst is an attractive strategy to take advantage of their potential in a photosynthetic microorganism. Here, we present a biological engineering approach for producing an active [FeFe] hydrogenase (HydA) from Clostridium acetobutylicum in the heterocysts of the filamentous cyanobacterium Nostoc PCC7120. To further decrease the O2 amount inside the heterocyst, the GlbN cyanoglobin from Nostoc commune was coproduced with HydA in the heterocyst. The engineered strain produced 400 µmol-H2 per mg Chlorophyll a, which represents 20-fold the amount produced by the wild type strain. This result is a clear demonstration that it is possible to associate oxygenic photosynthesis with H2 production by an O2-sensitive hydrogenase.

Glyceraldehyde-3-phosphate dehydrogenase is regulated by ferredoxin-NADP reductase in the diatom Asterionella formosa.

Diatoms are a widespread and ecologically important group of heterokont algae that contribute c. 20% to global productivity. Previous work has shown that regulation of their key Calvin cycle enzymes differs from that of the Plantae, and that in crude extracts, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) can be inhibited by nicotinamide adenine dinucleotide phosphate reduced (NADPH) under oxidizing conditions. The freshwater diatom, Asterionella formosa, was studied using enzyme kinetics, chromatography, surface plasmon resonance, mass spectrometry and sequence analysis to determine the mechanism behind this GAPDH inhibition. GAPDH interacted with ferredoxin-nicotinamide adenine dinucleotide phosphate (NADP) reductase (FNR) from the primary phase of photosynthesis, and the small chloroplast protein, CP12. Sequences of copurified GAPDH and FNR were highly homologous with published sequences. However, the widespread ternary complex among GAPDH, phosphoribulokinase and CP12 was absent. Activity measurements under oxidizing conditions showed that NADPH can inhibit GAPDH-CP12 in the presence of FNR, explaining the earlier observed inhibition within crude extracts. Diatom plastids have a distinctive metabolism, including the lack of the oxidative pentose phosphate pathway, and so cannot produce NADPH in the dark. The observed down-regulation of GAPDH in the dark may allow NADPH to be rerouted towards other reductive processes contributing to their ecological success.

Translocation of solutes and proteins across the glycosomal membrane of trypanosomes; possibilities and limitations for targeting with trypanocidal drugs.

Glycosomes are specialized peroxisomes found in all kinetoplastid organisms. The organelles are unique in harbouring most enzymes of the glycolytic pathway. Matrix proteins, synthesized in the cytosol, cofactors and metabolites have to be transported across the membrane. Recent research on Trypanosoma brucei has provided insight into how these translocations across the membrane occur, although many details remain to be elucidated. Proteins are imported by a cascade of reactions performed by specialized proteins, called peroxins, in which a cytosolic receptor with bound matrix protein inserts itself in the membrane to deliver its cargo into the organelle and is subsequently retrieved from the glycosome to perform further rounds of import. Bulky solutes, such as cofactors and acyl-CoAs, seem to be translocated by specific transporter molecules, whereas smaller solutes such as glycolytic intermediates probably cross the membrane through pore-forming channels. The presence of such channels is in apparent contradiction with previous results that suggested a low permeability of the glycosomal membrane. We propose 3 possible, not mutually exclusive, solutions for this paradox. Glycosomal glycolytic enzymes have been validated as drug targets against trypanosomatid-borne diseases. We discuss the possible implications of the new data for the design of drugs to be delivered into glycosomes.

Assembling Multiple Fragments: The Gibson Assembly.

The Gibson Assembly is a popular method for molecular cloning which has been developed specifically to join several fragments together in a specific order, without the constraint of restriction enzyme sites. This method is based on the assembly of overlapping fragments, generally produced by PCR, and then combining them using three enzymes: a 5' exonuclease, a DNA polymerase, and a DNA ligase, in an isothermal reaction. Here, we describe this method, including the design of primers for the generation of the overlapping fragments and the assembly; to this end, we provide an example involving joining two fragments in a single plasmid.

Location of the photosynthetic carbon metabolism in microcompartments and separated phases in microalgal cells.

Carbon acquisition, assimilation and storage in eukaryotic microalgae and cyanobacteria occur in multiple compartments that have been characterised by the location of the enzymes involved in these functions. These compartments can be delimited by bilayer membranes, such as the chloroplast, the lumen, the peroxisome, the mitochondria or monolayer membranes, such as lipid droplets or plastoglobules. They can also originate from liquid-liquid phase separation such as the pyrenoid. Multiple exchanges exist between the intracellular microcompartments, and these are reviewed for the CO2 concentration mechanism, the Calvin-Benson-Bassham cycle, the lipid metabolism and the cellular energetic balance. Progress in microscopy and spectroscopic methods opens new perspectives to characterise the molecular consequences of the location of the proteins involved, including intrinsically disordered proteins.

Concentration-Dependent Inhibition of Mesophilic PETases on Poly(ethylene terephthalate) Can Be Eliminated by Enzyme Engineering.

Enzyme-based depolymerization is a viable approach for recycling of poly(ethylene terephthalate) (PET). PETase from Ideonella sakaiensis (IsPETase) is capable of PET hydrolysis under mild conditions but suffers from concentration-dependent inhibition. In this study, this inhibition is found to be dependent on incubation time, the solution conditions, and PET surface area. Furthermore, this inhibition is evident in other mesophilic PET-degrading enzymes to varying degrees, independent of the level of PET depolymerization activity. The inhibition has no clear structural basis, but moderately thermostable IsPETase variants exhibit reduced inhibition, and the property is completely absent in the highly thermostable HotPETase, previously engineered by directed evolution, which simulations suggest results from reduced flexibility around the active site. This work highlights a limitation in applying natural mesophilic hydrolases for PET hydrolysis and reveals an unexpected positive outcome of engineering these enzymes for enhanced thermostability.

The NAD+ metabolism of Leishmania, notably the enzyme nicotinamidase involved in NAD+ salvage, offers prospects for development of anti-parasite chemotherapy.

NAD+ plays multiple, essential roles in the cell. As a cofactor in many redox reactions it is key in the cellular energy metabolism and as a substrate it participates in many reactions leading to a variety of covalent modifications of enzymes with major roles in regulation of expression and metabolism. Cells may have the ability to produce this metabolite either via alternative de novo synthesis pathways and/or by different salvage pathways. In this issue of Molecular Microbiology, Gazanion et al. (2011) demonstrate that Leishmania species can only rely on the salvage of NAD+ building blocks. One of the enzymes involved, nicotinamidase, is absent from human cells. The enzyme is important for growth of Leishmania infantum and essential for establishing an infection. The crystal structure of the parasite protein has been solved and shows prospects for design of inhibitors to be used as leads for development of new drugs. Indeed, NAD+ metabolism is currently being considered as a promising drug target in various diseases and the vulnerability of Leishmania for interference of this metabolism has been proved in previous work by the same group, by showing that administration of NAD+ precursors has detrimental effect on the pathogenic, amastigote stage of this parasite.

Naturally acquired immune responses to malaria vaccine candidate antigens MSP3 and GLURP in Guahibo and Piaroa indigenous communities of the Venezuelan Amazon.

BACKGROUND: Malaria transmission in most of Latin America can be considered as controlled. In such a scenario, parameters of baseline immunity to malaria antigens are of specific interest with respect to future malaria eradication efforts. METHODS: A cross-sectional study was carried out in two indigenous population groups in Amazonas/Venezuela. Data from the regional malaria documentation system were extracted and participants from the ethnic groups of the Guahibo (n = 180) and Piaroa (n = 295) were investigated for the presence of Plasmodium parasites and naturally acquired antibodies to Plasmodium falciparum antigens in serum. The GMZ2 vaccine candidate proteins MSP3 and GLURP were chosen as serological markers. RESULTS: The incidence of P. falciparum in both communities was found to be less than 2%, and none of the participants harboured P. falciparum at the time of the cross-sectional. Nearly a quarter of the participants (111/475; 23,4%) had positive antibody titres to at least one of the antigens. 53/475 participants (11.2%) were positive for MSP3, and 93/475 participants (19.6%) were positive for GLURP. High positive responses were detected in 36/475 participants (7.6%) and 61/475 participants (12.8%) for MSP3 and GLURP, respectively. Guahibo participants had significantly higher antibody titres than Piaroa participants. CONCLUSIONS: Considering the low incidence of P. falciparum, submicroscopical infections may explain the comparatively high anti-P. falciparum antibody concentrations.

Sourcing thermotolerant poly(ethylene terephthalate) hydrolase scaffolds from natural diversity.

Enzymatic deconstruction of poly(ethylene terephthalate) (PET) is under intense investigation, given the ability of hydrolase enzymes to depolymerize PET to its constituent monomers near the polymer glass transition temperature. To date, reported PET hydrolases have been sourced from a relatively narrow sequence space. Here, we identify additional PET-active biocatalysts from natural diversity by using bioinformatics and machine learning to mine 74 putative thermotolerant PET hydrolases. We successfully express, purify, and assay 51 enzymes from seven distinct phylogenetic groups; observing PET hydrolysis activity on amorphous PET film from 37 enzymes in reactions spanning pH from 4.5-9.0 and temperatures from 30-70 °C. We conduct PET hydrolysis time-course reactions with the best-performing enzymes, where we observe differences in substrate selectivity as function of PET morphology. We employed X-ray crystallography and AlphaFold to examine the enzyme architectures of all 74 candidates, revealing protein folds and accessory domains not previously associated with PET deconstruction. Overall, this study expands the number and diversity of thermotolerant scaffolds for enzymatic PET deconstruction.

Leishmania mexicana: LACK (Leishmania homolog of receptors for activated C-kinase) is a plasminogen binding protein.

Leishmania mexicana is able to interact with the fibrinolytic system through its component plasminogen, the zymogenic form of the protease plasmin. In this study a new plasminogen binding protein of this parasite was identified: LACK, the Leishmania homolog of receptors for activated C-kinase. Plasminogen binds recombinant LACK with a K(d) value of 1.6±0.4 µM, and binding is lysine-dependent since it is inhibited by the lysine analog ε-aminocaproic acid. Inhibition studies with specific peptides and plasminogen binding activity of a mutated recombinant LACK have highlighted the internal motif (260)VYDLESKAV(268), similar to those found in several enolases, as involved in plasminogen binding. Recombinant LACK and secreted proteins, in medium conditioned by parasites, enhance plasminogen activation to plasmin by the tissue plasminogen activator (t-PA). In addition to its localization in the cytosol, in the microsomal fraction and as secreted protein in conditioned medium, LACK was also localized on the external surface of the membrane. The results presented here suggest that LACK might bind and enhance plasminogen activation in vivo promoting the formation of plasmin. Plasminogen binding of LACK represents a new function for this protein and might contribute to the invasiveness of the parasite.

Trypanosoma cruzi: A kinetoplast-associated protein of the photolyase/cryptochrome family.

A photolyase-like protein gene found in the Trypanosoma cruzi genome database was cloned and expressed in Escherichia coli resulting in the formation of inclusion bodies. Antibodies against this protein were used to determine expression of the protein in the different forms of the parasite. It was visualized in the epimastigote form but not in amastigote or trypomastigote forms obtained from culture in Vero cells. In epimastigotes, this protein is located at the level of the mitochondrion associated to both sides of the kinetoplast. Sequence analyses indicated that this protein, as well as other photolyases from Leishmania spp. and Trypanosoma brucei are related to single-stranded photolyases or cryptochromes DASH.

Description of envenomation by the "gusano-pollo" caterpillar (Megalopyge opercularis) in Venezuela.

Lepidoptera is a large order of insects, with more than 180,000 species word-wide, showing larval stages of butterflies and moths known as wormlike caterpillars. Almost 12 families of butterflies around the world are capable of causing severe human injuries, varying from dermatitis, renal failure, hemostatic alterations, respiratory failure and neurotoxic symptoms. These caterpillars are coated in long, hair-like setae containing venom to protect themselves against aggressive predators. The setae cause a painful reaction, upon contact, due to presence of neurotoxins. These caterpillars are extensively dispersed all through North America and often, during the dry and wet seasons in tropical regions, being able to sustain two annual larval generations. There exist several species of Megalopyge caterpillars; however, Megalopyge opercularis is the most widely distributed species in Latin America and the United States. This work reports, to our knowledge, the first case of envenomation by the "gusano-pollo" (Megalopyge opercularis), a stinging caterpillar, described in Venezuela. The patient in this report presented severe symptoms, including systemic reactions such as intense hand pain irradiated to the upper arm, restricted swelling, headache, dizziness, serious chest distress and shock-like symptoms that required hospitalization. Symptoms improved upon treatment with opiaceous analgesic drugs.

Targeting TOR signaling for enhanced lipid productivity in algae.

Microalgae can produce large quantities of triacylglycerols (TAGs) and other neutral lipids that are suitable for making biofuels and as feedstocks for green chemistry. However, TAGs accumulate under stress conditions that also stop growth, leading to a trade-off between biomass production and TAG yield. Recently, in the model marine diatom Phaeodactylum tricornutum it was shown that inhibition of the target of rapamycin (TOR) kinase boosts lipid productivity by promoting TAG production without stopping growth. We believe that basic knowledge in this emerging field is required to develop innovative strategies to improve neutral lipid accumulation in oleaginous microalgae. In this minireview, we discuss current research on the TOR signaling pathway with a focus on its control on lipid homeostasis. We first provide an overview of the well characterized roles of TOR in mammalian lipogenesis, adipogenesis and lipolysis. We then present evidence of a role for TOR in controlling TAG accumulation in microalgae, and draw parallels between the situation in animals, plants and microalgae to propose a model of TOR signaling for TAG accumulation in microalgae.

Cloning, expression and biochemical characterization of mitochondrial and cytosolic malate dehydrogenase from Phytophthora infestans.

The genes of the mitochondrial and cytosolic malate dehydrogenase (mMDH and cMDH) of Phytophthora infestans were cloned and overexpressed in Escherichia coli as active enzymes. The catalytic properties of these proteins were determined: both enzymes have a similar specific activity. In addition, the natural mitochondrial isoenzyme was semi-purified from mycelia and its catalytic properties determined: the recombinant mitochondrial isoform behaved as the natural enzyme. A phylogenetic analysis indicated that mMDH, present in all stramenopiles studied, can be useful to study the relationships between these organisms. MDH with the conserved domain MDH_cytoplasmic_cytosolic is absent in some stramenopiles as well as in fungi. This enzyme seems to be less related within the stramenopile group. The Phytophthora cMDHs have an insertion of six amino acids that is also present in the stramenopile cMDHs studied, with the exception of Thalassiosira pseudonana cMDH, and is absent in other known eukaryotic cMDHs.

Hydrogenases and H2 metabolism in sulfate-reducing bacteria of the Desulfovibrio genus.

Hydrogen metabolism plays a central role in sulfate-reducing bacteria of the Desulfovibrio genus and is based on hydrogenases that catalyze the reversible conversion of protons into dihydrogen. These metabolically versatile microorganisms possess a complex hydrogenase system composed of several enzymes of both [FeFe]- and [NiFe]-type that can vary considerably from one Desulfovibrio species to another. This review covers the molecular and physiological aspects of hydrogenases and H2 metabolism in Desulfovibrio but focuses particularly on our model bacterium Desulfovibrio fructosovorans. The search of hydrogenase genes in more than 30 sequenced genomes provides an overview of the distribution of these enzymes in Desulfovibrio. Our discussion will consider the significance of the involvement of electron-bifurcation in H2 metabolism.