Self-Assembly of Nanofilaments in Cyanobacteria for Protein Co-localization.

Cyanobacteria offer great potential as alternative biotechnological hosts due to their photoautotrophic capacities. However, in comparison to established heterotrophic hosts, several key aspects, such as product titers, are still lagging behind. Nanobiotechnology is an emerging field with great potential to improve existing hosts, but so far, it has barely been explored in microbial photosynthetic systems. Here, we report the establishment of large proteinaceous nanofilaments in the unicellular model cyanobacterium Synechocystis sp. PCC 6803 and the fast-growing cyanobacterial strain Synechococcus elongatus UTEX 2973. Transmission electron microscopy and electron tomography demonstrated that expression of pduA*, encoding a modified bacterial microcompartment shell protein, led to the generation of bundles of longitudinally aligned nanofilaments in S. elongatus UTEX 2973 and shorter filamentous structures in Synechocystis sp. PCC 6803. Comparative proteomics showed that PduA* was at least 50 times more abundant than the second most abundant protein in the cell and that nanofilament assembly had only a minor impact on cellular metabolism. Finally, as a proof-of-concept for co-localization with the filaments, we targeted a fluorescent reporter protein, mCitrine, to PduA* by fusion with an encapsulation peptide that natively interacts with PduA. The establishment of nanofilaments in cyanobacterial cells is an important step toward cellular organization of heterologous pathways and the establishment of cyanobacteria as next-generation hosts.

Engineering mammalian cells to produce plant-specific N-glycosylation on proteins.

Protein N-glycosylation is an essential and highly conserved posttranslational modification found in all eukaryotic cells. Yeast, plants and mammalian cells, however, produce N-glycans with distinct structural features. These species-specific features not only pose challenges in selecting host cells for production of recombinant therapeutics for human medical use but also provide opportunities to explore and utilize species-specific glycosylation in design of vaccines. Here, we used reverse cross-species engineering to stably introduce plant core α3fucose (α3Fuc) and ß2xylose (ß2Xyl) N-glycosylation epitopes in the mammalian Chinese hamster ovary (CHO) cell line. We used directed knockin of plant core fucosylation and xylosylation genes (AtFucTA/AtFucTB and AtXylT) and targeted knockout of endogenous genes for core fucosylation (fut8) and elongation (B4galt1), for establishing CHO cells with plant N-glycosylation capacities. The engineering was evaluated through coexpression of two human therapeutic N-glycoproteins, erythropoietin (EPO) and an immunoglobulin G (IgG) antibody. Full conversion to the plant-type α3Fuc/ß2Xyl bi-antennary agalactosylated N-glycosylation (G0FX) was demonstrated for the IgG1 produced in CHO cells. These results demonstrate that N-glycosylation in mammalian cells is amenable for extensive cross-kingdom engineering and that engineered CHO cells may be used to produce glycoproteins with plant glycosylation.

The Role of the Glucosinolate-Myrosinase System in Mediating Greater Resistance of Barbarea verna than B. vulgaris to Mamestra brassicae Larvae.

We investigated the influences of two structurally similar glucosinolates, phenethylglucosinolate (gluconasturtiin, NAS) and its (S)-2-hydroxyl derivative glucobarbarin (BAR), as well as their hydrolysis products on larvae of the generalist Mamestra brassicae (Lepidoptera: Noctuidae). Previous results suggested a higher defensive activity of BAR than NAS based on resistance toward M. brassicae larvae of natural plant genotypes of Barbarea vulgaris R. Br. (Brassicaceae) dominated by BAR. In the present study, the hypothesis of a higher defensive activity of BAR than NAS was tested by comparing two Barbarea species similarly dominated either by BAR or by NAS and by testing effects of isolated BAR and NAS on larval survival and feeding preferences. Larvae reared on leaf disks of B. verna (Mill.) Asch. had a lower survival than those reared on B. vulgaris P- and G-chemotypes. Leaves of B. verna were dominated by NAS, whereas B. vulgaris chemotypes were dominated by BAR or its epimer. In addition, B. verna leaves showed a threefold higher activity of the glucosinolate-activating myrosinase enzymes. The main product of NAS from breakdown by endogenous enzymes including myrosinases ("autolysis") in B. verna leaves was phenethyl isothiocyanate, while the main products of BAR in autolyzed B. vulgaris leaves were a cyclized isothiocyanate product, namely an oxazolidine-2-thione, and a downstream metabolite, an oxazolidin-2-one. The glucosinolates BAR and NAS were isolated and offered to larvae on disks of cabbage. Both glucosinolates exerted similar negative effects on larval survival but effects of NAS tended to be more detrimental. Low concentrations of BAR, but not of NAS, stimulated larval feeding, whereas high BAR concentrations acted deterrent. NAS only tended to be deterrent at the highest concentration, but the difference was not significant. Recoveries of NAS and BAR on cabbage leaf disks were similar, and when hydrolyzed by mechanical leaf damage, the same isothiocyanate-type products as in Barbarea plants were formed with further conversion of BAR to cyclic products, (R)-5-phenyloxazolidine-2-thione [(R)-barbarin] and (R)-5-phenyloxazolidin-2-one [(R)-resedine]. We conclude that a previously proposed generally higher defensive activity of BAR than NAS to M. brassicae larvae could not be confirmed. Indeed, the higher resistance of NAS-containing B. verna plants may be due to a combined effect of rather high concentrations of NAS and a relatively high myrosinase activity or other plant traits not investigated yet.

Glucosinolate turnover in Brassicales species to an oxazolidin-2-one, formed via the 2-thione and without formation of thioamide.

Glucosinolates are found in plants of the order Brassicales and hydrolyzed to different breakdown products, particularly after tissue damage. In Barbarea vulgaris R.Br. (Brassicaceae), the dominant glucosinolate in the investigated "G-type" is glucobarbarin, (S)-2-hydroxy-2-phenylethylglucosinolate. Formation of the nitrile from glucobarbarin was observed in vitro, while a previously suggested thioamide (synonym thionamide) was not confirmed. Resedine (5-phenyl-1,3-oxazolidin-2-one) was detected after glucobarbarin hydrolysis in crushed B. vulgaris leaves and siliques, but not in intact parts. The abundance increased for several hours after completion of hydrolysis. The corresponding 1,3-oxazolidine-2-thione (OAT), with the common name barbarin, was also formed, and appeared to be the precursor of resedine. Addition of each of two non-endogenous OATs, (S)-5-ethyl-5-methylOAT and (R)-5-vinylOAT (R-goitrin), to a leaf homogenate resulted in formation of the corresponding 1,3-oxazolidin-2-ones (OAOs), confirming the metabolic connection of OAT to OAO. Formation of OAOs was inhibited by prior brief heating of the homogenate, suggesting enzyme involvement. We suggest the conversion of OATs to OAOs to be catalyzed by an enzyme ("oxazolidinethionase") responsible for turnover of OAT formed in intact plants. Resedine had been reported as an alkaloid from another species - Reseda luteola L. (Resedaceae) - naturally containing the glucosinolate glucobarbarin. However, resedine was not detected in intact R. luteola plants, but formed after tissue damage. The formation of resedine in two families suggests a broad distribution of putative OATases in the Brassicales; potentially involved in glucosinolate turnover that needs myrosinase activity as the committed step. In agreement with the proposed function of OATase, several candidate genes for myrosinases in glucosinolate turnover in intact plants were discovered in the B. vulgaris genome. We also suggest that biotechnological conversion of OATs to OAOs might improve the nutritional value of Brassicales protein. HPLC-MS/MS methods for detection of these glucobarbarin products are described.

CHLOROPLAST RIBOSOME ASSOCIATED Supports Translation under Stress and Interacts with the Ribosomal 30S Subunit.

Chloroplast ribosomes, which originated from cyanobacteria, comprise a large subunit (50S) and a small subunit (30S) containing ribosomal RNAs (rRNAs) and various ribosomal proteins. Genes for many chloroplast ribosomal proteins, as well as proteins with auxiliary roles in ribosome biogenesis or functioning, reside in the nucleus. Here, we identified Arabidopsis (Arabidopsis thaliana) CHLOROPLAST RIBOSOME ASSOCIATED (CRASS), a member of the latter class of proteins, based on the tight coexpression of its mRNA with transcripts for nucleus-encoded chloroplast ribosomal proteins. CRASS was acquired during the evolution of embryophytes and is localized to the chloroplast stroma. Loss of CRASS results in minor defects in development, photosynthetic efficiency, and chloroplast translation activity under controlled growth conditions, but these phenotypes are greatly exacerbated under stress conditions induced by the translational inhibitors lincomycin and chloramphenicol or by cold treatment. The CRASS protein comigrates with chloroplast ribosomal particles and coimmunoprecipitates with the 16S rRNA and several chloroplast ribosomal proteins, particularly the plastid ribosomal proteins of the 30S subunit (PRPS1 and PRPS5). The association of CRASS with PRPS1 and PRPS5 is independent of rRNA and is not detectable in yeast two-hybrid experiments, implying that either CRASS interacts indirectly with PRPS1 and PRPS5 via another component of the small ribosomal subunit or that it recognizes structural features of the multiprotein/rRNA particle. CRASS plays a role in the biogenesis and/or stability of the chloroplast ribosome that becomes critical under certain stressful conditions when ribosomal activity is compromised.

Tat proteins as novel thylakoid membrane anchors organize a biosynthetic pathway in chloroplasts and increase product yield 5-fold.

Photosynthesis drives the production of ATP and NADPH, and acts as a source of carbon for primary metabolism. NADPH is also used in the production of many natural bioactive compounds. These are usually synthesized in low quantities and are often difficult to produce by chemical synthesis due to their complex structures. Some of the crucial enzymes catalyzing their biosynthesis are the cytochromes P450 (P450s) situated in the endoplasmic reticulum (ER), powered by electron transfers from NADPH. Dhurrin is a cyanogenic glucoside and its biosynthesis involves a dynamic metabolon formed by two P450s, a UDP-glucosyltransferase (UGT) and a P450 oxidoreductase (POR). Its biosynthetic pathway has been relocated to the chloroplast where ferredoxin, reduced through the photosynthetic electron transport chain, serves as an efficient electron donor to the P450s, bypassing the involvement of POR. Nevertheless, translocation of the pathway from the ER to the chloroplast creates other difficulties, such as the loss of metabolon formation and intermediate diversion into other metabolic pathways. We show here that co-localization of these enzymes in the thylakoid membrane leads to a significant increase in product formation, with a concomitant decrease in off-pathway intermediates. This was achieved by exchanging the membrane anchors of the dhurrin pathway enzymes to components of the Twin-arginine translocation pathway, TatB and TatC, which have self-assembly properties. Consequently, we show 5-fold increased titers of dhurrin and a decrease in the amounts of intermediates and side products in Nicotiana benthamiana. Further, results suggest that targeting the UGT to the membrane is a key factor to achieve efficient substrate channeling.

Spatial separation of the cyanogenic ß-glucosidase ZfBGD2 and cyanogenic glucosides in the haemolymph of Zygaena larvae facilitates cyanide release.

Low molecular weight compounds are typically used by insects and plants for defence against predators. They are often stored as inactive ß-glucosides and kept separate from activating ß-glucosidases. When the two components are mixed, the ß-glucosides are hydrolysed releasing toxic aglucones. Cyanogenic plants contain cyanogenic glucosides and release hydrogen cyanide due to such a well-characterized two-component system. Some arthropods are also cyanogenic, but comparatively little is known about their system. Here, we identify a specific ß-glucosidase (ZfBGD2) involved in cyanogenesis from larvae of Zygaena filipendulae (Lepidoptera, Zygaenidae), and analyse the spatial organization of cyanide release in this specialized insect. High levels of ZfBGD2 mRNA and protein were found in haemocytes by transcriptomic and proteomic profiling. Heterologous expression in insect cells showed that ZfBGD2 hydrolyses linamarin and lotaustralin, the two cyanogenic glucosides present in Z. filipendulae. Linamarin and lotaustralin as well as cyanide release were found exclusively in the haemoplasma. Phylogenetic analyses revealed that ZfBGD2 clusters with other insect ß-glucosidases, and correspondingly, the ability to hydrolyse cyanogenic glucosides catalysed by a specific ß-glucosidase evolved convergently in insects and plants. The spatial separation of the ß-glucosidase ZfBGD2 and its cyanogenic substrates within the haemolymph provides the basis for cyanide release in Z. filipendulae. This spatial separation is similar to the compartmentalization of the two components found in cyanogenic plant species, and illustrates one similarity in cyanide-based defence in these two kingdoms of life.

Glucosinolate diversity within a phylogenetic framework of the tribe Cardamineae (Brassicaceae) unraveled with HPLC-MS/MS and NMR-based analytical distinction of 70 desulfoglucosinolates.

As a basis for future investigations of evolutionary trajectories and biosynthetic mechanisms underlying variations in glucosinolate structures, we screened members of the crucifer tribe Cardamineae by HPLC-MS/MS, isolated and identified glucosinolates by NMR, searched the literature for previous data for the tribe, and collected HPLC-MS/MS data for nearly all glucosinolates known from the tribe as well as some related structures (70 in total). This is a considerable proportion of the approximately 142 currently documented natural glucosinolates. Calibration with authentic references allowed distinction (or elucidation) of isomers in many cases, such as distinction of ß-hydroxyls, methylthios, methylsulfinyls and methylsulfonyls. A mechanism for fragmentation of secondary ß-hydroxyls in MS was elucidated, and two novel glucosinolates were discovered: 2-hydroxy-3-methylpentylglucosinolate in roots of Cardamine pratensis and 2-hydroxy-8-(methylsulfinyl)octylglucosinolate in seeds of Rorippa amphibia. A large number of glucosinolates (ca. 54 with high structural certainty and a further 28 or more suggested from tandem MS), representing a wide structural variation, is documented from the tribe. This included glucosinolates apparently derived from Met, Phe, Trp, Val/Leu, Ile and higher homologues. Normal side chain elongation and side chain decoration by oxidation or methylation was observed, as well as rare abnormal side chain decoration (hydroxylation of aliphatics at the δ rather than ß-position). Some species had diverse profiles, e.g. R. amphibia and C. pratensis (19 and 16 individual glucosinolates, respectively), comparable to total diversity in literature reports of Armoracia rusticana (17?), Barbarea vulgaris (20-24), and Rorippa indica (>20?). The ancestor or the tribe would appear to have used Trp, Met, and homoPhe as glucosinolate precursor amino acids, and to exhibit oxidation of thio to sulfinyl, formation of alkenyls, ß-hydroxylation of aliphatic chains and hydroxylation and methylation of indole glucosinolates. Two hotspots of apparent biochemical innovation and loss were identified: C. pratensis and the genus Barbarea. Diversity in other species mainly included structures also known from other crucifers. In addition to a role of gene duplication, two contrasting genetic/biochemical mechanisms for evolution of such combined diversity and redundancy are discussed: (i) involvement of widespread genes with expression varying during evolution, and (ii) mutational changes in substrate specificities of CYP79F and GS-OH enzymes.

SILAC and alternatives in studying cellular proteomes of plants.

Quantitative proteomics by metabolic labeling has a high impact on the growing field of plant systems biology. SILAC has been pioneered and optimized for plant cell culture systems allowing for SILAC-based quantitative experiments in specialized experimental setups. In comparison to other model organisms, the application of SILAC to whole plants is challenging. As autotrophic organisms, plants under their natural growth conditions can hardly be fully labeled with stable isotope-coded amino acids. The metabolic labeling with inorganic nitrogen is therefore the method of choice for most whole-plant physiological questions. Plants can easily metabolize different inorganic nitrogen isotopes. The incorporation of the labeled inorganic nitrogen then results in proteins and metabolites with distinct molecular mass, which can be detected on a mass spectrometer. In comparative quantitative experiments, similarly as in SILAC experiments, treated and untreated samples are differentially labeled by nitrogen isotopes and jointly processed, thereby minimizing sample-to-sample variation. In recent years, heavy nitrogen labeling has become a widely used strategy in quantitative proteomics and novel approaches were developed for metabolite identification. Here we present a typical hydroponics setup, the workflow for processing of samples, mass spectrometry and data analysis for large-scale metabolic labeling experiments of whole plants.

The contributions of wobbling and superwobbling to the reading of the genetic code.

Reduced bacterial genomes and most genomes of cell organelles (chloroplasts and mitochondria) do not encode the full set of 32 tRNA species required to read all triplets of the genetic code according to the conventional wobble rules. Superwobbling, in which a single tRNA species that contains a uridine in the wobble position of the anticodon reads an entire four-fold degenerate codon box, has been suggested as a possible mechanism for how tRNA sets can be reduced. However, the general feasibility of superwobbling and its efficiency in the various codon boxes have remained unknown. Here we report a complete experimental assessment of the decoding rules in a typical prokaryotic genetic system, the plastid genome. By constructing a large set of transplastomic knock-out mutants for pairs of isoaccepting tRNA species, we show that superwobbling occurs in all codon boxes where it is theoretically possible. Phenotypic characterization of the transplastomic mutant plants revealed that the efficiency of superwobbling varies in a codon box-dependent manner, but--contrary to previous suggestions--it is independent of the number of hydrogen bonds engaged in codon-anticodon interaction. Finally, our data provide experimental evidence of the minimum tRNA set comprising 25 tRNA species, a number lower than previously suggested. Our results demonstrate that all triplets with pyrimidines in third codon position are dually decoded: by a tRNA species utilizing standard base pairing or wobbling and by a second tRNA species employing superwobbling. This has important implications for the interpretation of the genetic code and will aid the construction of synthetic genomes with a minimum-size translational apparatus.

Downregulation of the δ-subunit reduces mitochondrial ATP synthase levels, alters respiration, and restricts growth and gametophyte development in Arabidopsis.

The mitochondrial ATP synthase (F(1)F(o) complex) is an evolutionary conserved multimeric protein complex that synthesizes the main bulk of cytosolic ATP, but the regulatory mechanisms of the subunits are only poorly understood in plants. In yeast, the δ-subunit links the membrane-embedded F(o) part to the matrix-facing central stalk of F(1). We used genetic interference and an inhibitor to investigate the molecular function and physiological impact of the δ-subunit in Arabidopsis thaliana. Delta mutants displayed both male and female gametophyte defects. RNA interference of delta resulted in growth retardation, reduced ATP synthase amounts, and increased alternative oxidase capacity and led to specific long-term increases in Ala and Gly levels. By contrast, inhibition of the complex using oligomycin triggered broad metabolic changes, affecting glycolysis and the tricarboxylic acid cycle, and led to a successive induction of transcripts for alternative respiratory pathways and for redox and biotic stress-related transcription factors. We conclude that (1) the δ-subunit is essential for male gametophyte development in Arabidopsis, (2) a disturbance of the ATP synthase appears to lead to an early transition phase and a long-term metabolic steady state, and (3) the observed long-term adjustments in mitochondrial metabolism are linked to reduced growth and deficiencies in gametophyte development.

Elemental formula annotation of polar and lipophilic metabolites using (13) C, (15) N and (34) S isotope labelling, in combination with high-resolution mass spectrometry.

The unbiased and comprehensive analysis of metabolites in any organism presents a major challenge if proper peak annotation and unambiguous assignment of the biological origin of the peaks are required. Here we provide a comprehensive multi-isotope labelling-based strategy using fully labelled (13) C, (15) N and (34) S plant tissues, in combination with a fractionated metabolite extraction protocol. The extraction procedure allows for the simultaneous extraction of polar, semi-polar and hydrophobic metabolites, as well as for the extraction of proteins and starch. After labelling and extraction, the metabolites and lipids were analysed using a high-resolution mass spectrometer providing accurate MS and all-ion fragmentation data, providing an unambiguous readout for every detectable isotope-labelled peak. The isotope labelling assisted peak annotation process employed can be applied in either an automated database-dependent or a database-independent analysis of the plant polar metabolome and lipidome. As a proof of concept, the developed methods and technologies were applied and validated using Arabidopsis thaliana leaf and root extracts. Along with a large repository of assigned elemental compositions, which is provided, we show, using selected examples, the accuracy and reliability of the developed workflow.

Alteration of mitochondrial protein complexes in relation to metabolic regulation under short-term oxidative stress in Arabidopsis seedlings.

Plants reconfigure their metabolic network under stress conditions. Changes of mitochondrial metabolism such as tricarboxylic acid (TCA) cycle and amino acid metabolism are reported in Arabidopsis roots but the exact molecular basis underlying this remains unknown. We here hypothesise the reassembly of enzyme protein complexes to be a molecular mechanism for metabolic regulation and tried in the present study to find out mitochondrial protein complexes which change their composition under oxidative stress by the combinatorial approach of proteomics and metabolomics. Arabidopsis seedlings were treated with menadione to induce oxidative stress. The inhibition of several TCA cycle enzymes and the oxidised NADPH pool indicated the onset of oxidative stress. In blue native/SDS-PAGE analysis of mitochondrial protein complexes the intensities of 18 spots increased and those of 13 spots decreased in menadione treated samples suggesting these proteins associate with, or dissociate from, protein complexes. Some spots were identified as metabolic enzymes related to central carbon metabolism such as malic enzyme, glyceraldehyde-3-phosphate dehydrogenase, monodehydroascorbate reductase and alanine aminotransferase. The change in spot intensity was not directly correlated to the total enzyme activity and mRNA level of the corresponding enzyme but closely related to the metabolite profile, suggesting the metabolism is regulated under oxidative stress at a higher level than translation. These results are somewhat preliminary but suggest the regulation of the TCA cycle, glycolysis, ascorbate and amino acid metabolism by reassembly of plant enzyme complexes.

Two DEAD-box proteins may be part of RNA-dependent high-molecular-mass protein complexes in Arabidopsis mitochondria.

Posttranscriptional processes are important for regulation of gene expression in plant mitochondria. DEAD-box proteins, which form a huge protein family with members from all kingdoms, are fundamental components in virtually all types of processes in RNA metabolism. Two members of this protein family, designated PMH1 and PMH2 (for PUTATIVE MITOCHONDRIAL RNA HELICASE), were analyzed and characterized in mitochondria of Arabidopsis (Arabidopsis thaliana). Green fluorescent protein tagging with N-terminal PMH1 and PMH2 sequences supports the mitochondrial localization of these proteins. Northern experiments, as well as histochemical beta-glucuronidase staining of transgenic plants carrying respective promoter:beta-glucuronidase fusion constructs, revealed differing transcription patterns for the two genes. In response to cold, however, transcript levels of both genes increased. Immunodetection analyses of mitochondrial protein complexes after two-dimensional blue native/urea SDS-PAGE and after fractionation on sucrose gradients strongly suggest that one or both proteins are part of RNA-dependent complexes. Cold treatment of cell cultures or solubilization of mitochondria in the presence of MgCl(2) favored the detection of high-molecular-mass complexes. This study paves the way for detailed analysis of high-molecular-mass complexes in mitochondria of higher plants.

The structure of carbonmonoxy neuroglobin reveals a heme-sliding mechanism for control of ligand affinity.

Neuroglobin (Ngb), a globular heme protein expressed in the brain of vertebrates, binds oxygen reversibly, with an affinity comparable to myoglobin (Mb). Despite low sequence identity, the overall 3D fold of Ngb and Mb is very similar. Unlike in Mb, in Ngb the sixth coordination position of the heme iron is occupied by the distal histidine, in the absence of an exogenous ligand. Endogenous ligation has been proposed as a unique mechanism for affinity regulation and ligand discrimination in heme proteins. This peculiarity might be related to the still-unknown physiological function of Ngb. Here, we present the x-ray structure of CO-bound ferrous murine Ngb at 1.7 A and a comparison with the 1.5-A structure of ferric bis-histidine Ngb. We have also used Fourier transform IR spectroscopy of WT and mutant CO-ligated Ngb to examine structural heterogeneity in the active site. Upon CO binding, the distal histidine retains (by and large) its position, whereas the heme group slides deeper into a preformed crevice, thereby reshaping the large cavity ( approximately 290 A(3)) connecting the distal and proximal heme sides with the bulk. The heme relocation is accompanied by a significant decrease of structural disorder, especially of the EF loop, which may be the signal whereby Ngb communicates hypoxic conditions. This unexpected structural change unveils a heme-sliding mechanism of affinity control that may be of significance to understanding Ngb's role in the pathophysiology of the brain.