Hyperthermophilic endoglucanase for in planta lignocellulose conversion

ABSTRACT: BACKGROUND: The enzymatic conversion of lignocellulosic plant biomass into fermentable sugars is a crucial step in the sustainable and environmentally friendly production of biofuels. However, a major drawback of enzymes from mesophilic sources is their suboptimal activity under established pretreatment conditions, e.g. high temperatures, extreme pH values and high salt concentrations. Enzymes from extremophiles are better adapted to these conditions and could be produced by heterologous expression in microbes, or even directly in the plant biomass. RESULTS: Here we show that a cellulase gene (sso1354) isolated from the hyperthermophilic archaeon Sulfolobus solfataricus can be expressed in plants, and that the recombinant enzyme is biologically active and exhibits the same properties as the wild type form. Since the enzyme is inactive under normal plant growth conditions, this potentially allows its expression in plants without negative effects on growth and development, and subsequent heat-inducible activation. Furthermore we demonstrate that the recombinant enzyme acts in high concentrations of ionic liquids and can therefore degrade alpha-cellulose or even complex cell wall preparations under those pretreatment conditions. CONCLUSION: The hyperthermophilic endoglucanase SSO1354 with its unique features is an excellent tool for advanced biomass conversion. Here we demonstrate its expression in planta and the possibility for post harvest activation. Moreover the enzyme is suitable for combined pretreatment and hydrolysis applications.     

Hagenia from the early Miocene of Ethiopia: Evidence for possible niche evolution?

Fossil pollen believed to be related to extant Hagenia abyssinica were discovered in the early Miocene (21.73 Ma) Mush Valley paleoflora, Ethiopia, Africa. Both the fossil and extant pollen grains of H. abyssinica were examined with combined light microscopy, scanning electron microscopy, and transmission electron microscopy to compare the pollen and establish their relationships. Based on this, the fossil pollen grains were attributed to Hagenia. The presence of Hagenia in the fossil assemblage raises the questions if its habitat has changed over time, and if the plants are/were wind pollinated. To shed light on these questions, the morphology of extant anthers was also studied, revealing specialized hairs inside the anthers, believed to aid in insect pollination. Pollen and anther morphology are discussed in relation to the age and origin of the genus within a molecular dated phylogenetic framework, the establishment of complex topography in East Africa, other evidence regarding pollination modes, and the palynological record. The evidence presented herein, and compiled from the literature, suggests that Hagenia was an insect‐pollinated lowland rainforest element during the early Miocene of the Mush Valley. The current Afromontane habitat and ambophilous (insect and wind) pollination must have evolved in post‐mid‐Miocene times.     

Chemical studies on the lipopolysaccharide of Rhizobium meliloti 10406 and its lipid A region

The structure of the lipopolysaccharide from Rhizobium meliloti 10406, a derivative of the wild-type strain MVII-1, was examined. The compositional analysis of its polysaccharide moiety demonstrated lack of heptose(s), but high contents in glucose, galacturonic acid and 2-keto-3-deoxy-octonate (dOclA) as characteristic features. The lipid A moiety consisted of a -1,6 linked glucosamine disaccharide carrying ester (at C-4) and glycosidically (at C-1) linked phosphate residues, both present exclusively as monoester phosphates but not as phosphodiesters. Ester- and amidelinked 3-hydroxy fatty acids were mostly present as non-3-O-acylated residues. Laser desorption mass spectrometry (LD-MS) revealed heterogeneity in the fatty acid substitution, as was also indicated by the non-stoichiometric ratios obtained by quantitative fatty acid analysis. The predominating lipid A structure contained at the reducing glucosamine residue ester-linked 3-hydroxy-tetradecanoic acid (3-OH-14:0) and amide-linked 3-OH-18:0, or 3-OH-18:1, respectively. The distal (non-reducing) glucosamine carried ester-bound the recently discovered 27-hydroxyoctacosanoic acid and 3-OH-14:0 and, as amide-linked fatty acid, mostly 3-hydroxy-stearic acid (3-OH-18:0).The isolated lipopolysaccharide exhibited a high extent of lethal toxicity in galactosamine-treated mice, comparable to that of enterobacterial lipopolysaccharide. The structural relationship of LPS and lipid A of Rhizobium meliloti to other rhizobial lipopolysaccharides and lipid A's with respect to questions of taxonomy and of phylogenetic relationships will be discussed.Abbreviations LPS lipopolysaccharide - dOclA 3-deoxy-D-mannooctulosonic acid (KDO) - GalA galacturonic acid - DOC sodium deoxycholate - PAGE polyacrylamide gel electrophoresis - LD-MS laser desorption-mass spectrometry     

The VASP-Spred-Sprouty domain puzzle

Sprouty-related proteins with an EVH1 domain (Spreds) belong to a new protein family harboring a conserved N-terminal EVH1 domain, which is related to the VASP (vasodilator-stimulated phosphoprotein) EVH1 domain (Enabled/VASP homology 1 domain) and a C-terminal Sprouty-related domain, typical for Sprouty proteins. Spreds were, like Sproutys, initially discovered as inhibitors of the Ras/MAPK pathway, and the SPR (Sprouty-related) domains of both protein families seem to be very important for many protein interactions and cellular processes. VASP was initially characterized as a proline-rich substrate of protein kinases A and G in human platelets and later shown to be a scaffold protein, regulating both signal transduction pathways and the actin filament system. The VASP-EVH1 domain is known to bind specifically to a FP(4) binding motif, which is, for example, present in the focal adhesion proteins vinculin and zyxin. In this review we give a structural and functional overview on these three protein families and ask whether nature plays a modular protein domain puzzle with stable exchangeable elements or if these closely related domains have various functions when pasted in a different protein context.     

Chloroplast phosphoglycerate kinase from Euglena gracilis: endosymbiotic gene replacement going against the tide.

Two chloroplast phosphoglycerate kinase isoforms from the photosynthetic flagellate Euglena gracilis were purified to homogeneity, partially sequenced, and subsequently cDNAs encoding phosphoglycerate kinase isoenzymes from both the chloroplast and cytosol of E. gracilis were cloned and sequenced. Chloroplast phosphoglycerate kinase, a monomeric enzyme, was encoded as a polyprotein precursor of at least four mature subunits that were separated by conserved tetrapeptides. In a Neighbor-Net analysis of sequence similarity with homologues from numerous prokaryotes and eukaryotes, cytosolic phosphoglycerate kinase of E. gracilis showed the highest similarity to cytosolic and glycosomal homologues from the Kinetoplastida. The chloroplast isoenzyme of E. gracilis did not show a close relationship to sequences from other photosynthetic organisms but was most closely related to cytosolic homologues from animals and fungi.     

“Candidatus Hepatoplasma crinochetorum,” a New, Stalk-Forming Lineage of Mollicutes Colonizing the Midgut Glands of a Terrestrial Isopod

Uncultivated bacteria that densely colonize the midgut glands (hepatopancreas) of the terrestrial isopod Porcellio scaber (Crustacea: Isopoda) were identified by cloning and sequencing of their 16S rRNA genes. Phylogenetic analysis revealed that these symbionts represent a novel lineage of the Mollicutes and are only distantly related (<82% sequence identity) to members of the Mycoplasmatales and Entomoplasmatales. Fluorescence in situ hybridization with a specific oligonucleotide probe confirmed that the amplified 16S rRNA gene sequences indeed originated from a homogeneous population of symbionts intimately associated with the epithelial surface of the hepatopancreas. The same probe also detected morphotypically identical symbionts in other crinochete isopods. Scanning and transmission electron microscopy revealed uniform spherical bacterial cells without a cell wall, sometimes interacting with the microvilli of the brush border by means of stalk-like cytoplasmic appendages, which also appeared to be involved in cell division through budding. Based on the isolated phylogenetic position and unique cytological properties, the provisional name “Candidatus Hepatoplasma crinochetorum” is proposed for this new taxon of Mollicutes colonizing the hepatopancreas of P. scaber.     

Protein identification and tracking in two-dimensional electrophoretic gels by minimal protein identifiers

Protein identification by matrix-assisted laser desorption/ionization mass-spectrometry peptide mass fingerprinting (MALDI-MS PMF) represents a cornerstone of proteomics. However, it often fails to identify low-molecular-mass proteins, protein fragments, and protein mixtures reliably. To overcome these limitations, PMF can be complemented by tandem mass spectrometry and other search strategies for unambiguous protein identification. The present study explores the advantages of using a MALDI-MS-based approach, designated minimal protein identifier (MPI) approach, for protein identification. This is illustrated for culture supernatant (CSN) proteins of Mycobacterium tuberculosis H37Rv after separation by two-dimensional gel electrophoresis (2-DE). The MPI approach takes into consideration that proteins yield characteristic peptides upon proteolytic cleavage. In this study, peptide mixtures derived from tryptic protein cleavage were analyzed by MALDI-MS and the resulting spectra were compared with template spectra of previously identified counterparts. The MPI approach allowed protein identification by few protein-specific signature peptide masses and revealed truncated variants of mycobacterial elongation factor EF-Tu, previously not identified by PMF. Furthermore, the MPI approach can be employed to track proteins in 2-DE gels, as demonstrated for the 14 kDa antigen, the 10 kDa chaperone, and the conserved hypothetical protein Rv0569 of M. tuberculosis H37Rv. Furthermore, it is shown that the power of the MPI approach strongly depends on distinct factors, most notably on the complexity of the proteome analyzed and accuracy of the mass spectrometer used for peptide mass determination.     

Pathways of chaperone-mediated protein folding in the cytosol

Cells are faced with the task of folding thousands of different polypeptides into a wide range of conformations. For many proteins, the folding process requires the action of molecular chaperones. In the cytosol of prokaryotic and eukaryotic cells, molecular chaperones of different structural classes form a network of pathways that can handle substrate polypeptides from the point of initial synthesis on ribosomes to the final stages of folding.     

Therapy of Venezuelan patients with severe mucocutaneous or early lesions of diffuse cutaneous leishmaniasis with a vaccine containing pasteurized Leishmania promastigotes and bacillus Calmette-Guerin: preliminary report

Severe mucocutaneous (MCL) and diffuse (DCL) forms of American cutaneous leishmaniasis (ACL) are infrequent in Venezuela. Chemotherapy produces only transitory remission in DCL, and occasional treatment failures are observed in MCL. We have evaluated therapy with an experimental vaccine in patients with severe leishmaniasis. Four patients with MCL and 3 with early DCL were treated with monthly intradermal injections of a vaccine containing promastigotes of Leishmania (Viannia) braziliensis killed by pasteurization and viable Bacillus Calmette- Guerin. Clinical and immunological responses were evaluated. Integrity of protein constituents in extracts of pasteurized promastigotes was evaluated by gel electrophoresis. Complete remission of lesions occurred after 5-9 injections in patients with MCL or 7-10 injections in patients with early DCL. DCL patients developed positive skin reactions, average size 18.7 mm. All have been free of active lesions for at least 10 months. Adverse effects of the vaccine were limited to local reactivity to BCG at the injection sites and fever in 2 patients. Extracts of pasteurized and fresh promastigotes did not reveal differences in the integrity of protein components detectable by gel electrophoresis. Immunotherapy with this modified vaccine offers an effective, safe option for the treatment of patients who do not respond to immunotherapy with vaccine containing autoclaved parasites or to chemotherapy.     

Brain choline concentrations may not be altered in euthymic bipolar disorder patients chronically treated with either lithium or sodium valproate

BACKGROUND: It has been suggested that lithium increases choline concentrations, although previous human studies examining this possibility using 1H magnetic resonance spectroscopy (1H MRS) have had mixed results: some found increases while most found no differences. METHODS: The present study utilized 1H MRS, in a 3 T scanner to examine the effects of both lithium and sodium valproate upon choline concentrations in treated euthymic bipolar patients utilizing two different methodologies. In the first part of the study healthy controls (n = 18) were compared with euthymic Bipolar Disorder patients (Type I and Type II) who were taking either lithium (n = 14) or sodium valproate (n = 11), and temporal lobe choline/creatine (Cho/Cr) ratios were determined. In the second part we examined a separate group of euthymic Bipolar Disorder Type I patients taking sodium valproate (n = 9) and compared these to controls (n = 11). Here we measured the absolute concentrations of choline in both temporal and frontal lobes. RESULTS: The results from the first part of the study showed that bipolar patients chronically treated with both lithium and sodium valproate had significantly reduced temporal lobe Cho/Cr ratios. In contrast, in the second part of the study, there were no effects of sodium valproate on either absolute choline concentrations or on Cho/Cr ratios in either temporal or frontal lobes. CONCLUSIONS: These findings suggest that measuring Cho/Cr ratios may not accurately reflect brain choline concentrations. In addition, the results do not support previous suggestions that either lithium or valproate increases choline concentrations in bipolar patients.