Characterization of the multiheme cytochromes involved in the extracellular electron transfer pathway of Thermincola ferriacetica

Bioelectrochemical systems (BES) are emerging as a suite of versatile sustainable technologies to produce electricity and added‐value compounds from renewable and carbon‐neutral sources using electroactive organisms. The incomplete knowledge on the molecular processes that allow electroactive organisms to exchange electrons with electrodes has prevented their real‐world implementation. In this manuscript we investigate the extracellular electron transfer processes performed by the thermophilic Gram‐positive bacteria belonging to the Thermincola genus, which were found to produce higher levels of current and tolerate higher temperatures in BES than mesophilic Gram‐negative bacteria. In our study, three multiheme c‐type cytochromes, Tfer_0070, Tfer_0075, and Tfer_1887, proposed to be involved in the extracellular electron transfer pathway of T. ferri-acetica, were cloned and over‐expressed in E. coli. Tfer_0070 (ImdcA) and Tfer_1887 (PdcA) were purified and biochemically characterized. The electrochemical characterization of these proteins supports a pathway of extracellular electron transfer via these two proteins. By contrast, Tfer_0075 (CwcA) could not be stabilized in solution, in agreement with its proposed insertion in the pepti-doglycan wall. However, based on the homology with the outer‐membrane cytochrome OmcS, a structural model for CwcA was developed, providing a molecular perspective into the mechanisms of electron transfer across the peptidoglycan layer in Thermincola.publishersversionpublishe

Exploring the molecular mechanisms of electron shuttling across the microbe/metal space

Dissimilatory metal reducing organisms play key roles in the biogeochemical cycle of metals as well as in the durability of submerged and buried metallic structures. The molecular mechanisms that support electron transfer across the microbe-metal interface in these organisms remain poorly explored. It is known that outer membrane proteins, in particular multiheme cytochromes, are essential for this type of metabolism, being responsible for direct and indirect, via electron shuttles, interaction with the insoluble electron acceptors. Soluble electron shuttles such as flavins, phenazines and humic acids are known to enhance extracellular electron transfer. In this work, this phenomenon was explored. All known outer membrane decaheme cytochromes from Shewanella oneidensis MR-1 with known metal terminal reductase activity and a undecaheme cytochrome from Shewanella sp. HRCR-6 were expressed and purified. Their interactions with soluble electron shuttles were studied using stopped-flow kinetics, NMR spectroscopy and molecular simulations. The results show that despite the structural similarities, expected from the available structural data and sequence homology, the detailed characteristics of their interactions with soluble electron shuttles are different. MtrC and OmcA appear to interact with a variety of different electron shuttles in the close vicinity of some of their hemes, and with affinities that are biologically relevant for the concentrations typical found in the medium for this type of compounds. All data support a view of a distant interaction between the hemes of MtrF and the electron shuttles. For UndA a clear structural characterization was achieved for the interaction with AQDS a humic acid analogue. These results provide guidance for future work of the manipulation of these proteins toward modulation of their role in metal attachment and reduction

Optimizing Electroactive Organisms: The Effect of Orthologous Proteins

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Enhanced bioproduction of poly-3-hydroxybutyrate from wheat straw lignocellulosic hydrolysates

Project: EC/FP7/246449 - New tailor-made PNB-based nanocomposites for high performance applications produced from environmentally friendly production routesNOTICE: this is the author’s version of a work that was accepted for publication in New Biotechnology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in New Biotechnology, [Vol. 31, Issue 1, (January 2014)] DOI 10.1016/j.nbt.2013.10.004"Polyhydroxyalkanoates (PHAs) are bioplastics that can replace conventional petroleum derived products in various applications. One of the major barriers for their widespread introduction in the market is the higher production costs when compared with their petrochemical counterparts. In this work, a process was successfully implemented with high productivity based on wheat straw, a cheap and readily available agricultural residue, as raw material. The strain Burkholderia sacchari DSM 17165 which is able to metabolize glucose, xylose and arabinose, the main sugars present in wheat straw hydrolysates (WSH), was used. Results in shake flask showed that B. sacchari cells accumulated ca 70 % g P(3HB)/g cell-dry-weight with a yield of polymer on sugars (YP/S) of 0.18 g/g when grown on a mixture of commercial C6 and C5 sugars (control), while these values reached ca 60 % g P(3HB)/g cell-dry-weight and 0.19 g/g, respectively, when WSHs were used as carbon source. In fed-batch cultures carried out in 2L stirred tank reactors on WSH, a maximum polymer concentration of 105 g/L was reached after 61 h of cultivation corresponding to an accumulation of 72% of CDW. Polymer yield and productivity were 0.22 g P(3HB)/g total sugar consumed and 1.6 g/L. h, respectively. The selected feeding strategy successfully overcame the carbon catabolite repression phenomenon observed in sugar mixtures containing hexoses and pentoses. This is the first work describing fed-batch cultivations aiming at PHA production using real lignocellulosic hydrolysates. Additionally, the P(3HB) volumetric productivities attained are, by far, the highest achieved ever on agricultural wastes hydrolysates."U

Spatio-temporal expression patterns of anandamide-binding receptors in rat implantation sites: evidence for a role of the endocannabinoid system during the period of placental development

<p>Abstract</p> <p>Background</p> <p>Although there is growing evidence that endocannabinoids play a critical role in early pregnancy, there are no studies describing the possible targets for this system after implantation. The endometrial stroma, which undergoes extensive proliferation and differentiation giving rise to the decidua and the trophoblast cells that invade after the initial stages of implantation, are potential targets. Since high anandamide (AEA) levels, the main endocannabinoid, are detrimental to implantation and in order to gain insight into the role of the endocannabinoid system in the development of the fetoplacental unit, the spatio-temporal pattern of expression of the anandamide-binding receptors, CB1, CB2 and the vanilloid receptor (TRPV1), were investigated by quantitative RT-PCR, western blot and immunohistochemistry.</p> <p>Methods</p> <p>Rat uterine maternal tissues from different days of pregnancy were used to investigate the expression of CB1, CB2 and vanilloid receptors by quantitative RT-PCR, western blot and immunohistochemistry.</p> <p>Results</p> <p>The data indicate that all the three receptors were expressed in decidualized cells and placenta. Interestingly, CB1 and CB2 were also expressed in smooth muscle cells of maternal blood vessels and in endovascular trophoblast cells, whereas TRPV1 was mainly expressed in uterine natural killer (uNK) cells and in the longitudinal muscle layer throughout pregnancy. In all tissues, CB2 protein was present at a lower level than CB1.</p> <p>Conclusion</p> <p>These observations support a role for the endocannabinoid system during the period of decidualization and placental development.</p

Protocol of the SOMNIA project : an observational study to create a neurophysiological database for advanced clinical sleep monitoring

Introduction Polysomnography (PSG) is the primary tool for sleep monitoring and the diagnosis of sleep disorders. Recent advances in signal analysis make it possible to reveal more information from this rich data source. Furthermore, many innovative sleep monitoring techniques are being developed that are less obtrusive, easier to use over long time periods and in the home situation. Here, we describe the methods of the Sleep and Obstructive Sleep Apnoea Monitoring with Non-Invasive Applications (SOMNIA) project, yielding a database combining clinical PSG with advanced unobtrusive sleep monitoring modalities in a large cohort of patients with various sleep disorders. The SOMNIA database will facilitate the validation and assessment of the diagnostic value of the new techniques, as well as the development of additional indices and biomarkers derived from new and/or traditional sleep monitoring methods. Methods and analysis We aim to include at least 2100 subjects (both adults and children) with a variety of sleep disorders who undergo a PSG as part of standard clinical care in a dedicated sleep centre. Full-video PSG will be performed according to the standards of the American Academy of Sleep Medicine. Each recording will be supplemented with one or more new monitoring systems, including wrist-worn photoplethysmography and actigraphy, pressure sensing mattresses, multimicrophone recording of respiratory sounds including snoring, suprasternal pressure monitoring and multielectrode electromyography of the diaphragm

Production of poly (3-hydroxybutyrate-co-4-hydroxybutyrate) by Burkholderia sacchari using wheat straw hydrolysates and gamma-butyrolactone

“NOTICE: this is the author’s version of a work that was accepted for publication in International Journal of Biological Macromolecules. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in International Journal of Biological Macromolecules, Vol. 71, Special issue, (November 2014). DOI 10.1016/j.ijbiomac.2014.04.054 ""Burkholderia sacchari DSM 17165 is able to grow and produce poly(3-hydroxybutyrate) both on hexoses and pentoses. In a previous study, wheat straw lignocellulosic hydrolysates (WSH) containing high C6 and C5 sugar concentrations were shown to be excellent carbon sources for P(3HB) production. Using a similar feeding strategy developed for P(3HB) production based on WSH, fedbatch cultures were developed aiming at the production of the copolymer P(3HB-co- 4HB) (poly(3-hydroxybutyrate-co-4-hydroxybutyrate)) by B. sacchari. The ability of this strain to synthesize P(3HB-co-4HB) was first shown in shake flasks using gammabutyrolactone (GBL) as precursor of the 4HB units. Fed-batch cultures using glucose as carbon source (control) and GBL were developed to achieve high copolymer productivities and 4HB incorporations. The attained P(3HBco- 4HB) productivity and 4HB molar % were 0.7 g/(L·h) and 4.7 molar %, respectively. The 4HB incorporation was improved to 6.3 and 11.8 molar % by addition of 2 g/L propionic and acetic acid, respectively. When WSH were used as carbon source under the same feeding conditions, the values achieved were 0.5 g/(L·h) and 5.0 molar %, respectively. Burkholderia sacchari, a strain able to produce biopolymers based on xylose-rich lignocellulosic hydrolysates, is for the first time reported to produce P(3HB-co-4HB) using gamma butyrolactone as precursor."This work has received funding from the European Union 7th Framework Programme (FP7/2007-2013) under Grant Agreement number 246449 / Project: Bugworkers - New tailor-made PHB-based nanocomposites for high performance applications produced from environmentally friendly production routes.Financial support: Fundação para a Ciência e Tecnologia, Portugal (fellowships SFRH/BPD/68587/2010 and SFRH/BPD/26678/2006)

Biodegradable polyesters from agro&industrial by-products

Poster presented at the 11th annual World Congress on Industrial Biotechnology. Studies on GRP were financed by the EU Integrated Project BIOPRODUCTION (contract nº 026515-2) and those on cellulosic hydrolysates are funded by the EU Collaborative Project BUGWORKERS (contract nº 246449). C. Almeida, J. Cavalheiro, T. Cesário and F. Ferreira were supported by Fundação para a Ciência e Tecnologia, Portugal (SFRH/BPD/26678/2006, SFRH/BD/45266/2008 and SFRH/BPD/68587/2010, IF/00442/2012). Waste glycerol was kindly provided by Torrejana, Fábrica de Biocombustíveis (Torres Vedras). Lignocellulosic hydrolysates were produced by biorefinery.de GmbH, Germany, in the framework of BUGWORKERS project.Institute for Biotechnology and Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Portugal / Biotrend S.A., Cantanhede, Portugal / Ecole Europeènne de Chimie, Polymères et Matériaux, Université de Strasbourg, Franc