Verbesserung der Produktivität von Shewanella oneidensis in bioelektrochemischen Systemen durch erhöhte Biofilmbildung

Die begrenzte Verfügbarkeit fossiler Ressourcen erfordert die Umstellung industrieller Prozesse hin zur Verwendung nachhaltiger Rohstoffe. Eine Möglichkeit der nachhaltigen Synthese von Basischemikalien bietet dabei die Anoden-assistierte Fermentation. Hierbei fungieren Bakterien als Ganzzellkatalysatoren, die den Elektronenüberschuss ihres Katabolismus auf eine Anode in einem bioelektrochemischen System (BES) übertragen. Der Modellorganismus für den extrazellulären Elektronentransfer, Shewanella oneidensis, ist ein vielversprechender Kandidat zur mikrobiellen Synthese von Basischemikalien in BES. S. oneidensis ist von Natur aus in der Lage, Elektronen auf Anoden zu übertragen und daher für die Anwendung in BES prädestiniert. Durch die einfache genetische Zugänglichkeit ist zudem die Anpassung an verschiedene Produktionsbedingungen möglich. Eine notwendige Optimierung des Organismus mit Blick auf eine industrielle Anwendung ist eine verbesserte Biofilmbildung. Dadurch ließen sich möglicherweise die erreichbaren Stromdichten un damit die Raum-Zeit-Ausbeuten von BES drastisch steigern. In dieser Arbeit wurde die Funktionsweise der Riboflavin-induzierten Biofilmbildung von S. oneidensis untersucht, um die molekularen Mechanismen der Biofilmbildung besser zu verstehen und eine gezielte Biofilminduktion zu ermöglichen. Dabei wurde die Funktion von Riboflavin als Quorum sensing-Molekül in S. oneidensis aufgeklärt. Riboflavin induziert konzentrationsabhängig die Expression des Gens der Ornithin-Decarboxylase SpeC. Die Überexpression von SpeC führt wiederum durch Protein-Protein Interaktion zu einer vermehrten Biofilmbildung einhergehend mit einer gesteigerten Stromdichte in BES. Des Weiteren wurde die natürliche Riboflavin-Produktion von S. oneidensis untersucht und festgestellt, dass die zur Induktion des Biofilms nötige Riboflavin-Konzentration unter nativen Bedingungen durch den Organismus synthetisiert werden kann. Neben der Biofilmbildung ist die Erweiterung des Substrat- und Produktspektrums von S. oneidensis Gegenstand der aktuellen Forschung. Die Erweiterung des Substratspektrums um Glukose sowie des Produktspektrums um Acetoin konnten bereits etabliert werden, gingen allerdings mit geringen Umsatzraten einher. Um diese zu erhöhen und damit die Raum-Zeit-Ausbeute zu verbessern, wurde in dieser Arbeit ein optimierter Produktionsstamm erstellt. Dieser sollte den Glukoseverbrauch mit der Acetoinproduktion vereinen und durch eine Langzeitadaptation erhöhte Raum-Zeit-Ausbeuten ermöglichen. Sowohl die Stromdichte, und damit der Glukoseverbrauch, als auch die Biofilmbildung konnten durch den neuen Produktionsstamm nach der Langzeitadaptation erfolgreich verbessert werden. Allerdings war die Produktion von Acetoin nicht möglich, da der Kohlenstoff in die Produktion von Laktat umgeleitet wurde. Durch die Genomanalyse zweier adaptierter Klone konnte ein detaillierter Einblick in die Regulation der Biofilmbildung unter den Adaptationsbedingungen gewonnen werden. Mithilfe dieser Informationen könnte bei einem zukünftigen Produktionsstamm gezielt die Biofilmbildung induziert werden und so durch eine verbesserte Anodeninteraktion eine industriell lohnenswerte Raum-Zeit-Ausbeute erzielt werden

Exploring the Effects of bolA in Biofilm Formation and Current Generation by Shewanella oneidensis MR-1

Microbial electrochemical technologies (METs) have emerged in recent years as a promising alternative green source of energy, with microbes consuming organic matter to produce energy or valuable byproducts. It is the ability of performing extracellular electron transfer that allows these microbes to exchange electrons with an electrode in these systems. The low levels of current achieved have been the limiting factor for the large-scale application of METs. Shewanella oneidensis MR-1 is one of the most studied electroactive organisms regarding extracellular electron transfer, and it has been shown that biofilm formation is a key factor for current generation. The transcription factor bolA has been identified as a central player in biofilm formation in other organisms, with ist overexpression leading to increased biofilm. In this work we explore the effect of this gene in biofilm formation and current production by S. oneidensis MR-1. Our results demonstrate that an increased biofilm formation and consequent current generation was achieved by the overexpression of this gene. This information is crucial to optimize electroactive organisms toward their practical application in METs

Electron transfer of extremophiles in bioelectrochemical systems

The interaction of bacteria and archaea with electrodes is a relatively new research field which spans from fundamental to applied research and influences interdisciplinary research in the fields of microbiology, biochemistry, biotechnology as well as process engineering. Although a substantial understanding of electron transfer processes between microbes and anodes and between microbes and cathodes has been achieved in mesophilic organisms, the mechanisms used by microbes under extremophilic conditions are still in the early stages of discovery. Here, we review our current knowledge on the biochemical solutions that evolved for the interaction of extremophilic organisms with electrodes. To this end, the available knowledge on pure cultures of extremophilic microorganisms has been compiled and the study has been extended with the help of bioinformatic analyses on the potential distribution of different electron transfer mechanisms in extremophilic microorganisms

Soluble versions of outer membrane cytochromes function as exporters for heterologously produced cargo proteins

This study reveals that it is possible to secrete truncated versions of outer membrane cytochromes into the culture supernatant and that these proteins can provide a basis for the export of heterologously produced proteins. Different soluble and truncated versions of the outer membrane cytochrome MtrF were analyzed for their suitability to be secreted. A protein version with a very short truncation of the N-terminus to remove the recognition sequence for the addition of a lipid anchor is secreted efficiently to the culture supernatant, and moreover this protein could be further truncated by a deletion of 160 amino acid and still is detectable in the supernatant. By coupling a cellulase to this soluble outer membrane cytochrome, the export efficiency was measured by means of relative cellulase activity. We conclude that outer membrane cytochromes of S. oneidensis can be applied as transporters for the export of target proteins into the medium using the type II secretion pathway

Extracellular riboflavin induces anaerobic biofilm formation in Shewanella oneidensis

Background Some microorganisms can respire with extracellular electron acceptors using an extended electron transport chain to the cell surface. This process can be applied in bioelectrochemical systems in which the organisms produce an electrical current by respiring with an anode as electron acceptor. These organisms apply flavin molecules as cofactors to facilitate one-electron transfer catalyzed by the terminal reductases and in some cases as endogenous electron shuttles. Results In the model organism Shewanella oneidensis, riboflavin production and excretion trigger a specific biofilm formation response that is initiated at a specific threshold concentration, similar to canonical quorum-sensing molecules. Riboflavin-mediated messaging is based on the overexpression of the gene encoding the putrescine decarboxylase speC which leads to posttranscriptional overproduction of proteins involved in biofilm formation. Using a model of growth-dependent riboflavin production under batch and biofilm growth conditions, the number of cells necessary to produce the threshold concentration per time was deduced. Furthermore, our results indicate that specific retention of riboflavin in the biofilm matrix leads to localized concentrations, which by far exceed the necessary threshold value. Conclusion This study describes a new quorum-sensing mechanism in S. oneidensis. Biofilm formation of S. oneidensis is induced by low concentrations of riboflavin resulting in an upregulation of the ornithine-decarboxylase speC. The results can be applied for the development of strains catalyzing increased current densities in bioelectrochemical systems

RNA sequencing and lipidomics uncovers novel pathomechanisms in recessive X-linked ichthyosis

Recessive X-linked ichthyosis (RXLI), a genetic disorder caused by deletion or point mutations of the steroid sulfatase (STS) gene, is the second most common form of ichthyosis. It is a disorder of keratinocyte cholesterol sulfate retention and the mechanism of extracutaneous phenotypes such as corneal opacities and attention deficit hyperactivity disorder are poorly understood. To understand the pathomechanisms of RXLI, the transcriptome of differentiated primary keratinocytes with STS knockdown was sequenced. The results were validated in a stable knockdown model of STS, to confirm STS specificity, and in RXLI skin. The results show that there was significantly reduced expression of genes related to epidermal differentiation and lipid metabolism, including ceramide and sphingolipid synthesis. In addition, there was significant downregulation of aldehyde dehydrogenase family members and the oxytocin receptor which have been linked to corneal transparency and behavioural disorders respectively, both of which are extracutaneous phenotypes of RXLI. These data provide a greater understanding of the causative mechanisms of RXLI’s cutaneous phenotype, and show that the keratinocyte transcriptome and lipidomics can give novel insights into the phenotype of patients with RXLI

Helios expression coordinates the development of a subset of striatopallidal medium spiny neurons

Here we unravel the mechanism of action of Helios (He) during the development of striatal medium spiny neurons (MSNs). He regulates the second wave of striatal neurogenesis involved in the generation of striatopallidal neurons that express dopamine 2 receptor (D2R) and enkephalin (ENK). To exert this effect He is expressed in neural progenitor cells (NPCs) retaining them into the G1/G0 phase of the cell cycle. Thus, the lack of He produces an increase of S-phase entry and S-phase length of NPCs which in turn impairs striatal neurogenesis and produces an accumulation of the number of cycling NPCs in the germinal zone (GZ) that end up dying at postnatal stages. Therefore, He-/- mice show a reduction in the number of Dorso-Medial Striatal MSNs in the adulthood that produces deficits in motor skills acquisition. In addition, overexpression of He in NPCs induce DARPP32 phenotype when transplanted in mouse striatum.Present findings demonstrate that He is involved in the correct development of a subset of striatopallidal MSNs and reveal new cellular mechanisms for neuronal development

ProMeQuaLab - Projeto de Melhoria da Qualidade Laboratorial para Países de Língua Portuguesa: trabalhos realizados e em curso 2017

Apresentação sobre os trabalhos realizados e em curso no âmbito do projeto de Melhoria da Qualidade Laboratorial para Países de Língua Portuguesa (ProMeQuaLab).N/

A time-resolved proteomic and prognostic map of COVID-19

COVID-19 is highly variable in its clinical presentation, ranging from asymptomatic infection to severe organ damage and death. We characterized the time-dependent progression of the disease in 139 COVID-19 inpatients by measuring 86 accredited diagnostic parameters, such as blood cell counts and enzyme activities, as well as untargeted plasma proteomes at 687 sampling points. We report an initial spike in a systemic inflammatory response, which is gradually alleviated and followed by a protein signature indicative of tissue repair, metabolic reconstitution, and immunomodulation. We identify prognostic marker signatures for devising risk-adapted treatment strategies and use machine learning to classify therapeutic needs. We show that the machine learning models based on the proteome are transferable to an independent cohort. Our study presents a map linking routinely used clinical diagnostic parameters to plasma proteomes and their dynamics in an infectious disease

Clinical and virological characteristics of hospitalised COVID-19 patients in a German tertiary care centre during the first wave of the SARS-CoV-2 pandemic: a prospective observational study

Purpose: Adequate patient allocation is pivotal for optimal resource management in strained healthcare systems, and requires detailed knowledge of clinical and virological disease trajectories. The purpose of this work was to identify risk factors associated with need for invasive mechanical ventilation (IMV), to analyse viral kinetics in patients with and without IMV and to provide a comprehensive description of clinical course. Methods: A cohort of 168 hospitalised adult COVID-19 patients enrolled in a prospective observational study at a large European tertiary care centre was analysed. Results: Forty-four per cent (71/161) of patients required invasive mechanical ventilation (IMV). Shorter duration of symptoms before admission (aOR 1.22 per day less, 95% CI 1.10-1.37, p < 0.01) and history of hypertension (aOR 5.55, 95% CI 2.00-16.82, p < 0.01) were associated with need for IMV. Patients on IMV had higher maximal concentrations, slower decline rates, and longer shedding of SARS-CoV-2 than non-IMV patients (33 days, IQR 26-46.75, vs 18 days, IQR 16-46.75, respectively, p < 0.01). Median duration of hospitalisation was 9 days (IQR 6-15.5) for non-IMV and 49.5 days (IQR 36.8-82.5) for IMV patients. Conclusions: Our results indicate a short duration of symptoms before admission as a risk factor for severe disease that merits further investigation and different viral load kinetics in severely affected patients. Median duration of hospitalisation of IMV patients was longer than described for acute respiratory distress syndrome unrelated to COVID-19