Biological inorganic carbon fixation proceeds through a number of fundamentally different autotrophic pathways that are defined by specific key enzymatic reactions. Detection of the enzymatic genes ...in (meta)genomes is widely used to estimate the contribution of individual organisms or communities to primary production. Here we show that the sulfur-reducing anaerobic deltaproteobacterium
is capable of both acetate oxidation and autotrophic carbon fixation, with the tricarboxylic acid cycle operating either in the oxidative or reductive direction, respectively. Under autotrophic conditions, the enzyme citrate synthase cleaves citrate adenosine triphosphate independently into acetyl coenzyme A and oxaloacetate, a reaction that has been regarded as impossible under physiological conditions. Because this overlooked, energetically efficient carbon fixation pathway lacks key enzymes, it may function unnoticed in many organisms, making bioinformatical predictions difficult, if not impossible.
Fidelity and selectivity of DNA polymerases are critical determinants for the biology of life, as well as important tools for biotechnological applications. DNA polymerases catalyze the formation of ...DNA strands by adding deoxynucleotides to a primer, which is complementarily bound to a template. To ensure the integrity of the genome, DNA polymerases select the correct nucleotide and further extend the nascent DNA strand. Thus, DNA polymerase fidelity is pivotal for ensuring that cells can replicate their genome with minimal error. DNA polymerases are, however, further optimized for more specific biotechnological or diagnostic applications. Here we report on the semi-rational design of mutant libraries derived by saturation mutagenesis at single sites of a 3'-5'-exonuclease deficient variant of Thermococcus kodakaraensis DNA polymerase (KOD pol) and the discovery for variants with enhanced mismatch extension selectivity by screening. Sites of potential interest for saturation mutagenesis were selected by their proximity to primer or template strands. The resulting libraries were screened via quantitative real-time PCR. We identified three variants with single amino acid exchanges-R501C, R606Q, and R606W-which exhibited increased mismatch extension selectivity. These variants were further characterized towards their potential in mismatch discrimination. Additionally, the identified enzymes were also able to differentiate between cytosine and 5-methylcytosine. Our results demonstrate the potential in characterizing and developing DNA polymerases for specific PCR based applications in DNA biotechnology and diagnostics.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Arbuscular mycorrhiza (AM) symbioses contribute to global carbon cycles as plant hosts divert up to 20% of photosynthate to the obligate biotrophic fungi. Previous studies suggested carbohydrates as ...the only form of carbon transferred to the fungi. However,
fatty acid (FA) synthesis has not been observed in AM fungi in absence of the plant. In a forward genetic approach, we identified two
mutants defective in AM-specific paralogs of lipid biosynthesis genes (
and
). These mutants perturb fungal development and accumulation of emblematic fungal 16:1ω5 FAs. Using isotopolog profiling we demonstrate that
C patterns of fungal FAs recapitulate those of wild-type hosts, indicating cross-kingdom lipid transfer from plants to fungi. This transfer of labelled FAs was not observed for the AM-specific lipid biosynthesis mutants. Thus, growth and development of beneficial AM fungi is not only fueled by sugars but depends on lipid transfer from plant hosts.
Mycobacterium tuberculosis (Mtb) uses alveolar macrophages as primary host cells during infection. In response to an infection, macrophages switch from pyruvate oxidation to reduction of pyruvate ...into lactate. Lactate might present an additional carbon substrate for Mtb. Here, we demonstrate that Mtb can utilize L-lactate as sole carbon source for in vitro growth. Lactate conversion is strictly dependent on one of two potential L-lactate dehydrogenases. A knock-out mutant lacking lldD2 (Rv1872c) was unable to utilize L-lactate. In contrast, the lldD1 (Rv0694) knock-out strain was not affected in growth on lactate and retained full enzymatic activity. On the basis of labelling experiments using U-
C
-L-lactate as a tracer the efficient uptake of lactate by Mtb and its conversion into pyruvate could be demonstrated. Moreover, carbon flux from lactate into the TCA cycle, and through gluconeogenesis was observed. Gluconeogenesis during lactate consumption depended on the phosphoenolpyruvate carboxykinase, a key enzyme for intracellular survival, showing that lactate utilization requires essential metabolic pathways. We observed that the ΔlldD2 mutant was impaired in replication in human macrophages, indicating a critical role for lactate oxidation during intracellular growth.
Campylobacter jejuni is one of the leading infectious causes of food-borne illness around the world. Its ability to persistently colonize the intestinal tract of a broad range of hosts, including ...food-producing animals, is central to its epidemiology since most infections are due to the consumption of contaminated food products. Using a highly saturated transposon insertion library combined with next-generation sequencing and a mouse model of infection, we have carried out a comprehensive genome-wide analysis of the fitness determinants for growth in vitro and in vivo of a highly pathogenic strain of C. jejuni. A comparison of the C. jejuni requirements to colonize the mouse intestine with those necessary to grow in different culture media in vitro, combined with isotopologue profiling and metabolic flow analysis, allowed us to identify its metabolic requirements to establish infection, including the ability to acquire certain nutrients, metabolize specific substrates, or maintain intracellular ion homeostasis. This comprehensive analysis has identified metabolic pathways that could provide the basis for the development of novel strategies to prevent C. jejuni colonization of food-producing animals or to treat human infections.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Terpene synthases are versatile catalysts in all domains of life, catalyzing the formation of an enormous variety of different terpenoid secondary metabolites. Due to their diverse bioactive ...properties, terpenoids are of great interest as innovative ingredients in pharmaceutical and cosmetic applications. Recent advances in genome sequencing have led to the discovery of numerous terpene synthases, in particular in Basidiomycota like the wood rotting fungus Coniophora puteana, which further enhances the scope for the manufacture of terpenes for industrial purposes.
In this study we describe the identification of two novel (+)-δ-cadinol synthases from C. puteana, Copu5 and Copu9. The sesquiterpene (+)-δ-cadinol was previously shown to exhibit cytotoxic activity therefore having an application as possible, new, and sustainably sourced anti-tumor agent. In an Escherichia coli strain, optimized for sesquiterpene production, titers of 225 mg l
and 395 mg l
, respectively, could be achieved. Remarkably, both enzymes share the same product profile thereby representing the first two terpene synthases from Basidiomycota with identical product profiles. We solved the crystal structure of Copu9 in its closed conformation, for the first time providing molecular details of sesquiterpene synthase from Basidiomycota. Based on the Copu9 structure, we conducted structure-based mutagenesis of amino acid residues lining the active site, thereby altering the product profile. Interestingly, the mutagenesis study also revealed that despite the conserved product profiles of Copu5 and Copu9 different conformational changes may accompany the catalytic cycle of the two enzymes. This observation suggests that the involvement of tertiary structure elements in the reaction mechanism(s) employed by terpene synthases may be more complex than commonly expected.
The presented product selectivity and titers of Copu5 and Copu9 may pave the way towards a sustainable, biotechnological production of the potentially new bioactive (+)-δ-cadinol. Furthermore, Copu5 and Copu9 may serve as model systems for further mechanistic studies of terpenoid catalysis.
Despite the importance of small and medium-sized enterprises (SMEs) for the growth and development of companies, the high failure rate of these companies persists, and this correspondingly demands ...the attention of managers. Thus, to boost the company success rate, we may deploy certain approaches, for example predictive models, specifically for the SME innovation. This study aims to examine the variables that positively shape and contribute towards innovation of SMEs. Based on the Spinner innovation model, we explore how to predict the innovation of SMEs by applying the variables, namely knowledge creation, knowledge transfer, public knowledge management, private knowledge management and innovation. This study applied the data mining technique according to the cross industry standard process for data mining (CRISP-DM) method while the Statistical Package for the Social Sciences (SPSS_Version28) served to analyze the data collected from 208 SME employees in Oporto, Portugal. The results demonstrate how the Spinner innovation model positively influences the contributions of the SMEs. This SME-dedicated model fosters the creation of knowledge between internal and external interactions and increases the capacity to predict the SME innovation by 56%.
Porphyrins, corrins, and tetrapyrroles constitute macrocycles in essential biomolecules such as heme, chlorophyll, cobalamin, and cofactor F430. The chemical synthesis as well as the enzymatic ...synthesis of these macrocycles starts from pyrrole derivatives. We here show that pyrrole and dimethyl pyrrole can be formed under the simulated volcanic, hydrothermal conditions of Early Earth, starting from acetylene, propyne, and ammonium salts in the presence of NiS or CoS as catalysts.
Abstract
Microscopic compartmentalization is beneficial in synthetic chemistry and indispensable for the evolution of life to separate a reactive “inside” from a hydrolyzing “outside”. Here, we show ...compartmentalization in aqueous solution containing mixtures of fatty acids up to 19 carbon atoms which were synthesized by one-pot reactions of acetylene and carbon monoxide in contact with nickel sulfide at 105 °C, reaction requirements which are compatible to Hadean Early Earth conditions. Based on confocal, dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements, vesicle-like structures with diameters of 10–150 nm are formed after solvent extraction and resolubilisation. Moreover fluorescent dye was encapsulated into the structures proving their vesicular properties. This self-assembly could also have occurred on Early Earth as a crucial step in establishing simple membranes of proto-cells as a prerequisite in the evolution of metabolism and life.
Objective Using a salutogenic approach, this study aimed to identify similarities in the protective factors of nurses’ psychological Quality of Life (QoL) and professional wellbeing (PWB) in four ...countries and to assess their variability over time during the COVID-19 pandemic. Methods This multicentric study used a longitudinal design with three measurements points: Autumn 2021, spring 2022, and autumn 2022. The study consisted in a self-administered online questionnaire addressed to nurses working in hospitals. Across all measurement times, 3,310 observations were collected in France, 603 in Switzerland, 458 in Portugal, and 278 in Canada. The outcomes were psychological QoL and PWB, and several potential protective factors were used as determinants. Results Analyses revealed few changes over time in the outcomes. Across all countries, psychological QoL was associated positively with resilience and perceived social support, whereas PWB was associated positively with the ability to provide quality work and support from colleagues and superiors. Conclusion The findings of this study highlighted the potential of several factors protective of nurses’ psychological QoL and PWB. These should be fostered through policies and measures to support nurses.
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