40,844 research outputs found

    Anti-leishmanial evaluation of C2-aryl quinolines: Mechanistic insight on bioenergetics and sterol biosynthetic pathway of Leishmania braziliensis

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    A series of diverse simple C2-aryl quinolines was synthesized de novo via a straightforward synthesis based on the acid-catalyzed multicomponent imino Diels–Alder reactions. Seven selected quinolines were evaluated at different stages of Leishmania braziliensis parasite. Among them, the 6-ethyl-2-phenylquinoline 5f was able to inhibit the growth of promastigotes of this parasite without affecting the mammalian cells viability and decreasing the number of intracellular L. braziliensis amastigotes on BMDM macrophages. The mechanism of action studied for the selected compound consisted in: (1) alteration of parasite bioenergetics, by disrupting mitochondrial electrochemical potential and alkalinisation of acidocalcisomes, and (2) inhibition of ergosterol biosynthetic pathway in promastigote forms. These results validate the efficiency of quinoline molecules as leishmanicide compounds.Departamento Administrativo de Ciencia, Tecnología e Innovación [CO] Colciencias5507-543-31904Programa: Bioprospección y desarrollo de ingredientes naturales para las industrias cosmética, farmacéutica y de productos de aseo con base en la biodiversidad colombianan

    Investigations of metalloenzymes involved in bacterial natural product biosynthesis

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    Just as the structures of bacterial natural products have long been a source of inspiration for new developments in drug discovery and organic synthesis, so have investigations of their biosynthetic pathways led to the discovery of countless new enzyme-catalyzed reactions that open new frontiers of enzymology by defying conventional chemical intuition. Many of the most perplexing and challenging transformations are catalyzed by enzymes that use multiple redox-active metal ions to initiate and control radical chemistry, breaking inert C-H bonds and rearranging carbon skeletons with exquisite specificity. This work describes the characterization of two such reactions in the biosynthesis of two different natural products: fosfomycin, a clinically used phosphonate antibiotic discovered in 1969, and 3-thiaglutamate, a novel amino acid analog of unknown function discovered in 2019. In Chapter 2, newly available techniques were used to heterologously express and purify the fosfomycin biosynthetic enzyme Fom3 from Streptomyces wedmorensis, a methyltransferase in the radical S-adenosylmethionine (SAM) superfamily, with its required iron-sulfur cluster and cobalamin (B12) cofactors. The methyl transfer reaction catalyzed by Fom3 was then investigated in vitro using a combination of isotope labeling, enzymatic synthesis, liquid chromatography-mass spectrometry, and nuclear magnetic resonance methods. The results of these studies resolved a conflict in the literature about the stereochemistry of the product and supported a longstanding mechanistic hypothesis in which B12 serves as an intermediate methyl carrier, receiving a methyl group from SAM via a polar mechanism and transferring it to a substrate-derived species via a radical mechanism. In the collaborative study described in Chapter 3, the products of in vitro Fom3 reactions performed using SAM derived from methionine that was chirally labeled at the methyl position with deuterium and tritium were derivatized and analyzed, revealing overall retention of methyl stereochemistry during the reaction and therefore inversion during the radical methyl transfer step. The second reaction, oxidative excision of the β carbon from a peptide C-terminal cysteine residue by the complex of nonheme iron oxygenase TglH and putative substrate recognition protein TglI, was previously identified as a step in the biosynthetic pathway of the sulfur-substituted glutamate analog 3-thiaglutamate in a strain of Pseudomonas syringae. Chapter 4 describes initial studies of recognition of the 51-residue substrate peptide, TglACys, by the TglHI complex. In vitro activity assays of TglHI using synthetic TglACys variants with stretches of eight alanine mutations as well as C-terminal fragments of TglACys showed that no more than eighteen residues at the C-terminus of TglACys are required for modification by TglHI. Additionally, replacement of the C-terminal cysteine of TglACys by selenocysteine yielded a potent inhibitor of TglHI activity. Based on these results, several short, synthetically accessible peptide fragments were identified as candidate substrates and inhibitors for use in further mechanistic and structural studies.LimitedAuthor requested closed access (OA after 2yrs) in Vireo ETD syste

    O papel da aquisição de ferro na formação de biofilme e virulência de Staphylococcus epidermidis

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    Tese de Doutoramento em BioengenhariaStaphylococcus epidermidis é um importante microrganismo comensal da pele e mucosas em humanos, sendo também uma causa frequente de infeções potencialmente fatais em pacientes imunocomprometidos. A sua capacidade assinalável de formar biofilmes é amplamente apontada como o seu principal determinante patogénico. Apesar de se terem alcançado significativos avanços no entendimento dos seus mecanismos de formação de biofilme, esta espécie bacteriana continua a ser responsável por uma proporção significativa de infeções associadas aos cuidados de saúde, particularmente aquelas relacionadas com o uso de dispositivos médicos implantáveis. Este facto enfatiza a importância de um melhor entendimento do processo de formação de biofilme em S. epidermidis e dos fatores que o modulam. Tendo em conta que S. epidermidis enfrenta uma severa privação de ferro assim que entra na corrente sanguínea, testou-se a hipótese de que a aquisição de ferro desempenha um papel importante na formação de biofilme por S. epidermidis e na sua evasão ao sistema imune inato do hospedeiro. Os primeiros dados experimentais revelaram uma capacidade comprometida de S. epidermidis em formar biofilmes sob condições de restrição de ferro, principalmente atribuíveis a uma redução da taxa de crescimento, da viabilidade celular e da produção de adesina polissacarídica intercelular (PIA/PNAG). No entanto, e não obstante as condições desfavoráveis, S. epidermidis apresenta capacidade de proliferar neste cenário, implicando que esta espécie tem mecanismos dedicados à aquisição de ferro. Uma inspeção dos genomas de S. epidermidis disponíveis, juntamente com experiências de transcrição, levou à identificação de um grupo de genes putativamente envolvidos na aquisição de ferro. Seguindo uma abordagem mutagénica, foi demonstrado que uma dessas regiões genéticas (subsequentemente denominada sfaABCD) codifica a única via biossintética de sideróforos em S. epidermidis. Surpreendentemente, a eliminação de sfaABCD ou de dois outros loci que codificam um putativo transportador ABC de complexos ferro-sideróforo (htsABC e fhuA) resultou em estirpes mutantes seriamente incapacitadas de formar biofilme em condições de restrição de ferro. A eliminação de sfaABCD foi igualmente associada a uma replicação bacteriana mais baixa ou nula em macrófagos humanos e de ratinho, a uma inibição da produção de espécies reativas de oxigénio por neutrófilos e uma maior suscetibilidade à morte mediada por peróxido de hidrogénio. Os dados deste estudo mostram que a aquisição de ferro mediada por sideróforo é um importante processo para a formação de biofilmes por S. epidermidis sob condições de deficiência de ferro, mas também para a modulação da interação desta bactéria com o sistema imune inato do hospedeiro. Em última instância, estes resultados sugerem que a inibição deste processo de aquisição de ferro pode ser eficaz no tratamento de infeções por S. epidermidis associadas à formação de biofilme.Staphylococcus epidermidis is one of the most important commensal microorganisms of human skin and mucosae, but additionally it is often the cause of potential life-threatening infections in immunocompromised patients. A remarkable ability to form biofilms is widely regarded as its major known pathogenic determinant. Although a significant amount of knowledge on its biofilm formation mechanisms has been achieved, this bacterial species still accounts for a significant proportion of hospital-acquired infections, particularly those related with the use of implantable biomedical devices. This emphasizes the importance of a better understanding of biofilm formation in S. epidermidis and of the factors that modulate this process. Given that S. epidermidis faces severe deprivation of iron after entering the bloodstream, it was tested the hypothesis that iron acquisition plays an important role in S. epidermidis biofilm development and escape from the host innate immune system. The first experimental data revealed a compromised ability of S. epidermidis to form biofilms under ironrestricted conditions, mainly attributable to a reduced growth rate, cell viability, and production of polysaccharide intercellular adhesin (PIA/PNAG). However, and despite the unfavorable conditions, S. epidermidis is still able to proliferate in this scenario, implying that this species has dedicated mechanisms to acquire iron. An inspection of available S. epidermidis genomes along with transcriptional experiments has led to the identification of a group of genes putatively involved in iron acquisition, such as siderophore biosynthesis and uptake of iron-siderophore complexes. By following a mutagenesis approach, it was demonstrated that one of those genetic regions (subsequently termed sfaABCD) encodes the sole siderophore biosynthetic pathway in S. epidermidis. Strikingly, deletion of sfaABCD or two other loci putatively encoding an iron-siderophore ABC transporter (htsABC and fhuA) resulted in mutant strains severely incapacitated for biofilm formation in iron-restricted conditions. Deletion of sfaABCD was also associated with lower to null bacterial replication within murine and human macrophages, inhibition of reactive oxygen species generation by neutrophils, and higher susceptibility to hydrogen peroxidemediated killing. The data collected in this study show that siderophore-mediated iron acquisition is an important process for S. epidermidis to form biofilms under conditions of iron starvation, but also for the modulation of the interaction of this bacterium with the host innate immune system. Ultimately, these results suggest that inhibiting this iron acquisition process may be effective in the treatment of biofilm-associated S. epidermidis infections.This study was supported by the Portuguese Foundation for Science and Technology (FCT) through an individual PhD scholarship (SFRH/BD/101399/2014), the funded project PTDC/BIAMOL/29553/2017, under the scope of COMPETE2020 (POCI-01-0145-FEDER-029553), and the strategic funding of UID/BIO/04469/2019 unit. This study was also supported through funds from the German Research Council (DFG) and the Damp Foundation

    Structure, Function, and Biosynthesis of the Coenzyme Methylofuran

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    Methylotrophy is a metabolic trait that allows certain organisms to grow on carbon substrates without any C-C bonds, such as methanol or methane. The conversion and oxidation of these compounds usually proceeds via the toxic intermediate formaldehyde and is assisted by coenzymes that act as carriers for one-carbon units. Most methylotrophic bacteria rely on the tetrahydromethanopterin(H4MPT)-linked pathway for the oxidation of formaldehyde. This pathway involves not only H4MPT but also requires an analog of methanofuran (MFR), a coenzyme originally thought to be unique to methanogenic archaea. Previous biochemical and genetic evidence suggested that the structure of bacterial MFR must be similar to the one from methanogens; however, its purification and structural analysis remained challenging. In this thesis, the isolation and structural elucidation of MFR from the well-studied methylotroph Methylorubrum extorquens is described. Using preparative chromatography combined with high-resolution mass spectrometry and NMR, the bacterial analog of MFR—which was termed methylofuran (MYFR)—was identified and characterized. The core structure of MYFR was found to be identical to archaeal MFR, except for the presence of a tyrosine instead of a tyramine residue. Surprisingly, an unprecedented polyglutamate side chain consisting of up to 24 glutamate residues was connected to the tyrosine residue. NMR analysis further revealed that the glutamates in MYFR showed both α- and γ-linkages. In the H4MPT-linked pathway, MYFR is required by the formyltransferase/hydrolase complex (Fhc), where the coenzyme functions as a carrier of formyl units. To investigate whether the unusually large polyglutamate side chain of MYFR plays a role in the interaction with Fhc, the structure of the enzyme-coenzyme complex was solved at 3.1 Å. Interestingly, MYFR is bound as a non-covalent prosthetic group and the polyglutamate side chain tightly interacts with a large patch of positively charged residues of Fhc. This binding site is centrally located between the two active sites for formyl transfer and hydrolysis, thus suggesting that the polyglutamate chain functions as a flexible linker that allows the formyl-carrying aminomethylfuran moiety to reach both active sites of the bifunctional enzyme complex. Formyl units can thus be efficiently shuttled between the two active sites, without the need for MYFR to dissociate from Fhc. The electron density of Fhc-bound MYFR additionally revealed that the polyglutamate side chain of MYFR is branched, i.e. some glutamates are involved in isopeptide bonds with other glutamates. The branched polyglutamate structure might be required to support the strong interaction with Fhc and seems to be a unique feature of MYFR that is not present in archaeal MFRs. Since the H4MPT-linked pathway is widespread in Bacteria, MYFR is expected to be present in many strains. To determine whether there is structural diversity of MYFR, a survey comprising 12 proteobacterial strains was performed. Only in two strains, MYFR in the form present in M. extorquens was found. In six strains, a second type of MYFR was discovered which contained a tyramine instead of the tyrosine residue. For four strains, no MYFR could be identified. Interestingly, the number of glutamates in MYFR was not conserved across strains. While some had similar numbers as found in M. extorquens (around 16–20), two strains contained MYFR with 12 or fewer glutamates. The complex structure of the polyglutamate side chain of MYFR posed the question of its biosynthetic origin. In Proteobacteria, many genes essential for H4MPT-linked methylotrophy have previously been identified. For several of them, the function remained unknown. To identify genes involved in MYFR biosynthesis, strains with deletions in three of these genes (orf5, orfY, and orf17) were analyzed. All three mutants were unable to produce functional MYFR; however, the Dorf5 strain was accumulating MYFR-Glu2, a short intermediate of MYFR. Overexpression of orf5 in M. extorquens led to a significant increase in the number of glutamates attached to MYFR, as up to 40 glutamates were detected. The enzyme was thus renamed to MyfA, highlighting that this is the first enzyme discovered to be specifically involved in MYFR biosynthesis. In vitro assays with purified MyfA revealed de novo polyglutamate synthesis activity using L/D-glutamate and L-glutamine as substrates. Unexpectedly, L-glutamine was found to be an essential component of the assay. Assays with labeled glutamine showed that glutamine was serving as a source of glutamyl units for the incorporation into polyglutamates. The incorporation presumably took place after conversion to glutamate, as MyfA also showed glutaminase activity. Additionally, MyfA was able to cleave short glutamate containing peptides, thus also acting as a peptidase. These findings trigger the question of how the in vitro activities relate to MYFR biosynthesis in vivo. Taken together, the results obtained in this thesis shed light on various aspects of the structure, function, and biosynthesis of MYFR. They provide an in-depth understanding of the role MYFR plays in theH4MPTlinked pathway, thus expanding our knowledge about the biochemical basis of methylotrophy. The complex structure of MYFR that was revealed in this thesis, combined with the enzymes involved in its biosynthesis, will provide exciting opportunities for future research

    Ecological successions throughout the desiccation of Tirez lagoon (Spain) as an astrobiological time-analog for wet-to-dry transitions on Mars

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    Tirez was a small and seasonal endorheic athalassohaline lagoon that was located in central Spain. In recent years, the lagoon has totally dried out, offering for the first time the opportunity to analyze its desiccation process as a “time-analog” to similar events occurred in paleolakes with varying salinity during the wet-to-dry transition on early Mars. On the martian cratered highlands, an early period of water ponding within enclosed basins evolved to a complete desiccation of the lakes, leading to deposition of evaporitic sequences during the Noachian and into the Late Hesperian. As Tirez also underwent a process of desiccation, here we describe (i) the microbial ecology of Tirez when the lagoon was still active 20 years ago, with prokaryotes adapted to extreme saline conditions; (ii) the composition of the microbial community in the dried lake sediments today, in many case groups that thrive in sediments of extreme environments; and (iii) the molecular and isotopic analysis of the lipid biomarkers that can be recovered from the sediments today. We discuss the implications of these results to better understanding the ecology of possible Martian microbial communities during the wet-to-dry transition at the end of the Hesperian, and how they may inform about research strategies to search for possible biomarkers in Mars after all the water was los

    Holistic analysis of lysine acetylation in aquaculture pathogenic bacteria Vibrio alginolyticus under bile salt stress

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    Lysine acetylation modification is a dynamic and reversible post-translational modification, which plays an important role in the metabolism and pathogenicity of pathogenic bacteria. Vibrio alginolyticus is a common pathogenic bacterium in aquaculture, and bile salt can trigger the expression of bacterial virulence. However, little is known about the function of lysine acetylation in V. alginolyticus under bile salt stress. In this study, 1,315 acetylated peptides on 689 proteins were identified in V. alginolyticus under bile salt stress by acetyl-lysine antibody enrichment and high-resolution mass spectrometry. Bioinformatics analysis found that the peptides motif ****A*Kac**** and *******Kac****A* were highly conserved, and protein lysine acetylation was involved in regulating various cellular biological processes and maintaining the normal life activities of bacteria, such as ribosome, aminoacyl-tRNA biosynthesis, fatty acid metabolism, two-component system, and bacterial secretion system. Further, 22 acetylated proteins were also found to be related to the virulence of V. alginolyticus under bile salt stress through secretion system, chemotaxis and motility, and adherence. Finally, comparing un-treated and treated with bile salt stress lysine acetylated proteins, it was found that there were 240 overlapping proteins, and found amino sugar and nucleotide sugar metabolism, beta-Lactam resistance, fatty acid degradation, carbon metabolism, and microbial metabolism in diverse environments pathways were significantly enriched in bile salt stress alone. In conclusion, this study is a holistic analysis of lysine acetylation in V. alginolyticus under bile salt stress, especially many virulence factors have also acetylated

    Pollution-induced community tolerance in freshwater biofilms – from molecular mechanisms to loss of community functions

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    Exposure to herbicides poses a threat to aquatic biofilms by affecting their community structure, physiology and function. These changes render biofilms to become more tolerant, but on the downside community tolerance has ecologic costs. A concept that addresses induced community tolerance to a pollutant (PICT) was introduced by Blanck and Wängberg (1988). The basic principle of the concept is that microbial communities undergo pollution-induced succession when exposed to a pollutant over a long period of time, which changes communities structurally and functionally and enhancing tolerance to the pollutant exposure. However, the mechanisms of tolerance and the ecologic consequences were hardly studied up to date. This thesis addresses the structural and functional changes in biofilm communities and applies modern molecular methods to unravel molecular tolerance mechanisms. Two different freshwater biofilm communities were cultivated for a period of five weeks, with one of the communities being contaminated with 4 μg L-1 diuron. Subsequently, the communities were characterized for structural and functional differences, especially focusing on their crucial role of photosynthesis. The community structure of the autotrophs was assessed using HPLC-based pigment analysis and their functional alterations were investigated using Imaging-PAM fluorometry to study photosynthesis and community oxygen profiling to determine net primary production. Then, the molecular fingerprints of the communities were measured with meta-transcriptomics (RNA-Seq) and GC-based community metabolomics approaches and analyzed with respect to changes in their molecular functions. The communities were acute exposed to diuron for one hour in a dose-response design, to reveal a potential PICT and uncover related adaptation to diuron exposure. The combination of apical and molecular methods in a dose-response design enabled the linkage of functional effects of diuron exposure and underlying molecular mechanisms based on a sensitivity analysis. Chronic exposure to diuron impaired freshwater biofilms in their biomass accrual. The contaminated communities particularly lost autotrophic biomass, reflected by the decrease in specific chlorophyll a content. This loss was associated with a change in the molecular fingerprint of the communities, which substantiates structural and physiological changes. The decline in autotrophic biomass could be due to a primary loss of sensitive autotrophic organisms caused by the selection of better adapted species in the course of chronic exposure. Related to this hypothesis, an increase in diuron tolerance has been detected in the contaminated communities and molecular mechanisms facilitating tolerance have been found. It was shown that genes of the photosystem, reductive-pentose phosphate cycle and arginine metabolism were differentially expressed among the communities and that an increased amount of potential antioxidant degradation products was found in the contaminated communities. This led to the hypothesis that contaminated communities may have adapted to oxidative stress, making them less sensitive to diuron exposure. Moreover, the photosynthetic light harvesting complex was altered and the photoprotective xanthophyll cycle was increased in the contaminated communities. Despite these adaptation strategies, the loss of autotrophic biomass has been shown to impair primary production. This impairment persisted even under repeated short-term exposure, so that the tolerance mechanisms cannot safeguard primary production as a key function in aquatic systems.:1. The effect of chemicals on organisms and their functions .............................. 1 1.1 Welcome to the anthropocene .......................................................................... 1 1.2 From cellular stress responses to ecosystem resilience ................................... 3 1.2.1 The individual pursuit for homeostasis ....................................................... 3 1.2.2 Stability from diversity ................................................................................. 5 1.3 Community ecotoxicology - a step forward in monitoring the effects of chemical pollution? ................................................................................................................. 6 1.4 Functional ecotoxicological assessment of microbial communities ................... 9 1.5 Molecular tools – the key to a mechanistic understanding of stressor effects from a functional perspective in microbial communities? ...................................... 12 2. Aims and Hypothesis ......................................................................................... 14 2.1 Research question .......................................................................................... 14 2.2 Hypothesis and outline .................................................................................... 15 2.3 Experimental approach & concept .................................................................. 16 2.3.1 Aquatic freshwater biofilms as model community ..................................... 16 2.3.2 Diuron as model herbicide ........................................................................ 17 2.3.3 Experimental design ................................................................................. 18 3. Structural and physiological changes in microbial communities after chronic exposure - PICT and altered functional capacity ................................................. 21 3.1 Introduction ..................................................................................................... 21 3.2 Methods .......................................................................................................... 23 3.2.1 Biofilm cultivation ...................................................................................... 23 3.2.2 Dry weight and autotrophic index ............................................................. 23 3.2.4 Pigment analysis of periphyton ................................................................. 23 3.2.4.1 In-vivo pigment analysis for community characterization ....................... 24 3.2.4.2 In-vivo pigment analysis based on Imaging-PAM fluorometry ............... 24 3.2.4.3 In-vivo pigment fluorescence for tolerance detection ............................. 26 3.2.4.4 Ex-vivo pigment analysis by high-pressure liquid-chromatography ....... 27 3.2.5 Community oxygen metabolism measurements ....................................... 28 3.3 Results and discussion ................................................................................... 29 3.3.1 Comparison of the structural community parameters ............................... 29 3.3.2 Photosynthetic activity and primary production of the communities after selection phase ................................................................................................. 33 3.3.3 Acquisition of photosynthetic tolerance .................................................... 34 3.3.4 Primary production at exposure conditions ............................................... 36 3.3.5 Tolerance detection in primary production ................................................ 37 3.4 Summary and Conclusion ........................................................................... 40 4. Community gene expression analysis by meta-transcriptomics ................... 41 4.1 Introduction to meta-transcriptomics ............................................................... 41 4.2. Methods ......................................................................................................... 43 4.2.1 Sampling and RNA extraction................................................................... 43 4.2.2 RNA sequencing analysis ......................................................................... 44 4.2.3 Data assembly and processing................................................................. 45 4.2.4 Prioritization of contigs and annotation ..................................................... 47 4.2.5 Sensitivity analysis of biological processes .............................................. 48 4.3 Results and discussion ................................................................................... 48 4.3.1 Characterization of the meta-transcriptomic fingerprints .......................... 49 4.3.2 Insights into community stress response mechanisms using trend analysis (DRomic’s) ......................................................................................................... 51 4.3.3 Response pattern in the isoform PS genes .............................................. 63 4.5 Summary and conclusion ................................................................................ 65 5. Community metabolome analysis ..................................................................... 66 5.1 Introduction to community metabolomics ........................................................ 66 5.2 Methods .......................................................................................................... 68 5.2.1 Sampling, metabolite extraction and derivatisation................................... 68 5.2.2 GC-TOF-MS analysis ............................................................................... 69 5.2.3 Data processing and statistical analysis ................................................... 69 5.3 Results and discussion ................................................................................... 70 5.3.1 Characterization of the metabolic fingerprints .......................................... 70 5.3.2 Difference in the metabolic fingerprints .................................................... 71 5.3.3 Differential metabolic responses of the communities to short-term exposure of diuron ............................................................................................................ 73 5.4 Summary and conclusion ................................................................................ 78 6. Synthesis ............................................................................................................. 79 6.1 Approaches and challenges for linking molecular data to functional measurements ...................................................................................................... 79 6.2 Methods .......................................................................................................... 83 6.2.1 Summary on the data ............................................................................... 83 6.2.2 Aggregation of molecular data to index values (TELI and MELI) .............. 83 6.2.3 Functional annotation of contigs and metabolites using KEGG ................ 83 6.3 Results and discussion ................................................................................... 85 6.3.1 Results of aggregation techniques ........................................................... 85 6.3.2 Sensitivity analysis of the different molecular approaches and endpoints 86 6.3.3 Mechanistic view of the molecular stress responses based on KEGG functions ............................................................................................................ 89 6.4 Consolidation of the results – holistic interpretation and discussion ............... 93 6.4.1 Adaptation to chronic diuron exposure - from molecular changes to community effects.............................................................................................. 93 6.4.2 Assessment of the ecological costs of Pollution-induced community tolerance based on primary production ............................................................. 94 6.5 Outlook ............................................................................................................ 9

    A combinatorial DNA assembly approach to biosynthesis of N-linked glycans in E. coli

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    Glycoengineering of recombinant glycans and glycoconjugates is a rapidly evolving field. However, the production and exploitation of glycans has lagged behind that of proteins and nucleic acids. Biosynthetic glycoconjugate production requires the coordinated cooperation of three key components within a bacterial cell: a substrate protein, a coupling oligosaccharyltransferase, and a glycan biosynthesis locus. While the acceptor protein and oligosaccharyltransferase are the products of single genes, the glycan is a product of a multigene metabolic pathway. Typically, the glycan biosynthesis locus is cloned and transferred en bloc from the native organism to a suitable Escherichia coli strain. However, gene expression within these pathways has been optimized by natural selection in the native host and is unlikely to be optimal for heterologous production in an unrelated organism. In recent years, synthetic biology has addressed the challenges in heterologous expression of multigene systems by deconstructing these pathways and rebuilding them from the bottom up. The use of DNA assembly methods allows the convenient assembly of such pathways by combining defined parts with the requisite coding sequences in a single step. In this study, we apply combinatorial assembly to the heterologous biosynthesis of the Campylobacter jejuni  N-glycosylation (pgl) pathway in E. coli. We engineered reconstructed biosynthesis clusters that faithfully reproduced the C. jejuni heptasaccharide glycan. Furthermore, following a single round of combinatorial assembly and screening, we identified pathway clones that outperform glycan and glycoconjugate production of the native unmodified pgl cluster. This platform offers a flexible method for optimal engineering of glycan structures in E. coli

    The enterovirus genome can be translated in an IRES-independent manner that requires the initiation factors eIF2A/eIF2D

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    RNA recombination in positive-strand RNA viruses is a molecular-genetic process, which permits the greatest evolution of the genome and may be essential to stabilizing the genome from the deleterious consequences of accumulated mutations. Enteroviruses represent a useful system to elucidate the details of this process. On the biochemical level, it is known that RNA recombination is catalyzed by the viral RNA-dependent RNA polymerase using a template-switching mechanism. For this mechanism to function in cells, the recombining genomes must be located in the same subcellular compartment. How a viral genome is trafficked to the site of genome replication and recombination, which is membrane associated and isolated from the cytoplasm, is not known. We hypothesized that genome translation was essential for colocalization of genomes for recombination. We show that complete inactivation of internal ribosome entry site (IRES)-mediated translation of a donor enteroviral genome enhanced recombination instead of impairing it. Recombination did not occur by a nonreplicative mechanism. Rather, sufficient translation of the nonstructural region of the genome occurred to support subsequent steps required for recombination. The noncanonical translation initiation factors, eIF2A and eIF2D, were required for IRES-independent translation. Our results support an eIF2A/eIF2D-dependent mechanism under conditions in which the eIF2-dependent mechanism is inactive. Detection of an IRES-independent mechanism for translation of the enterovirus genome provides an explanation for a variety of debated observations, including nonreplicative recombination and persistence of enteroviral RNA lacking an IRES. The existence of an eIF2A/eIF2D-dependent mechanism in enteroviruses predicts the existence of similar mechanisms in other viruses
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