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From dark to light
(2016)
Dendritic cells (DCs) are the cutting edge in innate and adaptive immunity. The major functions of these antigen presenting cells are the capture, endosomal processing and presentation of antigens, providing them an exclusive ability to provoke adaptive immune responses and to induce and control tolerance. Immature DCs capture and process antigens, migrate towards secondary lymphoid organs where they present antigens to naive T cells in a well synchronized sequence of procedures referred to as maturation. Indeed, recent research indicated that sphingolipids are modulators of essential steps in DC homeostasis. It has been recognized that sphingolipids not only modulate the development of DC subtypes from precursor cells but also influence functional activities of DCs such as antigen capture, and cytokine profiling. Thus, it is not astonishing that sphingolipids and sphingolipid metabolism play a substantial role in inflammatory diseases that are modulated by DCs. Here we highlight the function of sphingosine 1-phosphate (S1P) on DC homeostasis and the role of SIP and SW metabolism in inflammatory diseases.
Microplastics (MP) provide a unique and extensive surface for microbial colonization in aquatic ecosystems. The formation of microorganism-microplastic complexes, such as biofilms, maximizes the degradation of organic matter and horizontal gene transfer. In this context, MP affect the structure and function of microbial communities, which in turn render the physical and chemical fate of MP. This new paradigm generates challenges for microbiology, ecology, and ecotoxicology. Dispersal of MP is concomitant with that of their associated microorganisms and their mobile genetic elements, including antibiotic resistance genes, islands of pathogenicity, and diverse metabolic pathways. Functional changes in aquatic microbiomes can alter carbon metabolism and food webs, with unknown consequences on higher organisms or human microbiomes and hence health. Here, we examine a variety of effects of MP pollution from the microbial ecology perspective, whose repercussions on aquatic ecosystems begin to be unraveled. (C) 2018 Elsevier B.V. All rights reserved.
Pollution by microplastics in aquatic ecosystems is accumulating at an unprecedented scale, emerging as a new surface for biofilm formation and gene exchange. In this study, we determined the permissiveness of aquatic bacteria towards a model antibiotic resistance plasmid, comparing communities that form biofilms on microplastics vs. those that are free-living. We used an exogenous and red-fluorescent E. coli donor strain to introduce the green-fluorescent broad-host-range plasmid pKJKS which encodes for trimethoprim resistance. We demonstrate an increased frequency of plasmid transfer in bacteria associated with microplastics compared to bacteria that are free-living or in natural aggregates. Moreover, comparison of communities grown on polycarbonate filters showed that increased gene exchange occurs in a broad range of phylogenetically-diverse bacteria. Our results indicate horizontal gene transfer in this habitat could distinctly affect the ecology of aquatic microbial communities on a global scale. The spread of antibiotic resistance through microplastics could also have profound consequences for the evolution of aquatic bacteria and poses a neglected hazard for human health.
Heterotrophic microbes with the capability to process considerable amounts of organic matter can colonize microplastic particles (MP) in aquatic ecosystems. Weather colonization of microorganisms on MP will alter ecological niche and functioning of microbial communities remains still unanswered. Therefore, we compared the functional diversity of biofilms on microplastics when incubated in three lakes in northeastern Germany differing in trophy and limnological features. For all lakes, we compared heterotrophic activities of MP biofilms with those of microorganisms in the surrounding water by using Biolog (R) EcoPlates and assessed their oxygen consumption in microcosm assays with and without MP. The present study found that the total biofilm biomass was higher in the oligo-mesotrophic and dystrophic lakes than in the eutrophic lake. In all lakes, functional diversity profiles of MP biofilms consistently differed from those in the surrounding water. However, solely in the oligo-mesotrophic lake MP biofilms had a higher functional richness compared to the ambient water. These results demonstrate that the functionality and hence the ecological role of MP-associated microbial communities are context-dependent, i.e. different environments lead to substantial changes in biomass build up and heterotrophic activities of MP biofilms. We propose that MP surfaces act as new niches for aquatic microorganisms and that the constantly increasing MP pollution has the potential to globally impact carbon dynamics of pelagic environments by altering heterotrophic activities. (C) 2018 Elsevier B.V. All rights reserved.
Plastic pollution is ubiquitous on the planet since several millions of tons of plastic waste enter aquatic ecosystems each year. Furthermore, the amount of plastic produced is expected to increase exponentially shortly. The heterogeneity of materials, additives and physical characteristics of plastics are typical of these emerging contaminants and affect their environmental fate in marine and freshwaters. Consequently, plastics can be found in the water column, sediments or littoral habitats of all aquatic ecosystems. Most of this plastic debris will fragment as a product of physical, chemical and biological forces, producing particles of small size. These particles (< 5mm) are known as “microplastics” (MP). Given their high surface-to-volume ratio, MP stimulate biofouling and the formation of biofilms in aquatic systems.
As a result of their unique structure and composition, the microbial communities in MP biofilms are referred to as the “Plastisphere.” While there is increasing data regarding the distinctive composition and structure of the microbial communities that form part of the plastisphere, scarce information exists regarding the activity of microorganisms in MP biofilms. This surface-attached lifestyle is often associated with the increase in horizontal gene transfer (HGT) among bacteria. Therefore, this type of microbial activity represents a relevant function worth to be analyzed in MP biofilms. The horizontal exchange of mobile genetic elements (MGEs) is an essential feature of bacteria. It accounts for the rapid evolution of these prokaryotes and their adaptation to a wide variety of environments. The process of HGT is also crucial for spreading antibiotic resistance and for the evolution of pathogens, as many MGEs are known to contain antibiotic resistance genes (ARGs) and genetic determinants of pathogenicity.
In general, the research presented in this Ph.D. thesis focuses on the analysis of HGT and heterotrophic activity in MP biofilms in aquatic ecosystems. The primary objective was to analyze the potential of gene exchange between MP bacterial communities vs. that of the surrounding water, including bacteria from natural aggregates. Moreover, the thesis addressed the potential of MP biofilms for the proliferation of biohazardous bacteria and MGEs from wastewater treatment plants (WWTPs) and associated with antibiotic resistance. Finally, it seeks to prove if the physiological profile of MP biofilms under different limnological conditions is divergent from that of the water communities. Accordingly, the thesis is composed of three independent studies published in peer-reviewed journals. The two laboratory studies were performed using both model and environmental microbial communities. In the field experiment, natural communities from freshwater ecosystems were examined.
In Chapter I, the inflow of treated wastewater into a temperate lake was simulated with a concentration gradient of MP particles. The effects of MP on the microbial community structure and the occurrence of integrase 1 (int 1) were followed. The int 1 is a marker associated with mobile genetic elements and known as a proxy for anthropogenic effects on the spread of antimicrobial resistance genes. During the experiment, the abundance of int1 increased in the plastisphere with increasing MP particle concentration, but not in the surrounding water. In addition, the microbial community on MP was more similar to the original wastewater community with increasing microplastic concentrations. Our results show that microplastic particles indeed promote persistence of standard indicators of microbial anthropogenic pollution in natural waters.
In Chapter II, the experiments aimed to compare the permissiveness of aquatic bacteria towards model antibiotic resistance plasmid pKJK5, between communities that form biofilms on MP vs. those that are free-living. The frequency of plasmid transfer in bacteria associated with MP was higher when compared to bacteria that are free-living or in natural aggregates. Moreover, comparison increased gene exchange occurred in a broad range of phylogenetically-diverse bacteria. The results indicate a different activity of HGT in MP biofilms, which could affect the ecology of aquatic microbial communities on a global scale and the spread of antibiotic resistance.
Finally, in Chapter III, physiological measurements were performed to assess whether microorganisms on MP had a different functional diversity from those in water. General heterotrophic activity such as oxygen consumption was compared in microcosm assays with and without MP, while diversity and richness of heterotrophic activities were calculated by using Biolog® EcoPlates. Three lakes with different nutrient statuses presented differences in MP-associated biomass build up. Functional diversity profiles of MP biofilms in all lakes differed from those of the communities in the surrounding water, but only in the oligo-mesotrophic lake MP biofilms had a higher functional richness compared to the ambient water. The results support that MP surfaces act as new niches for aquatic microorganisms and can affect global carbon dynamics of pelagic environments.
Overall, the experimental works presented in Chapters I and II support a scenario where MP pollution affects HGT dynamics among aquatic bacteria. Among the consequences of this alteration is an increase in the mobilization and transfer efficiency of ARGs. Moreover, it supposes that changes in HGT can affect the evolution of bacteria and the processing of organic matter, leading to different catabolic profiles such as demonstrated in Chapter III. The results are discussed in the context of the fate and magnitude of plastic pollution and the importance of HGT for bacterial evolution and the microbial loop, i.e., at the base of aquatic food webs. The thesis supports a relevant role of MP biofilm communities for the changes observed in the aquatic microbiome as a product of intense human intervention.
Metabolic engineering of microalgae offers a promising solution for sustainable biofuel production, and rational design of engineering strategies can be improved by employing metabolic models that integrate enzyme turnover numbers. However, the coverage of turnover numbers for Chlamydomonas reinhardtii, a model eukaryotic microalga accessible to metabolic engineering, is 17-fold smaller compared to the heterotrophic cell factory Saccharomyces cerevisiae. Here we generate quantitative protein abundance data of Chlamydomonas covering 2337 to 3708 proteins in various growth conditions to estimate in vivo maximum apparent turnover numbers. Using constrained-based modeling we provide proxies for in vivo turnover numbers of 568 reactions, representing a 10-fold increase over the in vitro data for Chlamydomonas. Integration of the in vivo estimates instead of in vitro values in a metabolic model of Chlamydomonas improved the accuracy of enzyme usage predictions. Our results help in extending the knowledge on uncharacterized enzymes and improve biotechnological applications of Chlamydomonas.
Genome-scale metabolic models are mathematical representations of all known reactions occurring in a cell. Combined with constraints based on physiological measurements, these models have been used to accurately predict metabolic fluxes and effects of perturbations (e.g. knock-outs) and to inform metabolic engineering strategies. Recently, protein-constrained models have been shown to increase predictive potential (especially in overflow metabolism), while alleviating the need for measurement of nutrient uptake rates. The resulting modelling frameworks quantify the upkeep cost of a certain metabolic flux as the minimum amount of enzyme required for catalysis. These improvements are based on the use of in vitro turnover numbers or in vivo apparent catalytic rates of enzymes for model parameterization. In this thesis several tools for the estimation and refinement of these parameters based on in vivo proteomics data of Escherichia coli, Saccharomyces cerevisiae, and Chlamydomonas reinhardtii have been developed and applied. The difference between in vitro and in vivo catalytic rate measures for the three microorganisms was systematically analyzed. The results for the facultatively heterotrophic microalga C. reinhardtii considerably expanded the apparent catalytic rate estimates for photosynthetic organisms. Our general finding pointed at a global reduction of enzyme efficiency in heterotrophy compared to other growth scenarios. Independent of the modelled organism, in vivo estimates were shown to improve accuracy of predictions of protein abundances compared to in vitro values for turnover numbers. To further improve the protein abundance predictions, machine learning models were trained that integrate features derived from protein-constrained modelling and codon usage. Combining the two types of features outperformed single feature models and yielded good prediction results without relying on experimental transcriptomic data. The presented work reports valuable advances in the prediction of enzyme allocation in unseen scenarios using protein constrained metabolic models. It marks the first successful application of this modelling framework in the biotechnological important taxon of green microalgae, substantially increasing our knowledge of the enzyme catalytic landscape of phototrophic microorganisms.
The Aquatic Warbler Acrocephalus paludicola was once a common breeding bird in mesotrophic fen mires all over Central and Western Europe. In the last century large parts of its habitat have been destroyed by wetland drainage and agricultural intensification. Besides protecting the remaining breeding habitats, it is of great importance to preserve suitable migration stopover habitats and wintering grounds to avert the extinction of the species.
We determined home-range size and the use of vegetation associations of Aquatic Warblers on the wintering grounds in a flooded plain north of the Djoudj National Park in Senegal. Individual birds (11) were caught in mist nets and equipped with radio transmitters. Locations were assessed by radiotelemetry and a compositional analysis was conducted to determine which vegetation types were preferred within home ranges.
Similar to their behaviour on the breeding grounds, the Aquatic Warblers showed no territorial behaviour in their winter quarters. They used home ranges that averaged 4 ha in size, which they shared with conspecifics and other warblers. The home ranges overlapped 54% on average, with a maximum of 90% in an area used by four individuals. The vegetation structure of the wintering habitat is similar to breeding grounds and stopover sites of the species. Preferential vegetation had 80% to 100% cover and consisted of 60 to 90 cm high stands of Oryza longistaminata, Scirpus maritimus or Eleocharis mutata. Most birds stayed more often near the edge of open water, probably for foraging. A constant inundation seems essential, because Aquatic Warblers never occurred in desiccated parts of the study site.
The tricarboxylic acid (TCA) cycle is a crucial component of respiratory metabolism in both photosynthetic and heterotrophic plant organs. All of the major genes of the tomato TCA cycle have been cloned recently, allowing the generation of a suite of transgenic plants in which the majority of the enzymes in the pathway are progressively decreased. Investigations of these plants have provided an almost complete view of the distribution of control in this important pathway. Our studies suggest that citrate synthase, aconitase, isocitrate dehydrogenase, succinyl CoA ligase, succinate dehydrogenase, fumarase and malate dehydrogenase have control coefficients flux for respiration of -0.4, 0.964, -0.123, 0.0008, 0.289, 0.601 and 1.76, respectively; while 2-oxoglutarate dehydrogenase is estimated to have a control coefficient of 0.786 in potato tubers. These results thus indicate that the control of this pathway is distributed among malate dehydrogenase, aconitase, fumarase, succinate dehydrogenase and 2-oxoglutarate dehydrogenase. The unusual distribution of control estimated here is consistent with specific non-cyclic flux mode and cytosolic bypasses that operate in illuminated leaves. These observations are discussed in the context of known regulatory properties of the enzymes and some illustrative examples of how the pathway responds to environmental change are given.
Biochemical and physiological studies of Arabidopsis thaliana Diacylglycerol Kinase 7 (AtDGK7)
(2006)
A family of diacylglycerol kinases (DGK) phosphorylates the substrate diacylglycerol (DAG) to generate phosphatidic acid (PA) . Both molecules, DAG and PA, are involved in signal transduction pathways. In the model plant Arabidopsis thaliana, seven candidate genes (named AtDGK1 to AtDGK7) code for putative DGK isoforms. Here I report the molecular cloning and characterization of AtDGK7. Biochemical, molecular and physiological experiments of AtDGK7 and their corresponding enzyme are analyzed. Information from Genevestigator says that AtDGK7 gene is expressed in seedlings and adult Arabidopsis plants, especially in flowers. The AtDGK7 gene encodes the smallest functional DGK predicted in higher plants; but also, has an alternative coding sequence containing an extended AtDGK7 open reading frame, confirmed by PCR and submitted to the GenBank database (under the accession number DQ350135). The new cDNA has an extension of 439 nucleotides coding for 118 additional amino acids The former AtDGK7 enzyme has a predicted molecular mass of ~41 kDa and its activity is affected by pH and detergents. The DGK inhibitor R59022 also affects AtDGK7 activity, although at higher concentrations (i.e. IC50 ~380 µM). The AtDGK7 enzyme also shows a Michaelis-Menten type saturation curve for 1,2-DOG. Calculated Km and Vmax were 36 µM 1,2-DOG and 0.18 pmol PA min-1 mg of protein-1, respectively, under the assay conditions. Former protein AtDGK7 are able to phosphorylate different DAG analogs that are typically found in plants. The new deduced AtDGK7 protein harbors the catalytic DGKc and accessory domains DGKa, instead the truncated one as the former AtDGK7 protein (Gomez-Merino et al., 2005).
Elaeidobius kamerunicus Faust. (Coleoptera: Curculionidae) is an essential insect pollinator in oil palm plantations. Recently, researches have been undertaken to improve pollination efficiency using this species. A fundamental understanding of the genes related to this pollinator behavior is necessary to achieve this goal. Here, we present the draft genome sequence, annotation, and simple sequence repeat (SSR) marker data for this pollinator. In total, 34.97 Gb of sequence data from one male individual (monoisolate) were obtained using Illumina short-read platform NextSeq 500. The draft genome assembly was found to be 269.79 Mb and about 59.9% of completeness based on Benchmarking Universal Single-Copy Orthologs (BUSCO) assessment. Functional gene annotation predicted about 26.566 genes. Also, a total of 281.668 putative SSR markers were identified. This draft genome sequence is a valuable resource for understanding the population genetics, phylogenetics, dispersal patterns, and behavior of this species.
The complete mitochondrial genome of oil palm pollinating weevil, Elaeidobius kamerunicus Faust
(2020)
Elaeidobius kamerunicusis the most important insect pollinator in oil palm plantations. In this study, the mitochondrial genome (mitogenome) ofE. kamerunicus(17.729 bp), a member of the Curculionidae family, will be reported. The mitogenome consisted of 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and a putative control region (CR). Phylogenetic analysis based on 13 protein-coding genes (PCGs) using maximum Likelihood (ML) methods indicated thatE. kamerunicusbelongs to the Curculionidae family. This mitochondrial genome provides essential information for understanding genetic populations, phylogenetics, molecular evolution, and other biological applications in this species.
Oil palm (Elaeis guineensis Jacq.) is the most productive oil-producing crop per hectare of land. The oil that accumulates in the mesocarp tissue of the fruit is the highest observed among fruit-producing plants. A comparative analysis between high-, medium-, and low-yielding oil palms, particularly during fruit development, revealed unique characteristics. Metabolomics analysis was able to distinguish accumulation patterns defining of the various developmental stages and oil yield. Interestingly, high- and medium-yielding oil palms exhibited substantially increased sucrose levels compared to low-yielding palms. In addition, parameters such as starch granule morphology, granule size, total starch content, and starch chain length distribution (CLD) differed significantly among the oil yield categories with a clear correlation between oil yield and various starch parameters. These results provide new insights into carbohydrate and starch metabolism for biosynthesis of oil palm fruits, indicating that starch and sucrose can be used as novel, easy-to-analyze, and reliable biomarker for oil yield.
Starch is a complex carbohydrate polymer produced by plants and especially by crops in huge amounts. It consists of amylose and amylopectin, which have alpha-1,4-and alpha-1,6-linked glucose units. Despite this simple chemistry, the entire starch metabolism is complex, containing various (iso)enzymes/proteins. However, whose interplay is still not yet fully understood. Starch is essential for humans and animals as a source of nutrition and energy. Nowadays, starch is also commonly used in non-food industrial sectors for a variety of purposes. However, native starches do not always satisfy the needs of a wide range of (industrial) applications. This review summarizes the structural properties of starch, analytical methods for starch characterization, and in planta starch modifications.
The oil palm (Elaeis guineensis Jacq.) produces a large amount of oil from the fruit. However, increasing the oil production in this fruit is still challenging. A recent study has shown that starch metabolism is essential for oil synthesis in fruit-producing species. Therefore, the transcriptomic analysis by RNA-seq was performed to observe gene expression alteration related to starch metabolism genes throughout the maturity stages of oil palm fruit with different oil yields. Gene expression profiles were examined with three different oil yields group (low, medium, and high) at six fruit development phases (4, 8, 12, 16, 20, and 22 weeks after pollination). We successfully identified and analyzed differentially expressed genes in oil palm mesocarps during development. The results showed that the transcriptome profile for each developmental phase was unique. Sucrose flux to the mesocarp tissue, rapid starch turnover, and high glycolytic activity have been identified as critical factors for oil production in oil palms. For starch metabolism and the glycolytic pathway, we identified specific gene expressions of enzyme isoforms (isozymes) that correlated with oil production, which may determine the oil content. This study provides valuable information for creating new high-oil-yielding palm varieties via breeding programs or genome editing approaches.
Transcriptomic dataset for early inflorescence stages of oil palm in response to defoliation stress
(2022)
Oil palm breeding and seed development have been hindered due to the male parent's incapacity to produce male inflorescence as a source of pollen under normal conditions. On the other hand, a young oil palm plantation has a low pollination rate due to a lack of male flowers. These are the common problem of sex ratio in the oil palm industry. Nevertheless, the regulation of sex ratio in oil palm plants is a complex mechanism and remains an open question until now. Researchers have previously used complete defoliation to induce male inflorescences, but the biological and molecular mechanisms underlying this morphological change have yet to be discovered. Here, we present an RNA-seq dataset from three early stages of an oil palm inflorescence under normal conditions and complete defoliation stress. This transcriptomic dataset is a valuable resource to improve our understanding of sex determination mechanisms in oil palm inflorescence.