Cationic Solution and Solid-State Emitters - Robust Imaging Agents for Cells, Bacteria, and Protists.

A library of eight different cationic emitters with emission properties in solution and in solid-state (solution and solid-state emitters - SSSE) is presented. These compounds, bearing either ammonium or pyridinium groups, have been investigated regarding their photophysical properties as well as their potential application in biological imaging. Besides high quantum yields as well as a high degree of stability during the imaging process, it was additionally revealed that a broad range of biological targets can be addressed, such as different bacterial strains, human cells as well as protists. The reported SSSE approach employing the mentioned robust emitters for biological imaging, will contribute to a rapid and facile way to design and apply affordable emitters with outstanding properties. Additionally, these emitters will overcome the drawbacks of classical luminophores and agents featuring well-known aggregation-induced emission (AIE) or aggregation-caused quenching (ACQ) properties.

Indigenous microbial communities in heavy oil show a threshold response to salinity.

Microbial degradation influences the quality of oil resources. The environmental factors that shape the composition of oil microbial communities are largely unknown because most samples from oil fields are impacted by anthropogenic oil production, perturbing the native ecosystem with exogenous fluids and microorganisms. We investigated the relationship between formation water geochemistry and microbial community composition in undisturbed oil samples. We isolated 43 microliter-sized water droplets naturally enclosed in the heavy oil of the Pitch Lake, Trinidad and Tobago. The water chemistry and microbial community composition within the same water droplet were determined by ion chromatography and 16S rRNA gene amplicon sequencing, respectively. The results revealed a high variability in ion concentrations and community composition between water droplets. Microbial community composition was mostly affected by the chloride concentration, which ranged from freshwater to brackish-sea water. Remarkably, microbial communities did not respond gradually to increasing chloride concentration but showed a sudden change to less diverse and uneven communities when exceeding a chloride concentration of 57.3 mM. The results reveal a threshold-regulated response of microbial communities to salinity, offering new insights into the microbial ecology of oil reservoirs.

Colpodean ciliate phylogeny and reference alignments for phylogenetic placements.

The Colpodea form a major clade of ciliates that are often found in environmental DNA sequencing studies. They are united by similar somatic ciliature, but differentiated by complex oral structures. Although there are four well supported colpodean subclades, there is disagreement in molecular phylogenetic inferences about their branching order. Using available nuclear SSU-rRNA sequences, we evaluated if the bursariomorphids or the platyophryids are sister to the remaining colpodeans. We inferred the "platyophryids-early" topologies using different alignment and masking methods, but constrained analyses could not reject the "bursariomorphids-early" topology. Both bursariomorphids and platyophryids clades have a similar number of nucleotide positions shared with the outgroup, and both are interconnected with the outgroup in phylogenetic networks. Based on these discordant results, it is hard to determine which clade branched off first, although the "platyophryids-early topology" is also supported by mitochondrial SSU-rRNA data. We also offer different reference alignments that can be used to phylogenetically place short- and long-read data from environmental DNA sequencing studies, and we propose some tentative evolutionary and ecological interpretations of those placements.

Putatively asexual chrysophytes have meiotic genes: evidence from transcriptomic data.

Chrysophytes are a large group of heterotrophic, phototrophic, or even mixotrophic protists that are abundant in aquatic as well as terrestrial environments. Although much is known about chrysophyte biology and ecology, it is unknown if they are sexual or not. Here we use available transcriptomes of 18 isolates of 15 putatively asexual species to inventory the presence of genes used in meiosis. Since we were able to detect a set of nine meiosis-specific and 29 meiosis-related genes shared by the chrysophytes, we conclude that they are secretively sexual and therefore should be investigated further using genome sequencing to uncover any missed genes from the transcriptomes.

Evolution of heterotrophy in chrysophytes as reflected by comparative transcriptomics.

Shifts in the nutritional mode between phototrophy, mixotrophy and heterotrophy are a widespread phenomenon in the evolution of eukaryotic diversity. The transition between nutritional modes is particularly pronounced in chrysophytes and occurred independently several times through parallel evolution. Thus, chrysophytes provide a unique opportunity for studying the molecular basis of nutritional diversification and of the accompanying pathway reduction and degradation of plastid structures. In order to analyze the succession in switching the nutritional mode from mixotrophy to heterotrophy, we compared the transcriptome of the mixotrophic Poterioochromonas malhamensis with the transcriptomes of three obligate heterotrophic species of Ochromonadales. We used the transcriptome of P. malhamensis as a reference for plastid reduction in the heterotrophic taxa. The analyzed heterotrophic taxa were in different stages of plastid reduction. We investigated the reduction of several photosynthesis related pathways e.g. the xanthophyll cycle, the mevalonate pathway, the shikimate pathway and the tryptophan biosynthesis as well as the reduction of plastid structures and postulate a presumable succession of pathway reduction and degradation of accompanying structures.

Flooding Duration Affects the Structure of Terrestrial and Aquatic Microbial Eukaryotic Communities.

The increasing number and duration of inundations is reported to be a consequence of climate change and may severely compromise non-adapted macroorganisms. The effect of flooding events on terrestrial and aquatic microbial communities is, however, less well understood. They may respond to the changed abiotic properties of their native habitat, and the native community may change due to the introduction of alien species. We designed an experiment to investigate the effect of five different flooding durations on the terrestrial and aquatic communities of eukaryotic microorganism, using the AquaFlow mesocosms. With amplicon sequencing of the small subunit (SSU) and internal transcribed spacer (ITS) rRNA gene regions, we analyzed community compositions directly before and after flooding. Subsequently, they were monitored for another 28 days, to determine the sustainability of community changes. Our results revealed a temporary increase in similarity between terrestrial and aquatic communities according to OTU composition (operational taxonomic unit, serves as a proxy for species). Increased similarity was mainly caused by the transmission of OTUs from water to soil. A minority of these were able to persist in soil until the end of the experiment. By contrast, the vast majority of soil OTUs was not transmitted to water. Flooding duration affected the community structure (abundance) more than composition (occurrence). Terrestrial communities responded immediately to flooding and the flooding duration influenced the community changes. Independent from flooding duration, all terrestrial communities recovered largely after flooding, indicating a remarkable resilience to the applied disturbances. Aquatic communities responded immediately to the applied inundations too. At the end of the experiment, they grouped according to the applied flooding duration and the amount of ammonium and chloride that leached from the soil. This indicates a sustained long-term response of the aquatic communities to flooding events.

TaxMapper: an analysis tool, reference database and workflow for metatranscriptome analysis of eukaryotic microorganisms.

BACKGROUND: High-throughput sequencing (HTS) technologies are increasingly applied to analyse complex microbial ecosystems by mRNA sequencing of whole communities, also known as metatranscriptome sequencing. This approach is at the moment largely limited to prokaryotic communities and communities of few eukaryotic species with sequenced genomes. For eukaryotes the analysis is hindered mainly by a low and fragmented coverage of the reference databases to infer the community composition, but also by lack of automated workflows for the task. RESULTS: From the databases of the National Center for Biotechnology Information and Marine Microbial Eukaryote Transcriptome Sequencing Project, 142 references were selected in such a way that the taxa represent the main lineages within each of the seven supergroups of eukaryotes and possess predominantly complete transcriptomes or genomes. From these references, we created an annotated microeukaryotic reference database. We developed a tool called TaxMapper for a reliably mapping of sequencing reads against this database and filtering of unreliable assignments. For filtering, a classifier was trained and tested on each of the following: sequences of taxa in the database, sequences of taxa related to those in the database, and random sequences. Additionally, TaxMapper is part of a metatranscriptomic Snakemake workflow developed to perform quality assessment, functional and taxonomic annotation and (multivariate) statistical analysis including environmental data. The workflow is provided and described in detail to empower researchers to apply it for metatranscriptome analysis of any environmental sample. CONCLUSIONS: TaxMapper shows superior performance compared to standard approaches, resulting in a higher number of true positive taxonomic assignments. Both the TaxMapper tool and the workflow are available as open-source code at Bitbucket under the MIT license: https://bitbucket.org/dbeisser/taxmapper and as a Bioconda package: https://bioconda.github.io/recipes/taxmapper/README.html .

Comprehensive transcriptome analysis provides new insights into nutritional strategies and phylogenetic relationships of chrysophytes.

BACKGROUND: Chrysophytes are protist model species in ecology and ecophysiology and important grazers of bacteria-sized microorganisms and primary producers. However, they have not yet been investigated in detail at the molecular level, and no genomic and only little transcriptomic information is available. Chrysophytes exhibit different trophic modes: while phototrophic chrysophytes perform only photosynthesis, mixotrophs can gain carbon from bacterial food as well as from photosynthesis, and heterotrophs solely feed on bacteria-sized microorganisms. Recent phylogenies and megasystematics demonstrate an immense complexity of eukaryotic diversity with numerous transitions between phototrophic and heterotrophic organisms. The question we aim to answer is how the diverse nutritional strategies, accompanied or brought about by a reduction of the plasmid and size reduction in heterotrophic strains, affect physiology and molecular processes. RESULTS: We sequenced the mRNA of 18 chrysophyte strains on the Illumina HiSeq platform and analysed the transcriptomes to determine relations between the trophic mode (mixotrophic vs. heterotrophic) and gene expression. We observed an enrichment of genes for photosynthesis, porphyrin and chlorophyll metabolism for phototrophic and mixotrophic strains that can perform photosynthesis. Genes involved in nutrient absorption, environmental information processing and various transporters (e.g., monosaccharide, peptide, lipid transporters) were present or highly expressed only in heterotrophic strains that have to sense, digest and absorb bacterial food. We furthermore present a transcriptome-based alignment-free phylogeny construction approach using transcripts assembled from short reads to determine the evolutionary relationships between the strains and the possible influence of nutritional strategies on the reconstructed phylogeny. We discuss the resulting phylogenies in comparison to those from established approaches based on ribosomal RNA and orthologous genes. Finally, we make functionally annotated reference transcriptomes of each strain available to the community, significantly enhancing publicly available data on Chrysophyceae. CONCLUSIONS: Our study is the first comprehensive transcriptomic characterisation of a diverse set of Chrysophyceaen strains. In addition, we showcase the possibility of inferring phylogenies from assembled transcriptomes using an alignment-free approach. The raw and functionally annotated data we provide will prove beneficial for further examination of the diversity within this taxon. Our molecular characterisation of different trophic modes presents a first such example.

Quantitative Proteomics Reveals Ecophysiological Effects of Light and Silver Stress on the Mixotrophic Protist Poterioochromonas malhamensis.

Aquatic environments are heavily impacted by human activities including climate warming and the introduction of xenobiotics. Due to the application of silver nanoparticles as bactericidal agent the introduction of silver into the environment strongly has increased during the past years. Silver ions affect the primary metabolism of algae, in particular photosynthesis. Mixotrophic algae are an interesting test case as they do not exclusively rely on photosynthesis which may attenuate the harmful effect of silver. In order to study the effect of silver ions on mixotrophs, cultures of the chrysophyte Poterioochromonas malhamensis were treated in a replicate design in light and darkness with silver nitrate at a sub-lethal concentration. At five time points samples were taken for the identification and quantitation of proteins by mass spectrometry. In our analysis, relative quantitative protein mass spectrometry has shown to be a useful tool for functional analyses in conjunction with transcriptome reference sequences. A total of 3,952 proteins in 63 samples were identified and quantified, mapping to 4,829 transcripts of the sequenced and assembled transcriptome. Among them, 720 and 104 proteins performing various cellular functions were differentially expressed after eight days in light versus darkness and after three days of silver treatment, respectively. Specifically pathways of the energy and primary carbon metabolism were differentially affected by light and the utilization of expensive reactions hints to an energy surplus of P. malhamensis under light conditions. The excess energy is not invested in growth, but in the synthesis of storage metabolites. The effects of silver were less explicit, observable especially in the dark treatments where the light effect could not mask coinciding but weaker effects of silver. Photosynthesis, particularly the light harvesting complexes, and several sulphur containing enzymes were affected presumably due to a direct interference with the silver ions, mainly affecting energy supply.