29.10.2019 - Dr. Danny Ionescu - Leibniz Institut Stechlin

Achromatium oxaliferum - A compartmentalized super bacterium with multiple-personalities

Polyploid bacteria are common, but the genetic and functional diversity resulting from polyploidy is not fully understood. Achromatium sp. is the largest freshwater bacterium. Its cells contain multiple calcite bodies whose evolutionary role has not been determined. Like other large-sulfur bacteria, it has multiple chromosomes as seen by nucleic acid staining. Using single-cell genomics, metagenomics, single- cell amplicon sequencing and fluorescence in-situ hybridization, we show that individual cells Achromatium harbor genetic diversity typical of multi-species populations. Interestingly, the rRNA distribution inside the cells hints to spatially- differential gene expression. Broad-scale surveys of short-read archives show that Achromatium sp. is globally present in rivers, freshwater lakes, and marine environments but no environment-specific phylogenetic clustering of the 16S rRNA gene is observed. This is likely due to high intra-cellular and population-wide phylogenetic diversity further supporting our findings. We show that these cells contain and also express tens of transposable elements, which likely contribute the unprecedented diversity that we observe in the sequence and synteny of genes. Accordingly, we suggest that the multiple chromosomes of Achromatium do not represent copies of its genome. Nevertheless, our analysis shows that most proteins are under conservation pressure. Thus, given the high single-cell diversity of functional genes and the usually conserved 16S rRNA gene, we suggest that gene convergence is limited to chromosomal clusters formed by the large calcite bodies in the cell. We further suggest that upon cell division, these cluster are shuffled resulting in two daughter cells different from each other as well as from the mother cell. To obtain information on how many alleles of a gene are expressed from the complete repertoire available in each cell, we are developing a method to simultaneously obtain both the genome and transcriptome of each single cell. This allows overcoming the lack of a common genome sequence to the entire population.

05.11.2019 - Dr. Marina Chekulaeva - MDC Berlin

Mechanisms of RNA localization in neurons

The proper subcellular localization of RNAs and proteins is crucial for their function. It is particularly important for highly polarized cells, such as oocytes, migrating and growing cells, neurons. In mRNAs, they are mediated by specific cis-regulatory elements, so called zip-codes. These elements are bound by trans-acting factors (RBPs, miRNAs), which regulate transport, stability or translation.  So far, our knowledge is restricted to only a few examples of zip-codes and regulatory factors. We aim to identify these elements genome-wide and dissect molecular mechanisms underlying their function. To identify proteins and RNAs that are differentially localized and translated between neuronal subcellular compartments - neurites and soma - we developed a neurite/soma separation scheme in combination with mass spectrometry, RNA-seq, 3'-mRNAseq, Ribo-seq and bioinformatic analyses (1, 2, 3). Our results demonstrate that mRNA localization is the primary mechanism for protein localization in neurites and may account for more than a half of the neurite-localized proteome. Moreover, we identified multiple neurite-targeted non-coding RNAs and RBPs with potential regulatory roles. Using a combination of PAR-CLIP, RIP, and CRISPR/Cas mediated knockouts, we are dissecting the roles of selected neurite-targeted RBPs and miRNAs in establishment of neuronal polarity. Relying on the same neurite/soma separation scheme, we are mapping cis-regulatory elements mediating RNA localization and investigating architecture of local proteome, transcriptome and translatome in motor neuron disorders.

19.11.2019 Dr. Kristian Parey - MPI für Biophysik, Frankfurt

The Alpha and Omega of Biological Energy Conservation

The biogeochemical cycles of carbon, oxygen, nitrogen and sulphur constitute the life-supporting system for our planet, as they determine the composition of the atmosphere as well as the fertility of land and water. Archaea and bacteria play a crucial role in global biogeochemical cycles and are becoming more and more the focus of public attention as they affect greenhouse gas emissions. All currently known life forms gain free metabolic energy through enzymatically catalysed electron transfer reactions, which use electron donors and acceptors (redox pairs) to convert biochemical energy that is used either for assimilation purposes or in respiration processes for energy conservation, e.g. in respiration. They developed a great diversity of electron transfer chains for sustaining energy supply in an impressively broad range of environmental conditions. I have characterized several of the protein complexes involved in the first and last step of the carbon, oxygen, nitrogen and sulphur metabolism. I have determined their structures by X-ray crystallography or single-particle electron cryo-microscopy and investigated their function by complementary biophysical techniques. These studies provided fundamental insights into the mechanisms of electron and proton transfer within the investigated metabolic pathways.

14.01.2020 Prof. Dr.  Miltos Tsiantis - MPI Köln

The genetic basis for leaf development and diversity: from understanding to reconstructing

A key challenge in biology is to understand how diversity in organismal form is generated. While key regulators that shape the body plans of model organisms have been identified, less is known about how the balance of conservation versus divergence of relevant developmental pathways influences cell growth to generate morphological diversity. To help address this issue, we developed the Arabidopsis thaliana relative Cardamine hirsuta into a versatile system for studying morphological evolution. We use a combination of genetics, advanced imaging and computational modelling to understand the mechanisms through which leaf morphology evolved in these species, resulting in simple leaves in A. thaliana and complex leaves with leaflets in C. hirsuta. This presentation will describe progress on identifying such mechanisms and in conceptualizing how they regulate the number, position and timing of leaflet production.

28.01.2020 - Frau Jun.Prof. Neva Caliskan - Helmholtz Institute for RNA-based Infection Research: Recoding Mechanisms in Infections (REMI))

Single-Molecule and Ensemble Analysis of Protein-Mediated Frameshifting

Three bases encoding for an amino acid seem to represent the universal feature of the genetic code, yet ribosomes have evolved to read the code in different ways by altering the triplet periodicity of the reading frame. This phenomenon is called programmed ribosome frameshifting (PRF). PRF requires specific cis-acting elements - a slippery site followed by a stable RNA structure. PRF efficiency is also affected by trans-acting factors, including proteins, miRNAs and metabolites. While the general mechanisms of PRF and the involvement of cis-acting elements in this process are well understood, the regulation of these events is still vastly understudied. Additionally, the interactions of these factors with the RNA and the translation machinery have not yet been completely understood. Recent advances in single-molecule techniques allow to study these events at the molecular level and thus unveil hitherto unrecognised details. In this study, we chose the encephalomyocarditis virus (EMCV) 2A protein as a model to study PRF regulation. The expression of this protein is essential for frameshifting on the EMCV mRNA, and inhibition of PRF leads to severely reduced virulence. We investigated the interplay of the 2A protein with its frameshifting-RNA target. To do so, we combined single-molecule techniques, such as optical tweezers and confocal microscopy, together with HPLC-MS and microscale thermophoresis (MST). We anticipate these assays to be a starting point in analysing the translational kinetics of frameshifting and its interplay by RNA binding factors. Furthermore, recent examples of identification of such factors indicate that they play a major role in PRF regulation and understanding their mode of action will certainly uncover new fundamental principles of RNA-based gene regulation.

04.02.2020 - Prof. Dr. Peter Schönheit - Universität Kiel

Glycolysis at the boiling point

Life on our planet likely originated in boiling water. Organisms adapted to these conditions, hyperthermophiles, belong mostly to archaea and represent the most ancestral living organisms. These organisms are thus suitable objects to study metabolism at an early stage of evolution. Hyperthermophilic archaea can grow on anorganic compounds of volcanic origin, such as hydrogen, carbon dioxide and sulphur, but can also utilize organic compounds, e.g. sugars. Many sugar-utilizing hyperthermophilic archaea degrade glucose and glucose polymers to acetate as major fermentation product. In these archaea glucose degradation to pyruvate mainly proceeds via modified versions of the Embden-Meyerhof (EM) pathway, which implicate several novel enzymes and enzyme families catalysing for example the phosphorylation of glucose and of fructose 6-phosphate, the isomerization of glucose 6-phosphate, and the oxidation of glyceraldehyde-3-phosphate, and unusual pyruvate kinases (1,2). Further, the conversion of acetyl-CoA to acetate, which represents a major energy conserving site in archaeal sugar fermentation, is catalysed by an novel prokaryotic enzyme, ADP-forming acetyl-CoA synthetase (acetyl-CoA + ADP + Pi → acetate + ATP + CoA) (ACD) (3). The modified EM pathways and selected enzymes, e.g. pyruvate kinase (structure and novel allosteric activator), and the enzyme of acetate formation (mechanism and structure) in hyperthermophilic archaea will be discussed in comparison with the anaerobic hyperthermophilic bacterium Thermotoga, which also ferments glucose to acetate. The data indicate that the unusual features of archaeal glycolysis and of acetate formation are due to their phylogeny rather than to an adaptation to a hyperthermophilic lifestyle (4).