Annual Conference 2025 of the Association for General and Applied Microbiology - Jahrestagung 2025 der Vereinigung für Allgemeine und Angewandte Mikrobiologie

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Prof. Dr.

Mutational hotspots drive rapid evolution of bacterial defenses against protistan predation

SFB Session / HHU/FZ Jülich

Soils are a hotspot of microbial predation, with numerous bacterivorous protists scavenging for bacterial prey. Despite this predation pressure, most functional genomic studies on soil bacteria are performed in the absence of trophic interaction partners. This biases our view on the selection pressures that shape bacterial evolution. Here, we aim to overcome this bias by studying how, under predation, selection affects B. subtilis" growth and survival. By screening thousands of genetic mutants with and without predation, we reveal that tens of different genes affect predation resistance, most of which have never been associated with predation before. We also show that mutations can rapidly emerge de novo and mostly occur in three loci only. These mutational hotspots either cause filamentation or biofilm formation and prevent protists from engulfing bacterial cells. Resistance however comes with a major cost. Mutants grow slower than susceptible cells and are rapidly replaced in the absence of predation. This strong antagonistic selection favors genetic regulation. Indeed, in one of the loci, we discovered a putative genetic switch that allows cells to switch back-and-forth between a slow-growing resistant state and a fast-grow susceptible state. We speculate that other cases of phase variation in B. subtilis can be linked to predation as well. Free-living bacteria might therefore evade predation much like pathogens use phase variation for immune evasion.

Invited lecture

Establishment of a conditional knockout system in the endosymbiont-harboring trypanosomatid Angomonas deanei

Introduction: Mitochondria and plastids originate from endosymbiotic events more than one billion years ago in which free-living bacteria were taken up by a host cell and subsequently evolved into organelles that are now fully integrated into the cellular networks of the host. Since the acquisition and evolution of these organelles date far back in time, we use the trypanosomatid Angomonas deanei, which acquired a bacterial endosymbiont (ES) more recently, to study early steps of endosymbiosis. A. deanei harbors a single β-proteobacterial ES whose cell cycle is synchronized with and likely controlled by the host by so-called endosymbiont-targeted proteins (ETPs) that are encoded by the host but transported to the ES.Goal: Preliminary work revealed that homozygous deletions of several ETP-encoding genes cannot be obtained, suggesting that these genes are essential for survival of the symbiosis. Thus, to study how the ETPs effect the host-ES interaction, we aimed to develop genetic tools to generate inducible homozygous knockout (or knockdown) mutants.Methods & results: To this end, we generated an A. deanei strain that produces the T7 RNA polymerase (T7RNAP) from a highly expressed locus. To avoid interference with the endogenous expression machinery we identified at least seven potentially silent loci that appeared suitable for transgene insertion and were expected to be transcriptionally inactive under native conditions. Using a blasticidin resistance cassette with T7 promoter as a reporter gene in the T7RNAP background, we demonstrated T7RNAP-dependent gene expression from one of these silent loci. Finally, implementation of the transcriptional regulator TetR enabled us to induce expression in a ligand-depended manner. Furthermore, we are exploring the integration of a blue light-inducible T7RNAP in A. deanei.Summary: A. deanei is an emerging model system to study early steps of organellogenesis. The conditional knockout system, that enables now inducible perturbations of the system, will be key for the in-depth exploration of the host-ES interaction, and, in particular, to observe effects of ETP losses.

Short lecture

Intracellular networking: coupling of mitochondrial energy metabolism with microtubule-dependent mRNA logistics

Eukaryotic cells are particularly characterized by the integration of an endosymbiont, which has gradually led to the development of highly specialized organelles. During the adaptation process, almost the entire genetic material of the endosymbiont was transferred into the nucleus of the host, significantly increasing the complexity of cellular logistics. Consequently, not only complex protein translocation systems had to evolve in the envelope membranes of the organelles, but also intensive bidirectional communication between the organelle and the cell nucleus in order to fine-tune the availability and demand for specific compounds in the organelle and the surrounding cell at any given time.In the polar-growing fungus Ustilago maydis, which has emerged as a new model system for cell biology, we discovered a novel link between endosomal long-distance transport of mRNA and mitochondrial energy metabolism. The key RNA-binding protein Rrm4 binds numerous nuclear-encoded mitochondrial mRNAs and loss of rrm4 causes defects in the formation of mitochondrial respiratory chain complexes. We used genetic engineering coupled with life-cell imaging to track the distribution of Atp3, the γ subunit of ATP synthase, in polar-growing cells with regulatable Rrm4. Fusion of Atp3 with photoconvertible fluorescent protein (tdEosFP), which can be converted from green to red emission upon exposure to violet light, enabled us to demonstrate that mitochondrial localization of newly synthesized Atp3 is dependent on long-distance transport in polar hyphae. Genome-wide transcript analysis indicates that U. maydis hyphae with defective microtubule dependent long-distance transport undergo a significant metabolic shift, suggesting a compensatory response to impaired mitochondrial function. To assess metabolic changes on a temporal-quantitative scale we are implementing genetically encoded, ratio metric Matryoshka biosensors for ATP and NADH in our life-cell imaging coupled with sophisticated metabolomics.Overall, we show that in polar cells such as fungal hyphae or neurons, mRNA logistics post-transcriptionally regulate mitochondrial energy metabolism, revealing key insights into host-organelle networking. However, our understanding of the principles underlying the establishment, maintenance and evolution of intracellular microbial associations, such as endosymbiont-derived organelles, remains in its very early stage.

Race for Iron: Modulating siderophore-based social interactions via light

Microbial communities are essential to various global ecosystems, participating in crucial biological processes. The intricate social interactions within these communities, characterized by intra- and interspecies communication, significantly shape their behaviour and composition. In this context, exchanging various secondary metabolites is pivotal, influencing cooperation, competition, and survival strategies. Among these metabolites, siderophores stand out because of their importance in microbial iron acquisition and their involvement in mediating interactions that affect community dynamics and ecological balance.Although siderophore-driven interactions have been the subject of extensive research, the spatiotemporal dynamics of these processes remain poorly understood. To bridge this gap, we employ new technologies such as optogenetics and microfluidics, which allow noninvasive control of cellular functions with high spatiotemporal resolution and high precision in studying microbial interactions at the single-cell level.For instance, by applying light-responsive switches, we can dynamically alter the production of siderophores within microbial communities. This approach allowed a dynamic transition from siderophore production to non-production states and even to overproduction. Using this strain in co-cultures, we could show how different illumination conditions that modulate pyoverdine production impact community composition, revealing shifts in community structure linked to siderophore output.Ultimately, leveraging these technologies will shed light on how metabolite-based interactions influence community structure and function over time and space, providing new insights into microbial cooperation, competition, and resilience.

Let it sense! Biosensor creation using computational and structural approaches

Gaining insight into the physiology of individual microbes and their interactions within intricate networks necessitates a profound understanding of dynamic metabolic processes. Deciphering and quantifying these processes hinges on key factors, including pathways of metabolite transport, nutrient exchange, and signaling. High temporal and spatial resolution of metabolic pathways can be achieved through the use of genetically encoded biosensors in combination with advanced imaging techniques.Here, we establish a pipeline for biosensor development based on rational design, leveraging state-of-the-art molecular techniques integrated with computational structural biology to create novel biosensors for various metabolites, such as ATP, ferrous iron, and sucrose. This pipeline currently employs two distinct biosensor designs: the FRET-based and Matryoshka biosensor cassettes. FRET-based biosensors are relatively straightforward and largely empirical in their design, in contrast to the Matryoshka biosensors, which require a more sophisticated design process. In Matryoshka biosensors, fluorescent proteins must be embedded within the recognition protein in a way that enables them to report metabolite binding through changes in fluorescence intensity. Additionally, ratiometric Matryoshka biosensors not only track metabolite binding but also monitor the biosensor"s expression levels within cells, facilitating direct comparisons across experiments and various bacterial growth phases. Our design pipeline has significantly accelerated the development of biosensors, achieving high success rates, whereas previous empirical design approaches were often time-consuming and heavily reliant on trial and error approaches.Here we will highlight the crucial steps in the design and show how computional structural biology can help to change from emperical to rational sensor design. As an example, we developed a novel biosensor for ferrous iron, validated both in vitro and in vivo, which senses within the micromolar range, that is based on the DtxR recognition protein.

Prof

Unravelling the genomic complexity of Peltigera cyanolichens: Insights into a complex cross-kingdom community

Many fungi engage in diverse symbiotic relationships, ranging from pathogenic and commensal to mutualistic interactions. Lichens exemplify a highly successful mutualistic system, typically formed between a fungus (mycobiont) and one or more photosynthetic organisms (photobionts), including green algae and/or cyanobacteria. In addition to this core partnership, lichens host a variety of associated fungal and bacterial communities.The genus Peltigera is widespread across multiple continents and serves as a representative of cyanolichens due to its symbiotic relationship with the cyanobacterial photobiont Nostoc. Despite more than two centuries of lichen research, many aspects of lichen biology remain unresolved. However, recent advances in genomics are beginning to fill these gaps, providing new insights into their complex biology. This includes the discovery of a carbohydrate-active- enzyme from the glycoside hydrolase 8 (GH8) family, likely acquired through horizontal gene transfer, as a candidate for facilitating lichen symbiosis in Trebouxiophyceae [1].To contribute to this growing knowledge, we present over 30 metagenome assemblies from Peltigera lichens collected across Germany, the Netherlands, Switzerland, and Lithuania. These assemblies, which capture both photobiont and mycobiont genomes, were produced using state-of-the-art long-read sequencing. Our analyses include interspecies comparisons among Peltigera mycobionts and investigations of structural variations between symbiotic Nostoc and its free-living counterparts. Additionally, we analyze the metagenome assembled genomes (MAGs) of the Peltigera microbiome to identify core microbial members across species and sampling locations. To gain insights into the spatial organization of these core microbes within the lichen thallus, we aim to integrate our omics data with microscopy. For this, we established the LichenMetaImage Use Case, a collaborative effort among CRC 1535 MibiNet and the NFDIs NFDI4Microbiota, DataPLANT, and NFDI4BIOIMAGE.This study offers an in-depth analysis of the complex genomic structure of Peltigera lichens, comparing their primary components (myco- and photobiont) and examining the core members of the microbiome within the lichen community, contributing valuable insights to the growing field of lichen research.References: 1. Puginier et al. (2024): Phylogenomics reveals the evolutionary origins of lichenization in chlorophyte algae. Nat Commun 15, 445.

SFB session
SFB 1535 – Microbial Networking (HHU)

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Programm

SFB sessionSFB 1535 – Microbial Networking (HHU)

Termin

Chair

Programm

SFB session
SFB 1535 – Microbial Networking (HHU)