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

Chair

Illuminating protein interactions: Photo-Leucine crosslinking in Bacillus subtilis

Signal transduction & gene regulation

Understanding the intricate network of protein-protein interactions (PPIs) is essential for unraveling the complexities of cellular function. Photo-crosslinking has emerged as an exciting new method for probing PPIs in many organisms but has not yet found widespread application in bacteria. In this study, we aim to apply photo-crosslinking with photo-leucine in the well-studied gram-positive model organism Bacillus subtilis, laying the groundwork for its potential future applications.We show that photo-leucine becomes highly toxic for B. subtilis and investigated the mechanisms of photo-leucine uptake. We provide evidence that the branched-chain amino acid importers BcaP and BraB are involved in the uptake of photo-leucine. Additionally, we unveil the pivotal role of the bipartite exporter AzlCD in facilitating the efficient export of photo-leucine from the cell. Furthermore, our investigation uncovers a previously uncharacterized exporter, AexB, belonging to the novel "sleeping beauty" group of the EamA family [1]. Notably, AexB is found to be positively regulated by the transcription factor AerB, a member of the AraC family. Additionally, we isolated a mutant highly resistant to photo-leucine and identified a single amino acid exchange in the leucine-tRNA synthetase LeuS responsible for this resistance. Using a molecular docking approach, we show that the mutation alters the binding cavity for leucine in a manner that only allows leucine, but not photo-leucine, to bind productively. Furthermore, incorporation rate studies in the suppressor mutant with the LeuS mutation revealed that the photo-leucine incorporation rate was below the detection limit. This work presents the homeostasis of photo-leucine in B. subtilis, laying the groundwork for future application of photo-crosslinking and thus the possible identification of novel protein-protein interactions as well as structure refinement.[1] Warneke et al. (2023) mBio 15:e03456-23.

Short lecture

Baseline estimates for cell-to-cell interactions in marine and terrestrial habitats

Environmental microbiology & ecology

Microbial taxa in the environment do not exist in isolation but are highly connected to other microbial cells in the exchange of nutrients, toxins, and mobile genetic elements. The presence of auxotrophy in many taxa means it is essential that cells can bridge the spatial distances between them, but the exact distances are largely unknown. What is the number of cells that a microbe can realistically expect to interact with in natural environments? Here we use order-of-magnitude calculations to estimate a typical distance between cells, based on empirical estimates of cell abundance for terrestrial and marine habitats. Our estimates reveal that 30%-80% of the global microbial biomass occurs in spatially structured habitats, with expected cell-to-cell distances ranging from 10s of microns in the soil to ~200 microns for planktonic cells in the ocean water column. Despite these short distances, only a small fraction of the molecules secreted by one cell reaches the other cell due to the three-dimensional diffusion of molecules. In a careful comparison, we estimate the spatial distances that cells can bridge purely by the diffusion of nutrients and with additional cell motility. Overall, our work provides an estimate for cell distances that helps to better contrast surface and subsurface habitats and formulate a baseline expectation for the conditions that microbial communities are experiencing in nature.

Prof.

Heat shock coupled transduction: a fast and facile method to circumvent the staphylococcal restriction barrier

Phage biology

As common members of the human microbiota, staphylococcal species colonize humans at various body sites. While some of these staphylococci, like Staphylococcus aureus, have the potential to cause a variety of devastating diseases, most of them have adopted a commensal lifestyle. Many of these staphylococcal species have been shown to produce various secondary metabolites, excluding more pathogenic bacteria from their niches, or modulating the host immune system to overcome infections by different pathogens. However, studying staphylococci in the laboratory context remains challenging, as many strains possess a strong restriction barrier, which hinders genetic manipulation. Even though significant progress has been made in recent years, the ability to study these bacteria and their genetic potential is still severely limited.In this work, we describe a novel method combining heat shock and transduction to enable the genetic manipulation of previously inaccessible staphylococcal strains. We present evidence that the method is applicable to different plasmids, species, and different phages. Our experimental data indicates that different species show a varying temperature optimum for the heat shock and that restriction-competence is recovered over time, suggesting a temporary inactivation of restriction enzymes. We propose that this method is generally applicable to different bacteria, for which transducing phages are available, and is not limited to staphylococci. Overall, this method will enable the study of previously genetically inaccessible bacteria in a fast, easy, and cost-effective way.

Modeling of intravenous caspofungin administration using an intestine-on-chip reveals altered Candida albicans microcolonies and pathogenicity

Biotechnology

The human intestine is a major reservoir of the commensal fungus Candida albicans and the main source for life-threatening systemic candidiasis. To dissect the fungal-host interactions, conventional 2D in vitro models are commonly used. However, these models lack biological complexity and are less suitable for evaluating antifungal drug administration routes and the drug concentration gradients across multilayered tissue barriers as observed in vivo. Therefore, a 3D intestine-on-chip model was leveraged to investigate fungal-host interactions and evaluate the antifungal activity of caspofungin.This intestinal model includes a vascular compartment with endothelial and immune cells, an intestinal compartment with villus- and crypt-like structures, and peristaltic flow in both compartments. The model was infected with either a wild-type (SC5314) or caspofungin-resistant (110.12) strain of C. albicans. Caspofungin was vascularly perfused to simulate the intravenous treatment of candidemia. Complementary to biomolecular analyses, we developed an automated image analysis pipeline that provides 3D morphological characterization of fungal objects. Additionally, this pipeline provides quantification of the epithelial tissue architecture, the interaction between fungal objects and tissue structures, as well as the level of fungal invasion and translocation.We demonstrated that C. albicans invades the epithelial tissue and translocates to the vascular compartment. Moreover, C. albicans induced severe host tissue damage by disrupting cell-cell contacts, increasing intestinal permeability, and promoting inflammation. Caspofungin treatment restored vascular and intestinal host tissue integrity. Further, fungal burden, tissue invasion, and vascular translocation were concentration-dependently reduced and accompanied by decreased inflammation. Advanced image-based analysis revealed a reduction in fungal biomass associated with more compact microcolonies and a diminished surface-to-volume ratio, which indicated a decreased exposure to the epithelial tissue after caspofungin treatment. In contrast, caspofungin showed limited effects in the case of infection with the caspofungin-resistant clinical isolate.Collectively, the intestinal candidiasis-on-chip-model can be leveraged for in-depth in situ characterization of antifungal treatment efficacy, emerging fungal drug resistances, and associated changes in host-pathogen interactions.

Prof. Dr.

Enhancing cultivable microbial diversity from mouse gut microbiome using droplet microfluidics

To comprehend the complex dynamics of the gut microbiome and its influence on host health, it is essential to cultivate and isolate gut microorganisms. However, traditional culturing methods often struggle to capture the full diversity of gut microbiota, especially when it comes to rare and slow-growing species (1, 2). In this study, we introduce a droplet microfluidic platform as an efficient and high-throughput approach for cultivating and isolating microorganisms from mouse gut. Miniaturized droplets with volume ranging from few picoliters to nanoliters are generated at kilohertz frequencies targeting confinement of single gut bacteria per droplet (3–5). Millions of these droplets are incubated in parallel, with homogenous incubation conditions. We cultivated and isolated a diverse range of gut microorganisms from mouse fecal pellets. A comparative analysis with conventional agar plating techniques showed that our droplet cultivation method yielded a greater number of unique isolates, highlighting its superior ability to capture the cultivable fraction of the gut microbiome. Additionally, 16S rRNA amplicon sequencing demonstrated that our system reflects changes in microbial diversity. Droplet microfluidics serves as an innovative solution for expanding the cultivable fraction of the gut microbiota.ReferencesL. Mahler et al., Elife. 10, 1–23 (2021).D. K. Chaudhary, A. Khulan, J. Kim, Sci. Rep. 9, 6666 (2019).S. Hengoju, M. Tovar, D. K. W. Man, S. Buchheim, M. A. Rosenbaum, in Microfluidics in Biotechnology (2020), pp. 129–157.W. J. Watterson et al., Elife. 9, 1–22 (2020).T. Moragues et al., Nat. Rev. Methods Prim. 3, 32 (2023).

Prof., Dr.

Prof

Unveiling the carbon fixation potential of marine Prokaryotes using MarMAGs, a novel user-friendly database with over 130 thousand metagenome assemble genomes

Our team designed the MarMAGs, a user-friendly database containing over 130 thousand marine metagenome-assembled genomes (MAGs). Users can access this data from coordinates in a map or by filtering it based on its metadata (e.g., marine biome, biogeographic features, and sequencing platform). Further, we showcased carbon fixation as a use case. We recovered metagenome-assembled genomes (MAGs) from 3405 marine metagenomes selected from MarineMetagenomeDB (https://web.app.ufz.de/marmdb/) and added 52162 MAGs from the OceanDNA dataset and 8558 marine MAGs from the GEM catalog. We evaluated genome quality, taxonomy, and assembly. After metadata harmonization, we identified the genes for prokaryotes' carbon fixation pathways that included the Reductive pentose phosphate cycle (CBB), Reverse Citrate cycle, 3-Hydroxypropionate bicycle (3HPB), Dicarboxylate-hydroxybutyrate cycle (DCHB), Hydroxypropionate Hydroxybutyrate cycle, and Reducive Acetyl-CoA pathway. Due to the complexity of reversible enzymatic reactions and the versatility of the involved enzymes, the Reductive Glycine pathway and reverse oxidative TCA cycle were not annotated in our analysis. We recovered 73,944 novel MAGs that, together with the other two MAG datasets, comprised the MarMAGs with 134,644 MAGs. The MarMAGs contain 31.2%(42047) high-quality and 68.78%(92617) medium-quality MAGs. Bacteria dominate the taxonomic composition of the MAGs at 89.89% (121046), and archaea at 10.11% (13618) spread across 135 phyla. Our analyses revealed 3.96% (5333) of the MAGs with genetic potential for at least one carbon fixation pathway, with CBB comprising 79.75% (4253), while 3HPB 0.02% (1) and DCHB 0.08% (4) have the least of them. Also, 28 MAGs affiliated to 5 phyla have the genetic potential for two pathways. Additionally, we are conducting phylogenetic analyses to explore the relatedness of MAGs based on their genetic capacities for carbon fixation and provide insights into the evolutionary adaptations of marine prokaryotes. The MarMAGs database is a powerful tool for understanding marine prokaryotes' genetic potential. For example, investigating the genetic potential of carbon fixation within MarMAGs provided a detailed view of marine prokaryotes' diverse carbon fixation strategies. Our interactive web platform is a step forward in democratizing access to this valuable genome resource for researchers interested in microbial ecology, carbon cycling, and evolutionary biology in the marine environment.

Towards net zero land biotechnology – a potential assessment for selected bioprocesses in Germany

To stay within the planetary boundaries transformation to a more circular economy is key, necessitating the consequent utilization of residues as resources. The circular material use (CMU) in Germany is only 12.2% that is below EU average, with biomass CMU of 7.6% being particularly low. On the other hand, Germany has about 240 Mio Mg a-1 dry mass of biogenic residues accruing, which are not well valorized yet [1]. Biotechnological processes will play a crucial role to leverage biogenic residues for providing value-added products in a circular economy [2]. To accelerate increasing CMU the feasibility of biogenic residues for the wide range of biotechnological processes needs to be assessed.Biogenic residues are heterogeneous while established bioprocesses typically demand specific compounds as substrates. To bring together resource quality with the dedicated demand, we categorized the residues compiled in the DE biomass monitor regarding their biochemical composition, and thus feedstock potential. 95% of biogenic residues were organized into a resource matrix, detailing their fractions and the quality of available data. Three biotechnological processes, making use of lignin, non-fibrous carbohydrates, and oil, respectively, served as model processes to assess the biogenic production potential. Material flows based on state-of-the-art conversion routes will be presented, depicting that residue-based production via all three biotechnology examples can meet national demands of specific polymer precursors, medium chain carboxylates, and platform chemicals, respectively, when only 20-30% of possible raw materials is mobilized. The high share of (unused) by-product, underlines the importance of production networks and cluster approaches as early as possible in biotechnological process development. Specific challenges to fully exploiting the potential of biogenic residues, including legal and acceptance issues, the need for intelligent biomass decomposition in networked production, and residue availability and management will be discussed.[1] Mujtaba, M. et al., 2023. https://doi.org/10.1016/j.jclepro.2023.136815[2] Naegeli de Torres, F. et al., 2023, https://doi.org/10.5281/zenodo.10370137

Utilization of the small regulatory protein CP12 to engineer photosynthetic electron fluxes in cyanobacteria

Prokaryotic physiology and metabolism

Photoautotrophic microorganisms like cyanobacteria hold great potential for sustainable production of chemicals and fuels from light, CO2, and water. However, their economical applicability is limited, in part due to low product yields achieved to date. Recent insights into their metabolism, energy and metabolite fluxes, and metabolic regulation provide new opportunities for interventions to enhance product yields. For conversions that only require electrons – such as redox catalysis or the formation of hydrogen by hydrogenases - the partitioning of electrons from the photosynthetic electron transport chain (PETC) determines the yield. In these processes the Calvin-Benson-Bassham (CBB) cycle is the main competitor for electrons. Here, the small regulatory protein CP12 was used to reduce electron flux into the CBB cycle and redirect them into valuable products in the model cyanobacterium Synechocystis sp. PCC 6803. In most phototrophic organisms, CP12 is used to control the CBB cycle in dark-night cycles by binding and inhibiting the enzymes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and phosphofructokinase (PRK). The native CP12 of Synechocystis itself is redox-regulated, inhibiting the CBB cycle only in the dark and becoming deactivated by a reduced cell status when light is present and the PETC is running1,2. We therefore employed variants of CP12 either derived from cyanophages - which use them during infection in light as auxiliary metabolic genes yielding a more reduced status of their hosts3 - or rationally designed to be unaffected by the redox-status. By inducible expression of these CP12 variants, we were able to modulate photoautotrophic growth and shift the intracellular redox potential to a more reduced state. This altered metabolic state increased the electron flux from photosynthesis into hydrogen production and, in combination with disruption of cyclic electron flow and partial uncoupling of the proton-motif-force, enabled higher continuous hydrogen production. By utilizing CP12 variants to redirect electrons from the CBB cycle toward hydrogen production in cyanobacteria, we highlight the potential of small regulatory proteins for metabolic engineering to optimize resource allocation towards desired products.1 McFarlane, et al. 2019, PNAS 116(42): 20984-20990.2 Lucius et al. 2022, Front. Plant Sci. 13: 1028794.3 Thompson et al. 2011, PNAS 108: E757-E764

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