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

Chair

Prof. Dr.

Viral Take-over of Host Pigment Biosynthesis: Understanding the Role of Cyanophage-Encoded Tetrapyrrole Biosynthesis

MiCon session

Viruses and bacteriophages (viruses that infect bacteria) are the most abundant biological entities on our planet. In the marine environment, cyanophages (viruses infecting cyanobacteria of the genera Prochlorococcus and Synechococcus) have significant impact on ecology, evolution, and biogeochemical processes. Their self-replication is dependent on the molecular machinery of a host bacterium to generate viral progeny. Bacteriophages may have a lysogenic or lytic cycle, with the latter ultimately resulting in the lysis of the host cell. Infection by a lytic phage transforms the host bacterium into a so-called virocell. The virocell represents the intracellular state of the phage"s life cycle whose sole function is to produce virions. Phage infection induces a dramatic change in various host metabolic pathways, which is further expanded by the introduction of auxiliary metabolic genes (AMGs)1,2. Cyanophage encoded AMGs are often related to light harvesting, pigment biosynthesis and photosynthesis and are suggested to modulate and supplement the host bacterium"s metabolism to satisfy the elevated metabolic demand. Over the years, our research has focused on AMGs associated with tetrapyrrole pigment biosynthesis3,4. We have shown that phage-encoded homologs often mimic the activities of their cyanobacterial counterparts, with one notable exception: the phage's ferredoxin-dependent bilin reductase phycoerythrobilin synthase (PebS)3. This enzyme independently performs a reaction that normally requires two sequential enzymes in uninfected host cells. With the recently established recombinant phage technology5 we hope to gain insights into the role of cyanophage-encoded PebS, its importance for phage progeny production and to elucidate how cyanophage proteins interact with host metabolism.References:L.R. Thompson, Q. Zeng, L. Kelly, K.H. Huang, A.U. Singer, J. Stubbe, S.W. Chisholm. Proc Natl Acad Sci U S A., 2011, 108, E757-64.D. Lindell, J.D. Jaffe, Z.I. Johnson, G.M. Church, S.W. Chisholm. Nature, 2005, 438, 86-89.H. Wegner, S. Roitman, A. Kupczok, V. Braun, J.N. Woodhouse, H.P. Grossart, O. Beja, S. Zehner, N. Frankenberg-Dinkel., Nat. Comm., 2024, 15, 8783.T. Dammeyer, S. C. Bagby, M. B. Sullivan, S. W. Chisholm, N. Frankenberg-Dinkel. Curr. Bio., 2008, 18, 442-448.D. Shitrit, T. Hackl, R. Laurenceau, N. Raho, M.C.G Carlson, G. Sabehi, D. A. Schwartz, S. W. Chisholm, D. Lindell. ISME J.,2022, 16, 488–499.

Short lecture

From NMR to function: Mechanistic Insight into the Interaction between Ferredoxin and the Ferredoxin Dependent Bilin Reductase PebS

Ferredoxin-dependent bilin reductases (FDBRs) play a critical role in photosynthetic organisms by catalyzing the reduction of biliverdin (BV) to different open-chain tetrapyrroles, including phytochromobilin (PΦB), phycocyanobilin (PCB), and phycoerythrobilin (PEB)1. These open-chain tetrapyrroles serve as chromophores that facilitate light-depended processes, e.g. in development or regulation of photosynthesis. Different representatives of the class of FDBRs are very distinct in their stereo- and regioselective reduction of BV, yet they all rely on the efficient interaction with the electron donor Ferredoxin (Fd) during catalysis.In this study we investigated the interaction between the phycoerythrobilin synthase PebS1, encoded by a phage, and its host Fd. To determine the molecular interaction between PebS and Fd, NMR spectroscopy and computational docking were employed. Using [15N]-labelled gallium-substituted Fd from Thermosynechococcus elongatus, chemical shift perturbations were observed, allowing the identification of residues critical for protein-protein interactions2. Using these data, we performed HADDOCK docking simulations to predict the contact interface between both proteins and guide the design of PebS variants potentially disrupting the interaction. These variants lead to inhibition of protein activity and therefore support the location of the proposed interface. Based on a phylogenetic analysis, we expand this to the phage Fd and other Fd-FDBR interaction pairs to obtain a better understanding of this essential protein-protein interaction.References[1] Thorben Dammeyer, Sarah C. Bagby, Matthew B. Sullivan, Sallie W. Chisholm, Nicole Frankenberg-Dinkel: Efficient Phage-Mediated Pigment Biosynthesis in Oceanic Cyanobacteria; Current Biology; 2008, 442-448; ISSN 0960-9822; https://doi.org/10.1016/j.cub.2008.02.067.[2] Risa Mutoh, Norifumi Muraki, Kanako Shinmura, Hisako Kubota-Kawai, Young-Ho Lee, Marc M. Nowaczyk, Matthias Rögner, Toshiharu Hase, Takahisa Ikegami, and Genji Kurisu; X-ray Structure and Nuclear Magnetic Resonance Analysis of the Interaction Sites of the Ga-Substituted Cyanobacterial Ferredoxin; Biochemistry2015 54 (39), 6052-6061; DOI:10.1021/acs.biochem.5b00601

Prof.

Actinobacterial Glutathione S-transferases: Catalysts for racemic kinetic resolution of Epoxides?

Epoxides are important platform chemicals in industry but also present in nature. Particularly chiral epoxides hold significant industrial interest due to their application in agrochemical or drug synthesis. A major challenge in organic chemistry is achieving high-yield production while maintaining regio- and enantioselectivity. Enzyme-catalyzed epoxidation offers these features but is limited by their selectivity, restricting its universal applicability. Some enzymes such as epoxide hydrolases (EHs), CoM-transferases (CoMTs) or glutathione S-transferases (GSTs) have shown epoxide conversion with certain selectivity. As the selectivity of GSTs is not well studied, these enzymes became our focus.Lately, the bacterial GST GrStyI was found in the styrene degradation pathway of Gordonia rubripertincta CWB2. Unlike other styrene degraders, CWB2 can metabolize various styrene-related compounds, leading to the co-metabolic formation of ibuprofen for example. The presence of GSTs in bacterial styrene degradation is unusual but appears to broaden the substrate spectrum of this pathway. Similar gene clusters were found in styrene and isoprene-degrading bacteria, encoding GSTs styI and isoI. Consequently, we heterologously produced 5 GSTs in E. coli for characterization of substrate selectivity and enantiomeric preference. Herein, GrStyI appeared as the most promising enzyme for enzymatic kinetic resolution.To explore GrStyIs potential for enantioselective epoxide opening, chiral GC-FID analysis was conducted on various aromatic and aliphatic epoxides. For styrene oxide derivatives, GrStyI exhibited high selectivity for (S)-enantiomers, while the enantiomeric preference of more diverse structures like aliphatic epoxides showed reduced enantioselectivity. Given its high enantioselectivity towards (S)-epoxides, we performed enzymatic kinetic resolution. To proof the feasibility, the reaction volume was subsequently increased to 150 ml for large-scale production, yielding approx. 14 mg (R)-styrene oxide, validated by NMR.Summarizing, we identified GrStyI as highly enantioselective for (S)-styrene oxide and structural analogous. Our study highlights GrStyIs potential for large-scale production of enantiopure epoxides like styrene oxide. Moreover, GSTs from isoprene degradation appear as promising candidates for the production of enantiopure aliphatic epoxides.

Interrogating a PKS Ketosynthase Domain via Site-Directed Active-site Mutations

Polyketides, known for their diverse structures and potent pharmaceutical activities, underpin a wide array of clinical applications, including antibiotics, anticancer agents, and immunosuppressants. Their structural diversity arises from polyketide synthases (PKSs), modular enzymatic assembly lines that offer significant potential for genetic engineering to produce novel derivatives with improved therapeutic properties [1]. However, engineered PKSs often exhibit low activity, resulting in poor yields of the desired derivatives. In our previous work, we deactivated the reduction domain in module 4 of the monensin PKS, which enabled the detection of oxidized derivatives but revealed a biosynthetic bottleneck [2]. High intermediate accumulation suggested a substrate transfer inefficiency between modules 4 and 5, potentially linked to the substrate specificity of the ketosynthase (KS) domain in module 5.To address this, we inspected the KS domain sequences of monensin PKS in comparison to those of other polyether-forming PKSs, identifying fingerprint motifs likely influencing substrate specificity. Guided by multiple sequence alignments, we performed site-directed mutagenesis on the KS5 domain and introduced point mutations into two engineered mutants: the KR40-null mutant (with the ketoreductase domain in module 4 deactivated) and the DH40-null mutant (with the dehydratase domain in module 4 deactivated). The same mutations were introduced in the A495-WT mutant as a control [3]. Targeted LC-MS-based metabolomics was employed to evaluate the impact on product spectrum.Our findings highlight the critical role of KS domains in PKS assembly line efficiency. Strategic alterations at key active-site residues can improve overall productivity, modify substrate preference, or influence stereospecificity, paving the way for more efficient production of diverse polyketide derivatives.References:[1] Cremosnik, G. S., Liu, J., & Waldmann, H. (2020). Guided by evolution: from biology oriented synthesis to pseudo natural products. Natural Product Reports, 37(11), 1497-1510.[2] Grote, M., Kushnir, S., Pryk, N., Möller, D., Erver, J., Ismail-Ali, A., & Schulz, F. (2019). Identification of crucial bottlenecks in engineered polyketide biosynthesis. Organic & biomolecular chemistry, 17(26), 6374-6385.[3] Kushnir, S., Sundermann, U., Yahiaoui, S., Brockmeyer, A., Janning, P., & Schulz, F. (2012). Minimally invasive mutagenesis gives rise to a biosynthetic polyketide library. Angewandte Chemie International Edition, 51(42), 10664-10669.

Exploring the antibacterial properties of natural products from a soil-derived Streptomyces isolate: A comprehensive metabolomic and proteomic approach

Natural products are known for their diverse chemical structures and potent bioactivities. Thus, delving into natural product exploration has emerged as one of the most promising sources for drug discovery. Actinomycetes, particularly Streptomyces species, were identified as prolific producers of antibiotics and continue to offer significant potential. Recent studies revealed that the full biosynthetic potential of many Streptomyces strains has not yet been fully exploited1,2.In this study, we investigate the antibacterial potential of natural products produced by the soil-isolate CS39. Whole genome sequencing confirmed that this isolate belongs to the genus Streptomyces. By cultivating the isolate under various conditions, extracts were obtained, which were then assed for their antibacterial activity against model organisms and ESKAPE pathogens. Furthermore, metabolomic studies of active extracts were performed by LC-MS/MS. Using the GNPS workflow, a molecular network was created and detected masses annotated3. Selected natural products were isolated using mass spectrometry-guided purification. Interested in their unexplored mechanisms of action, biological characterization and mechanism of action studies supported by global proteomic analysis of the response of B. subtilis 168 were performed.Our results showed that the crude extracts from Streptomyces CS39 exhibit antibacterial activity against both gram-positive and gram-negative bacteria. Antibiotic compounds known for their efficacy against gram-positive bacteria were successfully annotated and three of them purified. Proteomic analysis of B. subtilis 168 to these natural products showed upregulation of marker proteins some of which related to energy metabolism, oxidative stress, and protein folding stress. Future research will focus on further characterization of the mechanisms of action of the natural products, as well as investigation of further compounds that may explain the observed activity against gram-negative bacteria.[1] Hutchings et al., Curr Opin Microbiol. 2019, 51:72-80.[2] Rutledge and Challis, Nat Rev Microbiol. 2015, 13, 509-523.[3] Wang et al., Nat Biotechnol. 2016, 9;34(8):828-837.

The anti-tubercular callyaerins target the Mycobacterium tuberculosis-specific non-essential membrane protein Rv2113

Tuberculosis remains one of the most threatening bacterial infections, particularly due to the increasing prevalence of antibiotic-resistant strains of the pathogen Mycobacterium tuberculosis. Therefore, alternative antibiotics with divergent mechanisms of action are urgently required. Recently, callyaerin A and B, two derivatives of the callyaerins, a group of cyclic peptides bearing an unusual (Z)-2,3-diaminoacrylic acid unit, were identified as promising anti-tubercular natural products. Through a combination of spontaneous resistant mutant screening and affinity-based protein profiling (AfBPP) the non-essential but Mycobacterium tuberculosis-specific membrane protein Rv2113 was identified as the biological target of the callyaerins. However, the function of Rv2113 has not been fully resolved. Fluorescent microscopy experiments showed that Rv2113 is not involved in the cellular uptake of the callyaerins. Instead, our data indicate that Rv2113 is involved in mycobacterial lipid metabolism.

MiCon Session
GRK 2341 – Microbial substrate conversion

Termin

Chair

Programm

MiCon SessionGRK 2341 – Microbial substrate conversion

Termin

Chair

Programm

MiCon Session
GRK 2341 – Microbial substrate conversion