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

Chair

Prof.

Insights into guanidine assimilation, its regulation and utilization to drive synthetic processes in cyanobacteria

Prokaryotic physiology and metabolism

Cyanobacteria receive increasing attention in bioeconomy research as they could be used as whole-cell biocatalysts for a light-driven, CO2-neutral and hence, sustainable production of chemicals and fuel components. The realization of this potential requires deep understanding of cyanobacterial metabolism and its regulation as well as the simultaneous development of molecular tools for metabolic engineering. Here, we provide insights into the assimilation of guanidine via a recently identified guanidine hydrolase (GdmH) [1]. The corresponding gdmH gene is widely distributed among cyanobacteria and allows them to grow on guanidine as sole nitrogen source. Consistently, strains lacking gdmH, either naturally or by gene deletion, do not grow on guanidine. In the model strain Synechocystis sp. PCC 6803, the expression of gdmH is controlled at the transcriptional level by NtcA that mediates transcriptional activation in response to nitrogen limitation. Moreover, a conserved RNA motif is present in the 5"UTR that forms the aptamer of a guanidine I riboswitch. Via in-line probing we demonstrate specific and high affinity binding of guanidine to the aptamer. In vivo, the riboswitch enabes guanidine-specific and titratable induction of gene expression. In addition, the gdmH gene is frequently co-localized with ABC transporter genes. Their mutation also interferes with guanidine-dependent growth of Synechocystis pointing towards a guanidine-specific substrate binding protein in the periplasm. However, this interference can only be observed in presence of rather low guanidine concentrations between 0.1 and 0.5 mM. At concentrations >1 mM the mutants grow similar to WT indicating the presence of additional, yet to be identified guanidine uptake systems. In addition, we made use of the guanidine riboswitch to achieve precise and dynamic regulation of heterologous gene expression in cyanobacteria. As guanidine is quite cheap and the required guanidine concentrations are rather low, guanidine riboswitches enable gene expression control at large scale without significant costs for inducers. Altogether, our findings not only contribute to the understanding of cyanobacterial metabolism, but also highlight the potential of guanidine riboswitches as valuable tool for synthetic biology applications in cyanobacteria.[1] Funck, D. […], Hartig, J.S., 2022. Discovery of a Ni2+-dependent guanidine hydrolase in bacteria. Nature 603, 515–521.

Short lecture

Prof. Dr.

Investigating the cryptic sulfur incorporation during biosynthesis of the marine antibiotic tropodithietic acid

Question: The structurally unique antibiotic tropodithietic acid (TDA) is produced by various gram-negative marine bacteria like Phaeobacter inhibens and contains two sulfur atoms, which appear to be incorporated non-canonically. Focus of this work was the investigation of the cryptic sulfur incorporation during TDA biosynthesis.Methods: Candidate enzymes were chosen based on bioinformatic predictions and knockout studies. These candidate enzymes were heterologously produced, and their biochemical functions assessed using in vitro assays as well as biophysical methods. To test biosynthetic hypotheses structural analogues of postulated intermediates were synthesized and used as surrogate substrates for these investigations. Complementary structural studies were conducted using X-ray crystallography to scrutinize the underlying enzymology.Results: We were able to successfully reconstitute a three‑enzyme cascade in vitro, leading to the integration of a thiol group into a model substrate. More precisely, it could be shown that a GSH moiety attached to an aromatic backbone via a C-S bond is sequentially degraded by a γ-glutamyl-cyclotransferase, metallopeptidase and cysteine-S-conjugate-β-lyase. Additionally, the structures of the γ-glutamyl-cyclotransferase aswell as the β‑lyase could be elucidated using X-ray crystallography, granting insight into the catalytic mechanism behind GSH degradation and C-S bond cleavage.Conclusion: These findings corroborate the notion of GSH as the source of sulfur during TDA biosynthesis and pave the way for the biotechnological production of analogous compounds. Interestingly, the degradation of GSH to a reactive thiol has been described in the biosynthesis of the infamous fungal epipolythiodiketopiperazine type toxins during formation of a transannular disulfide bridge, making this the second report of GSH involvement in microbial secondary metabolism in general and the first one specifically in bacteria [1].References[1] Scherlach K, Kuttenlochner W, Scharf DH, Brakhage AA, Hertweck C, Groll M, u. a. Structural and Mechanistic Insights into C−S Bond Formation in Gliotoxin. Angew Chem Int Ed Engl. 14. June 2021;60(25):14188–94.

Elucidating Polyhydroxybutyrate metabolism in Streptomycetes

QuestionPlastic pollution poses a significant environmental threat causing multiple problems harming life on earth. One potential approach to lessen plastic pollution is to replace conventional plastics with biodegradable plastics, such as polyhydroxybutyrate (PHB). PHB can be produced and broken down by various bacteria using PHB synthase (PhaC) and PHB depolymerase (PhaZ), making it an ideal candidate for a circular economy approach. The goal is to identify and characterise the enzymes involved in PHB metabolism in Streptomyces and to evaluate whether they can serve as biofactories for converting PHB into antibiotics, as they are known as prolific hydrolytic enzyme and antibiotic producers.Methodology S. venezuelae was utilised as a model strain and was tested in a growth assay for PHB degradation. Known PHB-metabolizing enzymes were used as queries to bioinformatically identify PHB-related proteins based on sequence or structural similarities. Knockout mutants were created for each of the putative candidates and were analysed phenotypically.Results S.venezuelae can grow in minimal media with PHB as the sole carbon source. We identified three potential enzymes involved in PHB metabolism in S. venezuelae: a putative PhaC enzyme (vnz_01345), an extracellular PHB depolymerase (PhaZ, vnz_35915), and a PHB dehydrogenase (BdhA, vnz_03225). Macrocolonies of the Δvnz_01345 strain (putative PhaC) and the Δvnz_03225 strain (putative BdhA) exhibited more difficulty in sporulation compared to the wild type (WT) when grown on high salt sporulation medium. In contrast, the strain Δvnz_35915 (putative PhaZ) demonstrated better growth than the WT under the same conditions.ConclusionWe showed that S.venezuelae can utilize PHB as a carbon source and identified several candidate enzymes responsible for PHB metabolism in Streptomyces. The phenotypic analysis suggests that PHB may have a protective role under osmotic stress conditions, as it has been shown for other bacteria. Further characterisation is necessary to understand these enzymes better and to prove their role in PHB metabolism. However, these results are promising and indicate Streptomyces as a suitable PHB upcycling platform.

Regulatory mechanisms controlling the catabolism of glycogen in cyanobacteria

Glycogen is a wide-spread reserve polymer important for the survival to environmental stresses of all kinds of organisms. In the cyanobacterium Synechocystis sp. PCC 6803, glycogen is necessary to overcome dark and starvation periods. Given the important role of this polysaccharide, its metabolism is subjected to a complex regulation. The activity of the enzymes involved in glycogen synthesis and degradation must be tightly controlled to ensure rapid access to the sugar stores when required. Our research focuses on how such a responsive and fine control is achieved via post-translational modifications of the glycogen catabolic enzymes. We found that modulation of the activity of glycogen phosphorylase (GlgP), phosphoglucomutase (PGM), and glucose-6-phosphate dehydrogenase (G6PDH) through phosphorylation, redox-induced modifications, allosteric interactions and metabolite-level regulation is essential for survival under fluctuating environmental conditions. Interestingly, some of these mechanisms are not specific of cyanobacteria, but evolutionary conserved from bacteria to humans.

Being lazy is not always bad: The effects of c-di-AMP signalling on glutamine toxicity in cyanobacteria

Cyanobacteria exhibit a distinctive lifestyle as photoautotrophic bacteria, necessitating rapid responses to environmental shifts that jeopardize their carbon/nitrogen (C/N) balance. To maintain homeostasis, cyanobacteria employ cyclic nucleotide second messengers, such as cyclic di-adenosine monophosphate (c-di-AMP)1. In cyanobacteria, c-di-AMP has now been implicated in a broader range of functions, including C/N balance regulation2, day-night acclimation3, and natural competence4. While mechanistic aspects of C-regulation have been elucidated3, N-regulation remains unexplored. In the search for novel c-di-AMP targets, we identified the glutamine (Gln) transporter BgtAB as a c-di-AMP target. Gln was reported to be toxic for cyanobacteria5, but its mode of action remained elusive. Surprisingly, the c-di-AMP-free mutant (ΔdacA) was able to grow on toxic Gln concentrations and use it as sole N-source, while wildtype (WT) died. We found that ΔdacA exhibited impaired Gln uptake compared to WT. Here, targeted metabolomics unveiled an overaccumulation of arginine and its intermediates in WT cells growing on Gln. In contrast, Gln metabolization was severely hampered in ΔdacA, highlighting a role for c-di-AMP in controlling N-metabolism. We further show that the high Gln uptake in WT induces dacA gene expression and c-di-AMP overproduction, which is known to be toxic for bacteria6. Overall, our findings suggest that c-di-AMP positively regulates BgtAB, and the resulting Gln uptake triggers c-di-AMP overproduction, establishing a lethal feedback loop. This work presents the first mechanistic explanation of Gln toxicity in cyanobacteria due to c-di-AMP signalling and showcases the role of c-di-AMP in N-metabolism.1. Mantovani et al. (2023). MicroLife, 4, uqad008. DOI: 10.1093/femsml/uqad0082. Haffner et al. (2023). BioRxiv. DOI: 10.1101/2023.11.14.5670063. Selim et al. (2021). Science Advances, 7(50), eabk0568. DOI: 10.1126/sciadv.abk05684. Samir et al. (2023). BioRxiv. DOI: 10.1101/2023.11.27.5688195. Flores & Muro-Pastor (1990). Archives of Microbiology, 154(6). DOI: 10.1007/BF002488316. Gundlach et al. (2015). Journal of Bacteriology, 197(20), 3265–3274. DOI: 10.1128/jb.00564-15

Novel approaches in genetics and systems biology of marine Roseobacter group bacteria

IntroductionRoseobacter group bacteria are a large and diverse group of Alphaproteobacteria that are abundant across marine ecosystems. They are known for their versatile metabolism and genome plasticity. Frequently, these bacteria interact with other marine organisms, especially algae. Therefore, they are thought to play an important role in mediating the marine carbon cycle. Since Roseobacter group bacteria have only been investigated in the past 30 years, many aspects of their physiology, ecology, and evolution are still unknown. Consequently, further studies on different Roseobacter group bacteria are required to better understand this important group of bacterioplankton.GoalsHere, we aim to establish novel genetic tools and to investigate the metabolic network of three model strains from the Roseobacter group, with the dual goal of 1) enabling more sophisticated approaches for genetic modification and 2) improving our understanding of the physiology of these abundant marine bacteria.Materials & methods We focused our investigations on the three model strains Ruegeria pomeroyi, Phaeobacter inhibens, and Dinoroseobacter shibae. Using molecular cloning, transformation or conjugation, and high-throughput screening experiments, we tested different genetic constructs in all three bacteria. Furthermore, we generated comprehensive proteomics datasets for D. shibae grown under different conditions. Finally, we compared this dataset to proteomics datasets of P. inhibens to identify shared and divergent traits in the proteomes of these Roseobacter group bacteria.ResultsWe generated a vector suite with different constitutive and inducible promoters that is functional across the three tested model strains. Furthermore, we characterized changes in the proteome of D. shibae on rich medium and minimal medium with different carbon sources. Finally, we identified the core and accessory proteomes of P. inhibens and report new insights on the regulation of catabolic and anabolic pathways in response to nutrient availability.SummaryThe establishment of novel genetic tools and the comparative analysis of comprehensive proteomics datasets highlight that Roseobacter group bacteria continue to be an exciting field of study and pave the way for further investigations on the fundamental biology and the application potential of these marine microorganisms.

Prokaryotic physiology and metabolism I
Advances in biosynthesis and bacterial applications

Appointment

Chair

Topic

Programme

Prokaryotic physiology and metabolism IAdvances in biosynthesis and bacterial applications

Appointment

Chair

Topic

Programme

Prokaryotic physiology and metabolism I
Advances in biosynthesis and bacterial applications