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

Prof. Dr.

Physiological interplay of iron and biotin metabolism in Corynebacterium glutamicum

Prokaryotic physiology and metabolism

Growth of Corynebacterium glutamicum in minimal medium relies on the supplementation of essential biotin and the siderophore protocatechuate (PCA) to compensate for transient iron limitation [1,2]. Previously, biotin-prototroph C. glutamicum was engineered [1] and iron-responsive, enforced PCA synthesis was implemented in strain C. glutamicum IRON+ to improve intracellular iron availability [2].Here, a prototrophic C. glutamicum strain was engineered that is independent of both, biotin and PCA. Therefore, C. glutamicum IRON+ was genomically equipped with heterologous bioF and bioI genes catalyzing the missing steps of biotin synthesis [1] which yielded the designated strain C. glutamicum IRON+BioFI. Growth properties were characterized in shake flask cultivations in biotin- and PCA-free CgXII minimal medium (CgXIIMM) with 20 g glucose L-1­.In the shake flask, C. glutamicum IRON+BioFI grew to a final biomass of 11.3 ± 0.8 g CDW L-1 in CgXIIMM, however, showed biphasic growth with reduced growth rates of 0.27 ± 0.00 h-1 and 0.34 ± 0.02 h-1 in each phase, respectively. In contrast, the wild type (WT) exhibited continuous exponential growth with a µ of 0.40 ± 0.01 h-1 reaching 10.2 ± 0.5 g CDW L-1 in fully supplemented CgXII medium. The biphasic growth was observed before for the WT grown in PCA-depleted CgXII medium which was traced back to a temporary iron limitation [2,3]. Thus, we assume that intracellular iron availability of C. glutamicum IRON+BioFI is impaired again.Upon bioFI integration, the regulator of the biotin metabolism bioQ was deleted which previously led to decreased transcription levels of genes involved in the iron metabolism, such as cg0924 [4] which encodes a component of a putative siderophore ABC transporter [5]. To investigate the underlying physiological effects of bioQ deletion on the iron metabolism, we deleted cg0924, integrated the bioFI genes into the neutral landing pad CgLP5 [6] and characterized growth of the engineered strains on different carbon sources.[1] Peters-Wendisch et al., J. Biotechnol. (2014)[2] Thoma et al., Microb. Biotechnol. (2023)[3] Müller et al., mBio (2020)[4] Brune et al., J. Biotechnol. (2012)[5] Küberl et al., Appl. Environ. Microbiol. (2020)[6] Lange et al., Microb. Biotechnol. (2018)

Poster

In-depth characterization of Bacillus subtilis mutants lacking the essential target of glyphosate

The best known and most widely used herbicide worldwide is glyphosate [1]. The non-selective, systemic broad-spectrum herbicide glyphosate (N-(phosphonomethyl)-glycine) is used against all types of weeds that compete with agricultural crops [1]. Glyphosate acts specifically on the 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase, which makes the EPSP synthase of bacteria an attractive target for antibiotics and herbicides. The EPSP synthase is involved in the synthesis of chorismate from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP), which is an essential precursor for the de novo synthesis of aromatic amino acids, folates (essential for purine and pyrimidine synthesis) and quinones (essential electron carrier in the respiratory chain) [2]. It has already been shown that the enzyme function of EPSP synthase in Bacillus subtilis is essential for growth in minimal medium and complex medium such as LB [3]. A B. subtilis deletion mutant in which the aroE gene coding for EPSP synthase has been deleted can still grow in complex BHI medium [3]. This is possible because B. subtilis apparently has unknown uptake systems for all chorismate derivatives and aromatic amino. Nevertheless, it has not yet been possible to cultivate an aroE deletion mutant stably in a minimal medium. For this purpose, the exact, minimal nutrient composition is to be determined. This can be achieved by characterizing suppressors of the aroE deletion mutant, which frequently contain mutations in genes encoding the ResED 2-component system, which is responsible for switching from aerobic to anaerobic metabolism [4]. This indicates that there is an imbalance in the menaquinone pool and the NADH+H+/NAD+ ratio during growth of the aroE mutant in minimal medium, which can be restored by suppressor mutations. Comprehensive suppressor analysis to understand the effect of aroE deletion on the physiology of the cell will be done. Also, stably growing aroE mutants will be established to identify additional glyphosate targets in B. subtilis. Subsequently, identification of aromatic amino acid transporters in B. subtilis will be performed.[1] Duke and Powles (2008) Pest Manag Sci 64: 319-325.[2] Hermann and Weaver (1999) Annu Rev Plant Physiol Plant Mol Biol 50: 473-503.[3] Schwedt et al. (2023) Environ Microbiol 25: 3604-3622.[4] Härtig and Jahn (2012) Adv Microb Physiol 61: 195-216.

The role of small proteins in Escherichia coli under acid stress

In the human gastrointestinal tract, bacteria have survival strategies in response to acidic environments. Escherichia coli, known for its high level of acid resistance, employs multiple acid defense mechanisms to ensure its survival under these harsh conditions. The main acid resistance mechanism involves three proton-consuming protection strategies, each consisting of an amino acid decarboxylase and an antiporter to take up the amino acid and release the more alkaline polyamine into the surrounding medium (Brameyer et al. 2022).In recent years, due to the advances in detection techniques, a significant number of previously uncharacterized small proteins have been identified in bacteria (Fuchs and Engelmann 2023). These small proteins encoded by small open reading frames (sORFs) are often overlooked during annotation and are difficult to characterize using conventional biochemical techniques.In 2023, ribosome profiling revealed 18 novel stress-induced sORFs in E. coli, found particularly under severe (pH 4.4) acid stress conditions (Schumacher et al. 2023). The current project aims to gain further insights into the subcellular location, properties, potential interactions, and the role in the acid resistance mechanism of the newly identified sORF candidates. sORF15 is located between gadW, encoding one of the major transcriptional regulators of the Gad acid resistance system, and mdtF, encoding a multidrug resistance protein. Interestingly, the last 64 bp of sORF15 overlaps with 3′ of mdtF. We found that E. coli MG1655 was more tolerant to stress imposed by organic acids and antibiotics than E. coli MG1655 ∆sORF15.References:Brameyer, Sophie, Kilian Schumacher, Sonja Kuppermann, and Kirsten Jung. 2022. "Division of Labor and Collective Functionality in Escherichia Coli under Acid Stress." Communications Biology 5 (1): 1–14. https://doi.org/10.1038/s42003-022-03281-4.Fuchs, Stephan, and Susanne Engelmann. 2023. "Small Proteins in Bacteria - Big Challenges in Prediction and Identification." Proteomics 23 (23–24): e2200421. https://doi.org/10.1002/pmic.202200421.Schumacher, Kilian, Rick Gelhausen, Willow Kion-Crosby, Lars Barquist, Rolf Backofen, and Kirsten Jung. 2023. "Ribosome Profiling Reveals the Fine-Tuned Response of Escherichia Coli to Mild and Severe Acid Stress." mSystems 8 (6): e0103723. https://doi.org/10.1128/msystems.01037-23.

Observation and control of sporulation failures in Bacillus subtilis

Spore formation in Bacilli is a complex developmental process in which vegetative cells form dormant endospores. However, not all produced spores are necessarily viable, yet high purity of spore preparations is crucial for many spore-based applications.Here, we characterized how such sporulation failures arise by tuning sporulation medium conditions in liquid batch culture in small-scale bioreactors. Supplementation of a chemically defined sporulation medium with L-alanine improved the sporulation outcome of Bacillus subtilis. We attribute this to alanine catabolism by alanine dehydrogenase, which prevents premature germination triggered by the major germinant receptor GerA. Pyruvate biosynthesis from alanine catabolism in turn supports spore formation via central metabolism.By using automated time-lapse microscopy, we further investigated spore morphogenesis during successful and failed sporulation events. We found that sporulation failures were associated with premature mother cell lysis and linked to an additional, understudied late step in the sporulation program: a centering motion of the forespore body within the mother cell.Our observations suggest a tight link between central metabolism and the outcome of the late stages of spore development. Furthermore, our new methods and insights offer new opportunities for improving spore production of Bacilli in applied contexts.

Biosynthesis and Bioorthogonal labelling of Cyclopeptides harboring reactive Furyl side chains

3-Furylalanine (Fua) is a reactive amino acid that occurs naturally in a limited number of cyclopeptides. For example, hepatotoxic rhizonins are produced by bacterial endosymbionts of Rhizopus microsporus,[1] and their toxicity critically depends on the Fua residues.[2] Fua moieties are also found in endolides, where the presence of Fua moieties confers receptor binding specificity.[3] Beyond, furyl-substituted peptides are amenable to bioorthogonal Diels-Alder-type derivatization.Despite its relevance, Fua biosynthesis and incorporation by nonribosomal peptide (NRP) synthetases has remained elusive. By genome sequencing and gene inactivation, we discovered the gene cluster responsible for rhizonin biosynthesis. Mutational analysis and heterologous reconstitution identified the dioxygenase RhzB as a Fua synthase, and isotope labeling pinpointed tyrosine and L-DOPA as Fua precursors.[4] Interestingly, RhzB-like Fua synthases form a novel clade of heme-dependent aromatic oxygenases and are encoded in the genomes of phylogenetically distant bacterial phyla.The prevalence of cryptic Fua-containing NRPs motivated us to devise a strategy to rapidly access novel clickable Fua-harboring bioactives. First, we screened various RhzB orthologs for Fua formation in a recombinant strain. After treating the native hosts" culture extracts with a dienophilic maleimide, LC-MS/MS analysis identified Diels-Alder-derivatized Fua-carrying compounds. This way, we discovered two groups of strongly antiproliferative Fua-containing cyclopeptides from Actinobacteria. We translated this approach to a tailor-made maleimide resin that allows the solid-support-based purification of Fua-harboring peptides.The presented work provides insight into the biosynthesis and incorporation of the Fua pharmacophore, which permits bioorthogonal labelling, and shows the power of combining genome mining with reactivity-based screening to discover novel antiproliferative cyclopeptides.[1] L. P. Partida-Martinez, C. F. D. Looß, K. Ishida, M. Ishida, M. Roth, K. Buder, C. Hertweck, Appl. Environ. Microbiol. 2007, 73, 793–797.[2] K. Shimokawa, K. Yamada, D. Uemura, Bioorg. Med. Chem. Lett. 2009, 19, 867–869.[3] C. Almeida, F. E. Maddah, S. Kehraus, G. Schnakenburg, G. M. König, Org. Lett. 2016, 18, 528–531.[4] F. J. Ehinger, S. P. Niehs, B. Dose, M. Dell, J. Krabbe, S. J. Pidot, T. P. Stinear, K. Scherlach, C. Ross, G. Lackner, C. Hertweck, Angew. Chem. Int. Ed. 2023, e202308540.

Ensuring anaerobic survival – the critical role of UspK in metabolic adaptation during pyruvate fermentation

Pseudomonas aeruginosa is a versatile opportunistic human pathogen associated with acute and chronic infections, particularly affecting immunocompromised individuals, including those with cystic fibrosis (CF). The bacterium's ability to thrive under diverse environmental conditions, such as oxygen limitation in the mucus of CF patients, is central to its pathogenicity and a key contributor to persistence in hostile settings. Under anaerobic conditions and the absence of other energy-generating systems, pyruvate fermentation serves as an alternative strategy ensuring long-term survival and basal metabolism. In these growth-limiting, nutrient-deficient conditions, the universal stress protein UspK was found to be highly upregulated. This study investigates the role of UspK in facilitating adaptation to anaerobic stress, revealing a significantly decreased anaerobic survival rate of the UspK mutant. Upon switching to pyruvate fermentation, we observed alterations in LPS levels and a significant increase in AMP levels. Further analysis showed that UspK displays a nucleotide-binding motif capable of binding AMP, suggesting high AMP levels may act as a signal for starvation beyond stringent response. Supported by phenotypic analysis, our results highlight the critical role of UspK in anaerobic stress adaptation via pyruvate fermentation and survival in such famine conditions. This study provides new insights into stress response and niche adaptation of P. aeruginosa, which are crucial for targeting chronic infections.

Prof., Dr.

Fosfomycin perception by CreBC: Novel insights into Escherichia coli's response to MurA blockage

Introduction:Antimicrobial resistance will be a leading cause of mortality within a few decades, due to the current antibiotic discovery pipeline crisis. Thus, it remains essential to optimize the use of existing antibiotics by better understanding their broad cellular impact on bacteria. Fosfomycin (FOS), first discovered in 1969, remains active against multi-drug resistant (MDR) bacteria and its excellent pharmacological properties and unique mode of action make it ideal for synergistic therapies. Within bacteria, it covalently inhibits the enzyme MurA, blocking the first committed step of peptidoglycan synthesis. However, its clinical use against Escherichia coli often leads to high rates of resistance development and infection relapse. Goal:A better understanding of FOS cellular effects and off-target actions in E. coli could enable improved treatments, reducing the risk of regrowth of resistant or adapted bacteria.Materials and methods:Susceptibility to FOS was tested with gradient strip tests and broth dilutions in LB medium, monitoring the growth with a microplate reader. Transcriptional analyses were conducted with Illumina RNA sequencing. E. coli BW25113 and KO-mutants of the genes of interest were tested. The induction conditions of these genes of interest were further assessed with a lacZ-reporter plasmid.Results:E. coli alters its transcriptional state in response to FOS, shortly before drug-induced lysis. Transcriptional analyses of exponentially growing E. coli treated with FOS showed the rapid up-regulation of genes of the CreBC core regulon (cbrA, cbrB, cbrC, creD). The CreBC two-component system (TCS) is known to be responsive to carbon source changes in E. coli, but its real purpose remains unclear. Knockout mutants of these genes showed heightened sensitivity to FOS, and a similar effect was observed in strains completely lacking the CreBC TCS. The link between FOS and the upregulation of the CreBC regulon core genes was further investigated with a lacZ-reporter of creD expression.Summary:FOS-treated E. coli upregulated CreBC-related genes, and their role in response to FOS bactericidal action was investigated. This work advances efforts to better exploit the potential of FOS in MDR therapy, while preventing resistance development.

Dormancy of the probiotic strain Escherichia coli Nissle 1917 after long-term storage: A physiological and molecular characterization

Mutaflor Suspension is a medical drug containing the probiotic strain Escherichia coli Nissle 1917 (EcN) as active ingredient. It is used to restore the human intestinal microbiota after e.g. diarrheal infection. After long-term storage (LTS) of Mutaflor Suspension at 4°C, EcN cells are impeded in their ability to form colonies on agar plates, while short-term storage (STS) does not affect culturability. The observed plating defect after LTS can be overcome by regeneration of suspensions at 37°C. Here, we use Mutaflor Suspension as a model system for bacterial dormancy and investigate the physiological and molecular differences. STS and LTS suspensions were either tested directly or after regeneration at 37°C using cultivation-based approaches in solid and liquid media. Furthermore, experiments with antibiotics, microscopic analysis, and ATP assays were performed and compared to exponential and stationary phase laboratory cultures. To elucidate the molecular response, total RNA was isolated and analyzed by RNA-sequencing. We found that Mutaflor suspensions showed an increased antibiotic tolerance as compared to exponential EcN cultures, clearly indicating the expected stress tolerance of dormant cells. The cultivation-based approaches further demonstrated that EcN in LTS suspensions regenerate in dependence of their cell density. Finally, the RNA-sequencing analysis revealed several candidates that might be either involved in establishing dormancy upon LTS or the regeneration process after LTS. Our study provides new insights into the dormancy and regeneration of bacteria, and represents an important basis for future research.

Prof.

Structural and mechanistic investigation of the key enzyme orchestrating backbone rearrangement in rishirilide biosynthesis

Rishirilide A and B, first isolated in 1984 during a α2-macroglobulin inhibitor screen, belong to the class of tricyclic aromatic polyketides and are produced by multiple Streptomyces strains (1). Labeling studies validated the polyketide synthase-dependent biosynthesis and already suggested an unusual backbone rearrangement of the isopentyl substituent (2). Knockout studies in the rishirilide producer Streptomyces bottropensis in combination with in vitro enzyme reactions revealed RslO9, a FAD-dependent Monooxygenase (FPMO), as the key player in the last steps of rishirilide biosynthesis (3). However, the catalytic mechanism of this biosynthesis leading to rishirilide A/B and lupinacidin A remains unknown. Unraveling the intriguing activity could give insights into the structural diversification of aromatic polyketides and FPMO chemistry.For in depth analysis, RslO9 and N-terminal deletion variants were cloned and heterologously expressed, followed by purification and structure elucidation by X-ray crystallography. The molecular structure of RslO9 was obtained at a resolution of 2.4 Å. Cocrystallization attempts with the native substrate prevented formation of crystals, while soaking experiments revealed a conformational change in the active site, although no clear electron density for the substrate could be observed. Activity assays with numerous substrate-like compounds showed conversion of multiple naphthoquinones and implied a facilitating effect of alkyl substituents. On the other hand, anthraquinone derivatives were not converted, suggesting a terminal paraquinone moiety as an important feature for catalysis. Furthermore, docking and molecular dynamics simulations as well as site-directed mutagenesis studies are ongoing to pin down essential residues for substrate binding and catalysis.In conclusion our studies give insights into the backbone rearrangement performed by RslO9, further deepening our understanding of flavoprotein chemistry and polyketide tailoring reactions.References[1] Hideyuki Iwaki et. al. Structures of rishirilides A and B, alpha 2-macroglobulin inhibitors produced by Streptomyces rishiriensis OFR-1056. J. of Antibiotics 1984; 34(9):1091–3.[2] Philipp Schwarzer et. al. Biosynthesis of rishirilide b. Antibiotics. 7. März 2018;7(1).[3] Olga Tsypik et al. Oxidative Carbon Backbone Rearrangement in Rishirilide Biosynthesis. J Am Chem Soc. 2020;142(13):5913–7.

Kinetic and mechanistic characterization of the carboxyvinyl transferase isozymes (MurAA & MurAB) of B. subtilis

Introduction: The carboxyvinyl-transferase MurA catalyses the first committed step of the peptidoglycan biosynthesis allowing bacteria to sustain a functional cell wall, thus, it is generally essential. MurA is of medical importance, since it is the molecular target for the broad-spectrum antibiotic fosfomycin (FOS). FOS binds to an active‑site cysteine of MurA, forming a highly stable thioether bond, which covalently blocks the enzyme, preventing substrate binding and keeping MurA in an inactivated state. While Gram‑negative have one isoform, Gram‑positive usually have two or more isoforms of the enzyme. Bacillus subtilis has two MurA isoforms: while MurAA is essential, MurAB is expandable. The reason for B. subtilis having two isoforms of MurA is unknown and their physiological roles, and kinetic and mechanistic differences have not been investigated, so far.Goal: Our aim is to gain a better understanding of the MurA isoforms of B. subtilis, in terms of catalytic properties, susceptibility to FOS, and physiological functions.Materials and methods: MurAA and MurAB of B. subtilis were cloned, heterologously expressed, purified and biochemically characterized. A malachite‑green based phosphate assay was used to determine their kinetic parameters and the IC50 for FOS. With native mass spectrometry (nMS) we investigated the occupation of the active site cysteine, after incubation with substrate or antibiotic at varying concentrations.Results: MurAA appears to have a higher affinity towards phosphoenolpyruvate (PEP) and FOS, while MurAB has a higher maximal velocity. Most interestingly, purified MurAA and MurAB both carried a PEP molecule covalently bound to the active-site cysteine residue, shown with nMS, and we were able to demonstrate that, in this form, both isozymes are no molecular targets for FOS. Addition of second substrate UDP-N-acetylglucosamine triggers the enzymatic reaction, relieves the active site and, in this form, enabled binding of FOS. Further, the two isozymes have differences in their active‑site occupation, where multiple substrates/products are bound to MurAB, but not to MurAA.Summary: Using nMS, we were able to directly show, for the first time, that MurA isozymes are isolated from cells in a FOS-insensitive form with a PEP covalently blocking the active site cysteine. Although the MurA isozymes of B. subtilis have rather similar kinetic properties, differences occur in reaction rate and the affinity for PEP and FOS.

Regulation of the carbon flux in Synechocystis using the PGAM-PirC Switch

Cyanobacteria are autotrophic organisms, which perform oxidative photosynthesis. Their ability to oxidize water and use the electrons for reducing carbon dioxide (CO2) to form organic matter by using solar energy makes them an interesting organism for biotechnological applications. Therefore, Synechocystis sp. PCC 6803 is studied as a model organism for the construction of green cell factories.An important control point of the carbon flux is the 2,3-bisphosphoglycerate-independent phosphoglycerate-mutase (PGAM), which converts the first CO2 fixation product 3-phosphoglycerate to 2-phosphoglycerate. This reaction directs carbon flow towards lower glycolysis for the production of amino acids, fatty acids, biopolymers, such as polyhydroxybutyrate (PHB) and more. PGAM activity is inhibited through binding of the small protein PirC, which itself is repressed by the PII protein, the sensor protein for the carbon/nitrogen status [1].The aim is to use this PGAM-PirC key hub to direct the metabolic flux towards lower glycolysis and the production of PHB and other feedstock chemicals. Therefore, a strain constitutively overexpressing pgam was constructed and the level of PGAM and glycogen, PHB production and metabolite excretion were analyzed. In addition, several deletion mutants were tested, e.g., a ΔphaEC mutant, which cannot produce PHB. Furthermore, we analyzed the PGAM enzyme biochemically and identified structural elements required for regulation by PirC [2]. Strains with the corresponding PGAM variants were also constructed.Our results revealed that PGAM overproduction led to lower glycogen levels, whereas PHB amounts were higher under nitrogen depletion compared to the WT. Deletion of pirC or phaEC resulted in higher concentrations of the tested metabolites pyruvate, succinate and 2-oxoglutarate extracellularly. The double mutant lacking pirC and phaEC showed the highest excretion of the tested metabolites.Our work shows the important role the PGAM-PirC switch is playing in the metabolism. By tuning this regulation further, a platform will be established to redirect carbon flow for enhanced valuable chemical production.[1] Orthwein et al., 2021 Proc. Natl. Acad. Sci. U. S. A., 10.1073/pnas.2019988118[2] Orthwein et al. 2024 www.biorxiv.org/content/10.1101/2024.10.28.619893v1

Investigating the regulation of the carbon switch in central metabolism of Synechocystis sp. PCC 6803

Dependent on the light/nutrient conditions Synechocystis is able to grow auto-, hetero-, and mixotrophically for which the reversibility of sugar metabolism particularly the Embden-Meyerhof-Parnas pathway (EMP) is essential. To switch between anabolic and catabolic direction sophisticated regulation is required and also the interplay of e.g. EMP and Calvin-Benson-Bassham (CBB) cycle for CO2 fixation must be tightly balanced. The antagonistic PFK and FBPase operate exclusively in the catabolic or anabolic direction of the EMP, respectively, and PFK is the classical control point of glycolysis. Two paralogous copies of both enzymes are present in Synechocystis. Furthermore, under mixotrophic conditions the reversible phosphoglucose isomerase (PGI) shunt has been shown to direct fluxes via the non-oxidative pentose phosphate pathway (NOPP) to the CBB cycle.1 However, the control points and the function and regulatory capacities of the enzymes are not established in Synechocystis.Detailed enzymatic characterization revealed that both PFKs are ADP dependent, thereby defining a new class of ADP-dependent PFKs within the PFK-A superfamily. PFK-A1 is inhibited by 3-phosphoglycerate (3PG), and PFK-A2 by ATP.2 FBPase is not regulated by various effectors whereas F/SBPase is bifunctional and inhibited by AMP and activated by DTT.3 The PGI displays a preference for the gluconeogenic direction and is inhibited by erythrose 4-phosphate (E4P) and 6-phosphogluconate (6PG).Our investigations reveal that in line with the ADP-dependence both PFKs are active under conditions of low energy-charge, facilitating the breakdown of glycogen/D-glucose. The inhibition of PFK-A1 and PFK-A2 by 3PG and ATP, respectively, helps in rapid generation of CO2 acceptor molecule. The sustained FBPase activity in presence of various effectors suggests its housekeeping function to enable reversibility of the EMP pathway in response to substrate availability/growth conditions. E4P and 6PG regulation enables PGI function as a valve to balance the flux via the PGI shunt into NOPP or the oxidative pentose phosphate pathway, respectively.References:Schulze et al., Microb Cell Fact (2022); 21:699.Shen et. Al., JBC (2024); 107868Feng et al., FEBS J (2014) 281.3: 916-926.

A naturally isolated co-culture of Streptomyces species displays antifungal synergy

Fungal pathogens pose unique threats to modern healthcare: current treatment is limited to four antifungal drug classes, and many produce adverse side effects in patients1. Exploration of new environments could lead to the isolation of novel antifungal-producing microorganisms.We report the isolationed and characterization ofed a mixed isolate of two Streptomyces species from dried moth larvae (Cirina forda) that exhibited antifungal synergy against filamentous fungi. Antimicrobial profiles were obtained through overlay assays with mature colonies. The observed antibacterial and antifungal activity was further characterized in regards to cellular targets of produced compounds using a panel of whole-cell biosensors containing the luciferase cassette under the control of an antibiotic-specific inducible promotor. Variation in colony morphology led to the subculturing of two morphologically distinct isolates, both of which displayed reduced activity against filamentous fungi. Whole genome sequencing identified these sub-strains as Streptomyces albidoflavus. Nucleotide variations in regulatory, metabolic genes, and biosynthetic gene clusters (BGCs) were found that may explain the observed differences in strain morphology, physiology, and antimicrobial activity. Biosensor tests indicated the production of antifungal polyene macrolide-like compounds in the strains and mixed isolate, and genome mining confirmed the presence of the BGC for the polyene macrolide candicidin. Further analysis revealed discrepancies between the antimicrobial profiles of candicidin and the mixed isolate. Determination of produced antimicrobial compounds via HPLC-MS is currently ongoing.This work demonstrates the co-isolation of antifungal compound-producing organisms from a niche habitat. Differences in antimicrobial activity between the sub-strains and the observed synergistic relationship in the mixed isolate indicate cross-regulation and activation of antifungal BGC expression in one or both strains. Further research towards these microbial interactions could aid the discovery and development of new antifungals as well as further our understanding of antifungal BGC regulation and the effects and mechanisms of strain interaction on secondary metabolite production in Streptomyces species.1WHO fungal priority pathogens list to guide research, development and public health action. Geneva: World Health Organization 2022; Licence: CC BY-NC-SA 3.0 IGO.

PD Dr.

Scavenging nitrogen in the gut: the next step of Nε-carboxymethyllysine metabolism in Escherichia coli

Amino acids are modified through a plethora of enzymatic and non-enzymatic reactions. Glycation, or Maillard reaction, takes place at high temperatures between reducing sugars and free amino groups of proteins, producing compounds commonly found in processed foods (Maillard et al. 1912). One of the best-known glycated amino acids, Nε-carboxymethyllysine(CML), is consumed widely by humans reaching a daily intake of 24.6 mg( Mark et al. 2014). Most of it remains undigested, and the limited absorption in the gastrointestinal tract suggests a major role in its degradation by colonic bacteria (Lassak et al. 2023). E. coli produces novel metabolites when exposed to CML, mainly carboxymethylcadaverine, however no enzyme responsible for this degradation has been identified (Hellwig et al. 2019). We have unveiled an underground metabolism proceeding through promiscuous enzymes that accept CML and its downstream metabolites as substrates. Ornithine decarboxylase SpeC is the first enzyme that generates CM-Cad. Furthermore, E. coli can utilize CML as a sole nitrogen source, and based on the identified metabolites, we discovered that GABA transaminase GabT accepts CM-Cad and CML as substrates, with the formation of glutamate. Our findings suggest that the degradation route proceeds similarly to other biogenic amines like cadaverine or putrescine, likely involving other enzymes with broad substrate specificity. This study reveals a new metabolic route within E. coli, with the generation of unique compounds that may influence both gut microbiota dynamics and host physiology. The unknown effects of these metabolites on other gut bacteria and the human host warrant further exploration.

Elucidating the mechanisms of symbiosis in bacterial consortia formed by cyanobacteria with proteobacterial epibionts

Cyanobacteria are key primary producers in almost every illuminated environment and often represent a significant proportion of natural microbial communities. Consequently, they serve as sources of organic carbon for many other surrounding microorganisms. While the resulting bacterial interactions are often antagonistic, commensal and mutualistic adaptations are also known. A cyanobacterium of the genus Anabaena has been shown to closely interact with the heterotrophic proteobacterium Hoeflea sp., constituting a conspicuous form of bacterial heterologous multicellularity. Within this multicellular consortium the Hoeflea epibiont adheres in high numbers specifically to the heterocysts of the filamentous cyanobacterial partner. This model system offers unique opportunities for the study of symbiosis as (1) it involves two bacteria with medium-sized genomes, (2) each partner can be grown axenically in the presence of oxygen, (3) combining pure cultures of both restores the consortium phenotype. We applied genomics, transcriptomics, metabolomics, metallomics, fluorescent and electron microscopic techniques to study the biotic interactions underlying this bacterial heterologous multicellularity. Following each partner in axenic culture, or as consortium, we demonstrated that specific metabolites and metals are sequestered by either species prior to being shared, which in turn enhances their combined growth. Cell signalling pathways were further identified via gene expression data that suggest this consortium to be related (and perhaps a predecessor) to the symbiosis of plants with nodulating rhizobia. Since the Anabaena and Hoeflea can be cultivated independently, and then mixed to form consortia, diversity assays were conducted to check the host and epibiont range by exchanging either partner for alternative species. In this way, we discovered that several members of Nostocales could form consortia, whereas each required the specific species of Hoeflea, providing greater context to the possible symbiosis detected in nature.

A cryptic acetyltransferase-like protein drives spiroketal ring contraction in rubromycin biosynthesis

Rubromycins are a class of aromatic polyketides with potent antibiotic properties and are produced by a wide variety of actinobacteria. A distinctive feature of rubromycins is the presence of a [5,6]‑spiroketal moiety at the core of the molecule. Previous work in our group elucidated the mechanism behind the initial formation of a [6,6]‑spiroketal unit during rubromycin maturation through the concerted action of GrhO1 and GrhO5.1 The resulting intermediate is subsequently transformed by the flavoprotein monooxygenase GrhO6, leading to the contraction of ring C to form the [5,6]‑spiroketal structure found in mature rubromycins. Interestingly, efficient ring contraction only takes place in presence of GrhJ, which belongs to the family of GCN5‑related N‑acetyltransferases (GNAT).2,3 However, the precise catalytic mechanism of GrhO6 and the role of GrhJ in this process remain to be elucidated. Here, we aimed to investigate the catalytic mechanism of GrhO6 and the role of GrhJ in the [5,6]‑spiroketal formation by recombinantly producing these proteins and employing a combination of biochemical assays, biophysical methods, and X‑ray crystallography. The crystal structure of GrhO6, together with biochemical and mutagenesis studies, indicates that a potential acetylation of GrhO6 by GrhJ is unlikely to be responsible for the spiroketal ring contraction. Furthermore, differential scanning fluorimetry data indicate that GrhJ binds to rubromycin-type molecules, despite the absence of N‑acetylation sites in rubromycins or their biosynthetic intermediates. We propose that GrhJ may not act as an acetyltransferase. Instead, it might facilitate the release of the product from GrhO6 or stabilize reaction intermediates to minimize side reactions during spiroketal ring contraction. These insights advance our understanding of rubromycin biosynthesis and suggest new functional roles for members of the GNAT family. Further investigation is required to elucidate the detailed mechanism by which GrhJ promotes this reaction.References[1] Toplak et al. Angew Chem Int Ed. 2021 Dec 20;60(52):26960–70.[2] Yunt et al. J Am Chem Soc. 2009 Feb 18;131(6):2297–305.[3] Toplak et al. Chem Sci. 2022;13(24):7157–64.

Acetone and acetophenone metabolism in Rhodococcus aetherivorans

Acetone and acetophenone are common intermediates of natural secondary metabolism. As these substrates are chemically inert, they must be activated prior to microbial degradation. In the known metabolic pathways, ketone activation proceeds either by oxygenation or by ATP-dependent carboxylation [1,2].Acetone carboxylase (Acx) and acetophenone carboxylase (Apc) are homologous enzymes, but have different subunit compositions. While Acx forms a hexameric complex out of three different types of subunits ([αβγ]2), the octameric Apc core complex comprises four distinct subunits ([αα′βγ]2). Furthermore, Apc activity requires a fifth subunit (Apcε), which is not present in Acx [2,3].The strictly aerobic actinobacterium Rhodococcus aetherivorans encodes an apparent apc operon. Surprisingly, acetone rather than acetophenone induces expression of this gene cluster. In contrast, a phenol monooxygenase homolog is up-regulated during growth on acetophenone. Under both conditions, we observe strong expression of isocitrate lyase, which is involved in acetyl-CoA assimilation.Our current data suggest that acetone and acetophenone degradation in R. aetherivorans is accomplished by different principles. We propose that acetone catabolism is initiated by a carboxylase with Apc-like subunit structure and then proceeds in the canonical pathway via acetoacetate and acetyl-CoA. In contrast, acetophenone may be activated by oxygenation. The responsible enzyme and the subsequent reaction steps are not known yet, but we hypothesize that phenol and acetate are intermediates of the acetophenone degradation pathway.[1] Heider et al., J Mol Microbiol Biotechnol 26:152-64 (2016).[2] Weidenweber et al., Sci Rep 7:39674 (2017).[3] Mus et al., Sci Rep 7:7234 (2017).

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Trace metal availability drives microbial metabolic shifts and shapes community functional diversity

Microbial life relies on redox reactions to power essential cellular and metabolic processes, in which oxidoreductases are the main players. These enzymes use trace elements, and especially transition metals, as their cofactors, crucial in controlling the electron flow from electron donors to final electron acceptors, and contribute to maintaining the redox balance and thermodynamic disequilibria of natural systems. Despite their low abundance in the environment, trace metals distribution and availability might play a key role in influencing microbial functional diversity, shaping microbial community composition, and modulating biogeochemical cycling processes.Here, we report findings from both field and laboratory experiments elucidating the impact of trace elements availability on microbial functional diversity. Metagenomic data obtained from environmental samples suggest that trace elements availability shapes the diversity of the oxidoreductases in microbial communities, driving functional diversity in natural ecosystems. Furthermore, through physiological, proteomic, and functional analyses on microbial model organisms, we show that trace elements deficiency imposes energetic costs on microbial growth, delays microbial respiration transitions, alters electron acceptor utilization order, and enhances metal scavenging mechanisms. Our findings provide valuable insights into how trace metals regulate microbial functions, offering new perspectives for optimizing microbial processes in both environmental and industrial applications.

Prokaryotic physiology and metabolism 2

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