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

Prof. Dr.

Investigation of a non-canonical fumarate-based glutamate biosynthetic pathway in Bacillus subtilis and Escherichia coli

Prokaryotic physiology and metabolism

The amino acid glutamate plays important roles in cellular processes in the Gram-positive and ‑negative model bacteria Bacillus subtilis and Escherichia coli, respectively. It is the major amino group donor in anabolic reactions and a key molecule connecting carbon with nitrogen metabolism (1,2). B. subtilis synthesises glutamate via the glutamate synthase (GOGAT)/glutamine synthetase (GS) pathway (1), while E. coli can utilize the GOGAT/GS pathway, as well as the glutamate dehydrogenase (GDH) for glutamate biosynthesis (1,2). The GDHs of B. subtilis are strictly catabolically active. Recently, a non-canonical, fumarate-based pathway – involving the ansB-encoded L-aspartase and the aspB- encoded aspartate transaminase – was found that restored glutamate-prototrophy of a glutamate-auxotrophic B. subtilis strain lacking the GOGAT-encoding genes gltAB (3). The aim of this project is to better understand the mechanism and regulations of the different glutamate synthesizing pathways. To further analyse the fumarate-based pathway for glutamate biosynthesis, we constructed plasmids allowing for ansB and aspB expression in B. subtilis and E. coli glutamate-auxotrophic strains and further we characterized the novel pathway by growth experiments under defined growth conditions with different carbon and nitrogen sources. Results show that the novel pathway is able to restore glutamate-prototrophy not only in B. subtilis but also in E. coli and highlight different needs of the novel-pathway to the canonical glutamate biosynthetic pathway.(1) Gunka, K., & Commichau, F.M. (2012) Mol Microbiol. 85 (2): 213–224. doi: 10.1111/j.1365-2958.2012.08105.x.(2) Schulz-Mirbach, H., et al. (2022) 11: e77492. doi: 10.7554/eLife.77492.(3) Mardoukhi, M.S.Y., et al. (2024) Microb Biotechnol. 17 (3): e14429. doi: 10.1111/1751-7915.14429.

Poster

Redirecting carbon flux towards products in cyanobacteria by engineering the regulatory mechanism of the 2,3-bisphosphoglycerate-independent phosphoglycerate mutase reaction

IntroductionAmong prokaryotes oxygenic photosynthesis is a unique feature in cyanobacteria. In the context of establishing a sustainable bioeconomy, they receive growing interest as biocatalysts in photo-biotechnological applications. To rationally engineer cyanobacteria and to direct metabolic fluxes towards chemical products, it is important to consider native molecular processes that control metabolism. Recently, the 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (PGAM) has been shown to serve as a central control point determining the carbon flux from the Calvin-Benson-Bassham cycle to lower glycolysis. In particular, PGAM activity is controlled by the small protein PirC that also binds to the central regulatory protein PII. Upon PirC-binding, PGAM is inactivated leading to glycogen accumulation within the cell.1Here we aim to target the PirC-PGAM switch in the model strain Synechocystis sp. PCC 6803. In particular, flux through PGAM shall be blocked to increase product yields that derive from gluconeogenic routes.Material and MethodsExpression of the genes encoding PGAM and PirC was manipulated by using controllable promoters that respond to copper ions. To reduce PGAM activity we aimed for an inducible downregulation of pgam and simultaneous upregulation of pirC. Similar cloning strategies were used to rationally design sucrose production strains, achieved by overexpression of the sps gene and by installing an invertase gene knockout (similar to Du et al.2). Glycogen and sucrose formation were analyzed in the obtained strains.ResultsRecombinant Synechocystis strains showing Cu2+-inducible downregulation of pgam and overexpression of pirC were cloned and glycogen accumulation was analyzed in the corresponding cultures. In both mutant strains an accumulation of glycogen after addition of copper ions was determinable. This glycogen accumulation did not occur in the wild type control or in the mutant strains before changing expression through addition of copper ions. These data confirm that PGAM activity can be controlled through downregulation of pgam expression and overexpression of pirC routing higher amounts of the fixed carbon into glycogen.1T. Orthwein, J. Scholl, P. Spät, S. Lucius, M. Koch, B. Macek, M. Hagemann, K. Forchhammer. PNAS, 2021, 118(6): e20199881182Du, W.; Liang, F.; Duan, Y.; Tan, X.; Lu, X. Exploring the photosynthetic production capacity of sucrose by cyanobacteria. Metabolic engineering 2013, 19, 17–25

PD Dr.

CRISPR Cas9-based genome editing of Hyphomicrobium denitrificans

Hyphomicrobium denitrificans XT is a chemoorganoheterotroph that grows on C1 and C2 compounds. It serves as an important model organism for the analysis of dissimilatory sulfur oxidation because it can oxidize thiosulfate as a supplementary electron donor [1]. Establishing gene deletions and insertions in its chromosome has been instrumental in elucidating the sulfur oxidation pathway. Currently, genomic mutagenesis in H. denitrificans is based on suicide plasmid-driven homologous recombination (HR) systems. These systems work through an initial single-crossover (SCO) event, which integrates a non-replicating vector like pK18mobsacB into the genome, followed by a second SCO event, which either restores the wild-type sequence or induces the intended modification [1, 2]. A major challenge is the high rate of wild-type reversion, especially when targeting important genes. This leads to time-intensive mutant screening with low success rates. In fact, the lack of efficient markerless genome editing tools for H. denitrificans is a bottleneck for fundamental studies and also for possible future biotechnological exploitation.The Cas9 RNA-guided DNA-endonuclease from the type II CRISPR-Cas system of Streptococcus pyogenes (SpCas9) has been extensively used to develop genome engineering tools for both prokaryotes and eukaryotes, but not for H. denitrificans, an Alphaproteobacterium of the order Hyphomicrobiales. Here, we present a SpCas9-based genome editing system for this organism, integrating plasmid-borne HR templates. We began with plasmid pBBR_Cas9_NT, which contains a cas9 gene codon-optimized for the Alphaproteobacterium Cereibacter sphaeroides (formerly Rhodobacter sphaeroides, order Rhodobacterales) [3]. We further optimized the guide RNA for H. denitrificans and constructed plasmids designed to delete and replace the shdrI gene within the H. denitrificans genome. The protein sHdrI, which plays an important role in sulfur oxidation, has a distinctive dynamite bundle structure with an unknown function, making it a particularly interesting target. Finally, we compared the performance of our new CRISPR-Cas system with the traditional suicide plasmid-driven HR method.[1] Koch, Dahl 2018 ISME J 12, 2479-2491[2] Schäfer et al. 1994 Gene, 145, 69-73[3] Mougiakos et al. 2019 Microb Cell Fact 18, 204

Elucidating the role of a cryptic small protein in the assimilation of inorganic carbon in cyanobacteria

Cyanobacteria have evolved a sophisticated carbon concentrating mechanism (CCM) to significantly enhance the CO2 concentration in close proximity to the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase and thereby diminish the frequency of its detrimental oxygenation reaction1. While previous research has extensively studied the sodium dependent bicarbonate transporter SbtA and its regulator SbtB2, both crucial for the CCM, a so far non-annotated gene for a small protein of 80 amino acids has been identified upstream of the sbtAB operon in many cyanobacterial genomes. We hypothezise a function linked to the CCM and SbtA or B in particular and hence termed the protein SbtC. The 3D structure of SbtC, conserved regions as well as potential binding sites for interactions with DNA or other proteins were identified in silico. Structural analysis of SbtC identified a central transmembrane helix and a downstream region displaying a protein-binding motif. Moreover, ΔsbtC mutants have been generated using the cyanobacterial model Synechocystis sp. PCC 6803 and comparative growth analyses have been conducted compared to wild type and further mutants deficient in other inorganic carbon transporters such as NDH-13, NDH‑14, and CmpA. We also performed transcriptomics with the ΔsbtC strain to uncover the impact on the gene regulatory network potentially associated with SbtC. Microarrays revealed a significant decrease in transcript abundance of ten protein encoding genes, including sigA, rbcS, and cpcG2. In combination with the reduced growth exhibited under diurnal illumination this may point towards an involvement of SbtC in the mediation of cyanobacterial photoacclimation, potentially through interaction with a transcription factor.1 Price, G. D.; Badger, M. R.; Woodger, F. J.; Long, B. M. (2008): Advances in understanding the cyanobacterial CO2-concentrating-mechanism (CCM): functional components, Ci transporters, diversity, genetic regulation and prospects for engineering into plants. In Journal of experimental botany 59 (7), pp. 1441–1461.2 Selim, Khaled A.; Haase, F.; Hartmann, M. D.; Hagemann, M.; Forchhammer, K. (2018): PII-like signaling protein SbtB links cAMP sensing with cyanobacterial inorganic carbon response. In Proceedings of the National Academy of Sciences of the United States of America 115 (21), E4861-E4869.

Investigation of temperature-regulated mechanisms in Yersinia pseudotuberculosis

Pathogenic bacteria are confronted with frequently changing conditions during infection of warm-blooded hosts and after excretion into the environment. They therefore must be able to respond to these changes rapidly and adapt their metabolism accordingly. Among various environmental factors, temperature plays an important role. Bacteria can react to changes in temperature at different levels of regulation. At the DNA level, transcription factors can up- or downregulate gene expression. At the RNA level, non-coding RNAs, such as small regulatory RNAs (sRNAs) and RNA thermometers (RNATs) can have a post-transcriptional effect on the translation efficiency of various mRNAs through temperature-induced conformational changes.Yersinia pseudotuberculosis is a food-borne human pathogen that can lead to diseases of the gastrointestinal tract and the intestinal lymphatic system. Its optimum growth temperature is ~28 °C, but the cells can quickly react to an increase in host temperature to 37 °C and adapt their metabolism and virulence. In addition, Y. pseudotuberculosis can survive cold temperatures, allowing growth on food in the refrigerator.While some regulatory mechanisms of Y. pseudotuberculosis are well described once the cells are taken up from the environment by a host, we aim to determine how the bacterium reacts when it is released from the host to low temperatures in the environment. RNA-Seq revealed numerous genes that might play an important role for Yersinia within 30 min after the temperature is reduced from 37 °C to 25 °C, 17 °C or 10 °C.We are also interested in the function of the outer membrane protein OmpX of Y. pseudotuberculosis. It is primarily produced at 25 °C due to the temperature-mediated regulation by the sRNA CyaR (1). Interestingly, Yersinia produces a structurally similar, virulence-related protein, Ail, at 37 °C, which is upregulated by an RNAT. To examine the reciprocal production of OmpX and Ail at 25 °C or 37 °C, respectively, we carry out phenotypic characterizations of mutant strains to understand a potential connection between these outer membrane proteins. We found that single or combined deletions of both genes lead to an increased sensitivity at elevated salt concentrations.(1) Guanzon et al. Two temperature-responsive RNAs act in concert: The small RNA CyaR and the mRNA ompX bioRxiv 2024

Aerobic gas fermentation with carboxydotrophic 'Knallgas' bacteria

Industrial waste gases emit large amounts of pollutants into the atmosphere and may contain significant amounts of CO2, CO, H2, and O2 depending on the source. Such gas streams represent an abundant feedstock for gas fermentation to produce chemicals with microbial catalysts. Current gas fermentation technologies enable the transformation of gaseous feedstocks into products such as ethanol, utilizing anaerobic acetogenic bacteria. However, the energy-limited anaerobic metabolism makes the synthesis of ATP-intensive products challenging.To overcome this limitation, we propose the utilization of carboxydotrophic 'Knallgas' bacteria as cell factories to convert gaseous streams into valuable chemicals. These organisms possess the ability to utilize CO as a sole energy source with O2 to produce CO2, which is subsequently used for biomass formation via the CBB cycle. Moreover, these organisms can also oxidize H2 with hydrogenases to drive the reduction of CO2 in the absence of CO. One representative is Hydrogenophaga pseudoflava DSM 1084, which shows comparably high growth rates under autotrophic conditions [1, 2].Several knowledge gaps remain regarding the essential genes for CO utilization and electron transport of carboxydotrophic 'Knallgas' bacteria. A comparative genomic analysis between the carboxydotrophic strain H. pseudoflava DSM 1084 and the H2-oxidizing, non-carboxydotrophic H. pseudoflava DSM 1034 elucidated genetic elements that might be the key for growth on CO. Therefore, we conducted a combined approach involving genetic engineering and adaptive laboratory evolution on H. pseudoflava DSM 1034, to produce the first-reported synthetic carboxydotrophic organism, able to grow with CO as its sole carbon and energy source. Genome sequencing and re-engineering of the key mutations found on the evolved strain, together with the knowledge gained from proteome-level analysis might allow the transferability of carboxydotrophy to other microbial hosts, thus broadening the scope of biocatalysts that can be utilized for converting gaseous feedstocks into value-added chemicals and biofuels.References:[1] Grenz et al., Metab. Eng., Vol 55 (2019)[2] Siebert et al., Adv. Biochem. Eng. Biotechnol., Vol 180 (2022)

In vitro characterisation of central glycogen synthesis enzymes in Synechocystis sp. PCC 6803 reveals complex regulatory mechanisms

In cyanobacteria such as the model strain Synechocystis sp. PCC 6803, ADP-Glucose (ADP-Glc) is produced from Glucose-1-phosphate (Glc-1P) by Glucose-1-phosphate adenylyltransferase (GlgC). ADP-Glc is subsequently added to a growing glycogen molecule via a 1,4-alpha bond by glycogen synthases, present as two isoenzymes in Synechocystis (GlgA1 and GlgA2). This reaction is assisted by the branching enzyme (GlgB) which introduces branches that increases the number of potential extension sites for glycogen synthases. In this work, we reveal the fine-tuned regulatory mechanisms behind these enzymes using a NADH coupled-enzyme colorimetric assay, expanding upon the previously documented regulation of GlgC by 3-phosphoglycerate and inorganic phosphate [1,2]. In addition to that, we reveal the primer specificity and redox regulation of glycogen synthases, as well as modulatory effects of GlgC and other regulatory proteins, shedding light on the interconnected nature of glycogen synthesis regulation.1 Ballicora MA, Iglesias AA & Preiss J (2004) ADP-Glucose Pyrophosphorylase: A Regulatory Enzyme for Plant Starch Synthesis. Photosynth Res 79, 1–24.2 Iglesias AA, Kakefuda G & Preiss J (1991) Regulatory and Structural Properties of the Cyanobacterial ADPglucose Pyrophosphorylases. Plant Physiol 97, 1187–1195.

The path of the iron-sulfur clusters into the proteins: Unveiling the Sulfur Mobilization Machinery

Introduction: Iron-sulfur cluster containing proteins are ubiquitous in nature and conserved across all kingdoms of life; however, iron-sulfur clusters are not assembled within their target proteins. Instead, iron-sulfur clusters are assembled on scaffold proteins assisted by specialized assembly machinery. Currently, four iron-sulfur cluster machineries are known: the NIF (nitrogen-fixation system), ISC (iron-sulfur cluster assembly machinery), CIA (cytosolic iron-sulfur cluster assembly machinery), and SUF (sulfur mobilization machinery) [1]. The SUF machinery is the most ancient of the identified iron-sulfur cluster biogenesis systems. The SUF machinery contains the proteins SufS, SufE or SufU, SufBCD or SufBC, and SufA. The SufBCD / SufBC protein complex"s postulated function is the assembly of iron-sulfur clusters on its SufBD subunits [2]. SufC belongs to the highly diverse ABC ATPase-family, whose function within the iron-sulfur cluster biogenesis pathway remains unresolved [3]. To overcome this, structures in the enzyme"s conformational cycles are required to understand how the ATPase movement is related to the formation of a nascent iron-sulfur cluster.Objective: We determined a high-resolution structure of the SufBC protein complex, concentrating on the proposed iron-sulfur cluster forming residues and the proposed ATPase activity-induced, conformational changes.Materials and methods: We utilised single-particle cryo-electron microscopy (cryo-EM) to create structural models of the purified SufBC protein complex.Results: The SUF machinery components were overexpressed, purified and cryo-EM sample preparation was carried out under redox controlled conditions. UV/Vis spectroscopy confirmed the presence of an iron-sulfur cluster in the anaerobically purified SufBC complex. Preliminary data processing revealed SufBC undergoes conformational changes, possibly in relation to the presence of an iron-sulfur cluster and bound substrate.Conclusion: Overall, preliminary structural information acquired by single particle cryo-EM shows that the SufBC protein complex adopts different structural conformations during iron-sulfur cluster assembly.[1] Braymer et al. (2021) BBA – Molecular cell research. 1868: 118863, [2] Wollers et al. (2010) J Biol Chem. 285: 23331-23341, [3] Hirabayashi et al. (2015) J Biol Chem. 290: 29717–29731

Prof.

Analysis of the antibacterial mechanism of Farnesyltransferase inhibitors

IntroductionFarnesylation is a posttranslational modification, involving the attachment of a C15 isoprene unit (farnesyl) to proteins with a specific CAAX motif. Farnesylation enables over 200 proteins to bind to the membrane and fulfil their function in processes like cell growth, differentiation or apoptosis. The inhibition by farnesyltransferase inhibitors (FTI), such as lonafarnib or tipifarnib, came in focus for the treatment of various diseases, e.g. cancer. Furthermore, we demonstrated antibacterial effects of lonafarnib and tipifarnib in gram-positive bacteria such as S. aureus or S. epidermidis and even in the multi-resistant strain MRSA [1]. However, the underlying molecular mechanism remains elusive.Material & MethodsWe determined the minimal inhibitory concentration (MIC) as well as the physiological effective concentration (PEK) of lonafarnib and tipifarnib on B. subtilis. To analyse the effect of FTIs on the proteome, we performed high-resolution liquid chromatography followed by mass spectrometry analysis.ResultsIn total, 1060 proteins were detected via mass spectrometric analysis. Treatment with tipifarnib resulted in the upregulation of 38 proteins in B. subtilis, whereas treatment with lonafarnib upregulated 89 proteins. 33 proteins were regulated by both FTI. Among the identified marker proteins, those involved in the biosynthesis and modification of cell wall components, such as DitE, YocH or PbpE, were particularly important. Additionally, marker proteins related to protein biosynthesis, general stress response, oxidative stress and various transport processes were detected.DiscussionIt is known that both lonafarnib as well as tipifarnib show antibacterial effects in different bacterial strains, however, the underlying mechanism remains elusive. Here, we could show that both lonafarnib and tipifarnib treatment resulted in a significant change in several proteins, whereas the treatment with lonafarnib led to a change in twice as many proteins as tipifarnib. Strikingly, many marker proteins were proteins involved in processes affecting the cell envelope. Therefore, we assume that the cell wall and cell membrane are the main targets for the FTI in B. subtilis.[1] Weber et al. 2019 Front. Microbiol. 12, 628283

Impact of flavor enhancers on the structure and function of the intestinal microbiome

Introduction: The human intestinal microbiome plays a vital role in human health and is influenced by both dietary and non-dietary factors. Food additives like monosodium glutamate (MSG) are commonly used in the food industry as taste enhancers; however, their impact on the gut microbiome is still not well understood. Glutamate acts as a signaling molecule in the enteric nervous system, affecting neuroendocrine responses, suggesting that the gut microbiome could also be responsive to MSG.Goals: Using an in vitro simulator of the human intestinal microbial ecosystem (SHIME®) this study investigated the effects of MSG on the colonic microbiome in a case-control design focusing on transcriptional and metabolic responses.Materials and Methods: SHIME® is a chemostat system that can simulate the luminal colon region via different bioreactors representing the stomach/small intestine, as well as proximal (PC) and distal colon (DC). The study was conducted using a defined community of eight bacterial strains (SIHUMIx) representing the important gut phyla Firmicutes (Blautia producta, Clostridium butyricum, Clostridium ramosum, Lactobacillus plantarum, Anaerostipes caccae), Bacteroidetes (Bacteroides thetaiotaomicron), Actinobacteria (Bifidobacterium longum), and Proteobacteria (Escherichia coli). We applied MSG in a mixture with a defined medium containing 25% starch at a concentration of 0.5 mg/mL, which was adjusted to pH 2 and then heated by autoclaving.Results: Over a three-week period (stabilization, treatment, and post-treatment phases), shifts in microbial community composition and gene expression patterns were analyzed. In addition, short-chain fatty acid (SCFA) profiling was performed. As expected, the relative abundance of members of the synthetic communities varied in the different simulated gut compartments. In the proximal colon, B. thetaiotaomicron, L. plantarum, E. coli and C. butyricum were predominant, while the distal colon was dominated by B. thetaiotaomicron, B. producta, E. coli and A. caccae. The application of MSG led to a reduction in the abundance of B. thetaiotaomicron, L. plantarum, and C. butyricum in the proximal colon, as well as a decrease in B. thetaiotaomicron and E. coli, and an increase in B. producta in the distal colon. A reduction in SCFA concentrations was detected across both colon regions. Further findings, including transcriptomic data and detailed results, will be presented in the frame of the poster.

The heterodisulfide reductase-like (sHdr) complex for dissimilatory sulfur oxidation

Many dissimilatory sulfur-oxidizing bacteria and archaea pursue a pathway for sulfur oxidation that involves heterodisulfide reductase-like (sHdr) enzymes. Its core proteins are encoded in a strictly conserved shdrC1B1AHC2B2 cluster in Pseudomonadota and Aquificae and are similar to the subunits of the heterodisulfide reductase from methanogenic archaea. In the Alphaproteobacterium Hyphomicrobium denitrificans the sHdr proteins are essential for the use of thiosulfate as an accessory electron donor [2].We aim at the structural and biochemical characterization of the sHdr complex, including the elucidation of its cofactors and the identification of possible interaction partners. The sHdrAB1B2C1 and -C2 proteins were co-purified from membrane fractions of the hyperthermophile Aquifex aeolicus [2]. In contrast, hyphomicrobial sHdrA is soluble [1,3]. Here, we show that all sHdr proteins in the obligately chemolithoautotrophic Gammaproteobacterium Thioalkalivibrio thiocyanoxidans are soluble, as evidenced by immunodetection. sHdrAB1B2C1C2 is co-purified from T. thiocyanoxidans as a heterocomplex, containing FAD and multiple iron-sulfur clusters after two anion exchange chromatography steps. SDS-PAGE, Blue-Native-PAGE followed by 2D-SDS-PAGE and mass spectrometry revealed that it not only interacts with several other proteins encoded in the vicinity of the core shdr genes, such as the sulfur transferase DsrE3B, but that the complex composition may be dynamic with regard to the number and identity of the core subunits present. The sHdrH protein, which is inevitably encoded within the shdr genes and has no counterpart in methanogens, does not appear to be a component of the sulfur-oxidizing sHdr complex although it is not only co-transcribed and transcriptionally co-regulated with the core shdr genes in H. denitrificans, but also indispensable for thiosulfate oxidation. Together, our results provide first glimpses into the reaction mechanism of the sHdr complex.[1] Koch & Dahl 2018 ISME J 10, 2479[2] Boughanemi et al. 2016 FEMS Microbiol Lett 363, fnw156[3] Ernst et al. 2021 FEBS J 288, 1664

Wadjet System Inactivation Boosts Plasmid Acquisition in Phylogenetically Distant Gram-positive Bacteria Clostridium cellulovorans and Micrococcus luteus

Innate defense mechanisms heavily influence the ability of some bacteria to take up and maintain plasmid DNA. The Wadjet system is a recently characterized defense system that targets and cleaves circular plasmid DNA. In this study, we report the consequences of the knockout of Wadjet system-encoding genes in two Gram-positive model bacteria, Clostridium cellulovorans (phylum Bacillota) and Micrococcus luteus (phylum Actinomycetota), for the uptake of plasmid DNA via transconjugation and natural transformation, respectively.Genome sequencing of a spontaneous C. cellulovorans mutant with a dramatically enhanced frequency of plasmid DNA acquisition through conjugation revealed three chromosomal deletions, one of them including a putative Wadjet system-encoding jetABCD gene cluster. After deletion of only this gene cluster in the wildtype, the efficiency of conjugative plasmid transfer from E. coli to C. cellulovorans ΔjetABCD increased by about five orders of magnitude, confirming that the Wadjet system represents an efficient barrier to plasmid transfer in C. cellulovorans. Additional in vivo methylation of the plasmid in the E. coli donor to protect it from restriction by a type II RM endonuclease present in C. cellulovorans further improved uptake by conjugation.In M. luteus strain TrpE16, a model organism for the study of DNA uptake by natural transformation in high-GC Gram-positive bacteria, deletion of the chromosomal jetABCD gene cluster likewise substantially increased the frequency of plasmid uptake, in this case by natural transformation. Via introduction of the ΔjetABCD deletion it was possible to overcome a known limitation of M. luteus TrpE16 to be transformed with circular plasmid DNA. Notably, reisolation and analysis of plasmids from the Wadjet-deficient and the parental M. luteus strains seemed to yield different topologies or concatemeric states of the plasmid.These findings collectively underscore the restrictive role of Wadjet systems in two phylogenetically distant Gram-positive bacteria from the Actinomycetota (high-GC) and Bacillota (low-GC) on plasmid stability and consequently on the efficiency of gene transfer via natural transformation and conjugation. This demonstrates the importance of considering Wadjet systems in addition to RM and CRISPR-Cas systems as defense systems that can seriously hinder the introduction of recombinant plasmids for genetic engineering purposes.

Characterization of the key enzyme of Isoprenoid Biosynthesis DXS from Corynebacterium glutamicum

In isoprenoid biosynthesis, 1-Deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the first step of the methyl erythritol phosphate (MEP) pathway by converting pyruvate and glyceraldehyde-3-phosphate to 1-deoxy-D-xylulose 5-phosphate (DXP). DXP serves as a primary intermediate in the biosynthesis of thiamine and terpenoids, such as carotenoids. Previously, overexpression of DXS in Corynebacterium glutamicum resulted in a twofold increase in carotenoid production [1], corroborating that DXS catalyzes a rate-limiting step in terpenoid biosynthesis. However, the available knowledge on the regulation of DXS in C. glutamicum is limited. Previously, the lysin residue 352 of DXS from C. glutamicum was shown to be pupylated by a prokaryotic ubiquitin-like regulatory mechanism [2], but the physiological consequences are unknown.In order to gain insight into the regulation of DXS in C. glutamicum, K352 was changed to either glutamic acid or alanine to obtain DXS variants that cannot be pupylated. These variants were expressed in E. coli, purified using a HisC tag and enzymatically characterized in vitro. Interestingly, the pupylation deficient variants DXSK352E and DXSK352A showed almost twice the enzymatic activity than the wild-type DXS (0.68 U/mg and 0.72 U/mg vs 0.37 U/mg, respectively). The overexpression of wild-type DXS in the C. glutamicum WT strain led to a shorter lag-phase, but reduced the decaprenoxanthin titer compared to the overexpression of the pupylation deficient mutants. By contrast, in the pupylation deficient strain C. glutamicum Δpup no such differences occurred. The mechanism of regulation of DXS by pupylation in C. glutamicum remains to be disclosed.1. Heider, S.A., Wolf, N., Hofemeier, A., Peters-Wendisch, P. and Wendisch, V.F., 2014. Optimization of the IPP precursor supply for the production of lycopene, decaprenoxanthin and astaxanthin by Corynebacterium glutamicum. Frontiers in bioengineering and biotechnology, 2:28.2. Küberl, A., Fränzel, B., Eggeling, L., Polen, T., Wolters, D.A. and Bott, M., 2014. Pupylated proteins in Corynebacterium glutamicum revealed by MudPIT analysis. Proteomics, 14(12):1531-1542.

Structure function analysis of ADP-dependent cyanobacterial phosphofructokinase reveals new phylogenetic grouping in the PFK-A family

In photosynthetic organisms, efficient carbohydrate metabolism is crucial for adapting to varying environmental conditions, particularly during transitions between light and dark cycles. Phosphofructokinase (PFK) plays a pivotal role in glycolysis, acting as one of two key regulatory points that govern the flow of metabolites via the Embden-Meyerhof-Parnas (EMP) pathway. Three different types of PFKs have been described that vary in their phosphate donor, i.e. ATP, ADP or PPi and regulatory properties [1]. Synechocystis PFK activity has been analysed in cell extracts [2], and it was classified as member of the PFK-B family, even though sequence comparisons indicated them to be members of the PFK-A family [3].We aimed to characterize the enzymatic properties, regulatory mechanisms, and evolutionary adaptations of the two PFK isoenzymes in Synechocystis, particularly their roles in adjusting to changing growth conditions and metabolic states.The genes encoding PFK-A1 and PFK-A2 from Synechocystis were cloned, expressed, and purified. Kinetic parameters were determined under varying substrate and effector concentrations, and the phylogenetic relationships were examined through sequence comparisons and structural modeling.Both PFK-A1 and PFK-A2 showed exclusive dependence on ADP for catalytic activity, with distinct regulatory profiles: PFK-A1 is inhibited by 3-phosphoglycerate, while PFK-A2 is inhibited by ATP. Both of these inhibitors accumulate during photosynthesis, facilitating enzyme inactivation during light. Phylogenetic analysis identified these ADP-dependent PFK-A isoenzymes as monophyletic group within the PFK-A family, comprising cyanobacterial evolutionary lineage and structural adaptation among cyanobacteria and homologs [4].The study provides new insights into the regulatory properties and evolution of PFK-A isoenzymes in cyanobacteria, emphasizing an adaptive mechanism for balancing glycolysis and gluconeogenesis in response to light and energy charge of the cell. In addition, their ADP dependence offers interesting potential for optimization of carbohydrate metabolism in metabolic engineering and synthetic biology approaches.Schirmer, T. et al., Nature, 1990. 343(6254): p. 140-145.Knowles, V.L. and W.C. Plaxton, Nature, 1990. 343(6254): p. 140-145.Dandekar, T., et al., Biochemical Journal, 1999. 343(1): p. 115--124.Shen, L., Peraglie, C., et al., J Biol Chem, 2024: p. 107868.

Triggering natural product synthesis in Streptomyces sp.

Streptomyces sp. are the most prolific source of antibiotics in nature. In face of the rise in antibiotic resistance of pathogens, the numerous biosynthetic gene clusters (BGCs) encoded in Streptomyces' genomes give rise to undiscovered natural products (NPs) and may be a treasure trove for antibiotic discovery. Nevertheless, searching for new NPs through variation of cultivation conditions (1. Bode et al.) suffers from high rediscovery rates and non-annotated BGCs often remain silent. Commonly, this approach involves the following steps: cultivation, detection of bioactive extracts, dereplication and compound isolation. Activity-based screening has the disadvantage that low-abundant, bioactive metabolites may not be discovered because their activity is masked by that of other more abundant compounds. Subsequent fractionation often fails to yield active fractions because low abundant substances are lost. Here, eight new Streptomyces isolates with up to 39 non-annotated silent BGCs are cultivated using small molecular elicitors to trigger production of bioactive compounds (2. Alwali et al.). Using liquid-chromatography mass-spectrometry and dose-dependent response experiments, rapid dereplication of antimicrobials in bioactive extracts is achieved, identifying previously discovered compounds and annotating the corresponding BGCs. With CRISPR-Cas9, knock-out mutants are then generated that are unable to produce these compounds. Hereby the masking effect of the dereplicated compound is eliminated in the mutant strains and low abundant bioactive molecules may in principle be detected using an identical workflow. Since in the mutant the functionality of the natural product is missing and no resources are spent on the production of the dereplicated molecule, it is also investigated whether resources are redirected to an increased production of other antimicrobials. The goal of this "knock-out and rescreen"-approach is to identify and isolate new and rare antimicrobial compounds.Bode, H. B. et al. 2002, ChemBioChem, 3, 619-627.Alwali, A. Y. et al. 2023, JIMB, 50, kuad019.

From mineral to microbe: lanthanide mobilization, uptake, and gene expression dynamics in a lanthanide-utilizing methylotroph

The lanthanides (elements 57–71, [La–Lu], Ln), along with the chemically similar elements scandium (Sc) and yttrium (Y), are known as "rare-earth elements" (REE). Ln are critical resources for the ongoing green energy transition, but mining and recycling them is expensive and environmentally harmful. They are omnipresent in our everyday lives and represent the most recently described biometals. Ln-utilizing microbes are mostly studied using soluble Ln chloride salts, which are a poor mimic for natural Ln bioavailability. We performed an in-depth characterization of selected Ln minerals and alloys (e.g. bastnaesite, gadolinite, monazite, xenotime, lighter flint) that differ by type (carbonate, oxide, silicate, phosphate) and Ln content through energy-dispersive X-ray spectroscopy (EDX), inductively coupled plasma mass spectrometry, and complementary methods. Using our model strain Beijerinckiaceae bacterium RH AL1, which naturally depends on light Ln (La - Nd) for methanol oxidation, we applied transcriptomics and transmission electron microscopy (TEM) to understand how this strain mobilizes, takes up, and discriminates Ln during methylotrophic growth. We could show Ln mobilization for methanol oxidation from all tested Ln sources. TEM combined with EDX revealed that Beijerinckiaceae bacterium RH AL1 takes up certain Ln and selectively accumulates them in the periplasm as Ln phosphates, confirming our previous hypothesis that periplasmic Ln accumulation contributes to intracellular Ln homeostasis. Transcriptomics indicated that gene expression dynamics—up to 53% of the encoded genes were differentially expressed—were strongly governed by the type of Ln source. These aspects were not restricted to genes known to be involved in Ln utilization but reached into many aspects of cellular metabolism. Ln mobilization is presumably enabled by Ln-based chemotaxis, bacteria attaching to the Ln source, and the concerted action of extracellular pH modulation, and the release of potential Ln-binding metallophores. We noted significant crosstalk with Fe metabolism and could pinpoint candidate genes encoding proteins involved in intracellular Ln discrimination. Adaptations to low Ln bioavailability and types of Ln sources make Ln-utilizing microbes a treasure chest for developing strategies for the sustainable use of Ln as key elements for our modern way of living in the 21st century.

Prokaryotic physiology and metabolism 1

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