Biology subjects

Bott, M.

Publications and source records attributed to Bott, M..

9 recordsLinked to original sources

bioRxiv2026

Context-dependent siderophore exploitability shapes microbial community structure

1)Siderophores are classically viewed as shared iron-scavenging public goods, yet their ecological roles in multispecies communities remain poorly defined. Here, we establish a synthetic microbial community to dissect how different siderophores, their uptake compatibility and spatial structure shape iron competition. Using Corynebacterium glutamicum as a model, we show that this siderophore non-producer accesses diverse xenosiderophores, including enterobactin secreted by Escherichia coli. However, exploitation was constrained and co-cultures converged to stable compositions. Dose-response experiments combined with mathematical modelling indicated that the producer retains more effective access to enterobactin than the exploiter. Presence of Pseudomonas putida altered this interaction, as it exploited enterobactin while producing pyoverdine, a siderophore inaccessible to the other community members that restricted their iron access. Across different cultivation scales, community dynamics was strongly influenced by spatial organization and initial composition. These findings identify siderophores as context-dependent iron-allocation agents that can promote microbial coexistence or exclusion.

microbiology↗

bioRxiv2025

Metabolic engineering of Corynebacterium glutamicum for production of the low-caloric natural sweetener D-allulose via phosphorylated intermediates

D-Allulose is a natural, low-calorie sweetener providing [~]70% of the sweetness of sucrose but [≤]10% of its caloric value, making it an attractive alternative to conventional sugars. Recently, a phosphorylation-dephosphorylation pathway for D-allulose production was established, involving the formation and irreversible dephosphorylation of D-allulose 6-phosphate. Although this pathway has been demonstrated in Escherichia coli, efficient production involved complex medium, complicating downstream processing. Here, we report D-allulose production in minimal medium by implementing the phosphorylation-dephosphorylation pathway in a Corynebacterium glutamicum strain unable to metabolize D-fructose. Growth- and production-based screenings identified fructokinase MakEC and D-allulose 6-phosphate 3-epimerase AlsEEC from E. coli, together with D-allulose 6-phosphate phosphatase AlsPCT from Clostridium thermocellum, as the most effective enzyme combination for D-allulose formation. Further metabolic engineering of C. glutamicum including deletion of zwf (D-glucose 6-phosphate dehydrogenase), overexpression of fbp (D-fructose 1,6-bisphosphatase), and downregulation of pgm (phosphoglucomutase) partially redirected central carbon flux toward D-allulose synthesis, resulting in a 2.3-fold increase in production. The engineered strain produced [~]3.6 g L-1 D-allulose from a D-glucose-D-fructose mixture with a yield of 9.1%.

bioengineering↗

bioRxiv2025

Growth of Corynebacterium glutamicum on 5-oxo-L-proline (pyroglutamate) as carbon and nitrogen source requires the PxpR-controlled pxpTABC genes

5-Oxo-L-proline (5-OP) is inevitably formed in all cells by spontaneous cyclization of L-glutamate, L-glutamine, or {gamma}-glutamyl phosphate. Its use as a substrate by bacteria has rarely been described. Here, we show that the actinobacterial species Corynebacterium glutamicum can grow well in minimal medium with 5-OP as sole carbon and nitrogen source. We identified the pxpTABC gene cluster as being essential for growth on 5-OP. The pxpT gene encodes a secondary transporter of the APC superfamily which most likely catalyzes 5-OP uptake into the cell. The pxpABC genes encode a recently identified ATP-dependent 5-oxoprolinase that converts 5-OP into L-glutamate for further metabolism. Both PxpT and PxpABC were required for growth with 5-OP. Upstream and divergent to pxpT, the gene pxpR is located, encoding a GntR-type transcriptional regulator. Deletion of pxpR improved growth on 5-OP suggesting that PxpR acts as repressor of the pxpTABC operon. This function was further supported by reporter gene studies. Purified PxpR was shown by isothermal titration calorimetry (ITC) to bind 5-OP with a KD of 726 {+/-} 23 nM. L-proline, L-glutamate, L-glutamine, and L-aspartate were not bound under the conditions tested, suggesting that PxpR is a specific 5-OP biosensor. IMPORTANCEThe utilization of 5-OP as a carbon and nitrogen source for bacteria has rarely been investigated in the past, although this metabolite is ubiquitous in cells due to its non-enzymatic formation from the amino acids L-glutamate and L-glutamine. In Corynebacterium glutamicum cells, but also in other bacteria such as Escherichia coli, L-glutamate is present at concentrations in the range of 100 mM, suggesting a substantial and continuous 5-OP formation. Our results demonstrate that 5-OP can serve both as carbon and nitrogen source for C. glutamicum and presumably also for other bacteria.

microbiology↗

bioRxiv2025

Genetic mechanisms of resistance to targeted KRAS inhibition

KRAS mutations are among the most prevalent oncogenic drivers in non-small cell lung cancer (NSCLC), yet the mechanisms of therapeutic resistance to KRAS inhibitors in these cancers remains poorly understood. Here, we deploy high-throughput CRISPR base editing screens to systematically map resistance mutations to three mechanistically distinct KRAS-targeted therapies, including KRAS-G12C(OFF) inhibitor (adagrasib), RAS(ON) G12C-selective tri-complex inhibitor (RMC-4998), and RAS(ON) multi-selective tri-complex inhibitor (RMC-7977). Using both a saturation Kras tiling approach and cancer-associated mutation library, we identify common and compound-selective second-site resistance mutations in Kras, as well as gain-of-function and loss-of-function variants across cancer-associated genes that rewire signaling networks in a context-dependent manner. Notably, we identify a recurrent missense mutation in capicua (Cic), that promotes resistance to RMC-7977 in vitro and in vivo. Moreover, we show that targeting NF{kappa}B signaling in CIC-mutant cells can resensitize them to RAS pathway inhibition and overcome resistance.

cancer biology↗

bioRxiv2025

A novel biosensor for ferrous iron developed via CoBiSe:A computational method for rapid biosensor design

Genetically encoded biosensors enable monitoring of metabolite dynamics in living organisms. We present CoBiSe, a computational approach using Constraint Network Analysis to identify optimal insertion sites for reporter modules in molecular recognition elements (MREs). Applied to the iron-binding protein DtxR from Corynebacterium glutamicum, CoBiSe identified a flexible connective loop (residues 138-150) for inserting the reporter module, resulting in IronSenseR, a novel ratiometric biosensor for ferrous iron (Fe{superscript 2}). IronSenseR demonstrates high specificity for Fe{superscript 2} with dissociation constants of 1.55 {+/-} 0.08 {micro}M (FeSO4) and 2.44 {+/-} 0.28 {micro}M (FeCl2), while showing no binding to Fe3 and other divalent cations. In vivo assessment in Escherichia coli, Pseudomonas putida and Corynebacterium glutamicum confirmed IronSenseRs capability to detect changes in the intracellular iron pool. The creation of IronSenseR underlines that, by reducing search space and eliminating labor-intensive screening, CoBiSe streamlines biosensor development and enables precise creation of next-generation biosensors for diverse metabolites.

bioengineering↗

bioRxiv2024

Efficient microbial glucose-fructose conversion at ambient temperature through adaptive laboratory evolution and application for D-allulose production

The low-calorie sugar D-allulose is a promising alternative to D-sucrose and high-fructose corn syrup, but its microbial production from D-glucose at mesophilic temperatures is limited by insufficient D-glucose isomerase (XylA) activity. Here, we overcome this bottleneck by evolving a Corynebacterium glutamicum selection strain whose growth strictly depends on XylA function. This strategy yielded a XylA variant with a nine-fold higher catalytic efficiency, sugar transporter variants (IolT1) with ten-fold increased activity for D-glucose and D-fructose, and hints for co-transport of these sugars by the D-sucrose transporter PtsS. Molecular dynamics simulations provided possible mechanistic explanations for the adaptive mutations. Combining the evolved enzymes with a suitable D-allulose 3-epimerase in a highly engineered chassis strain enabled whole-cell conversion of D-glucose to D-allulose with a 15% yield at 30 {degrees}C. This performance rivals immobilized enzyme processes performed at [~]60{degrees}C while avoiding enzyme purification and immobilization, offering an alternative for low-calorie sweetener production.

bioengineering↗

bioRxiv2021

The respiratory supercomplex from C. glutamicum

Corynebacterium glutamicum is a preferentially aerobic Gram-positive bacterium belonging to the Actinobacteria phylum, which also includes the pathogen Mycobacterium tuberculosis. In the respiratory chain of these bacteria, complexes III (CIII) and IV (CIV) form a CIII2CIV2 supercomplex that catalyzes oxidation of menaquinol and reduction of dioxygen to water. Electron transfer within the CIII2CIV2 supercomplex is linked to transmembrane proton translocation, which maintains an electrochemical proton gradient that drives ATP synthesis and transport processes. We isolated the C. glutamicum supercomplex and used cryo-EM to determine its structure at 2.9 [A] resolution. The structure shows a central CIII2 dimer flanked by a CIV on each side. One menaquinone is bound in each of the QN and QP sites in each CIII, near the cytoplasmic and periplasmic sides, respectively. In addition, we identified a menaquinone positioned ~14 [A] from heme bL on the periplasmic side. A di-heme cyt. cc subunit provides an electronic connection between each CIII monomer and the adjacent CIV. In CIII2, the Rieske iron-sulfur (FeS) proteins are positioned with the iron near heme bL. Multiple subunits interact to form a convoluted sub-structure at the cytoplasmic side of the supercomplex, which defines a novel path that conducts protons into CIV.

biochemistry↗

bioRxiv2021

A TPR scaffold couples signal detection to OdhI phosphorylation in metabolic control by the protein kinase PknG

Signal transduction is essential for bacteria to adapt to changing environmental conditions. Among many forms of post-translational modifications, reversible protein phosphorylation has evolved as a ubiquitous molecular mechanism of protein regulation in response to specific stimuli. The Ser/Thr protein kinase PknG modulates the fate of intracellular glutamate by controlling the phosphorylation status of the 2-oxoglutarate dehydrogenase regulator OdhI, a function that is conserved among diverse actinobacteria. PknG has a modular organization characterized by the presence of regulatory domains surrounding the catalytic domain. Here we present an investigation through in vivo experiments as well as biochemical and structural methods of the molecular bases of the regulation of PknG from C. glutamicum (CgPknG), in the light of previous knowledge available for the kinase from M. tuberculosis (MtbPknG). We found that OdhI phosphorylation by CgPknG is regulated by a conserved mechanism that depends on a C-terminal domain composed of tetratricopeptide repeats (TPR) essential for metabolic homeostasis. Furthermore, we identified a conserved structural motif that physically connects the TPR domain and a flexible N-terminal extension of the kinase that is involved in docking interactions with OdhI. Based on our results and previous reports, we propose a model in which the TPR domain of PknG couples signal detection to the specific phosphorylation of OdhI. Overall, the available data indicate that conserved PknG domains in distant actinobacteria retain their roles in kinase regulation in response to nutrient availability. IMPORTANCEBacteria control the metabolic processes by which they obtain nutrients and energy in order to adapt to the environment. In this way, the metabolic characteristics of a microorganism determine its ecological role and its usefulness in industrial processes. Here, we use genetic, biochemical, and structural approaches to study a key component in a system that regulates glutamate production in C. glutamicum, a species that is used for the industrial production of amino acids. We elucidated molecular mechanisms involved in metabolic control in C. glutamicum, which are conserved in related pathogenic bacteria. The findings have broader significance for diverse actinobacteria, including microorganisms that cause disease as well as environmental species used to produce billions of dollars of amino acids and antibiotics every year.

biochemistry↗

bioRxiv2021

Growth-rate dependency of ribosome abundance and translation elongation rate in Corynebacterium glutamicum differs from Escherichia coli

The growth rate {micro} of bacteria depends on the protein synthesis capacity of the cell and thus on the number of active ribosomes and their translation elongation rate. The relationship between these fundamental growth parameters have only been described for a few bacterial species, in particular Escherichia coli, but are missing for most bacterial phyla. In this study, we systematically analysed the growth-rate dependency of ribosome abundance and translation elongation rate for Corynebacterium glutamicum, a gram-positive model species differing from E. coli by a lower growth temperature optimum and a lower {micro}max. Ribosomes were quantified via single-molecule localization microscopy (SMLM) using fluorescently tagged ribosomal proteins and via RNA/protein ratio. Both methods revealed a non-linear relationship with little change in ribosome abundance below {micro} = 0.4 h-1 and a steep increase at higher {micro}. Unlike E. coli, C. glutamicum keeps a large pool of active ribosomes at low {micro}, but the translation elongation rate declines from [~]9 amino acids s-1 at {micro}max to <2 aa s-1 at {micro} < 0.1 h-1. A model-based approach shows that depletion of translation precursors at low growth rates can explain the observed decrease in translation elongation rate. Nutrient up-shift experiments support the hypothesis that maintenance of excess ribosomes during poor nutrient conditions enables C. glutamicum to quickly restart growth when conditions improve.

microbiology↗