Graduate Students Mini Symposium XI - 2024

13:15 h Tristan Reif-Trauttmansdorff - AG Schuller
Structural insights into the Mtr-complex of a methylotrophic methanogen: Implications for oxygen stress-response

Methanogenic archaea are widely distributed across anaerobic ecosystems, where they catalyze the final step of organic matter degradation by converting various substrates into methane. Central to this process is the membrane-bound protein complex N5-methyl-tetrahydromethanopterin-CoM methyltransferase (Mtr), which mediates the exergonic transfer of a methyl group from N5-methyl-tetrahydromethanopterin (Methyl-H4MPT, or H4HSPT in some species) to coenzyme M (HS-CoM), a reaction with a standard free energy change (ΔG°′) of −30 kJ/mol. This energy release drives the translocation of sodium ions across the membrane, establishing a sodium gradient that sodium-dependent ATP synthases subsequently exploit to generate ATP. Here, we present a 2.9Å cryo-electron microscopy (cryo-EM) structure of the Mtr complex from Methanosarcina mazei, detailing the architecture and interactions of all catalytic subunits. Additionally, we identify a novel protein, MtrI, bound to the complex exclusively under aerobic conditions, suggesting a role in oxygen stress response and repair mechanisms. These findings offer new insights into methanogen energy conservation mechanisms and uncover a potential adaptive response to oxygen exposure, expanding our understanding of methanogen survival strategies in fluctuating environments.

13:45 h Ekaterina Jalomo Khayrova - AG Bange
Characterization of newly discovered small alarmone hydrolases from a variety of bacteriophages

Bacteria and their viruses are in constant evolution. They can directly affect diverse cellular processes including replication, transcription, and translation. These bacterial mechanisms are also regulated by bacterial alarmones (p)ppGpp; signaling molecules during the stringent response (SR). Since the replication of phages largely relies on the functionality of the host machinery and processes that are targets of the (p)ppGpp alarmones, we are exploring if and how the stringent response and phage infection are interconnected. To investigate this, using bioinformatic analysis, we identified potential small alarmone-hydrolyzing (SAH) enzymes present in several lytic phages. Moreover, our biochemical results demonstrate that phages infecting P. aeruginosa (phiKZ and PAK_P4), P. chlororaphis (201phi2.1), E. coli (Av-05), and B. cereus (Izhevsk), encode for a small alarmone hydrolase (SAH) able to degrade (p)ppGpp, and (p)ppApp. Even when the biological role is not yet well elucidated, we found that the presence of phiKZ SAH in its host causes a deficiency in bacterial growth.

14:15 h Alina Schrodt - AG Erb
Engineering Methylorubrum extorquens PA1 for the production of intermediates from Ethyl-Malonyl-CoA Pathway from methanol

The development of methylotrophic microbial platforms for the production of value-added compounds is a crucial step towards a methanol-based bioindustry and a carbon-neutral bioeconomy. Methanol is a promising substrate for the synthesis of such compounds, as it is a cheap carbon source, which can be generated from syngas and CO₂, does not compete with the food industry and reduces the risk for contamination during fermentation. In this project, we aimed to engineer Methylorubrum extorquens PA1 for the production of crotonic acid during growth on methanol as a sole source of carbon and energy. For this, we selected a strain deleted in ccr (crotonyl-CoA reductase/carboxylase) to accumulate the precursor crotonyl-CoA, which is then converted to crotonic acid via the heterologously expressed thioesterase yciA from Escherichia coli. As the deletion of ccr would seize the ability of the strain to grow on C1 and C2 compounds due to a lack of glyoxylate, a glyoxylate-regeneration module was introduced on a plasmid. This strain was then evolved for improved growth on methanol through adaptive laboratory evolution, which yielded shorter doubling times and an increase of the final OD₆₀₀. Additionally, a CRISPR interference (CRISPRi) system was tested in the evolved strain to target polyydroxybutyrate (PHB) synthesis, a storage compound in many Alphaproteobacteria and a potential bottleneck in crotonic acid production. The CRISPRi system targeted phaC (PHB synthase) to reduce the accumulation of PHB to redirect the metabolic flux towards crotonyl-CoA.