Lean, green, heat-combating machine: a proteomics-based investigation

Lean, green, heat-combating machine: a proteomics-based investigation Josephine Boel Andresen 𝟏𝟏, Mansi Jain 𝟐𝟐, Eva Csosz 𝟐𝟐, Dino Zinovie Ciobanu 𝟑𝟑, Christine Carapito 𝟑𝟑, Kenneth Valerio Aguilar 𝟒𝟒, Harald Barsnes 𝟒𝟒, Eveline Peeters 𝟏𝟏 1Research Group of Microbiology (MICR), Department of Bioengineering, Vrije Universiteit Brussel, Belgium 2Proteomics Core Facility, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Hungary 3Laboratoire de Spectrométrie de Masse Bioorganique (LSMBO), Centre National de la Recherche Scientifique, University of Strasbourg, France 3Proteomics Unit at the University of Bergen (PROBE), Department of Biomedicine, University of Bergen, Norway The University of Debrecen has received the funding 2020-2.1.1-ED-2023-00269 from the Hungarian National Research, Development and Innovation Fund to cover the participation of DC10 in PROHITS PROHITS’ proteomics tools Towards increasing shot-gun proteomics coverage Thermal Protein Profiling the photosynthetic system Sigma factor regulation during heat shock References Figure 1: The consortium encompasses a range of methods to apply for proteomics-based research. Cultivating a thermophilic cyanobacterium Figure 5:sigma factors recruit the RNA polymerase to given promotors [4,5], and are regulated by the kaiABC operon, which initiates a sigma factor cascade (A). To map the sequence of the cascade and the effect of increasing temperatures (heat shock) on it, the gene products’ presence at different temperatures will be monitored with shot-gun proteomics (see current protein acquisitions with Method 2 in B). Significant heat shock is determined via spot test assays (C) and monitored with known heat shock-related genes with RT-qPCR (D), exemplified with C. thermodepolymerans DSM 15344 (see table 2 below). Figures 6-8: high-temperature cultivation instruments adapted to fit the thermophilic cyanobacterium’s optimal temp. at 55ºC for both dynamic (6& 7) and static (8) growth. UniProt ID Protein name Q8CM25 Photosystem II D2 protein P0A444 Photosystem II protein D1 1 Q8DKM3 Photosystem II reaction center protein Y P0A386 Photosystem II extrinsic protein V P0A401 Photosystem I reaction center subunit III P0A405 Photosystem I P700 chlorophyll aapoprotein A1 P0A407 Photosystem I P700 chlorophyll aapoprotein A2 P0A415 Photosystem I iron-sulfur center P0A420 Photosystem I reaction center subunit II P0A423 Photosystem I reaction center subunit IV P0A431 Photosystem II extrinsic protein O P0A444 Photosystem II protein D1 1 Q8DG60 Photosystem II assembly factor lipoprotein Psb27 Q8DGB3 Photosystem I assembly related protein Q8DGB4 Photosystem I reaction center subunit XI Q8DI95 Photosystem II assembly protein Ycf48 Q8DIF8 Photosystem II CP43 reaction center protein Q8DIQ1 Photosystem II CP47 reaction center protein Q8DLJ8 Photosystem II assembly factor Psb28 protein UniProt ID Protein name Q8DL79 RNA polymerase sigma factor SigA Q8DGF3 RpaB Q79V60 Circadian clock oscillator protein KaiC Q79V61 Circadian clock oscillator protein KaiB Thermophile portfolio Caldimonas thermodepolymerans DSM 15344 Haloferax volcanii H26 Parageobacillus thermoglucosidasius DSM 254 Sulfolobus acidocaldarius DSM 639 Sulfolobus acidocaldarius SK1 Thermosynechococcus elongatus BP-1 Table 1: current list of photosynthesis-related proteins acquired with Method 2 (lysis: 2% SDS and bead beating, protein digestion: S-trap). Table 2: list of the different thermophilic strains cultivated and investigated under PROHITS. Figure 2: Tests of different lysis/protein extraction methods to gain as much protein coverage, currently at 22% and 33% of the complete proteome. Figure 4: all plots are hypothesized values and purely conceptualising; the TPP assay once completed for T. elongatus BP-1 will enable characterizing the individual proteins of the photosynthetic system to identify which proteins are less thermo-stable than other and thereby find the “weaker” subunits. The findings could aid in optimizing the photosynthetic apparatus’ robustness. [1] Yamaoka, T., Satoh, K., & Katoh, S. (1978). Photosynthetic activities of a thermophilic blue-green alga. Plant and Cell Physiology, 19(6), 943–954. [2] Mateus, A., Kurzawa, N., Becher, I., Sridharan, S., Helm, D., Stein, F., Typas, A., & Savitski, M. M. (2020). Thermal proteome profiling for interrogating protein interactions. Molecular Systems Biology, 16(3), e9232. [3] Komárek, J., Johansen, J. R., Šmarda, J., & Strunecký, O. (2020). Phylogeny and taxonomy of Synechococcus-like cyanobacteria. Fottea, 20(2), 171–191 [4] Roncarati, D., & Scarlato, V. (2017). Regulation of heat-shock genes in bacteria: From signal sensing to gene expression output. FEMS Microbiology Reviews, 41(4), 549–574 [5] Rajaram, H., Chaurasia, A. K., & Apte, S. K. (2014). Cyanobacterial heat-shock response: Role and regulation of molecular chaperones. Microbiology, 160(4), 647–658. https://doi.org/10.1099/mic.0.073478-0 •Optimize shot-gun proteomics coverage, which will unlock the potential of the TPP assay •Perform TPP assay •Characterize heat shock moment for T. elongatus BP-1 •Make sigma factor mutants in T. elongatus BP-1 •Complete genome assembly from Oxford Nanopore sequencing for proteogenomics Figure 3: the performed TPP assay demonstrates the melting behaviour of the overall proteome [2], whereas when plotting specific proteins their unique thermal stability is visualised. On the right, highly stable β-subunit and less stable ɣ-subunit of the Thermosome complex (post-normalization). The temperature gradient is selected based on S. acidocaldarius’ optimal growth temperature (75ºC). Cyanobacteria’s imperative existence are due to their photosynthesis, rendering them able to fixate carbon and makes them an emerging group within sustainable bioproduction. Contrary to their predominantly mesophilic kin, some cyanobacteria thrive in warm areas, Thermosynechococcus elongatus BP-1 amongst other, thereby pushing to the boundaries of their ecological fitness as the photosynthetic apparatus is heat-sensitive (max. 72ºC) [1,3]. T. elongatus is part of a diverse portfolio of biotechnological relevant thermophiles being investigated in the EU-funded project PROHITS. Heat-related impact in T. elongatus, hereunder a regulatory response focusing on sigma factors (RNA polymerase recruiters), is the focus point of this research in which the proteomics methods available will be applied to illuminate the required adaptations for its habitat. The insights will feed back to the ongoing work of the consortium, as well as filling a knowledge gap that can unleash the potential of a thermophilic cyanobacterium as a future production chassis. A B D C 678 Abstract 5 4 2 13 Next steps 8 7 6