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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session E

Poster V3 - Influence of Protein Condensate Formation on Extracellular Electron Transfer

Saturday, October 26, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Lillian Hutchinson

Undergraduate
University of Arkansas
Little Rock, Arkansas, United States

Co-Author(s)

BT

Bailey Tibbet

Senior Graduate Assistant
University of Texas at Austin, United States

Last Author(s)

Keith Keitz, PhD

Principle Investigator
University of Texas at Austin, United States

Introduction: Shewanella oneidenesis is an electrochemically active microbe able to release electrons into its environment through an anaerobic respiratory mechanism, extracellular electron transfer (EET). Increased control of EET would allow S. oneidenesis to be used more efficiently in its various applications such as microbial fuel cells or bioremediation. This experiment sought to observe the effects biomolecular condensates on EET. Protein condensates are membraneless compartments that induce liquid-liquid phase separation (LLPS) within the cell. As a result, these condensates are capable of altering the electrochemical cellular environment which we hypothesize will perturb the cell’s native EET process. Overall, the goal of this experiment was to measure EET response in cells expressing different protein condensates.

Materials and

Methods: Inducible plasmids coding for three different protein condensates were obtained from Dr. Yifan Dai of Washington University at St. Louis, transformed via electroporation into S. oneidensis cells, sequence verified. Individual colonies containing DDx4 (UniProt: H0Y960), FUS LC (UniProt: P35637), and A1 LCD(UniProt: F8VZ49) were grown anaerobically overnight and washed with minimal media to have an optical density of 1.25. In an anaerobic chamber, the cells were diluted 10:250 into experimental media containing either 1 μg/mL antibiotic or 25 mM Fe(III) Citrate in a 96-well microplate, sealed, and placed into a plate reader where the absorbance at 600 nm was measured for 15-24 hours. The growth rates cells were then compared.

To directly track Fe(II) reduction, ferrozine-Fe(II) complex formation was tracked in a similar manner. However for this test, half of the cells were pre induced by 1:25 dilution into fresh media containing 1 mM Isopropyl ß-D-1-thiogalactopyranoside (IPTG) while the other half was diluted 1:25 into fresh media without IPTG and grown for 8 hours before being washed with minimal media to have an optical density of 0.25. For this experiment, the media contained 2.5 mM Ferrozine and 5 mM Fe(III) Citrate. Absorbance was measured at 562 nm every 10 minutes for 15-24 hours. Using Beer’s Law, the absorbance of ferrozine at 562 nm was converted into the concentration of Fe(II). Induced cells and uninduced cells’ respective kinetic rates were then compared.

Results, Conclusions, and Discussions: As condensates induce LLPS, they affect the properties of the cells therefore changing its interactions with the environment. This change can be demonstrated through the cell’s ability to uptake ions from its environment. To confirm that LLPS was being induced in vivo by the condensates, an antibiotic assay was performed. DDx4 and A1 LCD were found to resist the positively charged antibiotic, chloramphenicol, while FUS LC was found to deter the negatively charged antibiotic, carbenicillin. This was indicated by induced cells having increased growth rates in the presence of chloramphenicol or carbenicillin in comparison to uninduced cells. The k values representing the rate of the growth curves also indicate a faster growth rate. The increased growth rate demonstrates that LLPS is impacting the cell’s interactions with the antibiotics by preventing uptake. An Fe(III) Citrate reduction growth assay was used to further indicate the induction of LLPS and determine if the condensates would inhibit growth on iron and EET activity. Fe(III) reduction is mediated by EET, so a reduction driven growth assay was used to measure EET response in the presence of condensates. When their EET ability was tested, it was found that the protein condensate formation has an insignificant effect on EET when iron is present. It can be concluded that condensate formation does not inhibit cell growth on iron or EET. A ferrozine assay was used to directly measure the impact of condensates on EET. Ferrozine turns purple in the presence of Fe(II), so as cells undergo EET and the Fe(III) is being reduced to Fe(II), the absorbance of the media increases at 562 nm. When compared using a t-test, significant (p < 0.05)) differences in two out of three of the condensate types were shown as the rate of Fe(II) reduction was increased within induced cells in comparison to those without IPTG. In conclusion, DDx4, A1 LCD, and FUS LC were successfully formed in S. oneidenesis and the condensates demonstrated LLPS via the antibiotic assay. The Fe(III) and ferrozine assays demonstrated preliminary evidence that protein condensates have an impact on EET activity.

Acknowledgements (Optional): Support for this research was provided by the National Science Foundation through the Center for Dynamics and Control of Materials: an NSF MRSEC under Cooperative Agreement No. DMR-2308817.