Investigating the Control Mechanisms of Extracellular Electron Transfer in Gram-negative Bacteria

Morales Flores, Alejandro (2024) Investigating the Control Mechanisms of Extracellular Electron Transfer in Gram-negative Bacteria. Doctoral thesis, University of East Anglia.

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Abstract

Dissimilatory metal-reducing bacteria are capable of surviving in anoxic environments by transferring metabolic electrons to extracellular electron acceptors (EEA). One of these metal-reducing bacteria is Shewanella oneidensis, a Gram-negative facultative anaerobe, studied extensively as a model organism for extracellular electron transfer (EET). S. oneidensis uses the MtrCAB complex, a porin-cytochrome complex, to perform EET. Electrons pass from the periplasm through MtrA into MtrC, which is surface-exposed and facilitates electron transfer to EEA such as ferric oxides, flavins, and electrodes. However, the precise control mechanisms for EET in S. oneidensis remain largely unknown. In particular, the conserved domain arrangement of MtrC, where the 10 hemes are arranged into two domains separated by a β-barrel domain, is unusual and hypothesised to be involved in regulation of electron transfer. This thesis explores the control mechanisms by modifying the interactions between the domains of MtrC as well as the axial heme ligation of MtrA. Any consequent effects on electron transfer were investigated by biophysical characterisation and functional activity assays of MtrC/MtrA variants. The structures of the variant cytochromes were analysed to confirm that the changed amino acids were incorporated into the tertiary protein structure. The functional assays were performed to assess any impacts on EET rates. The results suggest that increasing the distance between the domains of MtrC resulted in minor differences to the EET rates. The increase in distance between domains I, II and domains III, IV was confirmed by SAXS. There was only a minimal difference in the UV-Vis spectra of the variants with increased inter-domain distance when compared to WT MtrC. Furthermore, there were no differences in reduction of extracellular FMN. However, removing domains III and IV of MtrC resulted in a significant decrease to the EET rates, depending directly on the electron acceptor. A structure for the MtrC variant comprised solely of domains I and II was obtained by X-ray crystallography. Finally, changing three heme distal histidines to methionines in MtrA also resulted in lowered EET rates. However, there were no major differences in the UV-Vis spectra of three single MtrA His/Met variants or a triple His/Met variant when compared to WT MtrA.These results highlight the essential role that the domains of MtrC play on the capacity of S. oneidensis to reduce extracellular substrates. They also show that replacing certain iii distal histidines for methionines in the hemes of MtrA is possible. These results have further characterised two decaheme c-type cytochromes of S. oneidensis, MtrA and MtrC, and could help improve existing biotechnological applications like microbial fuel cells and bioremediation that exploit the electrogenic properties of S. oneidensis.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Jennifer Whitaker
Date Deposited:	04 Apr 2025 15:11
Last Modified:	04 Apr 2025 15:11
URI:	https://ueaeprints.uea.ac.uk/id/eprint/98959
DOI:

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