Studies on the G. sulfurreducens PCA Porin Cytochrome Complex ExtABCD

Burton, Joshua A. J. (2024) Studies on the G. sulfurreducens PCA Porin Cytochrome Complex ExtABCD. Doctoral thesis, University of East Anglia.

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Abstract

Electroactive Gram-negative bacteria can respire on solid metals and minerals within anoxic environments by transferring electrons generated from intracellular metabolism to the bacterial cell surface. Many Gram-negative bacteria utilise porin cytochrome complexes (PCC) to traverse electrons across the bacterial outer membrane. The model electroactive bacterium Geobacter sulfurreducens PCA contains a total of five known PCC with the extABCD gene cluster being shown to enhance electron transfer. Strains of Geobacter expressing solely the extABCD cluster outperform WT strains with 1.4x higher current generated. Whilst Geobacter strains only expressing one of the other four PCC saw approximately 4x diminished electroactivity.

Work presented in this thesis has endeavoured to generate methods for the recombinant expression of the extA, extC and extD genes encoding a periplasmic dodecaheme and two outer membrane cytochromes respectively. UV-vis spectroscopy of ExtD and ExtA atypical cytochrome characteristics with a reduced split-Soret were uncovered, suggesting substantial differences in the heme environment to other multiheme cytochromes. Whilst ExtC displayed typical cytochrome spectral features. Through analytical sedimentation and gel filtration it was shown that ExtA forms a trimer with conjugation of the monomers predicted at the N-terminus. Generating a unique ‘heme junction’ which has not previously been shown in nature.

Interactions of the cytochromes from ExtABCD were additionally investigated with evidence suggesting that only ExtC and ExtD form a stable interaction. With the use of AlphaFold 2 and 3 potential PCC models of ExtABCD were analysed and compared to experimental evidence gathered in this thesis. Through this a new model for ExtABCD is proposed with ExtA acting as a trimeric periplasmic shuttle, ExtBC forming the porin-cytochrome interface and ExtD as the sole extracellular multiheme cytochrome.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Kitty Laine
Date Deposited:	11 Jun 2025 11:40
Last Modified:	11 Jun 2025 11:40
URI:	https://ueaeprints.uea.ac.uk/id/eprint/99461
DOI:

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