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Title: The physiological role of a novel flavocytochrome c from Shewanella putrefaciens
Author: Gordon, Euan H. J.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1996
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Flavocytochrome c (Fcc) is a novel, soluble periplasmic fumarate reductase from the Gram-negative bacterium Shewanella putrefaciens. A null mutant (Δfcc) which lacked the gene coding for the Fcc was constructed. The resultant strain was devoid of fumarate reductase activity and could not grow on fumarate as the sole terminal electron acceptor. Complementation of this mutation with recombinant Fcc restored the ability to grow anaerobically with fumarate. No other phenotype could be found for the Δfcc strain. This work demonstrates that flavocytochrome c is essential for fumarate respiration and furthermore is the sole terminal reductase for fumarate respiration. The fcc null mutant also allowed the expression of recombinant forms of fcc coding for the individual domains of the protein. The in vivo interaction between the haem and flavin domains of Fcc was investigated by expressing recombinant forms of the genes coding for these domains in the same cell. This indicated that the tether between the two domains, while not absolutely essential, was important for wild-type enzyme function. Studies using lacZ reporter genes fused to the fcc promoter demonstrated that the fcc gene is regulated primarily at the level of transcription and is regulated hierarchically in response to the redox potential of terminal electron acceptors. Regions of the fcc promoter sequence important to regulation of the fcc gene were identified by deletion analysis. The succinate dehydrogenase operon from S. putrefaciens was sequenced. The inferred amino acid sequences of the flavoprotein, and iron/sulphur subunits showed high sequence identity (77%) with the corresponding subunits from Escherichia coli succinate dehydrogenase.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available