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Title: Characterisation of cytochromes c3 and c5 from Shewanella frigidimarina NCIMB400
Author: Hill, Anne E.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1998
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The structural gene encoding cytochrome c5 has been cloned and sequenced, along with some surrounding sequence. The inferred amino acid sequence of the cloned gene, scyA, corresponds to a mature protein of 78 amino acids with a single haem attachment motif situated toward the N-terminal end of the protein; a methionine residue near the C-terminus serves as the sixth haem ligand. The scyA open reading frame contains a 21 amino acid N-terminal extension which is absent in purified cytochrome c5. This sequence conforms to the format of a typical periplasmic signal sequence. Two additional open reading frames were identified on analysis of the regions flanking the structural gene, neither of which is functionally related to cytochrome c5. Northern blott analysis confirmed that scyA is transcriptionally isolated. A null mutant which lacked the gene coding for cytochrome c5 was constructed. The anaerobic respiratory capacity of the resultant strain was assessed and compared to wild-type. No obvious mutant phenotype was identified. Gene disruption experiments were also used to characterise cytochrome c3. Deletion strains lacking the gene coding for cytochrome c3 (cctA) and also strains lacking both cytochrome c3 and flavocytochrome c3 were constructed. Comparison of the growth characteristics of the mutant strains with wild-type suggest the involvement of cytochrome c3 with respiratory iron (III) reduction. Ferrozine extraction experiments similarily demonstrated a decrease in iron (III) reduction activity by strains lacking the cytochrome c3 gene. In order to facilitate further study of cytochrome c5, and production of recombinant forms of the protein, an expression system was developed. Cytochrome c5 was successfully expressed in Shewanella frigidimarina NCIMB400 by using the expression vector pMMB503 which is inducible with isopropylthio-β-D-galactoside.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available