Flavocytochrome c, a multihaem cytochrome from <i>Shewanella putrefaciens</i> induced under anaerobic conditions, was studied to investigate the physiological function of this protein. These studies comprised two facets: the cloning and sequence analysis of the structural gene for flavocytochrome c and a biochemical study of the fumarate reductase activity associated with flavocytochrome c. A <i>S. putrefaciens</i> genomic library was constructed in the expression vector pEX3. This library was screened in <i>E. coli</i> MM294 by colony hybridization of induced recombinants with antibody raised to purified flavocytochrome c protein. One clone giving a strong signal to antibody was identified from this library. Restriction digests of this recombinant showed the insert DNA to be approximately 1.5 kb. Southern blot analysis of the clone gave hybridization of flavocytochrome c antibody to a protein of approximately 45 kDa which was encoded by the recombinant pEX3 vector. This cloned fragment was proposed to encode part of the flavocytochrome c gene and investigated in more detail. The 1.5 kb cloned fragment was partially sequenced and mapped. Sequencing yielded two non-overlapping contigs of 438 bp and 966 bp respectively. The DNA fragment joining the two contigs remained unsequenced. Database analysis showed that the second contig contained 4 conserved c-type haem binding site motifs CXYCH within the first 90 residues. A second region in this contig from bases 123-151 was found to be completely homologous with a highly conserved FAD-binding fingerprint common to many flavoproteins. This molecular analysis strongly suggested that the cloned DNA fragment encoded at least 322 residues of the N-terminal region of the flavocytochrome c protein. This was further confirmed by the finding that the first 8 residues of the second contig were completely homologous with residues 6-13 of the N-terminal sequence of flavocytochrome c. Cloning the entire flavocytochrome c gene was attempted also by functional complementation of an E. coli fumarate reductase mutation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:641665 |
| Date | January 1991 |
| Creators | Black, Ann Charlotte |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/10823 |
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