Characterisation of cytochromes c3 and c5 from Shewanella frigidimarina NCIMB400

• Main Author: Hill, Anne E.
• Published: University of Edinburgh 1998
• Subjects: 579
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.652431

The structural gene encoding cytochrome c<SUB>5</SUB> has been cloned and sequenced, along with some surrounding sequence. The inferred amino acid sequence of the cloned gene, <I>scyA</I>, 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 <I>scyA </I>open reading frame contains a 21 amino acid N-terminal extension which is absent in purified cytochrome <I>c<SUB>5</SUB></I>. 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 <I>c<SUB>5</SUB></I>. Northern blott analysis confirmed that <I>scyA </I>is transcriptionally isolated. A null mutant which lacked the gene coding for cytochrome <I>c<SUB>5</SUB></I> 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 <I>c<SUB>3</SUB></I>. Deletion strains lacking the gene coding for cytochrome <I>c<SUB>3</SUB></I> (<I>cctA</I>) and also strains lacking both cytochrome <I>c<SUB>3</SUB></I> and flavocytochrome <I>c<SUB>3</SUB></I> were constructed. Comparison of the growth characteristics of the mutant strains with wild-type suggest the involvement of cytochrome <I>c<SUB>3</SUB></I> with respiratory iron (III) reduction. Ferrozine extraction experiments similarily demonstrated a decrease in iron (III) reduction activity by strains lacking the cytochrome <I>c<SUB>3</SUB></I> gene. In order to facilitate further study of cytochrome <I>c<SUB>5</SUB></I>, and production of recombinant forms of the protein, an expression system was developed. Cytochrome <I>c<SUB>5</SUB></I> was successfully expressed in <I>Shewanella frigidimarina</I> NCIMB400 by using the expression vector pMMB503 which is inducible with isopropylthio-β-D-galactoside.