Degenerate oligonucloetide primers designed from sequence alignments of a number of well characterised nitrite reductase enzymes were used to amplify a 350bp fragment from wild type <I>N. subflava</I> DNA using PCR. Sequence analysis and database comparisons on this DNA led to the conclusion that the nitrite reductase gene had been identified. Upon translation the enzyme was found to contain copper in its active site and was proposed to be unusually associated with the outer membrane. Sequence and probing analysis on a Tn<I>5 </I>insertion mutant of <I>N. subflava</I>, lacking the ability to reduce nitrite, determined that Tn<I>5</I> had not inserted into a structural or regulatory gene for nitrite reduction but was affecting the nitrite reductase gene distally. Further evidence from nitrite reductase activity assays on native polyacrylamide gels determined that the gene affected by the Tn<I>5</I> insertion was responsible for electron donation to the outer membrane nitrite reductase enzyme. An outer membrane location for the electron donor protein was also postulated. The presence of an azurin like protein attached to the outer membrane has been identified in <I>N. gonorrhoeae</I> which is thought to act as donor to the gonococcal membrane bound nitrite reductase. However it is anticipated that the donor protein in <I>N. subflava</I> will differ from this as attempts to PCR an azurin gene from <I>N. subflava</I> were repeatedly unsuccessful (Lambert, 1997). The presence of outer membrane associated nitrite reductase and electron donor proteins in <I>N. subflava</I> indicate that a novel arrangement of the denitrification pathway may exist in this organism which spans the periplasmic space. This periplasmic arrangement is proposed to be unique to <I>Neisseria </I>spp.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:662852 |
| Date | January 1998 |
| Creators | Thomas, Joanne M. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/13109 |
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