Use this URL to cite or link to this record in EThOS:
Title: Functional and regulatory analysis of the 12-gene hyf operon of Escherichia coli
Author: Skibinski, Alexander George
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2002
Availability of Full Text:
Access from EThOS:
Access from Institution:
Sequence analysis of the 55.8-56.0 min region of the Escherichia coli genome has revealed a 12-gene operon designated the hyf operon (hyfABCDEFGHIR-focB). The hyf operon encodes a putative ten-subunit hydrogenlyase complex (hYdrogenase four or Hyf), a potential formate sensing 0'54_ dependent transcriptional activator, HyfR (related to FhIA), and a possible formate transporter, FocB (related to FocA). It has been proposed that Hyf in conjunction with Fdh-H forms a second formate hydrogenlyase pathway (Fhl-2) in Escherichia coli, which unlike the hyc operon encoded Fhl pathway (Fhl-l ) is a respiration-linked proton translocating Fhl complex. Initial experiments directly investigated these proposals and were conducted with hyf and hydrogenase-I, -2 and -3 mutants grown under hyf operon optimal transcriptional activation conditions. Radiolabelling experiments with 63Ni did not detect the proposed large subunit of hydrogenase-4, despite the detection of 63Ni_ associated polypeptides likely to correspond to the large subunits of hydrogenase-I, - 2 and -3. Also, Fdh-H, hydrogenase and hydrogen production assays detected no activity attributable to the hyf operon. Immunoblotting experiments with anti-HycE and anti-Hyf sera did not detect Hyf polypeptides, suggesting that expression of the hyf operon was very low under optimal transcriptional activation conditions. Transcriptional analysis of the hyf operon using a hyfA-lacZ transcriptional fusion showed that, like the hyc operon, the hyf operon is induced by formate at low pH via the formate sensing, 0'54-dependent transcriptional activator FhlA. The proposed transcriptional activator HyfR was also found to activate hyf operon transcription in a cr54-dependent manner. However the co-effector(s) used by HytR has yet to be identified. Finally bioreactors were used to analyse the growth and metabolism of hyf mutants. However, no differences in growth and metabolism attributable to the hyf operon were observed during anaerobic controlled batch cultivation and both aerobic and anaerobic glucose-limited chemostat cultivation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available