Abstract/Summary

In E. coli, four types of hydrogenases are recognized. Hydrogenases 1 (Hyd-1) and 2 (Hyd-2) are responsible for H2 consumption via respiration. Hydrogenase-3 (Hyd-3), which is encoded by the hycABCDEFGHI operon, combines with formate dehydrogenase H (Fdh-H) to form the formate hydrogen lyase (FHL) complex, which is responsible for formate disposal and H2 evolution during anaerobic fermentation under acidic pH. The hyfABCDEFGHIJR-focB operon of E. coli encodes a fourth potential hydrogenase (Hyd- 4) closely related to Hyd-3. The physiological purpose of the Hyf system is unclear although it is speculated to form a second, energy conserving, FHL with Fdh-H. However, current evidence suggests that the hyf operon is not expressed and may thus be cryptic. The aim here is to discover whether the Hyf/Hyd-4 system is indeed capable of hydrogenase activity and can thus be assigned a fourth hydrogenase. Analysis of the NCBI database showed that hyf is conserved in all E. coli/Shigella stains, except for the most basal phylogroup (B2) which is associated with avian- and uro-pathogenic strains; this might suggest a role for Hyf in colonisation of the mammalian intestine. To determine if hyf-encoded FocB acts as a second formate channel, a focA mutant was complemented with focB carried by pBADrha. Rhamnose induction of focB restored sensitivity to hypophosphite (formate analogue) supporting a role for FocB as a second formate channel. Similarly, induction of pBADrha-encoded focA also restored hypophosphite sensitivity. FocA supported H2 production at low pH, but had little impact at high pH. Low levels of FocB could compensate for absence of FocA. Plasmid induced focA restored the levels of formate released in a focA mutant at pH 6.5, to those seen in the wildtype, whereas plasmid induced focB increased such levels more modestly, although both FocA and FocB supported fermentative growth at pH 6.5. Expression of focB appeared toxic at pH 7.5. Thus, the results generally support a role for FocB in formate export and suggest roles in delivery of exogenous formate to FHL-1 indicating a role in formate import also under acidic pH conditions. When a hycE mutant strain was complemented with hyf carried by pBADrha at pH 6.5, H2 production was restored, and this was accompanied by restoration of formate consumption; a similar effect was seen upon plasmid-borne hyc induction. This indicates that hyf specifies an active hydrogen-evolving hydrogenase. Complementation of a ΔfdhF ΔhycE double mutant with both hyf and fdhF, carried by pBADrha and pBADara, respectively, re-enabled H2 production and formate consumption, thus showing that Hyf activity is Fdh-H dependent indicating that Hyf and Fdh-H combine to form a second FHL (FHL-2). FHL-2 activity was formate concentration dependent, with increased H2 production with increased formate provided. Induced-Hyf H2-production activity was enhanced by provision of nickel at up to 0.25 mM (but was inhibited at 0.5 mM nickel) supporting the presence of Ni in the proposed [NiFe] centre of the Hyf-hydrogenase. Molybdenum and selenium were also shown to be required for high Hyf-FdhH (FHL-2) H2-production activity. In conclusion, this thesis provides strong evidence showing that the hyf-focB locus encodes a second formate channel and a fourth hydrogenase (Hyd-4) in E. coli, with the Hyd-4 enzyme acting as an H2 producing and formate dependent enzyme that forms a second FdhF-dependent FHL complex. Further work is required to establish the environmental conditions under which the Hyf system is active, and offers an advantage, in the wildtype.