Functional and regulatory analysis of the 12-gene hyf operon of Escherichia coli

Sequence analysis of the 55.8-56.0 min region of the Escherichia coli \ngenome has revealed a 12-gene operon designated the hyf operon \n(hyfABCDEFGHIR-focB). The hyf operon encodes a putative ten-subunit \nhydrogenlyase complex (hYdrogenase four or Hyf), a potential formate sensing 0'54_ \ndependent transcriptional activator, HyfR (related to FhIA), and a possible formate \ntransporter, FocB (related to FocA). It has been proposed that Hyf in conjunction \nwith Fdh-H forms a second formate hydrogenlyase pathway (Fhl-2) in Escherichia \ncoli, which unlike the hyc operon encoded Fhl pathway (Fhl-l ) is a respiration-linked \nproton translocating Fhl complex. \nInitial experiments directly investigated these proposals and were conducted \nwith hyf and hydrogenase-I, -2 and -3 mutants grown under hyf operon optimal \ntranscriptional activation conditions. Radiolabelling experiments with 63Ni did not \ndetect the proposed large subunit of hydrogenase-4, despite the detection of 63Ni_ \nassociated polypeptides likely to correspond to the large subunits of hydrogenase-I, - \n2 and -3. Also, Fdh-H, hydrogenase and hydrogen production assays detected no \nactivity attributable to the hyf operon. Immunoblotting experiments with anti-HycE \nand anti-Hyf sera did not detect Hyf polypeptides, suggesting that expression of the \nhyf operon was very low under optimal transcriptional activation conditions. \nTranscriptional analysis of the hyf operon using a hyfA-lacZ transcriptional \nfusion showed that, like the hyc operon, the hyf operon is induced by formate at low \npH via the formate sensing, 0'54-dependent transcriptional activator FhlA. The \nproposed transcriptional activator HyfR was also found to activate hyf operon \ntranscription in a cr54-dependent manner. However the co-effector(s) used by HytR \nhas yet to be identified. \nFinally bioreactors were used to analyse the growth and metabolism of hyf \nmutants. However, no differences in growth and metabolism attributable to the hyf \noperon were observed during anaerobic controlled batch cultivation and both aerobic \nand anaerobic glucose-limited chemostat cultivation.