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
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.