The genes encoding the two structural subunits of Escherichia coli hydrogenase 2 (HYD2) have been cloned and sequenced. They occur in an operon (hyb) which contains seven open reading frames. An hyb deletion mutant (strain AP3) failed to grow on dihydrogen-fumarate medium and also produced very low levels of HIYD1. All seven open reading frames are required for restoration of wild-type levels of active HYD2 in AP3.The hyb operon was mapped at 65 min on the E. coli chromosome.Under anaerobic growth conditions, Escherichia coli produces three different nickel-containing hydrogenases (3, 39). Hydrogenase 3 (HYD3) is part of the formate hydrogenlyase (FHL) complex and is responsible for formate-dependent dihydrogen (H2) evolution. The operon encoding HYD3 and other accessory electron transport components of the FHL complex, hyc, has been identified and is located at 58 min on the E. coli chromosome (7). The highly oxygen-labile nature of HYD3 has precluded detailed biochemical characterization. HYD2 is involved in H2 uptake and can be differentially induced to high levels when cells are grown in medium containing H2 as an electron donor and fumarate as an electron acceptor (3,23,25,39). An active component of HYD2 has been purified and shown to be a heterodimeric enzyme with a 58-kDa large subunit and a 30-kDa small subunit (4). Although mutants defective in H2 uptake have been described (23,25), detailed analysis of the operon encoding HYD2 has not been carried out. HYDl has also been purified and shown to consist of a large (60 kDa) subunit and a small (30 kDa) subunit (16,40). An active form of HYD1 containing only the large subunit has also been purified and characterized (1, 16). The operon encoding the two structural subunits of HYD1 (hya) contains a total of six genes and has been mapped at 22 min on the E. coli chromosome (30,31). The function of HYD1 is not understood, but it is believed to have a role in hydrogen cycling during fermentative growth. In addition to the operons coding for the structural components of the three hydrogenases, a fourth operon, hyp, located at 58 min, is essential for activity of all three hydrogenases (20,26,38). At least one of the genes in this operon (hypB) is involved in nickel metabolism, most probably via nickel insertion into apoenzyme (27).In this paper, we present the DNA sequence of the operon encoding HYD2 (hyb), which contains seven open reading frames (ORFs). Cassette mutagenesis of the hyb operon on the chromosome resulted in a total loss of HYD2 expression and activity, as well as in significant reduction in HYD1 activity. MATERIALS AND METHODSBacterial strains. All bacterial strains used were E. coli K-12 derivatives and are listed in Table 1 [pH 7.0]), resuspended in the same buffer to an optical density of 0.5 at 600 nm, and used for whole-cell enzyme assays. Cell extracts were prepared by sonicating cell suspensions on ice with a model W385 sonicator (Heat Systems) for 20 5-s bursts. Triton X-100 was added to a final concentration of 2% (vol/vol), when req...

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