Cloning, sequencing, and mutational analysis of the hyb operon encoding Escherichia coli hydrogenase 2

Abstract

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...

Transcriptional regulation in response to oxygen and nitrate of the operons encoding the [NiFe] hydrogenases 1 and 2 of Escherichia coli

“…This led to the proposal that the function of hydrogenase 2 was in a respiratory capacity, allowing the cells to gain energy from the oxidation of molecular hydrogen. A subsequent mutational analysis of the hyb structural genes encoding the enzyme confirmed this hypothesis (Menon et al, 1994).…”

“…Primer extension analysis using total RNA extracted from anaerobically grown cultures revealed a single 5h end ( The DNA sequence in the neighbourhood of the hyb operon transcription start site. The start site of transcription is located 102 bp upstream of the ATG translation initiation codon of the hybO gene (Menon et al, 1994 ;Sargent et al, 1998) and is indicated by the bent arrow. The putative k10 sequence upstream of the start site is underlined.…”

“…Three immunologically and genetically distinct [NiFe] hydrogenases, termed hydrogenase 1, hydrogenase 2 and hydrogenase 3, have been identified and characterized in Escherichia coli , 1986Sawers et al, 1985 ;Bo$ hm et al, 1990 ;Menon et al, 1990Menon et al, , 1994Sauter et al, 1992). More recently, the existence of a fourth enzyme has been postulated based on a genetic analysis of the E. coli chromosome (Andrews et al, 1997).…”

“…The polycistronic operons encoding hydrogenases 1 and 2 are hyaABCDEF and hybOABCDEFG, respectively (Menon et al, 1990(Menon et al, , 1994. Recently, initiated an analysis of hya expression using a Φ(hya-lacZ) gene fusion and determined that anaerobic induction is dependent on the global transcriptional regulator ArcA (Lynch & Lin, 1996a) and a new anaerobic regulator termed AppY.…”

Requirement for nickel of the transmembrane translocation of NiFe‐hydrogenase 2 in Escherichia coli

“…Fax: (33) 91 71 89 14. E-mail: wu@ibsm.cnrs-mrs.fr HYD2 and HYD3 activities, respectively [2][3][4][5]. All three hydrogenases contain nickel [1,[6][7][8].…”