Cloning and sequencing of a putative Escherichia coli [NiFe] hydrogenase-1 operon containing six open reading frames

Abstract

DNA encompassing the structural genes of an Escherichia coli [NiFe] hydrogenase has been cloned and sequenced. The genes were identified as those encoding the large and small subunits of hydrogenase isozyme 1 based on NH2-terminal sequences of purified subunits (kindly provided by K. Francis and K. T. Shanmugam). The structural genes formed part of a putative operon that contained four additional open reading frames. We have designated the operon hya and the six open reading frames hyaA through F. hyaA and hyaB encode the small and large structural subunits, respectively. The nucleotide-derived amino acid sequence of hyaC has a calculated molecular mass of 27.6 kilodaltons, contains 20% aromatic residues, and has four potential membrane-spanning regions. Open reading frames hyaD through F could encode polypeptides of 21.5, 14.9, and 31.5 kilodaltons, respectively. These putative peptides have no homology to other reported protein sequences, and their functions are unknown.The anaerobic hydrogen metabolism of Escherichia coli and other enterobacteria is intricately regulated with regard to both hydrogen production during fermentation and hydrogen oxidation during anaerobic respiration (13,14). This complex regulatory system responds to specific substrates as well as to global regulatory signals and results in the biosynthesis of discrete hydrogenases specific to a given metabolic pathway. Three E. coli hydrogenases have been described which are synthesized in response to different physiological conditions. Hydrogenases 1 and 2 have been biochemically characterized and are immunologically distinct membranebound nickel-containing proteins (2, 3, 31). The existence of hydrogenase 3 was originally inferred from the fact that hydrogenases 1 and 2 immunoprecipitated from cell lysates did not account for total hydrogenase activity (30). Hydrogenase 3 activity is very labile and has been only partially characterized (34). Hydrogenases 1 and 3 are induced to higher levels by growth on glucose and formate. Hydrogenase 3 has also been shown to have a role in the formate hydrogenlyase pathway and accounts for about 60 to 70% of the total hydrogenase activity in the cell. The physiological role of hydrogenase 1 has not been defined. Hydrogenase 2 has been implicated as a respiratory uptake hydrogenase coupled to fumarate reduction and is induced to a higher level by growth in the presence of glycerol and fumarate (30).The presence of three different enzymes catalyzing the same reactions makes the biochemical, physiological, and genetic analyses of their metabolic roles technically difficult. Toward understanding the physiological roles of the different hydrogenases, a large number of E. coli mutants defective in hydrogenase activities have been analyzed. Most of these mutants lack all three hydrogenase activities; based on genetic analysis, the mutations appear to be exclusively at loci affecting regulation and do not encompass the hydrogenase structural genes. These genetic loci have been designated as hydA through F (8, 16...

Reassignment of the gene encoding the Escherichia coli hydrogenase 2 small subunit

“…6). HmcB, which has an N-terminal 'twin-arginine' signal sequence, is located at the periplasmic face of the cytoplasmic membrane to [25]; hups, hupS gene product from R. capsulatus [26]; hyaa, hyaA gene product of hydrogenase 1 from E. coli [7] ; hynb, hynB gene product from D. gigas [61; hyb0, small subunit of E. coli hydrogenase 2 [23]. Fig.…”

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].…”

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

“…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).…”

“…It is apparent from its subunit structure and from its overall amino acid sequence similarity with other uptake hydrogenases that it has a role in hydrogen oxidation (Menon et al, 1990). It has been proposed to be involved in recycling the hydrogen produced by hydrogenase 3 based on the fact that its synthesis shows a strong correlation with that of the FHL pathway (Sawers et al, 1985 ; however, such an activity for the enzyme has yet to be demonstrated unequivocally.…”

“…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.…”