Mutational analysis and characterization of the Escherichia coli hya operon, which encodes [NiFe] hydrogenase 1

Abstract

Deletion mutants of Escherichia coli specific for hydrogenase isoenzyme 1 (HYD1) have been constructed and characterized. The hya operon, which contains genes for the two HYD1 structural subunits and four additional genes, was mapped at 22 min on the E. coli chromosome. The total hydrogenase activities of the HYD1-negative mutant and wild-type strains were similar. However, the formate dehydrogenase activity associated with the formate hydrogen lyase pathway was lower in the mutant. The hya mutant (strain AP1), complemented with only the hydrogenase structural genes (hyaAB), produced antigenically identifiable but inactive HYD1 protein.The first five genes of hya (hyaA to hyaE) were required for the synthesis of active HYD1, but wild-type levels of HYDl activity were restored only when mutant cells were transformed with all six genes of the operon. When AP1 was complemented with hya carried on a high-copy-number plasmid, the HYD1 structural subunits were overexpressed, but the excess protein was unprocessed and localized in the soluble fraction of the cell. The products of hyaDEF are postulated to be involved in the processing of nascent structural subunits (HYAA and HYAB). This processing takes place only after the subunits are inserted into the cell membrane. It is concluded that the biosynthesis of active HYD1 is a complex biochemical process involving the cellular localization and processing of nascent structural subunits, which are in turn dependent on the insertion of nickel into the nascent HYD1 large subunit.Hydrogen metabolism in Escherichia coli is tightly regulated by parameters of growth (2, 27) and involves three discrete nickel-containing hydrogenases: two electrophoretically stable, membrane-bound heterodimeric enzymes, hydrogenase 1 (HYD1) (28) and hydrogenase 2 (HYD2) (3), and a labile hydrogen-evolving hydrogenase, hydrogenase 3, which is as yet uncharacterized (27). HYD1 has been purified from anaerobically grown cells and biochemically characterized (28). It has a molecular mass of 200 kDa, is composed of two large (60-kDa) and two small (32-kDa) subunits, and contains 11 nonheme iron atoms and 1 g-atom of nickel per mol of enzyme (28 (16,24,25,30,33); and (iii) mutants unable to utilize hydrogen in the presence of electron acceptors (16,32). None of the mutants, however, have been shown to specifically impair HYD1 activity, indicating that the mutations are not in the hya operon. In this paper, we report the construction, biochemical characterization, and genetic analysis of HYD1-specific deletion mutants. MATERIALS AND METHODSBacterial strains and culture conditions. All bacterial strains used in this study are derivatives of E. coli K-12 and are listed in Table 1. Bacteria were cultured in Luria broth with 0.4% glucose as the carbon source (LBG). Antibiotics were added at final concentrations of 50 jig/ml (kanamycin), 100 jig/ml (ampicillin), and 20 ,ug/ml (chloramphenicol). For studying the incorporation of nickel into HYD1, cells were grown anaerobically in LBG in the presence of 1.2 ,iM...

Heterologous expression of Alteromonas macleodii and Thiocapsa roseopersicina [NiFe] hydrogenases in Escherichia coli

“…The AltDE HupH protein also shares 10 % identity with HyaF from E. coli. Interestingly, in a case similar to our findings in this study, an E. coli hyaF mutant produced a functional hydrogenase in the absence of hyaF, but activity increased when this gene was reintroduced (Menon et al, 1991). HupH and HoxQ from Rhizobium leguminosarum and Ralstonia eutropha, respectively, were found to be essential for expression of an active hydrogenase and it was suggested that they may be involved in proper maturation of the hydrogenase small subunit (Bernhard et al, 1996;Manyani et al, 2005).…”

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

“…It is still unknown in which oT der these processes take place and whether further reactions a e required. It has been shown that nickel insertion into the p:ecursors is a prerequisite for the large subunit C-terminal p~ ocessing of the hydrogenase of A. vinelandii [23], HYD1 [24] a~Ld HYD3 of E. coli [25]. In this communication, we show tlat the intracellular availability of nickel is essential for I-i YD2 translocation to the periplasmic side of the membrane.…”

Novel regulation targets of the metal-response BasS–BasR two-component system of Escherichia coli

“…Here we found that some genes encoding enzymes for energy metabolism under acid-and/or anaerobic conditions were under the direct control of BasSR (see Tables 1 and 2): (1) both aceF and aceE are members of the pyruvate dehydrogenase operon, which is involved in acetate metabolism (Quail et al, 1994); (2) the hyaA and hyaB genes encode the small and large subunits of hydrogenase 1, respectively (Menon et al, 1991); and (3) the cydAB operon encodes cytochrome bd-I terminal oxidase (Green et al, 1984), which is expressed under oxygen-limited conditions and needed for growth under anaerobic conditions (Cotter et al, 1990). The expression levels of these genes were increased under acidic and/or anaerobic growth conditions.…”