Pleiotropic hydrogenase mutants of Escherichia coli K12: growth in the presence of nickel can restore hydrogenase activity

Waugh, Robert; Boxer, David H.

doi:10.1016/s0300-9084(86)81080-x

Cited by 126 publications

(111 citation statements)

References 25 publications

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“…An implication of HypB in nickel metabolism for hydrogenase synthesis had first been proposed on the basis of the phenotype of E. coli HypB-mutants; these mutants can be phenotypically reverted by growth in medium containing high nickel ion concentrations (27,48). A similar result has recently been reported forA.…”

Section: (X)-t-v-(x)-g-(x)-g-t-s-a-i-g Correspond To the Sequence Exsupporting

confidence: 54%

“…Co2+ and Cu2' displaced ca. 25 6 (lanes 1 and 5), 16 (lanes 2 and 6), 42 (lanes 3 and 7), and 48 (lanes 4 from R. leguminosarum cultures (Fig. 5).…”

Section: (X)-t-v-(x)-g-(x)-g-t-s-a-i-g Correspond To the Sequence Exmentioning

confidence: 97%

“…The hypB gene is particularly interesting. Early experiments showed that mutations in the E. coli hydB locus (identical to hypB) are phenotypically reverted by high nickel concentrations in the medium (48). Specific AhypB mutants were later shown to behave similarly (17).…”

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Purification of Rhizobium leguminosarum HypB, a nickel-binding protein required for hydrogenase synthesis

et al. 1994

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The products of the Rhizobium leguminosarum hyp gene cluster are necessary for synthesis of a functional uptake [NiFe] hydrogenase system in symbiosis with pea plants, and at least for HypB and HypF, a role in hydrogenase-specific nickel metabolism has been postulated (L. Rey, J. Murillo, Y. Hernando, E. Hidalgo, E. Cabrera, J. Imperial, and T. Ruiz-Argiieso, Mol. Microbiol. 8:471-481, 1993). The R. leguminosarum hypB gene product has been overexpressed in Escherichia coli and purified by immobilized nickel chelate affinity chromatography in a single step. The purified recombinant HypB protein was able to bind 3.9 + 0.1 Ni2+ ions per HypB monomer in solution. Co2+, Cu2+, and Zn2+ ions competed with Ni2+ with increasing efficiency.Monospecific HypB antibodies were raised and used to show that HypB is synthesized in R. eguminosarum microaerobic vegetative cells and pea bacteroids but not in R. leguminosarum aerobic cells. HypB protein synthesized by R. kguminosarum microaerobic vegetative cells could also be isolated by immobilized nickel chelate affinity chromatography. A histidine-rich region at the amino terminus of the protein (23-HGHHHH DGHHDHDHDHDHHRGDHEHDDHHH-54) is proposed to play a role in nickel binding, both in solution and in chelated form.Rhizobium leguminosarum bv. viciae possesses an H2 uptake system that is able to oxidize H2 generated by the nitrogenase complex as a byproduct of the N2 reduction reaction (8,40). This system consists of an uptake [NiFe] hydrogenase and accessory proteins, and it is only expressed in the plant symbiotic state. The main features of the system have been studied by our laboratory in the Pisum-pea bacteroid symbiosis. The genetic determinants for the H2 uptake system are clustered in a 15-kb DNA region (hup region) in the symbiotic plasmid (21,22). This region has been sequenced, and 17 potential genes have been identified. The first six genes constitute the hydrogenase structural operon and include the genes hupS and hupL, encoding the hydrogenase polypeptides (13), and four additional genes, hupCDEF (14). A five-gene cluster containing hupGHIJK has been identified downstream the hydrogenase structural operon (38 Purification of the HypB protein by Ni(II)-NTA-agarose chromatography. The Ni(II)-nitrilotriacetic acid (NTA)-agarose matrix was obtained from Diagen (Dusseldorf, Germany), and the manufacturer's recommendations for its use (12) were followed, with minor modifications as follows.(i) Denaturing conditions. Frozen cells from a 100-ml induced culture were lysed in the presence of 6 M guanidineHCl (3.5 ml), and cell extracts were applied to an Ni(II)-NTAagarose column (2 by 0.8 cm). Proteins were stepwise eluted by means of buffers of decreasing pH, 8.0, 6.3, 5.9, and 4.5, all of which contained 8 M urea, at a flow rate of 0.5 ml min-1. Fractions (1 ml) were collected, and portions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining (18).(ii) Nondenaturing conditions. All the manipulations were carri...

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Section: (X)-t-v-(x)-g-(x)-g-t-s-a-i-g Correspond To the Sequence Exsupporting

confidence: 54%

“…Co2+ and Cu2' displaced ca. 25 6 (lanes 1 and 5), 16 (lanes 2 and 6), 42 (lanes 3 and 7), and 48 (lanes 4 from R. leguminosarum cultures (Fig. 5).…”

Section: (X)-t-v-(x)-g-(x)-g-t-s-a-i-g Correspond To the Sequence Exmentioning

confidence: 97%

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Purification of Rhizobium leguminosarum HypB, a nickel-binding protein required for hydrogenase synthesis

et al. 1994

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“…The clear identification of the hydrogenase 2 small subunit has allowed its study in a hypB mutant, which is defective in the ability to insert nickel into the large subunit during biosynthesis [31,34]). The small subunit, in its precursor form, is found exclusively in the soluble fraction in this mutant, as is the large subunit.…”

Section: Discussionmentioning

confidence: 99%

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

Sargent¹,

Ballantine²,

Rugman³

et al. 1998

European Journal of Biochemistry

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An active tryptic fragment of hydrogenase 2 from Escherichia coli has been isolated from the periplasmic face of the cytoplasmic membrane, and the large and small subunits N-terminally sequenced. The large subunit is encoded by the hybC gene and shows no N-terminal processing, other than removal of the initiator methionine during its biosynthesis. Both N-terminal and the subsequent internal trypticfragment amino acid sequence indicate that the small subunit is neither encoded by hybA, a gene previously identified as encoding the small subunit [Menon et al. (1994) J. Bacteriol. 176, 4416Ϫ4423], nor any of the remaining genes in the hyb operon. Genome sequence analysis revealed the presence of an open reading frame which could potentially encode the peptide sequences of the proteolysed small subunit. The gene, designated hyb0, lies directly upstream of, and is separated by two nucleotides from, the start of the hybA gene. Hyb0, which shares an approximate 40% identity with other hydrogenase small subunit amino acid sequences, is synthesised with an N-terminal signal sequence containing a twin-arginine motif which is probably required for export of the enzyme. In the mature enzyme the small subunit is proteolytically cleaved after Ala37. Immunological analysis of strains overproducing either recombinant Hyb0 or HybA using antibodies specific for hydrogenase 2, readily identified Hyb0 as the small subunit. In a pleiotropic hypB mutant, which is unable to insert nickel into the active site, both the large and small subunits accumulate as unprocessed, soluble forms, consistent with the two subunits being assembled and processed in a coordinated manner during biosynthesis.

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“…Each operon encodes four to five ORFs in addition to the structural genes. A fourth operon containing the hyp genes (31) encompasses the previously described hydE, hydF, and hydB genes (52)(53)(54)65) vinelandii, the functions of only hoxK and hoxG are known (40). Mutagenesis of these genes not only confirms their role but also establishes that, at least under the growth conditions tested, this organism has only one enzyme capable of oxidizing H2.…”

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confidence: 86%

Nucleotide sequences and genetic analysis of hydrogen oxidation (hox) genes in Azotobacter vinelandii

1992

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Azotobacter vinelandii contains a heterodimeric, membrane-bound [NiFe]hydrogenase capable of catalyzing the reversible oxidation of H2. The beta and alpha subunits of the enzyme are encoded by the structural genes hoxK and hoxG, respectively, which appear to form part of an operon that contains at least one further potential gene (open reading frame 3 [ORF3]). In this study, determination of the nucleotide sequence of a region of 2,344 bp downstream of ORF3 revealed four additional closely spaced or overlapping ORFs. These ORFs, ORF4 through ORF7, potentially encode polypeptides with predicted masses of 22.8, 11.4, 16.3, and 31 kDa, respectively. Mutagenesis of the chromosome of A. vinelandii in the area sequenced was carried out by introduction of antibiotic resistance gene cassettes. Disruption of hoxK and hoxG by a kanamycin resistance gene abolished whole-cell hydrogenase activity coupled to O2 and led to loss of the hydrogenase alpha subunit. Insertional mutagenesis of ORF3 through ORF7 with a promoterless lacZ-Kmr cassette established that the region is transcriptionally active and involved in H2 oxidation. We propose to call ORF3 through ORF7 hoxZ, hoxM, hoxL, hoxO, and hoxQ, respectively. The predicted hox gene products resemble those encoded by genes from hydrogenase-related operons in other bacteria, including Escherichia coli and Alcaligenes eutrophus.

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Pleiotropic hydrogenase mutants of Escherichia coli K12: growth in the presence of nickel can restore hydrogenase activity

Cited by 126 publications

References 25 publications

Purification of Rhizobium leguminosarum HypB, a nickel-binding protein required for hydrogenase synthesis

Purification of Rhizobium leguminosarum HypB, a nickel-binding protein required for hydrogenase synthesis

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

Nucleotide sequences and genetic analysis of hydrogen oxidation (hox) genes in Azotobacter vinelandii

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