The identification, characterization, sequencing and mutagenesis of the genes (hupSL) encoding the small and large subunits of the H2-uptake hydrogenase of Azotobacter chroococcum

C. M. Ford, Nisha Garg, R. P. Garg, K. H. Tibelius, M. G. Yates, D. J. Arp, L. C. Seefeldt

Research output: Contribution to journalArticle

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Abstract

The structural genes (hupSL) of the membrane-bound NiFe-containing H2-uptake hydrogenase (Hup) of Azotobacter chroococcum were identified by oligonucleotide screening and sequenced. The small subunit gene (hupS) encodes a signal sequence of 34 amino acids followed by a 310-amino-acid, 34156 D protein containing 12 cysteine residues. The large subunit gene (hupL) overlaps hupS by one base and codes for a predicted 601-amino-acid, 66433 D protein. There are two regions of strong homology with other Ni hydrogenases: a Cys-Thr-Cys-Cys-Ser motif near the N-terminus of HupS and an Asp-Pro-Cys-Leu-Ala-Cys motif near the carboxy-terminus of HupL. Strong overall homology exists between Azotobacter, Bradyrhizobium japonicum and Rhodobacter capsulatus Hup proteins but less exists between the Azotobacter proteins and hydrogenases from Desulfovibrio strains. Mutagenesis of either hupS or hupL genes of A. chroococcum yielded Hup phenotypes but some of these mutants retained a partial H2-evolving activity. Hybridization experiments at different stages of gene segregation confirmed the multicopy nature of the Azotobacter genome.

Original languageEnglish (US)
Pages (from-to)999-1008
Number of pages10
JournalMolecular Microbiology
Volume4
Issue number6
StatePublished - Jun 1990
Externally publishedYes

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Azotobacter
Mutagenesis
Hydrogenase
Genes
Amino Acids
Proteins
Desulfovibrio
Rhodobacter capsulatus
Bradyrhizobium
Protein Sorting Signals
Oligonucleotides
Cysteine
uptake hydrogenase
Genome
Phenotype
Membranes

ASJC Scopus subject areas

  • Molecular Biology
  • Microbiology

Cite this

The identification, characterization, sequencing and mutagenesis of the genes (hupSL) encoding the small and large subunits of the H2-uptake hydrogenase of Azotobacter chroococcum. / Ford, C. M.; Garg, Nisha; Garg, R. P.; Tibelius, K. H.; Yates, M. G.; Arp, D. J.; Seefeldt, L. C.

In: Molecular Microbiology, Vol. 4, No. 6, 06.1990, p. 999-1008.

Research output: Contribution to journalArticle

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abstract = "The structural genes (hupSL) of the membrane-bound NiFe-containing H2-uptake hydrogenase (Hup) of Azotobacter chroococcum were identified by oligonucleotide screening and sequenced. The small subunit gene (hupS) encodes a signal sequence of 34 amino acids followed by a 310-amino-acid, 34156 D protein containing 12 cysteine residues. The large subunit gene (hupL) overlaps hupS by one base and codes for a predicted 601-amino-acid, 66433 D protein. There are two regions of strong homology with other Ni hydrogenases: a Cys-Thr-Cys-Cys-Ser motif near the N-terminus of HupS and an Asp-Pro-Cys-Leu-Ala-Cys motif near the carboxy-terminus of HupL. Strong overall homology exists between Azotobacter, Bradyrhizobium japonicum and Rhodobacter capsulatus Hup proteins but less exists between the Azotobacter proteins and hydrogenases from Desulfovibrio strains. Mutagenesis of either hupS or hupL genes of A. chroococcum yielded Hup phenotypes but some of these mutants retained a partial H2-evolving activity. Hybridization experiments at different stages of gene segregation confirmed the multicopy nature of the Azotobacter genome.",
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AU - Garg, R. P.

AU - Tibelius, K. H.

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