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

Abstract

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.

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

“…ORF4 (hybD). hybD could encode a 162-amino-acid polypeptide (17.5 kDa) having 40% identity to E. coli HYAD and less but significant homology to the predicted polypeptide products of A. vinelandii (28) and Azotobacter eutrophus (22) hoxM and R capsulatus (11) and Rhizobium leguminosarum (34) hupD. The hyaD gene product seems to be involved in the processing of the HYD1 large subunit (25), but the mechanism by which this is accomplished is not known.…”

“…eutrophus (13,22), A. vinelandii (10,17,28,29), A. chroococcum (15,42), R. leguminosarum (34,35), and R. capsulatus (9,11) all contain single membrane-bound [NiFe] hydrogenases which have been shown to be encoded as parts of large operons which may contain 15 or more contiguous ORFs. E. coli, on the other hand, has three membrane-bound hydrogenases encoded in separate operons: hya (HYD1), hyb (HYD2), and hyc (HYD3).…”

Carboxyl‐terminal processing may be essential for production of active NiFe hydrogenase in Azotobacter vinelandii

“…Complementation of a l~ya operon deletion mutant with a plasmid containing hyaA.E resuited in 30% wild.type HYDI activity, Addition of high concentrations of nickel to the culture media re. stored wild-type levels of activity, i,e, the hyaF gene product can be phcnotypigally replaced with high concentrations of Ni, Furthermore, there is a correlation between Ni incorporation, membrane localization, electrophoretic mobility, and activity [35], Since A. t,inel.ndii hoxQ is homologous to hyaF [4] A, vinelandii is a complex process requiring at least fifteen different gene products. These gene products are potentially involved in metal uptake and insertion, formation of metal centers, and localization and processins of this enzyme, as well as the coupling of Ha oxidation to O.. reduction.…”

“…The deduced molecular weight of thi.~ subunit is 65,60.5 De, Mutations in some of the accessory genes r¢sult in toss of whole.cell. O_,-de~ndent hydrogenase activity and production of a form of the ,~ subunit with altered clectrophoretic mobility [4]. Two classes of mutations were described: mutations in hoxZ.O and Q result in two forms of the a subunit, one with ¢[ectrophoretic mobility equivalent to the wild-tyl:m ¢z subunit, and a second with a mobility slightly lower than wild-tyi~ ~t subunit, white mutations in hoxM and L. result in a single form with lower than wild-tylz: mobility.…”

“…The structural genes, hoxK and hoxG encoding th= ,B and ~ subunits of hydrogcnas¢, as w¢11 as thirteen contiguous accessory genes involved in O:-dcl~nd,nt hydrogtnas, activity, have been cloned and sequenced [3][4][5][6] (Fig. I).…”