The genes hoxF, -U, -Y, and -H which encode the four subunit polypeptides a, -y, 8, and 1 of the NADreducing hydrogenase (HoxS) ofAkaligenes eutrophus H16, were cloned, expressed in PseudomonasfacUis, and sequenced. On the basis of the nucleotide sequence, the predicted amino acid sequences, and the N-terminal amino acid sequences, it was concluded that the structural genes are tightly linked and presumably organized as an operon, denoted hoxS. Two pairs of -24 and -12 consensus sequences resembling RpoN-activatable promoters lie upstream of hoxF, the first of the four genes. Primer extension experiments indicate that the second promoter is responsible for hoxS transcription. hoxF and hoxU code for the flavin-containing dimer (a and y subunits) of HoxS which exhibits NADH:oxidoreductase activity. A putative flavin-binding region is discussed. The 26.0-kilodalton (kDa) y subunit contains two cysteine clusters which may participate in the coordination of two [4Fe-4S] centers. The genes hoxY and hoxH code for the small 22.9-kDa 8 subunit and the nickel-containing 54.8-kDa ,B subunit, respectively, of the hydrogenase dimer of HoxS. The latter dimer exhibits several conserved regions found in all nickel-containing hydrogenases. The roles of these regions in coordinating iron and nickel are discussed. Although the deduced amino acid sequences of the 8 and ,1 subunits share some conserved regions with the corresponding polypeptides of other [NiFe] hydrogenases, the overall amino acid homology is marginal. Nevertheless, significant sequence homology (35%) to the corresponding polypeptides of the soluble methylviologen-reducing hydrogenase of Methanobacterium thermoautotrophicum was found. Unlike the small subunits of the membrane-bound and soluble periplasmic hydrogenases, the HoxS protein does not appear to be synthesized with an N-terminal leader peptide.Alcaligenes eutrophus H16 is a gram-negative facultatively lithoautotrophic bacterium which can assimilate CO2 and utilize H2 as an energy source (reviewed in reference 5). Hydrogen oxidation is catalyzed by hydrogenases. These enzymes are found in phylogenetically diverse microorganisms, where they are responsible for both consumption and production of H2 (reviewed in reference 13). Two distinct hydrogenases have been purified and characterized from extracts of A. eutrophus (39, 44). These two enzymes differ in cellular localization, cofactor content, polypeptide composition, and apparent molecular weight. [NiFeSe]. An enzyme of this type has been described for Desulfovibrio and Methanococcus strains (reviewed in reference 13).The genes coding for the two hydrogenases of A. eutrophus H16 lie in a cluster of genes on a 450-kilobase-pair (kb) conjugative megaplasmid (15,24). Molecular cloning of megaplasmid DNA in Escherichia coli and screening of the resultant hybrid plasmids for complementation of hydrogenase-deficient mutants led to the identification of hydrogenase (hox) genes. Subsequent studies revealed that the two structural gene loci hoxS and hoxP coding for...

Heterologous complementation studies using Alcaligenes eutrophus H16 as a recipient identified a hydrogenase-specific regulatory DNA region on megaplasmid pHG21-a of the related species Alcaligenes hydrogenophilus. Nucleotide sequence analysis revealed four open reading frames on the subcloned DNA, designated hoxA, hoxB, hoxC, and hoxJ. The product of hoxA is homologous to a transcriptional activator of the family of twocomponent regulatory systems present in a number of H 2 -oxidizing bacteria. hoxB and hoxC predict polypeptides of 34.5 and 52.5 kDa, respectively, which resemble the small and the large subunits of [NiFe] hydrogenases and correlate with putative regulatory proteins of Bradyrhizobium japonicum (HupU and HupV) and Rhodobacter capsulatus (HupU). hoxJ encodes a protein with typical consensus motifs of histidine protein kinases. Introduction of the complete set of genes on a broad-host-range plasmid into A. eutrophus H16 caused severe repression of soluble and membrane-bound hydrogenase (SH and MBH, respectively) synthesis in the absence of H 2 . This repression was released by truncation of hoxJ. H 2 -dependent hydrogenase gene transcription is a typical feature of A. hydrogenophilus and differs from the energy and carbon source-responding, H 2 -independent mode of control characteristic of A. eutrophus H16. Disruption of the A. hydrogenophilus hoxJ gene by an in-frame deletion on megaplasmid pHG21-a led to conversion of the regulatory phenotype: SH and MBH of the mutant were expressed in the absence of H 2 in response to the availability of the carbon and energy source. RNA dot blot analysis showed that HoxJ functions on the transcriptional level. These results suggest that the putative histidine protein kinase HoxJ is involved in sensing molecular hydrogen, possibly in conjunction with the hydrogenase-like polypeptides HoxB and HoxC.Alcaligenes hydrogenophilus is an aerobic, facultatively lithoautotrophic proteobacterium capable of obtaining energy from the oxidation of molecular hydrogen (5). Hydrogen oxidation in this strain is catalyzed by two hydrogenases (18). The soluble hydrogenase (SH) is closely related to the NAD-reducing hydrogenases present in Alcaligenes eutrophus, in the gram-positive Rhodococcus sp. strain 1b (5), and in the cyanobacterium Anabaena variabilis (42). The second hydrogenase of A. hydrogenophilus is a membrane-bound hydrogenase (MBH) coupled to the respiratory chain and involved in electron transportdependent phosphorylation (18). The large and the small subunits of this enzyme show 75 and 90% identity, respectively, to the corresponding polypeptides of the A. eutrophus MBH (28,53). Moreover, in both Alcaligenes species H 2 -oxidizing ability is genetically linked to a megaplasmid (18) and dependent on the function of an RpoN-like sigma factor of RNA polymerase which is encoded on the chromosome (32, 38).Despite the high degree of similarity, hydrogenase regulation in A. hydrogenophilus appears to be quite different from the mode of control observed in A. eutrophus. Whi...

No abstract

Expression of the soluble (SH) and membrane-bound (MBH) hydrogenases in the facultatively lithoautotrophic bacterium Alcaligenes eutrophus is dependent on the transcriptional activator HoxA and the alternative sigma factor 54 . Deletion analysis revealed that a region 170 bp upstream of the transcriptional start of the SH operon is necessary for high-level promoter activity. Mobility shift assays with DNA fragments containing the SH upstream region and purified ␤-galactosidase-HoxA fusion protein isolated from Escherichia coli or authentic HoxA isolated by immunoaffinity chromatography from A. eutrophus failed to detect specific binding. In contrast, A. eutrophus extracts enriched for HoxA by heparin-Sepharose chromatography and ammonium sulfate fractionation produced a weak but discrete shift in the mobility of the target DNA. This effect was not observed with comparable extracts prepared from hoxA mutants. A similar experiment using antibodies against HoxA confirmed that HoxA was responsible for the observed mobility shift. Extracts prepared from a temperature-tolerant mutant of A. eutrophus gave a stronger retardation than did those from the wild type. Unlike the wild type, the hox(Tr) mutant is able to grow with hydrogen at temperatures above 33؇C because of a mutation in the regulatory gene hoxA. In this paper, we show that a single amino acid substitution (Gly-4683Val) in the C-terminal part of HoxA is responsible for temperature tolerance. The SH upstream region also contains sequence motifs resembling the E. coli integration host factor (IHF) binding site, and purified E. coli IHF protein shifted the corresponding indicator fragment.Alcaligenes eutrophus, a member of the ␤ subgroup of the class Proteobacteria, can grow lithoautotrophically with hydrogen as the sole energy source and carbon dioxide as the carbon source (reviewed in reference 3). The bacterium harbors a large plasmid on which the genes required for hydrogen oxidation are located. The hox region encodes two hydrogenases: a cytoplasmic NAD-reducing enzyme (SH) and a membranebound electron transport-coupled protein (MBH). Both hydrogenases belong to the family of nickel-iron-containing proteins. The formation of these metalloproteins is an extremely complex and apparently highly conserved process requiring sets of ancillary functions in addition to the hydrogenase enzymes (reviewed in reference 17). The genes coding for these proteins are part of the hox gene complex in A. eutrophus. Their products are involved in nickel uptake (14), metal processing (9), and hydrogenase maturation (24).Physiological experiments have demonstrated that the synthesis of the two hydrogenases of A. eutrophus is coordinately regulated. Tight repression is observed during growth of the cells on preferentially utilized carbon sources, whereas derepression occurs on poor substrates even in the absence of hydrogen (18). Moreover, hydrogenase synthesis in A. eutrophus is temperature sensitive and abolished above 33ЊC (19). Mutant analysis has shown that the expression ...