These enzymes are used either to split hydrogen gas for use as energy, or to create hydrogen gas as a product of their reaction. Full text get a printable copy pdf file of the complete article 27k, or click on a page image below to browse page by page. A locus involved in the expression of the uptake hydrogenase system of bradyrhizobium japonicum was identified adjacent to genes encoding the hydrogenase subunits. Pdf on jan 1, 1989, michele leclerc and others published cloning and sequencing of the genes encoding the large and the small subunits of the h 2 uptake hydrogenase hup of rhodobacter.
Effects of the deletion of hup genes encoding the uptake hydrogenase on the activity of hydrogen production in the purple. Dna with homology to the hup structural genes of bradyrhizobium japonicum was present in 100 of 248 strains examined. The symbiotic requirement for hydrogenase expression in r. Cloning and sequencing of the genes encoding the large and the small subunits of the h. Heterogeneous composition of key metabolic gene clusters in a. Here we show that cosmid pal618 contains all of the genetic information required to confer high levels of hydrogenase activity on the naturally hup strains r. In this paper, we characterize the hydrogenase related protein hupe as a nickel transporter required for hydrogenase activity in microaerobic freeliving cells and bacteroids of r. Nucleotide sequence analysis of bradyrhizobium japonicum chromosomal dna, 9 kb downstream of the hup genes hydrogenase structural genes, revealed one incomplete and three complete open reading frames. A cloned fragment of dna was used to complement to autotrophy a hup.
Insights into the redox sensitivity of chloroflexi huphydrogenase. Membrane linked hydrogenase activity was absent in the mutant mc. Pdf identification and sequence analysis of hupr1 gene. Hydrogenase hup systems in bradyrhizobium japonicum and rhizobium leguminosarum bv. Get a printable copy pdf file of the complete article 980k, or click on a. Characterization of bradyrhizobium japonicum chromosomal. Download fulltext pdf identification and sequence analysis of hupr1 gene which encodes a response regulator of the ntrc family required for hydrogenase expression in rhodobacter capsulatus. The escherichia coli betagalactosidase enzyme was used as a reporter molecule for genetic fusions in rhodobacter capsulatus. Ijms free fulltext huptype hydrogenases of purple bacteria. Isolation of genes nifhup cosmids involved in hydrogenase and nitrogenase activities in rhizobium japonicum. The hup genes play an important role in plant productivity by recycling the consider able quantities of reducing equivaents in the form of h2 wasted during symbiotic nitrogen fixation of leguminous crops such as soybean. Hydrogen uptake is coupled to the reduction of electron acceptors such as oxygen, nitrate, sulfate, carbon dioxide co2, and fumarate.
Complementation of azotobacter chroococcum hydrogenase. Extrapolation suggests that 1 mole of hydrogenase could produce enough hydrogen to fill the airship graf zeppelin in ten minutes, or the main liquidhydrogen tank of the space shuttle in two hours this fanciful calculation assumes a sufficient supply of reductant. Transconjugants containing the nif hup cosmid were identified by their resistance to tetracycline tcr and ability to grow. The hydrogenase gene expression is also regulated by the level of o 2 present in the cell environment. The hydrogenase structural operon in rhodobacter capsulatus. Sep 29, 2015 in the bsepe genome, we found a membranebound uptake hydrogenase hup vignais and billoud, 2007 gene cluster consisting of the key structural genes hupl and hups and other accessory genes. Thus, these hydrogenases could draw interest for exploring their oxygen. The structural genes hup of the h2 uptake hydrogenase of rhodobacter capsulatus were isolated from a cosmid gene library of r. Pdf molecular characterization of structural genes. Effects of the deletion of hup genes encoding the uptake. The expression of hydrogenase genes was investigated in d. Identification of a locus upstream from the hydrogenase. Isolation of genes nifhup cosmids involved in hydrogenase. Every phu53 transconjugant was less active than the.
Mutants of azotobacter chroococcum defective in h 2uptake hup hydrogenase activity were complemented by plasmids containing hup genes from bradyrhizobium japonicum and a. Differential roles of hypc and hupf proteins for hydrogenase. Nucleotide sequence analysis of four genes, hupc, hupd, hupf. Hupsgfp fusion protein demonstrates a heterocystspecific. Dna fragments that were from the upstream region of the hydrogenase structural operon hupslm and contained 5 hups sequences were fused in frame to a promoterless lacz gene, yielding fusion proteins comprising the putative signal sequence and the first. The hupsl hydrogenase, encoded in the hupslcdhir operon, probably pla. Attempts are being made to introduce the energysaving hup genes into root nodule bacteria. These were designated as hypd, hype, hoxx and hox a, respectively, based on the strong homology of the deduced amino acid sequences with those encoded by genes. Dual role of hupf in the biosynthesis of nife hydrogenase in. Pdf recombinant cosmids containing a rhizobium japonicum gene involved in both hydrogenase hup and nitrogenase nif activities were. In this study, we determined the effect of adding nickel to the medium used for the growth ofhup mutants. Cloning and sequencing of the genes encoding the large and the small subunits of the h2 uptake hydrogenase hup of rhodobacter capsulatus.
The thin arrows indicate promoters that control symbiotic expression of hydrogenase structural genes p 1 19 or microaerobic. Strains of rhizobium forming nitrogenfixing symbioses with common bean were systematically examined for the presence of the uptake hydrogenase hup structural genes and expression of uptake hydrogenase hup activity. Cloning and expression of bradyrhizobium japonicum uptake hydrogenase structural genes in escherichia coli. Complementation of the hupv and hupuv mutants brought about a decrease in hydrogenase activity up to 10fold, to the level of the wildtype strain b10, indicating that hupu and hupv participate in negative regulation of hydrogenase expression in concert with hupt, a sensor histidine kinase involved in the repression process. The huptuv operon is involved in negative control of. These results indicate that the nifhup gene cosmids contain a gene involved in both nitrogenase and hydrogenase activities and at least one and perhaps other hup genes. The low number of ionic pairs shown in the present study for the catalytic. These results indicate that the nif hup gene cosmids contain a gene involved in both nitrogenase and hydrogenase activities and at least one and perhaps other hup genes which are exclusively. Among the photosynthetic bacteria, the organization of the hup and hyp genes has been the most studied in rhodobacter capsulatus, and the nucleotide sequence of the entire huphyp gene cluster has been determined. Dna with homology to the hup structural genes of bradyrhizobium japonicum was. It is also evident from this work that some genes located in pal618 insert dna are inducible in freeliving cells and may be involved in regulation of hup genes expression. These results indicate that the nifhup gene cosmids contain a gene involved in both nitrogenase and hydrogenase activities and at least one and perhaps other hup genes which are exclusively involved in h2 uptake activity. A manual for the practical study of rootnodule bacteria.
Genome characterization revealed the presence of two hydrogenase genes. Pdf characterization of a new internal promoter p3 for. Hydrogen independent expression of hupsl genes in thiocapsa. Characterization of a new internal promoter p3 for rhizobium leguminosarum hydrogenase accessory genes hupghij. Identification and sequence analysis of hupr1 gene which encodes a response regulator of the ntrc family required for hydrogenase expression in rhodobacter capsulatus. The hupb gene of the azotobacter chroococcum hydrogenase gene. The nucleotide sequence of a 22 kb region downstream of the hydrogenase structural genes in bradyrhizobium japonicum was determined. Methylacidiphilum fumariolicum solv, a thermoacidophilic. The small and large subunits of the hydrogenase are encoded by the structural genes hups and hupl. On the other hand, proton reduction is coupled to the oxidation of electron donors such as ferredoxin fnr, and serves to dispose excess electrons in cells essential in pyruvate fermentation. In a genetic background lacking the three main hydrogenases of e.
Four genes encoding predicted polypeptides of 278 hupc, 214 hupd, 106 hupf and 158 hupg kda were identified, of which the first three probably belong to the same operon as the hup structural genes, hups and hupl. At the top is a nickeliron hydrogenase pdb entry 2frv, in the center is an ironiron hydrogenase pdb entry 1feh, and at the bottom is an iron hydrogenase pdb entry 3dag and 3f47. Adjacent to the structural genes, three hy nal regions of the nife hydrogenases 18. In warma a ed, mycorrhiza manual, springerverlag, berlin. Biosynthesis of rhizobium leguminosarum feni hydrogenase requires a number of accessory proteins products of hup and hyp genes that mediate the incorporation of ni and fe into the active site. To study hup gene organization, sitedirected transposon. In bacteria, the genes for hydrogenase hup and hyp are generally clustered, and the sequence ofthese clustered genes has been determined in several species. Symbiotic expression of cosmidborne bradyrhizobium japonicum hydrogenase genes. Characterization of a new internal promoter p3 for. The first molecular biology study on the purple sulfur photosynthetic bacterium thiocapsa roseopersicina is reported, namely, the construction of cosmid libraries and isolation of a hydrogenase gene cluster by hybridization with hydrogenase structural genes from the purple nonsulfur bacterium, rhodobacter capsulatus. Among them, hypcparalog hupf and hupk are present in bacteria that synthesize hydrogenase in the presence of oxygen. Our data from immunological assays and our analysis of hup gene expression highlighted two main factors that affect the efficient expression of the hydrogenase system in heterologous rhizobial hosts.
Attempts are being made to introduce the energysaving hup genes. The structure of the actinobacterial hydrogenase is obviously severely different from the hup type hydrogenases which bind to a membraneintegral cytochrome b. A screening for h ydrogen up take hup genes in rhizobium leguminosarum bv. Pdf molecular characterization of structural genes coding. The genes coding for the small and large subunits,hupsl, are located at the 5. Nucleotide sequence analysis of four genes, hupc, hupd. The highly oxygensensitive hydrogen uptake hup hydrogenase from. Research article open access dual role of hupf in the. To obtain hydrogenase activity in freeliving cells of r. In this study we analyzed the expression of cosmidborne hup genes from r. It was not clear, however, whether the clone isolated contained all the genes necessary for uptake hydrogenase activity. The mutant strain lacked hydrogenase activity and synthesized low levels of the large subunit of hydrogenase. Cloning and sequences of the structural hupslc and. Effects of the deletion of hup genes encoding the uptake hydrogenase on the activity of hydrogen.
Read cloning and sequencing of the membranebound hydrogenase encoding genes hups and hupl from pseudomonas hydrogenovora, gene on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. The hupsl genes are part of a cluster of hup for hydrogen uptake and hyp for hydrogenase pleiotropic genes necessary for the biosynthesis of the hupslencoded hydrogenase 7. These results indicate that the nif hup gene cosmids contain a gene involved in both nitrogenase and hydrogenase activities and at least one and perhaps other hup genes which are exclusively involved in h2 uptake activity. Regulation of hydrogenase gene expression springerlink. Dec 01, 2005 a screening for h ydrogen up take hup genes in rhizobium leguminosarum bv. Amutationin arhodobactercapsulatus geneencodingan integration. Overproduction of the flv3b flavodiiron, enhances the. Construction of a hup gene system expressing hydrogenase activity in microaerobic freeliving cells of r. The uptake hydrogenase genebased phylogenetic tree of more than bacterial species showing both thermophilic and mesophilic strains contain a hydrogenase encoded by the genes hups small. It carried the aut4 mutation, which could be comple. The genetic determinants responsible for h 2uptake hup genes in rhizobium leguminosarum are organized in six transcriptional units, designated regions hupi to hupvi, with region hupi coding for the hydrogenase structural genes leyva et al. The sequenced gene cluster contains six open reading frames, the products of. The catalytic site on the enzyme provides simple hydrogenmetabolizing microorganisms a redox mechanism by which to store and utilize energy via the reaction shown in figure 1. This plasmid and its deletion derivatives, along with the hupdeleted r.
The putative products of the hydrogenase gene cluster of mc. Effects of the deletion of hup genes encoding the uptake hydrogenase on the activity of hydrogen production in the purple photosynthetic. These results demonstrate that we have created lac fusions within hup genes cloned in pal618, and that these genes are expressed in a leftward to rightward orientation. Molecular characterization of structural genes coding for a.
We conclude that the overproduced flv3b protein might have enhanced o2consumption, thus creating conditions inducing hox genes and facilitating h2 production. Unusual organization of the genes coding for hydsl, the. Differential expression of hydrogen uptake hup genes in. Transconjugants containing the nifhup cosmid were identified by their resistance to tetracycline tcr and ability to grow. This plasmid and its deletion derivatives, along with the hup deleted r. The hupsl genes are part of a cluster of hup for hydrogen uptake and hyp for hydrogenase pleiotropic genes necessary for the biosynthesis of the hupslencoded hydrogenase. We have also identified hupe2, a hupehomolog that displays a similar role in this strain. Nickel availability and hupsl activation by heterologous. A gene complex coding for the membranebound hydrogenase of alcaligenes eutrophus h16. The hydrogenase structural operon in rhodobacter capsulatus contains a third gene, hupm, necessary for the formation of a physiologically competent hydrogenase b. Rhizobium leguminosarum hupe encodes a nickel transporter.
Pmc free article leclerc m, colbeau a, cauvin b, vignais pm. Use of hupslacz gene fusion to study regulation of. Cloning and sequencing of the membranebound hydrogenase. Uptake hydrogenase hup in common bean phaseolus vulgaris. Diversity and evolution of hydrogenase systems in rhizobia. Full paper effects of the deletion of hup genes encoding. The potential physiological roles of the hydrogenase and hupl genes fig. This corrects the article isolation of genes nifhup cosmids involved in hydrogenase and nitrogenase activities in rhizobium japonicum. Adjacent to the structural genes, three hydrogenase accessory genes were found downstream from hupl. Pdf cloning and sequencing of the genes encoding the. Pdf isolation of genes nifhup cosmids involved in hydrogenase. Mar 10, 2020 interestingly, the transcription of the hox genes, encoding the nife hox hydrogenase, was significantly elevated, as shown by the quantitative rtpcr analyses. Molecular characterization of structural genes coding for.
Genetic organization of the hydrogen uptake hup cluster. The genetic determinants responsible for h2uptake hup genes in. The shaded arrows indicate genes whose encoded products were detected by an immunoblot analysis in this study. The mutant strain lacked hydrogenase activity and synthesized low levels of the large subunit of hydrogenase as. The first factor that controls hydrogenase activity is efficient transcription of hydrogenase structural genes in heterologous rhizobia. Transconjugants containing the nifhup cosmid were identified by their resistance to tetracycline tcr. Hydrogenase genes are uncommon and highly conserved in. A screening for hydrogen uptake hup genes in rhizobium leguminosarum bv. The hup and hyp gene products bear significant structural identity to hydrogenase gene products from escherichia coli, ralstonia eutropha formerly alcaligenes eutrophus. Although the data obtained by kakuno and colleagues do not evidence that it was exactly hup hydrogenase, later data by manness clarified that, among the three hydrogenase activities, two are inducible, and hup hydrogenase has the highest hydrogen uptake activity.
A gene cluster, encoding the hupslc hydrogenase structural genes and various hydrogenase accessory genes, had been identified in t. The hydrogenase specific transcriptional activator gene hoxa described in other species was shown to have been inactivated in r. Isolation of genes nifhup cosmids involved in hydrogenase and nitrogenase activities in rhizobium japonicum article pdf available in journal of bacteriology 16. Conserved plasmid hydrogenuptake hupspecific sequences. The hupb gene of the azotobacter chroococcum hydrogenase. Dual role of hupf in the biosynthesis of nife hydrogenase. The azotobacter chroococcum chromosome contains a region spanning about 14 kb associated with hydrogenuptake hup activity. Furthermore, there is a structure available by volbeda et al. The azotobacter chroococcum hydrogenase gene cluster. Nife hydrogenase is a type of hydrogenase, which is an oxidative enzyme that reversibly convert molecular hydrogen in prokaryotes including bacteria and archaea. The expression of many membrane bound nife hydrogenases is regulated by their substrate molecule, hydrogen. Inazotobacter chroococcum the hydrogenase gene hup cluster spans about 14 kb of dna.
Pdf genetic organization of the hydrogen uptake hup cluster. Download pdf 292k download meta ris compatible with endnote, reference manager, procite, refworks. Characterization of the hydrogendeuterium exchange. Recombinant cosmids containing a rhizobium japonicum gene involved in both hydrogenase hup and nitrogenase nif activities were isolated.
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