Use of hupS::lacZ gene fusion to study regulation of hydrogenase expression in Rhodobacter capsulatus: stimulation by H2.
The Escherichia coli beta-galactosidase enzyme was used as a reporter molecule for genetic fusions in Rhodobacter capsulatus. 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 22 amino acids of the HupS protein joined to the eight amino acid of beta-galactosidase. We demonstrate the usefulness of the hupS::lacZ fusion in monitoring regulation of hydrogenase gene expression. The activities of plasmid-determined beta-galactosidase and chromosome-encoded hydrogenase changed in parallel in response to various growth conditions (light or dark, aerobiosis or anaerobiosis, and presence or absence of ammonia or of H2), showing that changes in hydrogenase activity were due to changes in enzyme synthesis. Molecular hydrogen stimulated hydrogenase synthesis in dark, aerobic cultures and in illuminated, anaerobic cultures. Analysis of hupS::lacZ expression in various mutants indicated that neither the hydrogenase structural genes nor NifR4 (sigma 54) was essential for hydrogen regulation of hydrogenase synthesis.
Colbeau
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Laboratoire de Biochimie Microbienne (CNRS Unité 1130 alliée à l'INSERM), Département de Biologie Moléculaire et Structurale, Centre d'Etudes Nucléaires 85 X, Grenoble, France.
Vignais
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D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005798:Genes, Bacterial; D005838:Genotype; D006859:Hydrogen / Q000494:pharmacology*; D006864:Hydrogenase / Q000096:biosynthesis / Q000235:genetics*; D007700:Kinetics; D009876:Operon; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016354:Rhodobacter capsulatus / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D006859:Hydrogen; D006864:Hydrogenase; D001616:beta-Galactosidase