Open AccessCloning and expression of Bradyrhizobium japonicum uptake hydrogenase structural genes in Escherichia coli.
Author(s) -
Mohammed Zuber,
Alan R. Harker,
Mazhar sultana,
Harold J. Evans
Publication year - 1986
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.83.20.7668
Subject(s) - cosmid , biology , operon , escherichia coli , hydrogenase , bradyrhizobium japonicum , plasmid , microbiology and biotechnology , structural gene , gene , cloning (programming) , promoter , molecular cloning , gene expression , biochemistry , genetics , bacteria , rhizobiaceae , symbiosis , computer science , programming language
To identify the structural genes for the components of Bradyrhizobium japonicum uptake hydrogenase (Mr 60,000 and 30,000), we have expressed these genes in Escherichia coli and shown that the products cross-react with antibodies to the respective hydrogenase subunits. We constructed subclones of overlapping DNA fragments from an uptake hydrogenase-complementing cosmid, pHU52 [Lambert, G. R., Cantrell, M. A., Hanus, F. J., Russell, S. A., Haddad, K. R. & Evans, H. J. (1985) Proc. Natl. Acad. Sci. USA 82, 3232-3236], in pMZ 545, a plasmid expression vector. DNA fragments inserted into one or more of the four cloning sites downstream from the E. coli lac operon promoter (Plac) on pMZ 545 generate transcriptional, but not translational, fusions. Two subclones that directed the synthesis of Mr 60,000 and 30,000 proteins in E. coli "maxicells" were identified. The DNA inserts from these subclones were then inserted down-stream of the bacteriophage lambda PL promoter on a transcriptional fusion vector. When the PL promoter was activated in vivo by heat inactivation of the temperature sensitive cI repressor of lambda in an appropriate E. coli strain, the respective fragments expressed higher levels of Mr 60,000 and 30,000 proteins that could be detected in immunoblots. These data provide direct evidence for the presence of uptake hydrogenase structural genes on the uptake hydrogenase-complementing cosmid pHU52.