Cloning and Expression in E. coli of the Whole nif Genes of Klebsiella oxytoca, a Nitrogen Fixer in the Rhizosphere of Rice

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We cloned in E. coli the whole 17 nif genes (nifQ-J) of Klebsiella oxytoca NG13 using pBR322 as a vector, and constructed a recombinant plasmid, pNOW25 (nif⁺, Ap^r, 42.6kb). A non nif DNA fragment was deleted from the plasmid with Xhol, and a smaller plasmid, pNOK31 (nif⁺, Ap^r, 31.1 kb), was reconstructed.
We constructed the restriction map of the cloned nif genes. The map was the same as that of the K. pneumoniae M5al nif genes as to the EcoRI, HindIII, BamHI and Xhol sites, but differed considerably in the Pstl, SalI and BglII sites.
E. coli KO60 containing pNOW25 or pNOK31 can grow on a N-free medium. The acetylene reduction activities of KO60 (pNOW25) and KO60 (pNOK31) were 280nmol and 390nmol/48hr per 7ml of N-free liquid medium, whereas the activity of K. oxytoca NG13 was 3800 nmol. Thus, the expressed activity of the nif system of K. oxytoca is rather low in E. coli even if the nif genes are cloned on a multicopy plasmid.