Cloning of the entire region for nitrogen fixation from Klebsiella pneumoniae on a multicopy plasmid vehicle in Escherichia coli

Summary

Two deletion mutants pAP1 (MW 82 Mdals) and pAP2 (MW 64 Mdals) were isolated by P1 transduction of the plasmid pRD1 (MW 101 (Mdals). These plasmid mutants still carry the his-nif region of K. pneumoniae. They are selftransmissible and mediate resistance to ampicillin, kanamycin and tetracycline. Comparing the HindIII maps of pRD1, pAP1 and pAP2 showed that pAP1 was derived from pRD1 by an 8 μm deletion and pAP2 by two deletions — the same 8 μm deletion and a further 9 μm deletion. The plasmids pAP1 and pAP2 helped us to locate the his-nif region of pRD1 on 3 adjacent HindIII fragments (number 5, 4 and 3 according to gelelectrophoresis). The molecular weights of these fragments were 8.2, 10 and 15 Mdals. These 3 fragments were cloned separately on the multicopy plasmid vehicle pWL625 giving rise to the hybrid plasmids pWK1 (pWL625+HindIII fragment 4), pWK2 (pWL625+HindIII fragment 5). None of these hybrid plasmids conferred nitrogen fixation capacity on E. coli C cells. By combining HindIII fragment 4 and 3 in the same alignment as in pRD1 and cloning them together on pWL625 the hybrid plasmid pWK120 (pLW625+HindIII fragments 4 and 3) was found to carry the entire nif region. An E. coli C strain harbouring the plasmid pWK120 grew on nitrogen free medium and reduced acetylene. The plasmid pWK 120 had a contourlength of 17 μm, a buoyant density of 1.715 g/ml and a copy number up to 65.