PRESENCE OF A VANADIUM NITROGENASE IN AZOTOBACTER-PASPALI

There have been no previous studies on the genetics of Azotobacter pas pali, an aerobic bacterium which forms a highly specific diazotrophic association with Bahia grass (Paspalum notatum). We constructed A. pas pali strains defective in the molybdenum nitrogenase so that alternati ve N2ases could be studied. The cosmid vector pTBE and genomic DNA fra gments (approximately 50 kb) of A. paspali ATCC 23367 were used to con struct a gene library in Escherichia coli. Recombinant cosmids contain ing sequences homologous to molybdenum nitrogenase nifDK structural ge nes were identified by hybridization. A 2.9-kb fragment bearing the pu tative nifDK genes of A. paspali was subcloned and mutagenized in vitr o by the insertion of a kanamycin resistance gene cassette. The mutati on was recombined into the chromosome of A. paspali with the suicide v ector pCU101. One resultant mutant strain, AP2, was incapable of diazo trophic growth in a molybdenum-containing medium (Nif-) without vanadi um but grew well in a molybdenum-deficient medium with vanadium. The n itrogenase system in AP2 reduced acetylene to ethylene and produced et hane as 2.4% of the total products. Molybdenum levels as low as 10 nM prevented the diazotrophic growth of AP2, even in the presence of vana dium at levels up to 10 muM. These results are consistent with the exi stence of a vanadium nitrogenase system in A. paspali.