Construction of a working restriction map of Frankia cosmid clone pFAH1 and mapping of putative nif genes

Cosmid clone pFAH1 is believed to contain Frankia nif genes and open reading frames. It was subjected to a variety of single and double restriction enzyme digestions, blotted, and hybridized to various Azotobacter nif genes and an unidentified open reading frame orf10. Results from these experiments were used to construct a working restriction map of cosmid clone pFAH1.

The data obtained indicate that orf10, nifM and X are adjacent to each other and nifB is less than 1kb from nifX on an 18kb EcoRI/HindIII fragment. NifE is located on an 8.6kb EcoRI/HindIII fragment and is separated from nifB by a distance of approximately 10kb.

These data indicate that Frankia nif gene arrangement in cosmid clone PFAH1 is unique when compared with other nitrogen-fixing organisms. Further mapping of Frankia nif genes will enable researchers to make phylogenetic comparisons between Frankia and other nitrogen-fixing bacteria.