Isolation and Characterization of Frankia sp. Strain FaC1 Genes Involved in Nitrogen Fixation

Abstract

Genomic DNA was isolated from Frankia sp. strain FaC1, an Alnus root nodule endophyte, and used to construct a genomic library in the cosmid vector pHC79. The genomic library was screened by in situ colony hybridization to identify clones of Frankia nitrogenase ( nif ) genes based on DNA sequence homology to structural nitrogenase genes from Klebsiella pneumoniae. Several Frankia nif clones were isolated, and hybridization with individual structural nitrogenase gene fragments ( nifH, nifD , and nifK ) from K. pneumoniae revealed that they all contain the nifD and nifK genes, but lack the nifH gene. Restriction endonuclease mapping of the nifD and nifK hybridizing region from one clone revealed that the nifD and nifK genes in Frankia sp. are contiguous, while the nifH gene is absent from a large region of DNA on either side of the nifDK gene cluster. Additional hybridizations with gene fragments derived from K. pneumoniae as probes and containing other genes involved in nitrogen fixation demonstrated that the Frankia nifE and nifN genes, which play a role in the biosynthesis of the iron-molybdenum cofactor, are located adjacent to the nifDK gene cluster.