Physical map of chromosomal nitrogen fixation ( nif ) genes of Klebsiella pneumoniae

Abstract

A method for the rapid determination of the physical location of mutations caused by insertion of transposable elements is described. This method was used to construct a detailed physical map of the N2 fixation (nif) gene cluster of K. pneumoniae and to correlate it with the genetic map. Total cellular DNA was isolated from individual strains, each carrying an insertion in 1 of 15 different nif genes. The DNA was digested with a restriction endonuclease, fractionated by agarose gel electrophoresis, denatured and blotted onto nitrocellulose filter paper. The DNA on the filters was hybridized with 32P-labeled DNA fragments derived from amplifiable plasmids carrying cloned nif DNA fragments from K. pneumoniae. Altered hybridization patterns caused by insertions into nif genes allowed the mapping of nif mutations with respect to the previously mapped cleavage sites for various restriction endonucleases. The same method was used to map the end points of nif deletions. Using this procedure, physical locations on the K. pneumoniae chromosome to 86 nif insertion mutations and 13 nif deletion end points were assigned. This mapping procedure provides a convenient alternative to deletion mapping as a definitive method for mapping insertion mutations within a gene or for ordering genes within a gene cluster. This procedure will be especially useful for mapping mutations conferring phenotypes that are difficult to monitor and for mapping mutations in bacterial species in which techniques for conducting deletion mapping were not devised.