Molecular construction and characterization of nif mutants of the obligate methanotroph Methylosinus sp. strain 6.

We describe here a method for constructing mutants in bacteria that are not amenable to mutant isolation by conventional means. A one-step marker exchange procedure was used to construct nitrogen fixation (nif) mutants of the obligate methane-utilizing bacterium Methylosinus sp. strain 6, using transposon 5 (Tn5)-containing nif genes cloned into pBR325. The resultant mutants appeared to contain defects in nif structural genes, and DNA hybridization analysis showed that although one out of five had apparently been produced as a result of double-crossover homologous recombination, a variety of molecular events had led to the production of the other four mutants.