Constitutive expression of nitrogenase system in Klebsiella oxytoca by gene targeting mutation to the chromosomal nifLA operon

Abstract

K. oxytoca is a nitrogen-fixing bacterium living in the rhizosphere of rice plants. A strain with derepressed nitrogen fixing ability was constructed by insertion of a tetracycline-resistant (Tc r) gene into the coding sequence of the repressor gene ( nifL) and another insertion of a synthetic promoter upstream from the activator gene ( nifA) on the chromosome of K. oxytoca. The strain, R16, showed constitutive transcriptional activity of not only nifA gene but also nifB (iron-molybdenum cofactor) gene, and nitrogenase activity was derepressed to a level of 42% of that detected in the ammonia-less condition even in the presence of ammonia. R16, carrying a multicopy plasmid containing the constitutive nifA, acquired complete derepression from the control by ammonia and showed a high nitrogenase activity constitutively.