Mechanism of coordinated synthesis of the antagonistic regulatory proteins NifL and NifA of Klebsiella pneumoniae

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Abstract
The nifLA operon of Klebsiella pneumoniae codes for the two antagonistic regulatory proteins which control expression of all other nitrogen fixation genes. NifA is a transcriptional activator, and NifL inhibits NifA. The importance of a correct NifL-NifA stoichiometry for efficient regulation of nitrogen fixation genes has been investigated by constructing a strain with an altered nifL-nifA gene dosage ratio, resulting from the integration of an extra copy of nifA. Results showed that a balanced synthesis of both gene products is essential for correct regulation. Effects of mutations provoking translation termination of nifL upstream or downstream of its natural stop codon, combined with overproduction of both proteins when the genes are transcribed and translated from signals of the phi10 gene of the phage T7, showed that, in addition to the previously reported transcriptional polarity, there is translational coupling between nifL and nifA. In spite of the apparently efficient ribosome binding site of nifA, its rate of independent translation is very low. This is due to a secondary structure masking the Shine-Dalgarno sequence of nifA, which could be melted by ribosomes translating nifL. Mutational analysis confirmed the functional significance of the secondary structure in preventing independent translation of nifA. Translational coupling between the two cistrons is proposed as an efficient mechanism to prevent production of an excess of NifA, which would affect the normal regulation of nitrogen fixation genes.