Characterisation of the Klebsiella pneumoniae nitrogen-fixation regulatory proteins NIFA and NIFL in vitro

Abstract

Transcriptional activation by the Klebsiella pneumoniae nitrogen-fixation-specific positive control protein, NIFA, (nifA gene product) has been demonstrated in vitro in S30 extracts from cells which overproduce this protein. The activity of NIFA was dramatically reduced in vitro in the presence of the negative regulatory protein NIFL (nifL gene product) but was not inhibited by the presence of a mutant NIFL protein, NIFL2404. Transcriptional activation from the nifH promoter by NIFA was dependent on the alternative sigma factor, sigma 54, and also required the presence of an upstream activator sequence. NIFA activity was temperature-sensitive in vitro (as it is in vivo) which is due, at least in part, to the intrinsic lability of the protein itself. The majority of overproduced NIFA and NIFL was insoluble after low-speed centrifugation and was inactive in vitro. A low level of less aggregated NIFA protein present in cell extracts was responsible for in vitro activity and this fraction was partially purified.