SEQUENCE AND MOLECULAR ANALYSIS OF THE NIFL GENE OF AZOTOBACTER-VINELANDII

In both Klebsiella pneumoniae and Azotobacter vinelandii the nifL gene , which encodes a negative regulator of nitrogen fixation, lies immedi ately upstream of nifA. We have sequenced the A. vinelandii nifL gene and found that it is more homologous in its C-terminal domain to the h istidine protein kinases (HPKs) than is K. pneumoniae NifL. In particu lar A. vinelandii NifL contains a conserved histidine at a position sh own to be phosphorylated in other systems. Both NifL proteins are homo logous in their N-termini to a part of the Halobacterium halobium bat gene product; Bat is involved in regulation of bacterio-opsin, the exp ression of which is oxygen sensitive. The same region showed homology to the haem-binding N-terminal domain of the Rhizobium meliloti fixL g ene product, an oxygen-sensing protein. Like K. pneumoniae NifL, A. vi nelandii NifL is shown here to prevent expression of nif genes in the presence of NH4+ or oxygen. The sequences found homologous in the C-te rminal regions of NifL, FixL and Bat might therefore be involved in ox ygen binding or sensing. An in-frame deletion mutation in the nifL cod ing region resulted in loss of repression by NH4+ and the mutant excre ted high amounts of ammonia during nitrogen fixation, thus confirming a phenotype reported earlier for an insertion mutation. In addition, n ifLA are cotranscribed in A. vinelandii as in K. pneumoniae, but expre ssion from the A. vinelandii promoter requires neither RpoN nor NtrC.