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A mutation in the gene upstream of nifA in Azotobacter vinelandii was introduced into the chromosome to replace the corresponding wild-type region. The resulting mutant, MV376, produced nitrogenase constitutively in the presence of 15 mM ammonium. When introduced into a niJH-lacZ fusion strain, the mutation permitted 1-galactosidase production in the presence of ammonium. The gene upstream of nifA is therefore designated nifL because of its similarity to the Klebsiella pneumoniae nifL gene in proximity to nifA, in mutant phenotype, and in amino acid sequence of the gene product. The A. vinelandii nifL mutant MV376 excreted significant quantities of ammonium (-10 mM) during diazotrophic growth. In contrast, ammonium excretion during diazotrophy was much lower in a K. pneumoniae niJL deletion mutant (maximum, 0.15 mM) but significantly higher than in NifL+ K. pneumoniae. The expression of the A. vinelandii nifA gene, unlike that of K. pneumoniae, was not repressed by ammonium.

We sequenced the nitrogen fixation regulatory gene nfrX from Azotobacter vinelandi, mutations in which cause a Nif-phenotype, and found that it encodes a 105-kDa protein (NfrX), the N terminus of which is highly homologous to that of the uridylyltransferase-uridylyl-removing enzyme encoded by ginD in Escherichia coli.In Enteric bacteria modulate their pattern of gene expression and the activities of certain enzymes in response to the quality and quantity of their nitrogen source. The availability of fixed nitrogen is sensed by the glnD product, a uridylyltransferase-uridylyl-removing enzyme (UT-UR), which responds to the 2-ketoglutarate/glutamine ratio in the cell. In conditions of N deficiency, UT-UR uridylylates the next component of the regulatory pathway, the tetrameric protein PI, encoded by glnB, and conversely removes the modifying groups in N sufficiency. The unmodified, active form of PI, signals N sufficiency to the ntrBC system, which regulates gene expression. It also stimulates adenylylation and consequent inactivation of glutamine synthetase (GS) by an adenylyltransferase (42).In N deficiency, when PII is uridylylated, NtrB (also called NRII) phosphorylates the transcriptional activator protein NtrC (also called NRI) (27,38). The phosphorylated form of NtrC, NtrC-P, directly or indirectly activates the expression of a variety of genes, including those for utilization of histidine (hut) or arginine (aut), the structuiral gene for glutamine synthetase (glnA), and, in Klebsiella pneumoniae, the nitrogen fixation (nil) genes (3, 34, 38). In N excess, P11UMP becomes deuridylylated and unmodified PII stimulates dephosphorylation and consequent inactivation of NtrC by NtrB.In K. pneumoniae, NtrC-P activates the niTLA promoter. NifA is a transcriptional activator that is both structurally and functionally similar to NtrC but is specific for activation * Corresponding author. t Present address:

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