REDUNDANCY OF THE CONSERVED HIS RESIDUE IN AZOTOBACTER-VINELANDII NIFL, A HISTIDINE AUTOKINASE HOMOLOG WHICH REGULATES TRANSCRIPTION OF NITROGEN-FIXATION GENES

The NifL protein of Azotobacter vinelandii inhibits NifA, the activato r of nif(nitrogen fixation) transcription, in response to oxygen and f ixed nitrogen. NifL shows strong homology in its C-terminal domain to the histidine autokinase domains of the canonical two-component sensor proteins, including the region around His-304, which corresponds to t he residue known to be phosphorylated in other systems. To examine the mechanism of sensory transduction by NifL, mutations encoding 10 subs titutions for His-304 were introduced into the A. vinelandii chromosom e. Regulation of nif transcription was measured using acetylene reduct ion and RNA blots. The substitutions His-304 --> Arg and His-304 --> P ro impaired regulation by both fixed nitrogen and oxygen, but substitu tion of Ala, Phe, lie, Lys, Asn, Ser, Thr, Val had no effect. None of the mutants, including His-->Arg and His-304-->Pro, excreted ammonium during diazotrophy, a phenotype of nifL deletion mutants, suggesting t hat the molecular basis of this effect differs from that responsible f or the inhibition of nif transcription. The data show conclusively tha t phosphorylation of His-304 is not essential for any of the known fun ctions of A. vinelandii NifL. Homology to the family of histidine auto kinases is therefore inadequate evidence for a mechanism of sensory tr ansduction involving phosphorylation of the conserved histidine residu e.