Redundancy of the conserved His residue in Azotobacter vinelandii NifL, a histidine autokinase homologue which regulates transcription of nitrogen fixation genes.
The NifL protein of Azotobacter vinelandii inhibits NifA, the activator of nif (nitrogen fixation) transcription, in response to oxygen and fixed 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 the residue known to be phosphorylated in other systems. To examine the mechanism of sensory transduction by NifL, mutations encoding 10 substitutions for His-304 were introduced into the A. vinelandii chromosome. Regulation of nif transcription was measured using acetylene reduction and RNA blots. The substitutions His-304-->Arg and His-304-->Pro impaired regulation by both fixed nitrogen and oxygen, but substitution of Ala, Phe, Ile, Lys, Asn, Ser, Thr, Val had no effect. None of the mutants, including His-304-->Arg and His-304-->Pro, excreted ammonium during diazotrophy, a phenotype of nifL deletion mutants, suggesting that the molecular basis of this effect differs from that responsible for the inhibition of nif transcription. The data show conclusively that phosphorylation of His-304 is not essential for any of the known functions of A. vinelandii NifL. Homology to the family of histidine autokinases is therefore inadequate evidence for a mechanism of sensory transduction involving phosphorylation of the conserved histidine residue.
Woodley
P
P
AFRC IPSR Nitrogen Fixation Laboratory, University of Sussex, Brighton, UK.
Drummond
M
M
D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D017124:Conserved Sequence; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006639:Histidine / Q000235:genetics; D000071677:Histidine Kinase; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017672:Nitrogen Compounds / Q000494:pharmacology; D009586:Nitrogen Fixation / Q000235:genetics; D009591:Nitrogenase / Q000096:biosynthesis; D010088:Oxidoreductases; D010100:Oxygen / Q000494:pharmacology; D011494:Protein Kinases / Q000235:genetics; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000235:genetics; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic / Q000187:drug effects
D001426:Bacterial Proteins; D017672:Nitrogen Compounds; D000644:Quaternary Ammonium Compounds; C084296:nifL protein, Bacteria; D006639:Histidine; D010088:Oxidoreductases; D009591:Nitrogenase; C029130:nitrogenase reductase; D011494:Protein Kinases; D000071677:Histidine Kinase; D010100:Oxygen