Molecular analysis of the Azotobacter vinelandii glnA gene encoding glutamine synthetase
1990
Toukdarian, A. | Saunders, G. | Selman-Sosa, G. | Santero, E. | Woodley, P. | Kennedy, C.
The gene encoding glutamine synthetase (GS), glnA, wascloned from Azotobacter vinelandii on a 6-kb EcoRI fragment that also carries the ntrBC genes. The DNA sequence of1,952 bp including the GS-coding region was determined. An open reading frame of 467 amino acids indicated agene product of Mr 51,747. Transcription of glnA occurred from a C residue located 32 bases upstream of anATG considered to be the initiator codon because (i) it had a nearby potential ribosome-binding site and(ii) an open reading frame translated from this site indicated good N-terminal homology to 10 other procaryoticGSs. Sequences similar to the consensus RNA polymerase recognition sites at -10 and -35 were present at the appropriate distance upstream of the transcription initiation site. As expected from earlier geneticstudies indicating that expression of A. vinelandii glnA did not depend on the rpoN (ntrA; sigma 54) gene product, no sigma 54 recognition sequences were present, nor was there significant regulation of glnA expression by fixed nitrogen. Repeated attempts to construct glutamine auxotrophs by recombination of glnA insertion mutations wereunsuccessful. Although the mutated DNA could be found by hybridization experiments in drug-resistant A.vinelandii transformants, the wild-type glnA region was always present. These results suggest that glnA mutations arelethal in A. vinelandii. In [14C]glutamine uptake experiments, very little glutamine was incorporated intocells, suggesting that glutamine auxotrophs are nonviable because they cannot be supplied with sufficient glutamineto support growth.
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