This study focuses on the gene (glnA) encoding glutamine synthetase from the cyanobacterium Synechococcus sp. strain PCC 7942. A molecular analysis of the gene was initiated to test the hypothesis that expression of glnA is regulated at the level of transcription and that this regulation is reflective of global control of nitrogen metabolism in Synechococcus 7942. The method used for testing the hypothesis involved first isolating and sequencing the glnA gene and its flanking regions, and second, analyzing the RNA produced by the glnA gene by transcript mapping. Sequence analysis of the glnA gene revealed high homology within the open reading frame at the nucleotide level when compared with glnA from the cyanobacterium Anabaena 7120, and lower homology with glnA from other bacteria. Comparisons of the deduced amino acid sequences showed a similar pattern of highest homology between the cyanobacterial glutamine synthetases, with lower homologies in comparison with other bacteria. Northern analysis using the Synechococcus glnA gene as a hybridizing probe revealed a transcript of 1.6 kb, verifying expression of glnA. It was upon these three criteria--heterologous hybridization with the glnA gene of Anabaena 7120, sequence comparisons with several other bacterial glnA genes, and identification of a 1.6 kb transcript--that the open reading frame was identified as the structural gene (glnA) of glutamine synthetase. The results of transcript mapping indicate that transcription begins at a start site 142 nucleotides upstream of the translational start when cells are grown under either nitrogen sufficient or nitrogen deficient (N for 10 hours) conditions. When cells are grown in a medium lacking nitrogen for only 4.5 hours, the transcriptional start site maps to a position 139 nucleotides upstream of the translational start. The identification of two transcriptional start sites, 3 base pairs apart, might be an artifact inherent in the experimental strategy, or it may imply transcriptional control. A sequence consisting of TAGGAT is present 14 or 17 bases upstream of the two transcriptional starts, respectively, and is similar to other Synechococcus promoters (TAGAAT in psbA1 and TATTAT in psbA2) as well as the $-$10 promoter, (TATAAT), found in enteric bacterial $\sigma\sp{70}$ promoters. Two overlapping sequences, GTTACA and CAAAAG, are positioned in the $-$35 promoter region. The first of these resembles the $-$35 region for three light responsive genes (TTTACA for psbA1 and psbA2, and TTCACA for psbA3) from Synechococcus. It also resembles, TTGACA, found at $-$35 in unregulated promoters of enteric bacteria. The other sequence, CAAAAG, resembles two Anabaena 7120 $-$35 promoter regions, CAAAAC in glnA, and CATAAC in nifH, which are nitrogen regulated. Taken together, the presence of two potential $-$35 promoter regions and two transcriptional start sites, indicates transcriptional regulation of glnA in Synechococcus 7942.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-7725 |
| Date | 01 January 1990 |
| Creators | Curry, Jeanne |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
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