Molecular characteristics of glnA linked mutations in the nitrogen-fixing cyanobacterium Nostoc muscorum.
The glutamine synthetase (GS)-impaired mutants of Nostoc muscorum, isolated as ethylenediamine (EDA) resistant phenotype, were characterized for glnA linked processes to investigate the molecular mechanism of GS impairment. The GS enzyme was purified to homogeneity from the wild and mutant strains of N. muscorum employing ion-exchange followed by affinity chromatography. The final yield and specific activity of the enzyme were low in the mutant strains as compared to the wild type. The molecular weight of the native enzyme and the subunit size of GS polypeptide in all the strains were identical. Compared to the wild type, the GS purified from the mutant strains showed refractory response to its structural analogues like methionine sulfone (MSO), methionine sulfoximine (MSX), EDA, and metabolic substrates, such as ammonium and glutamine. The Km and Vmax for the substrates of transferase and biosynthetic reactions were altered in the mutant strains. The immunological assays performed with partially purified IgG molecules raised against wild-type GS revealed quantitative and qualitative heterogeneity for cross reactivity among the wild-type and EDA-resistant strains. Southern hybridization of genomic DNA in all the strains of N. muscorum restricted with different enzymes showed identical pattern with Anabaena 7120 glnA probe and localization of complete gene for GS on 7.5 kb HindIII fragment. Northern blot analysis indicated differential levels of GS specific mRNA and abundance of glnA transcripts of 1.7 and 1.9 kb in the mutant strains. Results suggested that point mutation(s) inflicted in glnA gene of N. muscorum are responsible for differential production of catalytically modified GS in EDA-resistant phenotypes.
Srivastava
Renu
R
Plant Molecular Biology Lab, National Botanical Research Institute, P.B. No. 436, Rana Pratap Marg, Lucknow-226 001, India.
Amla
Devindra Vijay
DV
D001426:Bacterial Proteins / Q000032:analysis; D015152:Blotting, Northern; D015139:Blotting, Southern; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay; D005974:Glutamate-Ammonia Ligase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005779:Immunodiffusion; D007700:Kinetics; D008970:Molecular Weight; D009154:Mutation; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012329:RNA, Bacterial; C081173:glutamine synthetase I; D005974:Glutamate-Ammonia Ligase