Bacterial type I glutamine synthetase of the rifamycin SV producing actinomycete, Amycolatopsis mediterranei U32, is the only enzyme responsible for glutamine synthesis under physiological conditions.
The structural gene for glutamine synthetase, glnA, from Amycolatopsis mediterranei U32 was cloned via screening a genomic library using the analog gene from Streptomyces coelicolor. The clone was functionally verified by complementing for glutamine requirement of an Escherichia coli glnA null mutant under the control of a lac promoter. Sequence analysis showed an open reading frame encoding a protein of 466 amino acid residues. The deduced amino acid sequence bears significant homologies to other bacterial type I glutamine synthetases, specifically, 71% and 72% identical to the enzymes of S. coelicolor and Mycobacterium tuberculosis, respectively. Disruption of this glnA gene in A. mediterranei U32 led to glutamine auxotrophy with no detectable glutamine synthetase activity in vivo. In contrast, the cloned glnA gene can complement for both phenotypes in trans. It thus suggested that in A. mediterranei U32, the glnA gene encoding glutamine synthetase is uniquely responsible for in vivo glutamine synthesis under our laboratory defined physiological conditions.
Peng
Wen-Tao
WT
Laboratory of Molecular Microbiology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
Wang
Jin
J
Wu
Ting
T
Huang
Jian-Qiang
JQ
Chiao
Jui-Shen
JS
Zhao
Guo-Ping
GP
D039903:Actinobacteria / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000378:metabolism; D005816:Genetic Complementation Test; D016680:Genome, Bacterial; D015698:Genomic Library; D005974:Glutamate-Ammonia Ligase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D012294:Rifamycins / Q000494:pharmacology*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D012294:Rifamycins; C023808:rifamycin SV; C081173:glutamine synthetase I; D005974:Glutamate-Ammonia Ligase