Nitrogen metabolism in Streptomyces coelicolor A3(2): modification of glutamine synthetase I by an adenylyltransferase.
An internal adenylyltransferase gene (glnE) fragment from Streptomyces coelicolor was amplified using heterologous PCR primers derived from consensus motifs. The sequence had significant similarity to bacterial glnE genes, and included a motif typical of the C-terminal adenylyltransferase domain of glnE. glnE from S. coelicolor lies on the Asel-C fragment of the chromosome and is localized near glnA (encoding glutamine synthetase I, GSI) and glnII (encoding GSII). To analyse the function of glnE in S. coelicolor, glnE (S. coelicolor E4) and glnA (S. coelicolor HT107) gene replacement mutants were constructed. The GSI activity of the glnE mutant was not down-regulated after an ammonium shock. However, the GSI activity of the wild-type cells decreased to 60% of the original activity. The glnA mutant is not glutamine auxotrophic, but in the gamma-glutamyltransferase assay no GSI activity was detected in unshifted and shifted HT107 cells. By snake venom phosphodiesterase treatment the GSI activity in the wild-type can be reconstituted, whereas no alteration is observed in the E4 mutant. Additionally, the loss of short-term GSI regulation in the E4 mutant was accompanied by an increased glutamine:glutamate ratio.
Fink
D
D
Mikrobiologie/Biotechnologie, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany1.
Falke
D
D
Mikrobiologie/Biotechnologie, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany1.
Wohlleben
W
W
Mikrobiologie/Biotechnologie, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany1.
Engels
A
A
Mikrobiologie/Biotechnologie, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany1.
D000595:Amino Acid Sequence; D015139:Blotting, Southern; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development
D004269:DNA, Bacterial; D005973:Glutamine; D018698:Glutamic Acid; D009713:Nucleotidyltransferases; C020658:glutamine-synthetase adenylyltransferase; C081173:glutamine synthetase I; D005974:Glutamate-Ammonia Ligase; D009584:Nitrogen