Nitrogen metabolism in Streptomyces coelicolor A3(2): modification of glutamine synthetase I by an adenylyltransferase

An internal adenylyltransferase gene (glnE) fragment from Streptomyces coel icolor was amplified using heterologous PCR primers derived from consensus motifs. The sequence had significant similarity to bacterial glnE genes, an d 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 glnI I (encoding GSII). To analyse the function of GlnE in S. coelicolor, glnE ( S. coelicolor E4) and glnA (S. coelicolor HT107) gene replacement mutants w ere 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 g-glutamyltransferase assay no GSI activity was de tected in unshifted and shifted HT107 cells. By snake venom phosphodiestera se treatment the GSI activity in the wild-type can be reconstituted, wherea s no alteration is observed in the E4 mutant. Additionally, the loss of sho rt-term GSI regulation in the E4 mutant was accompanied by an increased glu tamine:glutamate ratio.