SummaryStreptomyces coelicolor has an unusually large arsenal of glutamine synthetase (GS) enzymes: a prokaryotic GSI-b b b b -subtype enzyme (encoded by glnA ), three annotated glnA -like genes of the GSI-a a a a -subtype and a eukaryote-like glutamine synthetase II (encoded by glnII ). Under all tested conditions, GSI was found to represent the dominant glutamine synthetase activity. A significant heat-labile GSII activity, which is very low to undetectable in liquid-grown cultures, was only detected in morphologically differentiating S. coelicolor cultures. Analysis of glnA and glnII transcription by S1 nuclease mapping and egfp fusions revealed that, on nitrogen-limiting solid medium, glnII but not glnA expression is upregulated. An OmpR-like regulator protein, GlnR, has previously been implicated in transcriptional control of glnA expression. Gel retardation analysis revealed that GlnR is a DNA-binding protein, which interacts with the glnA promoter. It is not autoregulatory and does not bind to the upstream regions of the glnA -like genes of the a a a a -subfamily, nor to the glnII promoter in vitro . A second GlnR target was identified upstream of the amtB gene, encoding a putative ammonium transporter. amtB forms an operon with glnK (encoding a PII protein) and glnD (encoding a putative PII nucleotidylyltransferase) shown by S1 nuclease protection analysis and reverse transcription-polymerase chain reaction (RT-PCR). An amtB and glnA promoter alignment revealed a putative GlnR operator structure. Downstream of glnII , a gene encoding for another OmpR-like regulator, GlnRII, was identified, with strong similarity to GlnR. Gel shifts with GlnRII showed that the promoters recognized by GlnR are also targets of GlnRII. However, GlnRII also interacted with the glnII upstream region. Only inactivation of glnR resulted in a glutamine auxotrophic phenotype, whereas the glnRII mutant can grow on minimal medium without glutamine.

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The glutamine synthetase II (GSII, encoded by glnII) activity detectable in crude extracts from Streptomyces coelicolor is low compared to the activity of glutamine synthetase I (GSI, encoded by glnA) and to that of GSII from S. viridochromogenes. We have identified and sequenced a 3.9-kb BglII-BamHI fragment carrying the glutamine synthetase II gene (glnII) from S. coelicolor. Besides glnII, this region contains four ORFs (orf1-orf4). While homologues of orf1 and orf2 were also found in the glnII region of the S. viridochromogenes chromosome, this was not the case for orf3 and orf4, which encode a putative hydrolase and a transcriptional regulator (Ptr) of the MarR family, respectively. High-resolution S1 nuclease mapping showed that the S. coelicolor glnII gene is expressed from two overlapping promoters. The first comprises a vegetative promoter sequence and the second contains sequence elements that are recognized by Esigma31. Similar promoter structures were found upstream of the S. viridochromogenes glnII gene. The involvement of ptr in glnII regulation was studied by gel retardation assays. Recombinant Ptr interacted with the upstream region of ptr, but not with the promoter region of glnII. A ptr gene replacement mutant (S. coelicolor IP) was also constructed. RT-PCR analysis of RNA from wild-type S. coelicolor and the IP mutant demonstrated that expression of orf3 depends on Ptr. Thus, the difference in gene organization between S. coelicolor and S. viridochromogenes is not responsible for the difference in GSII activity.