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Summary

Streptomyces coelicolor has an unusually large arsenal of glutamine synthetase (GS) enzymes: a prokaryotic GSI-β-subtype enzyme (encoded by glnA ), three annotated glnA -like genes of the GSI-α-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 α-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.