Two transcriptional regulators GlnR and GlnRII are involved in regulation of nitrogen metabolism in Streptomyces coelicolor A3(2)

Abstract

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.

Three of Four GlnR Binding Sites Are Essential for GlnR-Mediated Activation of Transcription of the Amycolatopsis mediterranei nas Operon

“…Although the GlnR binding consensus sequences of the a site and the b site were obtained on the basis of a dozen studies in S. coelicolor (12)(13)(14)18), the DNA sequences of the GlnR binding sites were not conserved among the GlnR target genes, e.g., the a sites previously defined (see Fig. 2A in reference 13) and the a1 site in this study, which thus raises the question of whether the bioinformatically predicted GlnR binding site is accurate.…”

“…Instead of the Ntr system, global regulators in actinomycetes are responsible for the regulation of the nitrate assimilation as well as the whole nitrogen metabolism. In Streptomyces coelicolor, the nasA gene (encoding the nitrate reductase) and nirB1B2C genes (encoding the nitrite reductase subunits), locating separately on the chromosome, are all positively regulated by the global nitrogen regulator GlnR and a newly identified GlnR target, NnaR (11)(12)(13)(14). In Mycobacterium tuberculosis, the causative agent of tuberculosis, GlnR is also characterized as an activator for the nitrite reductase-encoding genes (nirBD) (15), although the regulation for the nitrate reductase-encoding genes still remains unknown.…”

The TetR Family of Regulators

“…AmtR homologs are found in Actinobacteria, including some species of Mycobacterium, Nocardia, Rhodococcus, and Streptomyces (25,151). In Streptomyces, two OmpR-like regulators, GlnR and GlnRII, are the master regulators of nitrogen metabolism (152). Although not present in Corynebacterium, GlnR homologs are more conserved in actinobacteria than AmtR homologs; however, there are a few species that encode both (151).…”

Regulation of nitrogen metabolism in Mycobacterium tuberculosis: A comparison with mechanisms in Corynebacterium glutamicum and Streptomyces coelicolor

“…An OmpR-type response regulator, GlnR, was shown to control glnA1 expression (41) and GlnRII was shown to regulate glnII transcription (5). In addition to controlling transcription of glnA1, gel retardation assays showed that GlnR was able to bind upstream of amtB and thereby regulate transcription of glnK and glnD as well, as all three genes are located in an operon.…”