The developmental regulator MtrA binds GlnR boxes and represses nitrogen metabolism genes inStreptomyces coelicolor

Abstract

Summary In Streptomyces, GlnR is an activator protein that activates nitrogen‐assimilation genes under nitrogen‐limiting conditions. However, less is known regarding the regulation of these genes under nitrogen‐rich conditions. We determined that the developmental regulator MtrA represses nitrogen‐assimilation genes in nitrogen‐rich media and that it competes with GlnR for binding to GlnR boxes. The GlnR boxes upstream of multiple nitrogen genes, such as amtB, were confirmed as MtrA binding sites in vitro by electrophoretic mobility shift assays and in vivo by ChIP‐qPCR analysis. Transcriptional analysis indicated that, on nutrient‐rich medium, MtrA profoundly repressed expression of nitrogen‐associated genes, indicating opposing roles for MtrA and GlnR in the control of nitrogen metabolism. Using in vitro and in vivo analysis, we also showed that glnR is itself a direct target of MtrA and that MtrA represses glnR transcription. We further demonstrated functional conservation of MtrA homologues in the recognition of GlnR boxes upstream of nitrogen genes from different actinobacterial species. As mtrA and glnR are widespread among actinomycetes, this mechanism of potential competitive control over nitrogen metabolism genes may be common in this group, adding a major new layer of complexity to the known regulatory network for nitrogen metabolism in Streptomyces and related species.

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“…Recently, it was experimentally demonstrated that MtrA binds to one of the three putative GlnR binding sequences upstream of the amtB-glnK-glnD cluster (Zhu et al, 2019). Previously, three putative GlnR binding sites (named a1-b1, a2-b2, and a3-b3), each consisting in two binding repeats had been identified in the amtB promoter region (Wang et al, 2012;Li et al, 2018).…”

“…Previously, three putative GlnR binding sites (named a1-b1, a2-b2, and a3-b3), each consisting in two binding repeats had been identified in the amtB promoter region (Wang et al, 2012;Li et al, 2018). More recently, Zhu et al (2019) showed that the developmental regulator MtrA recognizes the a3-b3 binding sequence which also binds GlnR efficiently. This was confirmed by EMSA DNA binding assays using the a3-b3 sequence as probe (Figure 2).…”

“…In vivo binding of MtrA to GlnR recognition sequences in different nitrogen metabolism genes was shown by ChIP-qPCR analysis using anti-Flag antibodies and a S. coelicolor mtrA mutant transformed with the Mtr-Flag fused gene (Zhu et al, 2019). Comparative transcriptional analysis of gene expression in the parental strain and the mtrAdeleted mutant showed that several nitrogen metabolism genes including the amtB-glnk-glnD cluster, glnII, nasA, and nirB1-nirB2-nirC/D were highly overexpressed in the mtrAdeleted mutant under nitrogen rich medium whereas the urea utilization cluster was not significantly upregulated under these conditions.…”

“…These results clearly indicate that under nitrogen rich conditions MtrA represses nitrogen metabolism genes, i.e., this is a regulation opposed to that of GlnR in minimal medium. Interestingly, comparative transcriptional experiments were also performed under nitrogen limiting conditions using the mtrAor the glnR-deleted mutant, compared to the parental strain (Zhu et al, 2019); the results clearly indicated that expression of several nitrogen metabolism genes respond to the GlnR concentration in the cells but not to the MtrA levels. In other words under nitrogen limiting conditions the positive regulatory effect of GlnR dominates over the MtrA repression.…”

“…Probably other proteins interact with MtrA and modify its affinity compared to that of GlnR. Similarly, NtrA of S. lividans regulates expression of several nitrogen metabolism genes under nitrogen rich conditions, as occurs in S. coelicolor (Zhu et al, 2019). Furthermore, the ntrA gene has been characterized in M. tuberculosis and C. glutamicum.…”