The transcription of amtB in Streptomyces coelicolor has been proposed to be counter-regulated by GlnR (a global regulator for nitrogen metabolism) and PhoP (a global regulator for phosphate metabolism). However, the GlnR-protected region, which was deduced to be two 22-bp GlnR binding boxes (gTnAc-n6-GaAAc-n6-GtnAC-n6-GAAAc-n6, abbreviated as a1-b1 and a2-b2), was separated from the PhoP-protected region in the promoter of amtB, leaving the mechanism for this regulation undefined. In this study, another 22-bp GlnR binding box, which consisted of a3-site-n6-b3-site (a3-b3) overlapping with the PhoP-binding sequences, was identified in the promoter region of amtB by a DNase I footprinting assay. An electrophoretic mobility shift assay (EMSA) using purified recombinant GlnR and the synthetic amtB promoter fragments with the three GlnR binding boxes individually mutated demonstrated that every box was involved in GlnR binding in vitro. Further in vivo assays using the egfp reporter gene fused to various kinds of mutated promoter regions of amtB demonstrated that all of the three GlnR binding boxes were required for GlnR-mediated activation of amtB transcription under the nitrogen-limited condition. The results of EMSA using the amtB promoter with mixtures of recombinant His-tagged GlnR and Trx-His-S-tagged PhoP inferred that PhoP might compete against GlnR from binding at the a3-b3 site, attributable to the PhoP/GlnR counter-regulatory function subjected to further experimental proof. The in vivo ratio of nitrogen versus phosphate affects bacterial growth and metabolism, including the biosynthesis of secondary metabolites (6), and the metabolism of nitrogen and phosphate is coordinated via complex regulatory networks (14). In Streptomyces coelicolor, nitrogen metabolism is globally regulated by an orphan response regulator, GlnR (22), while the expression of phosphate-regulated genes is controlled by the PhoR-PhoP two-component system (18). When phosphate is insufficient, the sensor kinase PhoR is self-phosphorylated and subsequently transfers the high-energy phosphate group to its cognate response regulator PhoP. The phosphorylated PhoP then binds to the PHO boxes, which are comprised of several 11-nucleotide (nt) direct repeat units (DRus) in the promoter regions of its target genes, and regulates their expression (20). Based on microarray data (13), a connection between the phosphate metabolism controlled by PhoP and the nitrogen metabolism regulated by GlnR was proposed. Rodriguez-Garcia et al. (14) subsequently proved that PhoP repressed the expression of both glnR and the GlnR target genes, including glnA, glnII, and the amount operon (amtB-glnKglnD), through directly binding to the DRus in their promoters.The proposed cis-element for GlnR binding is complex and is comprised of two GlnR binding boxes, each consisting of one a site and one b site separated by 6 nucleotides (i.e., a1-site-n6-b1-site-n6-a2-site-n6-b2-site-n6 [a1-b1 and a2-b2]) located upstream of most GlnR target genes (including glnA, glnII, an...
Amycolatopsis mediterranei is used for industry-scale production of rifamycin, which plays a vital role in antimycobacterial therapy. As the first sequenced genome of the genus Amycolatopsis, the chromosome of strain U32 comprising 10 236 715 base pairs, is one of the largest prokaryotic genomes ever sequenced so far. Unlike the linear topology found in streptomycetes, this chromosome is circular, particularly similar to that of Saccharopolyspora erythraea and Nocardia farcinica, representing their close relationship in phylogeny and taxonomy. Although the predicted 9 228 protein-coding genes in the A. mediterranei genome shared the greatest number of orthologs with those of S. erythraea, it was unexpectedly followed by Streptomyces coelicolor rather than N. farcinica, indicating the distinct metabolic characteristics evolved via adaptation to diverse ecological niches. Besides a core region analogous to that common in streptomycetes, a novel 'quasi-core' with typical core characteristics is defined within the non-core region, where 21 out of the total 26 gene clusters for secondary metabolite production are located. The rifamycin biosynthesis gene cluster located in the core encodes a cytochrome P450 enzyme essential for the conversion of rifamycin SV to B, revealed by comparing to the highly homologous cluster of the rifamycin B-producing strain S699 and further confirmed by genetic complementation. The genomic information of A. mediterranei demonstrates a metabolic network orchestrated not only for extensive utilization of various carbon sources and inorganic nitrogen compounds but also for effective funneling of metabolic intermediates into the secondary antibiotic synthesis process under the control of a seemingly complex regulatory mechanism.
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