Abstract
ArgR is the regulator of arginine biosynthesis genes in Streptomyces species. Transcriptomic comparison by microarrays has been made between Streptomyces coelicolor M145 and its mutant S. coelicolor ΔargR under control, unsupplemented conditions, and in the presence of arginine. Expression of 459 genes was different in transcriptomic assays, but only 27 genes were affected by arginine supplementation. Arginine and pyrimidine biosynthesis genes were derepressed by the lack of ArgR, while no strong effect on expression resulted on arginine supplementation. Several nitrogen metabolism genes expression as glnK, glnA and glnII, were downregulated in S. coelicolor ΔargR. In addition, downregulation of genes for the yellow type I polyketide CPK antibiotic and for the antibiotic regulatory genes afsS and scbR was observed. The transcriptomic data were validated by either reverse transcription-PCR, expression of the gene-promoter coupled to the luciferase gene, proteomic or by electrophoresis mobility shift assay (EMSA) using pure Strep-tagged ArgR. Two ARG-boxes in the arginine operon genes suggest that these genes are more tightly controlled. Other genes, including genes encoding regulatory proteins, possess a DNA sequence formed by a single ARG-box which responds to ArgR, as validated by EMSA.
Highlights
Arginine metabolism is feedback repressed by arginine in different Gram-positive and Gram-negative bacteria
This effect is mediated by ArgR, a hexameric protein that represses arginine biosynthesis genes, using L-arginine as co-repressor, in Escherichia coli [1] and Pseudomonas [2]
Thirty DNA fragments containing putative ARG boxes were used in binding assays in the presence of Strep-ArgR in electrophoresis mobility shift assay (EMSA) experiments. They correspond to upstream regions of: (i) genes related to amino acids biosynthesis such as SCO4293 for a putative threonine synthase, leuA or the bidirectional promoter region hisD–SCO2055; (ii) genes for nucleotide biosynthesis or regulation as nrdA, SCO2015 or rstP; (iii) genes related to nitrogen or carbon metabolism: glnII, amtB, glnR, SCO1086, ureA (SCO1236) or the bidirectional promoter region pyk1–SCO2015; (iv) genes for transcriptional regulators: whiB, malE–malR, SCO2686, SCO7302– 7303, SCO1220–1221, SCO3978–3979, SCO0800–0801, or for sigma or anti-sigma factors: SCO3067–sig15, SCO7314; (v) genes encoding hypothetical conserved proteins (SCO5864); and (vi) genes related to arginine or pyrimidine biosynthesis as positive controls
Summary
Arginine metabolism is feedback repressed by arginine in different Gram-positive and Gram-negative bacteria This effect is mediated by ArgR, a hexameric protein that represses arginine biosynthesis genes, using L-arginine as co-repressor, in Escherichia coli [1] and Pseudomonas [2]. Characterization of argR, encoding the ArgR repressor [6] and the use of AhrC protein permitted to understand the arginine biosynthesis cluster regulation in Streptomyces clavuligerus and to locate ARG-boxes, for ArgR binding, upstream of several arginine biosynthesis genes [7]. This provided the basis to study arginine regulation in other Streptomyces species. It was convenient to analyze gene expression under control and arginine-supplemented conditions, to obtain better knowledge about arginine transport and catabolism in this model microorganism, and to determine whether ArgR is a regulatory protein involved only in arginine control, or if it has wider regulatory functions
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