FIG 4.
The response regulator GluR directly activates glutamate uptake through binding to the intergenic region between gluABCD and gluR-K. (A) Cotranscriptional analysis of the gluABCD gene cluster. RT-PCR was performed using cDNA as the template and RNA as the negative control. (B) EMSA of GluR binding to the intergenic region between gluABCD and gluR-K (gluA-Rint). The assay was performed using the indicated (micromolar) amounts of purified His6-GluR protein and Cy5 fluorescence-labeled gluA-Rint probe. EMSAs in the presence of 100-fold unlabeled, specific probe (S) or nonspecific (NS) competitor DNA (salmon sperm DNA) were performed as controls. (C) RT-qPCR analysis of the gluABCD gene cluster in the wild-type strain M145 and the ΔgluR-K mutant at 48 and 96 h grown on MM with 75 mM glutamate. Gene expression is represented as fold change after being normalized to M145. (D) Analysis of the gluA promoter activity in M145 and the ΔgluR-K mutant using xylE as a reporter. Promoter activities were assayed after the S. coelicolor strains were grown on MM with 75 mM glutamate for 48 and 96 h, respectively. The error bars indicate the standard deviations from three independent experiments. (E) An illustration of GluR-binding sequence in the intergenic region of gluABCD and gluR-K. The sequence protected by GluR is shown in red. The −10 and −35 elements of the gluA promoter are boxed, and the transcription start site (TSS) is indicated by a bent arrow, which was determined by 5′ RACE.