FIG 1.

The SACE_3880 operon is regulated directly by PccD. (A) Genetic organization of the bkd operon in the S. erythraea genome. aldH (SACE_3880), gdhA1 (SACE_3882), and SACE_3884 encode aldehyde dehydrogenase (NAD⁺), glutamate dehydrogenase [NAD(P)⁺], and the 2-oxoisovalerate dehydrogenase E1 component, respectively. (B) Identification of the cotranscription of the operon (primers were designed for cross subunit genomic DNA as indicated in panel A). The cDNA was reverse transcribed from the RNA extracted from WT cultured in TSB and subjected to semiquantitative PCR. Genome DNA of S. erythraea and no reverse transcriptase PCR products were used as positive and negative controls, respectively. Semiquantitative PCR products were evaluated on 1% agarose gels. (C) Upstream promoter regions of the bkd operon. Black lines indicate the PccD-binding site. (D) EMSAs of His-PccD protein with upstream promoter regions of gdhA1. The DNA probe (about 15 ng in a 10-μl reaction system) was incubated with a protein concentration gradient (0, 1, 3, and 5 μM). Unlabeled specific probe (200-fold) or nonspecific competitor DNA (200-fold, sonicated salmon sperm DNA) was used as the control. The free probes that did not bind with protein are denoted by an arrowhead. (E) qRT-PCR analysis of the transcription profiles of SACE_3880, SACE_3882, and SACE_3884 in WT, WT/pIB-pccD and ΔpccD strains cultured in balanced Evans medium (basic Evans medium with 140 mM glucose, 100 mM NaNO₃). (F) Deduction of the PccD-binding motif in S. erythraea using MEME. The standard code of the Weblogo server is shown at the top. The GTG in wireframe is the initiation codon of SACE_3880.