Volume 25, Issue 2, Pages 143-153.e4 (February 2018) NosP-Regulated Nosiheptide Production Responds to Both Peptidyl and Small-Molecule Ligands Derived from the Precursor Peptide  Jingjing Li, Yue Li, Guoqing Niu, Heng Guo, Yanping Qiu, Zhi Lin, Wen Liu, Huarong Tan  Cell Chemical Biology  Volume 25, Issue 2, Pages 143-153.e4 (February 2018) DOI: 10.1016/j.chembiol.2017.10.012 Copyright © 2018 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2018 25, 143-153. e4DOI: (10. 1016/j. chembiol Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 1 Thiopeptide Structures, Biosynthetic Pathway, and Related Gene Clusters (A) Nosiheptide (NOS), nocathiacin I (NOC-I), and thiostrepton (TSR). (B) Conversion of the precursor peptide NosM into NOS (along with the co-product LP-carboxamide) through intermediate NOS-AC. The core peptide (CP) sequence that follows the leader sequence (LP) in NosM is colored blue, with associated common post-translational modifications (PTMs) and the main peptidyl skeleton of NOS. The specific PTMs, which are separated from common PTMs by the double dashed line, are colored purple with related structures. (C) Gene organizations of the nos (top) and noc (bottom) clusters for NOS and NOC-I, respectively. The functions of genes are labeled and annotated by rectangular blocks with different colors at the bottom. Cell Chemical Biology 2018 25, 143-153.e4DOI: (10.1016/j.chembiol.2017.10.012) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 2 Effects of nosP Disruption and Overexpression on NOS Production WT, S. actuosus wild-type strain; ΔnosP, nosP disruption mutant; ΔnosPc, nosP complementary strain; nosPOE, nosP overexpression strain; ΔnosP/pSET152 or WT/pSET152, the control strains by integrating pSET152 vector into the chromosome of ΔnosP or WT. (A) High-performance liquid chromatography analysis of NOS production. (B) Bioassays of NOS production. Bacillus subtilis was used as the indicator strain. Cell Chemical Biology 2018 25, 143-153.e4DOI: (10.1016/j.chembiol.2017.10.012) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 3 Binding of NosP to Its Target Region (A) EMSAs of the binding of NosP to PLM. Each lane contains 10 ng of DNA probe. (B) DNase I footprinting of the NosP-binding site on the intergenic region between nosL and nosM. The DNA probe was labeled with γ-32P and incubated with different concentrations of NosP (0, 1.2, 1.5, 2.0, and 0 μM from lanes 0 to 4). The bracket indicates the NosP-protected region. (C) Nucleotide sequences of the nosL-nosM promoter region and the NosP-binding site. Nucleotides of the NosP-binding site are underlined. The 7-bp direct repeat is colored purple. The transcription start points (tsp) and promoter elements (−10 and −35 sequences) are marked by angled arrows and boxes, respectively. The height of each letter in the sequence logo is proportional to the frequency of the base appearance. (D) GUS activity in S. coelicolor M1146 and its derivatives containing various promoter-gusA fusions. “no gusA” indicates that S. coelicolor M1146 does not contain gusA. See also Figures S1 and S2 for related information. Cell Chemical Biology 2018 25, 143-153.e4DOI: (10.1016/j.chembiol.2017.10.012) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 4 Real-Time qRT-PCR Analysis of the Transcription of Selected nos Genes in the Wild-Type Strain and ΔnosP RNAs were isolated from S. actuosus after incubation for 24, 36, 48, 60, and 72 hr. The transcriptional levels of the nos genes were normalized to that of the 16S rRNA homolog of S. actuosus. The transcriptional levels of genes are presented relative to those of the wild-type strain collected after incubation for 24 hr. Data are presented as the averages of three independent experiments conducted in triplicate. Error bars show SDs. See also Figure S3 for related information. Cell Chemical Biology 2018 25, 143-153.e4DOI: (10.1016/j.chembiol.2017.10.012) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 5 Comparative Analysis of NosP and NocP activities in the Activation of PLM or PLM-noc (A) EMSAs of the binding of NosP and NocP to target genes, respectively. Each lane contains 10 ng of DNA probe. (B) Effects of nocP overexpression on NOS production in S. actuosus. ΔnosP/nocP or WT/nocP, ΔnosP or WT derivatives with a copy of nocP controlled by its native promoter; ΔnosP/hnocP or WT/hnocP, ΔnosP or WT derivatives with a copy of nocP controlled by PhrdB. (C) Construction of the mutant S. actuosus strain in which PLM was replaced by PLM-noc. (D) Effects of changing PLM to PLM-noc on NOS production in S. actuosus. WT/PLM-noc, the S. actuosus (WT) derivative in which PLM is replaced by PLM-noc; ΔnosP/PLM-noc, the ΔnosP derivative in which PLM is replaced by PLM-noc. See also Figures S1 and S4 for related information. Cell Chemical Biology 2018 25, 143-153.e4DOI: (10.1016/j.chembiol.2017.10.012) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 6 Effects of Small-Molecule and Peptidyl Ligands on the Binding Affinity of NosP (A) Evaluation of small-molecule ligands. EMSAs were performed with 2 μM NosP and the concentrations of ligands were as indicated. “0” indicates that DMSO was used as a solvent control. (B) Evaluation of the peptidyl ligand. The concentration of NosP was held constant at 2 μM. DMSO was used as a solvent control. (C) DNase I footprinting in the presence of LP-carboxylate. The amount of NosP used in lanes 1–3 was 2 μM, and the concentrations of LP-carboxylate were 0, 100, and 200 μM from lanes 1 to 3, respectively. Lanes 0 and 4 were used as the controls in the absence of NosP and LP-carboxylate. (D) Real-time qRT-PCR analysis of the transcription of selected nos genes in WT and ΔnosM. Data are presented as the averages of three independent experiments conducted in triplicate. Error bars show SDs. See also Figures S5 and S6 for related information. Cell Chemical Biology 2018 25, 143-153.e4DOI: (10.1016/j.chembiol.2017.10.012) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 7 Time-Course Quantification of NOS Production and Transcription of Selected nos Genes (A) Quantification of NOS production in S. actuosus wild-type strain. (B) Transcriptional analysis of nosF, nosL, nosM and nosN in the NOS production process in S. actuosus wild-type strain. Data are presented as the averages of three independent experiments conducted in triplicate. Error bars show SDs. See also Figure S5 for related information. Cell Chemical Biology 2018 25, 143-153.e4DOI: (10.1016/j.chembiol.2017.10.012) Copyright © 2018 Elsevier Ltd Terms and Conditions