AvaR1, a Butenolide-Type Autoregulator Receptor in Streptomyces avermitilis, Directly Represses Avenolide and Avermectin Biosynthesis and Multiple Physiological Responses

Abstract

Avermectins are commercially important anthelmintic antibiotics produced by Streptomyces avermitilis. The homologous TetR-family transcriptional regulators AvaR1 and AvaR2 in this species were identified previously as receptors of avenolide, a novel butenolide-type autoregulator signal required for triggering avermectin biosynthesis. AvaR2 was found to be an important pleiotropic regulator in repression of avermectin and avenolide production and cell growth, whereas the regulatory role of AvaR1 remains unclear. Investigation of AvaR1 function in the present study showed that it had no effect on cell growth or morphological differentiation, but inhibited avenolide and avermectin production mainly through direct repression of aco (the key enzyme gene for avenolide biosynthesis) and aveR (the cluster-situated activator gene). AvaR1 also directly repressed its own gene (avaR1) and two adjacent homologous genes (avaR2 and avaR3). Binding sites of AvaR1 on these five target promoter regions completely overlapped those of AvaR2, leading to the same consensus binding motif. However, AvaR1 and AvaR2 had both common and exclusive target genes, indicating that they cross-regulate diverse physiological processes. Ten novel identified AvaR1 targets are involved in primary metabolism, stress responses, ribosomal protein synthesis, and cyclic nucleotide degration, reflecting a pleiotropic role of AvaR1. Competitive EMSAs and GST pull-down assays showed that AvaR1 and AvaR2 competed for the same binding regions, and could form a heterodimer and homodimers, suggesting that AvaR1 and AvaR2 compete and cooperate to regulate their common target genes. These findings provide a more comprehensive picture of the cellular responses mediated by AvaR1 and AvaR2 regulatory networks in S. avermitilis.

Keywords: AvaR1; AvaR2; Streptomyces avermitilis; avenolide; avermectins.

Figure 1

Effects of deletion and overexpression of avaR1 on avermectin production and cell growth in S. avermitilis. (A) Comparison of avermectin production in WT, avaR1 deletion mutant (ΔavaR1), complemented strain (CavaR1), and overexpression strain (OavaR1) cultured in FM-I for 10 days. WT/pKC1139 and WT/pSET152: vector control strains. Error bars: standard deviation (SD) from three replicate experiments. NS, not significant; ^*, P < 0.05; ^***, P < 0.001 for comparison with WT (Student's t-test). (B) Growth curves of WT, ΔavaR1, and CavaR1 in FM-II. Error bars: SD from three replicates.

Figure 2

Expression analysis of avaR1 and related genes. (A) Transcriptional profile of avaR1 during avermectin production process in WT grown in FM-I. Relative value of avaR1 on day 1 was assigned as 1. Error bars: SD from three replicates. (B) Western blotting analysis of AvaR1 protein expression profile during fermentation process. AvaR1 temporal expression in strain ΔavaR1/avaR1-3FLAG grown in FM-I was analyzed using ANTI-FLAG mAb. 100 μg total protein was added in each lane. Loading control: Coomassie Blue staining of total protein. (C) qRT-PCR analysis of aveR, aveA1, cyp17, aco, and three avaR genes in WT and ΔavaR1 grown in FM-I. Value for each gene was expressed relative to that of WT on day 2, which was assigned as 1. avaR1, 125-bp transcript amplified from the remainder avaR1 ORF in ΔavaR1 with primers ZJY129 and ZJY130. Error bars: SD from three replicates. NS, not significant; ^*, P < 0.05; ^**, P < 0.01; ^***, P < 0.001 (Student's t-test).

Figure 3

Interaction of AvaR1 with target promoters in vitro and in vivo. (A) In vitro EMSAs of His₆-AvaR1 with probes acop, avaR1p, avaR2p, avaR3p, aveRp, and aveA1p described previously (Zhu et al., 2016). Each reaction mixture contained 0.3 nM labeled probe. Specific (lanes S) and nonspecific (lanes N) competition assays were performed using ~200-fold excess of unlabeled competitor DNAs. Lanes 2–5 contained 10, 20, 50, and 100 nM His₆-AvaR1, respectively. Lanes –: EMSAs without His₆-AvaR1. 100 nM His₆-AvaR1 was used for competition assays and negative control probe 1 (sig25 promoter region) (Lanes +). Arrowheads: free probes. Brackets: AvaR1-DNA complexes. (B) In vivo ChIP-qPCR assays. ANTI-FLAG mAb against AvaR1-3FLAG was used to immunoprecipitate AvaR1-3FLAG-DNA complexes from 16-, 24-, 48-, 96-, 144-, and 192-h cultures treated with formaldehyde. IgG-coprecipitated complexes were used as negative control. Enrichment level of target DNA in control at each time point was assigned as 1. The y axis represents relative fold binding of target DNA compared with control. Error bars: SD from three replicate experiments. (C) Bioluminescence levels of E. coli reporter cultures containing various plasmid combinations. pCS26-Pac and pACYC184 were used as vector controls. Values were expressed as relative light units (RLU). Error bars: SD from three replicates. NS, not significant; ^***, P < 0.001 (Student's t-test).

Figure 4

Relative affinities of AvaR1 for various target promoters. (A) EMSA of His₆-AvaR1 with labeled probe aveRp and unlabeled probes (aveRp, avaR2p, avaR3p, acop, avaR1p). (B) EMSA of His₆-AvaR1 with labeled probe avaR2p and unlabeled probes. (C) EMSA of His₆-AvaR1 with labeled probe avaR3p and unlabeled probes. (D) EMSA of His₆-AvaR1 with labeled probe acop and unlabeled probes. (E) EMSA of His₆-AvaR1 with labeled probe avaR1p and unlabeled probes. For competition assays, labeled probe (0.3 nM) and unlabeled competitor probe (50- and 250-fold) were added with His₆-AvaR1 (50 nM). Arrowheads: free labeled probes.

Figure 5

Identification of AvaR1 binding sites. (A) DNase I footprinting assay of AvaR1 on target promoter regions. Protection fluorograms were acquired with increasing amounts of His₆-AvaR1. Top fluorograms: control reactions with 10 μM BSA. (B) Nucleotide sequences of target promoter regions and AvaR1 binding sites. Numbers: distance (nt) from respective TSS. Shaded areas: translational start codons. Bent arrows: TSSs. Boxes: potential −10 and −35 regions. Solid lines: AvaR1 binding sites. Boldface: ARE-like sequences. (C) Analysis of consensus AvaR1 binding sequence using the WebLogo program (http://weblogo.berkeley.edu). Asterisks: consensus bases. Arrows: inverted repeats. Height of each letter is proportional to appearance frequency of corresponding base.

Figure 6

Confirmation of new AvaR1 target genes. (A) EMSAs of His₆-AvaR1 protein with 17 putative binding promoter regions. Each lane contained 0.3 nM labeled probe. Lanes –, EMSAs without His₆-AvaR1. Lanes 2 to 3 contained 50 and 200 nM His₆-AvaR1, respectively. (B) qRT-PCR analysis of newly identified AvaR1 target genes in WT and ΔavaR1 strains. WT value of each gene on day 2 was assigned as 1. Error bars: SD from three replicates. NS, not significant; ^**, P < 0.01; ^***, P < 0.001 (Student's t-test).

Figure 7

Relationships between AvaR1 and AvaR2. (A) Competitive EMSAs of probe aveRp₂ with His₆-AvaR1 and GST-AvaR2 proteins. 0.3 nM labeled probe aveRp₂ was incubated with the indicated concentrations of His₆-AvaR1 and GST-AvaR2. (B) GST pull-down assays of AvaR1 and AvaR2 from E. coli whole cell lysate. His₆- and GST-tagged proteins were co-expressed in E. coli, recovered by sonication and centrifugation, and subjected to GST pull-down and Western blotting analysis with anti-GST and anti-His antibodies, respectively. Lanes 1: cell lysate before induction by IPTG. Lanes 2: cell lysate after induction. Lanes 3: GST pull-down.

Figure 8

Working model for the regulatory roles of AvaR1 and AvaR2 in control of avenolide and avermectin production. Solid-line bars: direct repression. Solid-line arrow: direct activation. Dashed-line arrows: production of avermectin or avenolide.