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

Jianya Zhu,Zhi Chen,Jilun Li,Ying Wen

doi:10.3389/fmicb.2017.02577

Jianya Zhu, Zhi Chen + Show 2 more

Open Access

https://doi.org/10.3389/fmicb.2017.02577

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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.

Highlights

Streptomycetes have the useful capability of producing a variety of antibiotics that have broad applications in medicine and agriculture
When an intact AvaR1 also directly repressed its own gene (avaR1) gene in integrative vector pSET152 was introduced into avaR1, avermectin yield in complemented strain CavaR1 was restored to WT level
Vector control strains WT/pSET152 and WT/pKC1139 produced closely amount of avermectins to that of WT (Figure 1A). These findings indicate that AvaR1 has an inhibitory effect on avermectin production

Summary

Introduction

Streptomycetes have the useful capability of producing a variety of antibiotics that have broad applications in medicine and agriculture. Interaction of an autoregulator with its receptor alters the receptor’s DNA-binding activity, leading to derepression of target genes involved in antibiotic biosynthesis and/or morphological differentiation (Folcher et al, 2001; Willey and Gaskell, 2011). Twenty-four autoregulators have been described to date in 12 Streptomyces species, and have been classified into five groups. Many Streptomyces genomes contain multiple genes encoding ArpA-like GBL receptor homologs, e.g., ScbR, ScbR2, CprA, and CprB in model species S. coelicolor; BarA and BarB in S. virginiae; JadR2 and JadR3 in S. venezuelae (Niu et al, 2016). ScbR2 and JadR2 are designated “pseudo” GBL receptors because they do not recognize GBLs; rather, they bind and respond to antibiotics as ligands to coordinate antibiotic biosynthesis (Xu et al, 2010; Wang W. et al, 2014). The signals recognized by CprA, CprB, and BarB remain to be clarified

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