Transcriptional Regulation of the Daptomycin Gene Cluster in Streptomyces roseosporus by an Autoregulator, AtrA

Xu-Ming Mao,Shuai Luo,Ri-Cheng Zhou,Feng Wang,Pin Yu,Ning Sun,Xiao-Xia Chen,Yi Tang,Yong-Quan Li

doi:10.1074/jbc.m114.608273

Abstract

Daptomycin is a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus. To reveal the transcriptional regulatory mechanism of daptomycin biosynthesis, we used the biotinylated dptE promoter (dptEp) as a probe to affinity isolate the dptEp-interactive protein AtrA, a TetR family transcriptional regulator, from the proteome of mycelia. AtrA bound directly to dptEp to positively regulate gene cluster expression and daptomycin production. Meanwhile, both ΔatrA and ΔadpA mutants showed bald phenotype and null production of daptomycin. AdpA positively regulated atrA expression by direct interaction with atrA promoter (atrAp), and removal of ArpA in S. roseosporus, a homolog of the A-factor receptor, resulted in accelerated morphological development and increased daptomycin production, suggesting that atrA was the target of AdpA to mediate the A-factor signaling pathway. Furthermore, AtrA was positively autoregulated by binding to its own promoter atrAp. Thus, for the first time at the transcriptional level, we have identified an autoregulator, AtrA, that directly mediates the A-factor signaling pathway to regulate the proper production of daptomycin.

Highlights

No investigation on daptomycin production at the transcriptional regulatory level has been reported
Temperature-sensitive vector for gene knock-out atrA in-frame deletion plasmid adpA in-frame deletion plasmid arpA in-frame deletion plasmid Integrative plasmid for complementation in Streptomyces atrA gene complementation plasmid adpA gene complementation plasmid arpA gene complementation plasmid T vector dptE promoter cloned in pTA2 vector atrA promoter cloned in pTA2 vector Cloning vector dptEp cloned in BamHI site of pUC18 vector E. coli expression vector atrA ORF cloned in BamHI/HindIII site of pET32a adpA ORF cloned in BamHI/HindIII site of pET32a Promoter probing vector with xylE reporter dptEp cloned in BamHI site of pIPP1 atrAp cloned in BamHI site of pIPP1
AtrA was required for morphological development and secondary metabolism in S. roseosporus, suggesting that AtrA is

Summary

Background

No investigation on daptomycin production at the transcriptional regulatory level has been reported. Results: The autoregulator AtrA directly regulates daptomycin gene cluster expression, and atrA is the transcriptional target of AdpA. AtrA bound directly to dptEp to positively regulate gene cluster expression and daptomycin production. AdpA positively regulated atrA expression by direct interaction with atrA promoter (atrAp), and removal of ArpA in S. roseosporus, a homolog of the A-factor receptor, resulted in accelerated morphological development and increased daptomycin production, suggesting that atrA was the target of AdpA to mediate the A-factor signaling pathway. AtrA was positively autoregulated by binding to its own promoter atrAp. for the first time at the transcriptional level, we have identified an autoregulator, AtrA, that directly mediates the A-factor signaling pathway to regulate the proper production of daptomycin. Temperature-sensitive vector for gene knock-out atrA in-frame deletion plasmid adpA in-frame deletion plasmid arpA in-frame deletion plasmid Integrative plasmid for complementation in Streptomyces atrA gene complementation plasmid adpA gene complementation plasmid arpA gene complementation plasmid T vector dptE promoter (dptEp) cloned in pTA2 vector atrA promoter (atrAp) cloned in pTA2 vector Cloning vector dptEp cloned in BamHI site of pUC18 vector E. coli expression vector atrA ORF cloned in BamHI/HindIII site of pET32a adpA ORF cloned in BamHI/HindIII site of pET32a Promoter probing vector with xylE reporter dptEp cloned in BamHI site of pIPP1 atrAp cloned in BamHI site of pIPP1

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION