Transcriptional Regulator DasR Represses Daptomycin Production through Both Direct and Cascade Mechanisms in Streptomyces roseosporus

Abstract

Daptomycin, produced by Streptomyces roseosporus, is a clinically important cyclic lipopeptide antibiotic used for the treatment of human infections caused by drug-resistant Gram-positive pathogens. In contrast to most Streptomyces antibiotic biosynthetic gene clusters (BGCs), daptomycin BGC has no cluster-situated regulator (CSR) genes. DasR, a GntR-family transcriptional regulator (TR) widely present in the genus, was shown to regulate antibiotic production in model species S. coelicolor by binding to promoter regions of CSR genes. New findings reported here reveal that DasR pleiotropically regulates production of daptomycin and reddish pigment, and morphological development in S. roseosporus. dasR deletion enhanced daptomycin production and morphological development, but reduced pigment production. DasR inhibited daptomycin production by directly repressing dpt structural genes and global regulatory gene adpA (whose product AdpA protein activates daptomycin production and morphological development). DasR-protected regions on dptEp and adpAp contained a 16 nt sequence similar to the consensus DasR-binding site dre in S. coelicolor. AdpA was shown to target dpt structural genes and dptR2 (which encodes a DeoR-family TR required for daptomycin production). A 10 nt sequence similar to the consensus AdpA-binding site was found on target promoter regions dptAp and dptR2p. This is the first demonstration that DasR regulates antibiotic production both directly and through a cascade mechanism. The findings expand our limited knowledge of the regulatory network underlying daptomycin production, and will facilitate methods for construction of daptomycin overproducers.

Keywords: AdpA; DasR; Streptomyces roseosporus; daptomycin; morphological development.

Figure 1

Effects of DasR on daptomycin and pigment production, cell growth, and morphological development in S. roseosporus. (A) Daptomycin production in WT, dasR-deletion mutant DdasR, complemented strain CdasR, overexpression strain OdasR, and plasmid control strains WT/pKC1139 and WT/pSET152 cultured in fermentation medium for 10 days. Statistical notations: NS, not significant; **, p < 0.01 for comparison with WT (t-test). (B) Growth curves for WT, DdasR, and OdasR. Biomass is presented as dry cell weight. (C) Daptomycin production curves for WT, DdasR, and OdasR. Error bars in panels (A–C): SD for three replicates. (D) Phenotypes of WT, DdasR, CdasR, and OdasR grown on DA1 plates at 28 °C.

Figure 2

RT-qPCR analysis of dpt genes in WT and DdasR grown in fermentation medium for 2, 4, or 6 days. WT transcription level for each gene on day 2 was defined as 1. NS, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001 (t-test). Error bars: SD for three replicates.

Figure 3

Binding of DasR to dptE-promoter region. (A) Promoter probes used for EMSAs (schematic). Long solid lines: transcriptional units. (B) EMSAs of interactions of His₆-DasR with promoter probes of dpt genes. Negative control probe: hrdB. Concentrations of His₆-DasR for probe dptEp: 1, 1.5, 3, and 4.5 μM; for other dpt promoter probes: 1 and 4.5 μM. Lanes –: EMSAs without His₆-DasR. A measure of 4.5 μM His₆-DasR was used for competition assays and control probe hrdB (lanes +). Lanes N and S: competition assays with ~100-fold excess of unlabeled nonspecific probe hrdB (N) and specific probe dptEp (S). Bracket (upper left): DasR-DNA complex. Arrowheads: free probe.

Figure 4

Determination of DasR-binding site on dptEp. (A) DNase I footprinting assay of DasR on dptEp. Protection patterns were acquired with increasing His₆-DasR concentrations. Control: reaction without His₆-DasR. (B) Nucleotide sequences of dptE-promoter region and DasR-binding site. Numbers: distance (nt) from dptE TSS. Bent arrow: dptE TSS. Boxes: putative −10 and −35 regions. Yellow highlight: dptE TSC. Solid line: DasR-binding site. Bold font: dre-like sequence.

Figure 5

Identification of DasR target adpA. (A) EMSAs of His₆-DasR with probe adpAp. Lanes 2–5: 1, 1.5, 3, and 4.5 μM His₆-DasR. Notations are as in Figure 3B. (B) RT-qPCR analysis of adpA in WT and DdasR grown in fermentation medium. *, p < 0.05; ***, p < 0.001 (t-test). Error bars: SD for three replicates. (C) DNase I footprinting assay of DasR on adpAp. (D) Nucleotide sequences of adpA promoter region and DasR-binding site. Formatting conventions as in Figure 4B.

Figure 6

Effects of AdpA on daptomycin and pigment production, cell growth, and morphological development in S. roseosporus. (A) Daptomycin production in WT, DadpA, CadpA, and OadpA cultured in fermentation medium for 10 days. NS, not significant; ***, p < 0.001 (t-test). (B) Growth curves for WT, DadpA, and OadpA. (C) Daptomycin production curves for WT, DadpA, and OadpA. Error bars in panels (A–C): SD for three replicates. (D) Phenotypes of WT, DadpA, CadpA, and OadpA grown on DA1 plates at 28 °C.

Figure 7

Identification of AdpA targets dptA and dptR2. (A) EMSAs of His₆-AdpA with indicated promoter probes. Concentrations of His₆-AdpA for probes dptAp and dptR2p: 50, 100, 150, and 200 nM; for control probe hrdB: 200 nM; for other probes: 50 and 200 nM. A measure of 200 nM His₆-AdpA was used for competition assays. Notations are same as in Figure 3B. (B) RT-qPCR analysis of dptR2 and dpt structural genes within dptA operon in WT and DadpA grown in fermentation medium. NS, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001 (t-test). Error bars: SD for three replicates.

Figure 8

DNase I footprinting assays and nucleotide sequences of AdpA-binding sites on dptAp (A) and dptR2p (B). Numbers: distance (nt) from respective TSSs. Bent arrows: TSSs. Boxes: putative −10 and −35 regions. Yellow highlight: TSCs. Solid lines: AdpA protected regions. Bold font: sequence similar to the consensus AdpA-binding site.

Figure 9

Proposed model of regulatory role of DasR in control of daptomycin production in S. roseosporus. Solid arrows: activation. Bars: repression. Solid lines: direct control. Dashed lines: indirect control.