SAV4189, a MarR-Family Regulator in Streptomyces avermitilis, Activates Avermectin Biosynthesis

Abstract

The bacterial species Streptomyces avermitilis is an important industrial producer of avermectins, which are widely utilized as effective anthelmintic and insecticidal drugs. We used gene deletion, complementation, and overexpression experiments to identify SAV4189, a MarR-family transcriptional regulator (MFR) in this species, as an activator of avermectin biosynthesis. SAV4189 indirectly stimulated avermectin production by altering expression of cluster-situated activator gene aveR, and directly repressed the transcription of its own gene (sav_4189) and adjacent cotranscribed gene sav_4190 (which encodes an unknown transmembrane efflux protein). A consensus 13-bp palindromic sequence, 5'-TTGCCYKHRSCAA-3' (Y = T/C; K = T/G; H = A/C/T; R = A/G; S = C/G), was found within the SAV4189-binding sites of its own promoter region, and shown to be essential for binding. The SAV4189 regulon was thus predicted based on bioinformatic analysis. Night new identified SAV4189 targets are involved in transcriptional regulation, primary metabolism, secondary metabolism, and stress response, reflecting a pleiotropic role of SAV4189. sav_4190, the important target gene of SAV4189, exerted a negative effect on avermectin production. sav_4189 overexpression and sav_4190 deletion in S. avermitilis wild-type and industrial strains significantly increased avermectin production. SAV4189 homologs are widespread in other Streptomyces species. sav_4189 overexpression in the model species S. coelicolor also enhanced antibiotic production. The strategy of increasing yield of important antibiotics by engineering of SAV4189 homologs and target gene may potentially be extended to other industrial Streptomyces species. In addition, SAV4189 bound and responded to exogenous antibiotics hygromycin B and thiostrepton to modulate its DNA-binding activity and transcription of target genes. SAV4189 is the first reported exogenous antibiotic receptor among Streptomyces MFRs.

Keywords: MarR-family regulator; SAV4189; Streptomyces avermitilis; avermectins; sav_4190.

FIGURE 1

Effects of SAV4189 on avermectin production and growth in Streptomyces avermitilis. (A) Organization of sav_4189 and its adjacent genes. (B) Avermectin yield in WT, sav_4189 deletion mutant (D4189), complemented strain (C4189), and overexpression strain (O4189) cultured in FM-I. Avermectin yield contains total yield of all eight components A1a, A1b, A2a, A2b, B1a, B1b, B2a, and B2b. NS, not significant; ^∗∗P< 0.01 (comparison with WT by Student’s t-test). (C) Growth curves of WT, D4189, and O4189 cultured in FM-II. In this and subsequent figures, error bars represent SD of three replicates.

FIGURE 2

Transcriptional analysis of aveR, sav_4189, and sav_4190 by qRT-PCR. (A) Transcriptional profile of sav_4189 during the avermectin fermentation process in WT grown in FM-I. Value of sav_4189 on day 1 was defined as 1. (B) aveR, (C) sav_4189, and (D) sav_4190 transcription levels in WT, D4189, and O4189 grown in FM-I. Value of each gene was calculated relative to WT value on day 2, defined as 1. sav_4189: 91-bp transcript amplified from remainder sav_4189 ORF in D4189 with primers ZX9 and ZX10. ^∗P< 0.05, ^∗∗P< 0.01, ^∗∗∗P< 0.001.

FIGURE 3

Electrophoretic mobility shift assays (EMSAs) of SAV4189 binding to its own promoter region. (A) Schematic diagram of probes used for EMSAs. Probe 4189p: 148-bp DNA fragment, positions –145 to +3 relative to sav_4189 translational start codon. Probe aveRp: 501-bp DNA fragment, positions –476 to +25 relative to aveR translational start codon. (B) Interaction of His₆-SAV4189 with probes 4189p and aveRp. Negative probe: hrdB, 118-bp DNA fragment within hrdB ORF. Negative protein control: 750 nM BSA. 0.15 nM labeled probe was added in each reaction. Concentrations of His₆-SAV4189 for probes: for 4189p, 12.5, 25, 50, and 62.5 nM; for aveRp and hrdB, 125 and 250 nM. 62.5 nM His₆-SAV4189 was used for competition experiments (lanes +). Lanes –: EMSAs without His₆-SAV4189. Lanes N and S: competition assays with ∼100-fold excess of unlabeled non-specific competitor DNA hrdB (N) and specific probe 4189p (S). Arrows: free probes. Bracket: SAV4189-DNA complex.

FIGURE 4

sav_4189 promoter structure and SAV4189-binding sites. (A) Determination of sav_4189 TSS by 5′-RACE PCR. Box: complementary sequence of oligo(dT) anchor primer. Arrow: complementary base of TSS. (B) DNase I footprinting assay of SAV4189 on its own promoter region. Top fluorogram: reaction with 10 μM BSA (control). Protection patterns were acquired with increasing His₆-SAV4189 concentrations. (C) Nucleotide sequences of sav_4189 promoter region and SAV4189-binding sites. Numbers: distance (nt) from sav_4189 TSS. Shading: translational start codons. Bent arrow: sav_4189 TSS and transcription orientation. Boxes: potential –10 and –35 regions. Underlining: SAV4189-binding sites. Straight arrows: inverted repeats. (D) EMSAs of His₆-SAV4189 with probes A, B1, and B2. Each lane contained 0.3 nM labeled probe. Each probe was 59 bp. Non-specific DNA sequences from hrdB ORF (shaded) were fused with intact 13-bp palindromic sequences a, b1, and b2 (boldfaced) to generate probes A, B1, and B2, respectively. Lanes –: EMSAs without His₆-SAV4189. Lanes 2, 3, and 4: 62.5, 125, and 250 nM His₆-4189. (E) Consensus sequence analysis of SAV4189-binding sites by WebLogo program. Asterisks: consensus bases. Arrows: inverted repeats. Height of each letter is proportional to appearance frequency of corresponding base.

FIGURE 5

Comparison of the relative affinities of SAV4189 for different target sequences. (A) EMSA of His₆-SAV4189 with labeled probe A and unlabeled probes (A, B1, B2). (B) EMSA of His₆-SAV4189 with labeled probe B1 and unlabeled probes. (C) EMSA of His₆-SAV4189 with labeled probe B2 and unlabeled probes. 0.15 nM labeled probe and ∼50- and 150-fold amounts of unlabeled specific competitor probe were used in competition assays. SAV4189 concentration: 125 nM. Arrows: free labeled probes. Brackets: SAV4189-DNA complexes.

FIGURE 6

Confirmation of new SAV4189 target genes. EMSAs of His₆-SAV4189 with promoter regions of 14 predicted target genes. Each lane contained 0.15 nM labeled probe. Lanes –, EMSAs without His₆-SAV4189. Lanes 2–3 contained 200 and 400 nM His₆-SAV4189, respectively.

FIGURE 7

Avermectin yield in various S. avermitilis strains grown in FM-I for 10 days. (A) WT and sav_4190 deletion mutant strains D4190-1, -2, and -3. (B) Industrial strain A-144 and its derivatives (O4189/A-144: sav_4189 overexpression strain; D4190/A-144: sav_4190 deletion strain). ^∗∗P< 0.01.

FIGURE 8

Effect of sav_4189 overexpression on antibiotic production in S. coelicolor M145. S. coelicolor strains were grown on YBP agar at 28°C. O4189/M145: sav_4189 overexpression strain of M145. pKC1139/M145: M145 carrying control plasmid pKC1139.

FIGURE 9

Effects of ligands on SAV4189 DNA-binding activity. (A) EMSAs of His₆-SAV4189 with various antibiotics. 125 nM His₆-SAV4189 was used for Apr and Kan; 62.5 nM His₆-SAV4189 was used for other antibiotics. DMSO was used as solvent control. (B) Effects of HygB (10 μg/ml) and Thi (diluted in DMSO; final concentration 5 μg/ml) on transcription of SAV4189 target genes in vivo. Control: no antibiotic or DMSO added. DMSO: DMSO (but no antibiotic) added. Value of each gene was shown as fold change relative to control value, defined as 1. NS, not significant; ^∗∗∗P< 0.001.