Characterization and manipulation of the pathway-specific late regulator AlpW reveals Streptomyces ambofaciens as a new producer of Kinamycins

Abstract

The genome sequence of Streptomyces ambofaciens, a species known to produce the congocidine and spiramycin antibiotics, has revealed the presence of numerous gene clusters predicted to be involved in the biosynthesis of secondary metabolites. Among them, the type II polyketide synthase-encoding alp cluster was shown to be responsible for the biosynthesis of a compound with antibacterial activity. Here, by means of a deregulation approach, we gained access to workable amounts of the antibiotics for structure elucidation. These compounds, previously designated as alpomycin, were shown to be known members of kinamycin family of antibiotics. Indeed, a mutant lacking AlpW, a member of the TetR regulator family, was shown to constitutively produce kinamycins. Comparative transcriptional analyses showed that expression of alpV, the essential regulator gene required for activation of the biosynthetic genes, is strongly maintained during the stationary growth phase in the alpW mutant, a stage at which alpV transcripts and thereby transcripts of the biosynthetic genes normally drop off. Recombinant AlpW displayed DNA binding activity toward specific motifs in the promoter region of its own gene and that of alpV and alpZ. These recognition sequences are also targets for AlpZ, the γ-butyrolactone-like receptor involved in the regulation of the alp cluster. However, unlike that of AlpZ, the AlpW DNA-binding ability seemed to be insensitive to the signaling molecules controlling antibiotic biosynthesis. Together, the results presented in this study reveal S. ambofaciens to be a new producer of kinamycins and AlpW to be a key late repressor of the cellular control of kinamycin biosynthesis.

FIG. 1.

Amino acid sequence alignment of AlpW and characterized homologues from the protein databases. The DNA-binding and regulatory domains were assigned by comparison with the sequence of CprB, a homologue of known structure (34). The low-complexity region of AlpW is indicated.

FIG. 2.

Effect of alpW deletion on production of diffusible pigment and antibiotic activity. (A) Pigment (Pig) and antibiotic (Bio) syntheses were assessed on R2 plates over 7 days in the wild-type (WT) and alpW double deletion (ΔΔalpW) strains. The photos were taken from below the plate. Inhibition of B. subtilis growth was visualized by the dark halo surrounding the agar plug. (B) Antibiotic production assays in the complemented ΔΔalpW strain carrying the pSET152 derivative pSET-alpW in comparison with the WT/pSET152 and ΔΔalpW/pSET152 control strains (C) Growth curves of the WT and ΔΔalpW strains in R2 liquid cultures. (D) Bioactivity and pigment production associated with the liquid fermentation in panel C.

FIG. 3.

Effect of overexpression of alpW in the wild-type (WT) and ΔΔalpW strains. (A) Two clones of the WT and ΔΔalpW strains carrying the overexpression construct pIJW (designated pIJW1 and pIJW2) are shown (photos are taken from below for the WT strain and from above for the ΔΔalpW strain) after 8 days of growth at 30°C. −tsr, no addition of thiostrepton to the medium; +tsr, 12.5 μg/ml of thiostrepton included as an inducer. (B) The antibiotic production by the WT and ΔΔalpW strains was tested against B. subtilis from R2 culture plates supplemented with 50 ng/ml of thiostrepton. The control plug was taken from a noninoculated R2 plate supplemented with 50 ng/ml of thiostrepton. (C) ΔΔalpW strains carrying the overexpression plasmids pIB139 (control), pIBW, and pIBW-loop were assessed for pigment production (left) and antibiotic production (right). The picture (left) was taken from above after 5 days of incubation at 30°C. Agar plugs (right) were taken from plates inoculated with the ΔΔalpW strain containing pIB139, pIBW, or pIBW-loop after 5 days of incubation at 30°C. (D) Structures of the known kinamycins identified as metabolic products of the alp cluster.

FIG. 4.

Comparative transcriptional studies of selected alp genes in the wild-type and ΔΔalpW strains by reverse transcriptase PCR. Transcripts of hrdB (major and essential sigma factor gene; positive control), alpV (essential SARP activator gene), alpA (β-ketoacyl synthase gene involved in both orange pigment and kinamycin biosynthesis), alpZ (repressor gene), alpT (SARP gene), alpU (SARP gene), and alpW from the WT and alpW mutant strains were analyzed by RT-PCR with 25 cycles of PCR on cDNA generated from RNA isolated at various times during growth (represented by the curves shown in Fig. 2C). Negative (−) and positive (+) controls for the PCR are indicated. The bioactivity (Bio) against B. subtilis observed at each time point is shown below each lane.

FIG. 5.

DNA binding activity of AlpW examined by EMSA. (A) Twenty femtomoles of the 40-bp DIG-labeled oligonucleotide probe alpV-ARE encompassing the ARE sequence located in the alpV promoter region was incubated with increasing amounts of recombinant AlpW. (B) Effect of addition of unlabeled probe and concentrated culture supernatant extract containing the AlpZ-binding ligand (“Ligand extract”) on DNA-bound AlpW. AlpZ was used as a positive control to demonstrate the disrupting activity of the extract containing the AlpZ-binding ligand. This extract was obtained from late-transition-phase culture of the WT strain as described in reference . “R2 extract” corresponds to solvent extract of noninoculated R2 medium. Competition with excess of unlabeled probe (100×; 2 pmol) confirmed specificity of AlpW DNA binding activity.

FIG. 6.

Analysis of the additional low-complexity sequence in AlpW. (A) Superposition of the 3D structure representation of the native AlpW (yellow; monomer) and the homologous pseudoautoregulator receptor CprB (blue; dimer), the structure of which was determined by X-ray crystallography (34). (B) Comparative DNA binding analysis of AlpW and AlpW-loop by EMSA. The DIG-labeled probe alpVp (386 bp) bearing the AlpW recognition sequence AREV was incubated in the absence (lane 1) or presence of soluble extracts containing recombinant AlpW (lanes 2 and 4) or AlpW-loop (lanes 3 and 5). Concentrated extracts containing the AlpZ-binding ligand were added to the mixture (lanes 4 and 5).