Characterization of rrdA, a TetR family protein gene involved in the regulation of secondary metabolism in Streptomyces coelicolor

Abstract

Streptomyces not only exhibits complex morphological differentiation but also produces a plethora of secondary metabolites, particularly antibiotics. To improve our general understanding of the complex network of undecylprodigiosin (Red) biosynthesis regulation, we used an in vivo transposition system to identify novel regulators that influence Red production in Streptomyces coelicolor M145. Using this screening system, we obtained 25 Red-deficient mutants. Twenty-four of these mutants had a transposon inserted in the previously described Red biosynthetic gene cluster and produced different amounts of another secondary metabolite, actinorhodin (Act). One mutant was shown to have an insertion in a different region of the chromosome upstream of the previously uncharacterized gene rrdA (regulator of redD, sco1104), which encodes a putative TetR family transcription factor. Compared with wild-type strain M145, the rrdA null mutant exhibited increased Red production and decreased Act production. A high level of rrdA expression resulted in a severe reduction in Red production and Act overproduction. Reverse transcription-PCR analysis showed that RrdA negatively regulated Red production by controlling redD mRNA abundance, while no change was observed at the transcript level of the Act-specific activator gene, actII-orf4. The effects on Act biosynthesis might arise from competition for precursors that are common to both pathways.

FIG. 1.

Phenotypes of Red-deficient mutants and locations of the insertions. (A) Locations of the mutations in the Red biosynthetic gene cluster. The black arrows indicate genes that are required for Red biosynthesis, while the open arrows indicate genes that are not essential. The gray arrows indicate the redD and redZ regulatory genes. The bent open arrows indicate mutations that influence only Red production, while the filled bent arrows indicate mutations that affect both Red production and Act production. The directions of the bent arrows indicate the transcript orientation of P_ermE in Tn315 in the mutants. (B) Red biosynthetic pathway. RedU is required for activation of the acyl carrier protein domains of RedO. The function of RedY is unknown (34). (C) Mutants and wild-type strain M145 were grown on YBP agar at 30°C for 2 days and 5 days. The bottoms of the plates are shown. WT, wild type. (D) Antibiotic production assay. Act production and Red production by the mutants and wild-type strain M145 were assayed in cultures grown in YBP liquid medium at 30°C for 88 h. The bars indicate the averages of three independent determinations, and the error bars indicate the standard errors. OD₅₃₀, optical density at 530 nm; OD640, optical density at 640 nm.

FIG. 2.

RrdA is highly conserved in Streptomyces. (A) rrdA (sco1104) locus in the S. coelicolor genome. The insertion site of transposon Tn315 in mutant K74 is indicated. The aac(3)-IV fragment (1.5 kb, conferring apramycin resistance) used to disrupt the rrdA gene is indicated below the map. (B) Multiple-amino-acid-sequence alignment of RrdA from S. coelicolor and the homologous proteins from S. avermitilis MA-4680 (SAV1503), S. ambofaciens ATCC 23877 (accession number CAJ90078.1), and Streptomyces sp. strain Mg1 (accession number EDX26764.1) obtained using BioEdit 7.0.0 (http://www.mbio.ncsu.edu/BioEdit/bioedit.html/).

FIG. 3.

Phenotypes of the rrdA null mutant and high-expression strain. The bottoms of the plates are shown. (A) Colonies of the parent strain S. coelicolor M145 (WT), the rrdA null mutant (ΔrrdA), the rrdA mutant harboring the empty vector pFDZ16 (ΔrrdA/pFDZ16), and the complemented strain (ΔrrdA/pFDZ16*-rrdA) were grown on YBP medium at 30°C for 2 days and 5 days. (B) Colonies of the parent strain M145 harboring the empty vector pFDZ16 (WT/pFDZ16) and the rrdA high-expression strain (WT/pFDZ16-rrdA) were grown on YBP medium at 30°C for 2 days and 5 days.

FIG. 4.

Antibiotic production by the M145 strain harboring the empty vector pFDZ16, the rrdA null mutant harboring the empty vector pFDZ16, the complemented strain, and the rrdA high-expression strain. Incubation was carried out in the YBP liquid medium at 30°C. The symbols indicate the averages of three independent determinations, and the error bars indicate the standard errors. OD₅₃₀, optical density at 530 nm; OD₆₄₀, optical density at 640 nm.

FIG. 5.

RT-PCR results for wild-type strain M145, the rrdA null mutant, the rrdA high-expression strain, and redD null mutant K68. (A) Transcriptional levels of the pathway-specific activators, including redD (primers Oxj201 and Oxj202, 368 bp), redZ (primers Oxj203 and Oxj204, 322 bp), actII-orf4 (primers Oxj205 and Oxj206, 320 bp), rrdA [primers Oxj211 and Oxj212x, 166 bp; the primer sequences are located in the upstream region of the insertion site of the aac(3)-IV fragment used for gene disruption], and hrdB (control) (primers Oxj237 and Oxj238, 207 bp). (B) RT-PCR results for the redD null mutant K68 and wild-type strain M145 for transcriptional detection of actII-orf4. WT, wild type.