BldG and SCO3548 interact antagonistically to control key developmental processes in Streptomyces coelicolor

Abstract

The similarity of BldG and the downstream coexpressed protein SCO3548 to anti-anti-sigma and anti-sigma factors, respectively, together with the phenotype of a bldG mutant, suggests that BldG and SCO3548 interact as part of a regulatory system to control both antibiotic production and morphological differentiation in Streptomyces coelicolor. A combination of bacterial two-hybrid, affinity purification, and far-Western analyses demonstrated that there was self-interaction of both BldG and SCO3548, as well as a direct interaction between the two proteins. Furthermore, a genetic complementation experiment demonstrated that SCO3548 antagonizes the function of BldG, similar to other anti-anti-sigma/anti-sigma factor pairs. It is therefore proposed that BldG and SCO3548 form a partner-switching pair that regulates the function of one or more sigma factors in S. coelicolor. The conservation of bldG and sco3548 in other streptomycetes demonstrates that this system is likely a key regulatory switch controlling developmental processes throughout the genus Streptomyces.

FIG. 1.

BldG-SCO3548 interaction was detected by bacterial two-hybrid analysis. The bldG (αασ) and sco3548 (ασ) ORFs were cloned into plasmids pKT25, pUT18, and pUT18C and were cotransformed into E. coli BTH101 in all possible combinations. Either two empty vectors (v) or one empty vector and one recombinant plasmid were used as negative controls. Plasmids containing leucine zipper fragments (zip) were used as the positive controls. The β-galactosidase activities of five individually isolated replicate liquid cultures of each cotransformant were determined by a microtiter plate assay using ONPG as a substrate. β-Galactosidase activity was expressed as (units per unit of optical density at 600 nm × ml of cell suspension × 10³), where 1 U was defined as 1 μmol of o-nitrophenol formed per min. The error bars indicate the standard deviation from the mean.

FIG. 2.

BldG-SCO3548 interaction was detected in vitro by affinity purification and far-Western analysis. (A) Cell lysate from E. coli BL21(DE3) containing pGEX-2TK (GST control protein) (lane 1) or containing pAU375 (GST-BldG) (lane 2) was mixed with cell lysate from E. coli BL21(DE3) containing pAU318 (His₁₀-SCO3548) at a ratio of 1:1 (vol/vol). Glutathione-Sepharose 4B beads were added to capture GST-containing protein complexes, which were then eluted with 20 mM reduced glutathione. Eluted proteins were analyzed by SDS-PAGE and Western analysis using anti-His antibody. (B) Cell lysates from S. coelicolor ΔbldG 1DB containing pIJ6902 (vector control) (lane 1) or pAU316 (bldG expression) (lane 2) were subjected to SDS-PAGE, and the separated proteins were transferred to a PVDF membrane. For far-Western analysis, the membrane was incubated with purified GST-SCO3548, washed thoroughly, and probed with anti-GST antibody. The positions of molecular mass markers are indicated on the left.

FIG. 3.

Inducible coexpression of BldG and SCO3548 does not complement the S. coelicolor bldG mutant phenotype. (A) S. coelicolor ΔbldG 1DB containing pIJ6902 (vector control), pAU316 (bldG expression), or pAU365 (bldG-sco3548 coexpression) was grown on R2YE agar containing 0 or 100 μg/ml thiostrepton (Thio) at 30°C for 40 h. Crude cell lysates were prepared and examined by SDS-PAGE, followed by Western analysis with anti-BldG antibody. (B) S. coelicolor ΔbldG 1DB containing pIJ6902 (top left region of each plate), pAU316 (top right region), or pAU365 (bottom center region) was grown at 30°C on R2YE agar containing 0, 10, 50, or 100 μg/ml thiostrepton and photographed after 80 h.

FIG. 4.

BldG and SCO3548 self-interactions were detected by bacterial two-hybrid analyses. The bldG ORF (αασ) (A) or the sco3548 ORF (ασ) (B) was cloned into plasmids pKT25, pUT18, and pUT18C, which were cotransformed into E. coli BTH101 in all possible combinations. Either two empty vectors (v) or one empty vector and one recombinant plasmid were used as negative controls. Plasmids containing leucine zipper fragments (zip) were used as the positive control. The β-galactosidase activities of five individually isolated replicate liquid cultures of each cotransformant were determined by a microtiter plate assay using ONPG as a substrate, as described in the legend to Fig. 1.

FIG. 5.

BldG and SCO3548 self-interactions were detected by far-Western analysis and affinity purification, respectively. (A) For far-Western analysis, cell lysates from S. coelicolor ΔbldG 1DB containing pIJ6902 (vector control) (lane 1) or pAU316 (bldG expression) (lane 2) were subjected to SDS-PAGE, and the separated proteins were transferred to a PVDF membrane. The membrane was incubated with purified GST-BldG, washed thoroughly, and probed with anti-GST antibody. (B) For affinity purification, cell lysate from E. coli BL21(DE3) containing pET30(a)⁺ (vector control) (lane 1) or containing pAU318 (His₁₀-SCO3548) (lane 2) was mixed with cell lysate from E. coli BL21(DE3) containing pAU376 (GST-SCO3548) at a ratio of 1:1 (vol/vol). Ni-NTA agarose was added to capture His₁₀-containing protein complexes, which were then eluted with 250 mM imidazole. Eluted proteins were analyzed by SDS-PAGE and Western analysis using anti-GST antibody. The positions of molecular mass markers are indicated on the left.