We have shown that the bldD gene of Streptomyces coelicolor, while required for antibiotic production and morphological differentiation, is not essential for viability. We have also demonstrated that BldD forms a higher order complex both in solution and when bound to target DNA. Purified BldD exists in three forms in solution, as a tetramer, dimer and monomer, but only in the dimeric form when bound to its own promoter/operator.
The life cycle of the filamentous bacterium Streptomyces coelicolor involves a series of metabolically and structurally distinct states. The life cycle begins with germination of a spore and the growth of a mat of vegetative, substrate mycelium that later undergoes both physiological differentiation to an antibiotic-producing state, and morphological differentiation to erect spore-forming, aerial hyphae. Antibiotic production in the substrate mycelium and the formation of aerial hyphae coincide during colony development and this, together with the existence of mutations that block both of these developmental processes, suggests that they share regulatory elements. Among the so-called bld (for ‘bald’, lacking aerial hyphae) mutants with pleiotropic defects in both antibiotic production and morphological differentiation is the single, known bldD mutant . On minimal medium containing glucose as carbon source, the bldD53 mutant produces none of the four well-characterized antibiotics known to be produced by S. coelicolor, and the colonies have a fragmented surface lacking aerial structures. As is the case for many of the bld mutants, the morphological defect, but not the loss of antibiotic production, is overcome by growth on minimal medium containing mannitol as carbon source.
The bldD gene encodes a 167 amino acid protein with a deduced molecular mass of 18 167 Da. The only known mutant allele has an A→G point mutation changing a tyrosine residue at amino acid 62 to a cysteine . The biochemical role for BldD has been addressed using electrophoretic mobility shift assays in which purified BldD was shown to be a DNA binding protein capable of binding its own promoter/operator, as well as the promoter/operators of several key developmental genes , including the sporulation-specific sigma factor, σWhiG, and the extracytoplasmic function sigma factor, σBldN. Independent studies showed that a promoter/operator fragment from the S. coelicolor sigH gene, encoding the stress response sigma factor, σH, could also be retarded in electrophoretic mobility shift assays using extracts from a bldD+ but not a bldD− strain, and BldD was later shown to be the sigH promoter binding factor. The use of DNase I footprinting analysis to delimit the DNA binding site for some of the BldD target genes suggested the imperfect inverted repeat, AGTgA (n)m TCACc, as the consensus sequence for BldD binding , and the location of the binding site, together with a comparison of the putative bldD target gene transcript levels in the wild-type and bldD mutant strains, were consistent with BldD functioning as a repressor during vegetative growth. How BldD repression of its various target genes might be relieved during development is currently unknown. To gain a further understanding of how BldD exerts its regulatory effects, we have constructed a chromosomal deletion of the bldD open reading frame (ORF) in S. coelicolor M600, and we have begun to address how BldD binds to its target DNAs.
2Materials and methods
2.1Bacterial strains, plasmids and culture conditions
The strains used in this work are listed in Table 1. The plasmids used in this work are listed in Table 2. For routine purposes, Escherichia coli strains were grown in Luria–Bertani (LB) medium at 37°C and S. coelicolor was grown at 30°C on R2YE agar . Ampicillin (Sigma), apralan (50% apramycin (wt/wt), Provel), and spectinomycin (Sigma) were all used at 100 μg ml−1, and kanamycin (Sigma) and chloramphenicol (Sigma) were used at 50 and 25 μg ml−1, respectively.
BldD was overexpressed as an amino-terminal His6 fusion protein as previously described . The His6-tagged BldD was initially purified using nickel affinity chromatography  and then subjected to a further round of purification by ion exchange chromatography. The nickel affinity-purified His6-tagged BldD protein was applied to a Pharmacia Mono Q HR 5/5 ion exchange column equilibrated with 20 mM Tris–HCl (pH 8.5) and then eluted with a linear gradient of 0–0.5 M NaCl. The BldD-containing fractions were then analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and BldD was judged to be >99% pure (not shown). The BldD-containing fractions were pooled, dialyzed against 20 mM Tris–HCl (pH 8.5), and then applied to a Pharmacia Superose 12 HR 10.30 gel filtration column that had been equilibrated in the same buffer. The elution profile of BldD was then compared to the profile of protein standards (bovine serum albumin (66 kDa), β-lactamase (29.5 kDa) and cytochrome c (12 kDa)) run under the same conditions.
BldD with an amino-terminal glutathione S transferase (GST) tag was overexpressed as previously described . The soluble protein fraction was purified by affinity chromatography using glutathione agarose (Sigma) . The column was washed with 1×phosphate buffered saline (PBS) and the protein was eluted with reduced glutathione (10 mM, Sigma). The purity of the GST-BldD fusion protein was assessed by SDS–PAGE analysis of aliquots from each fraction. In order to concentrate the protein and to replace the column buffer with 20 mM Tris–HCl (pH 8.5) buffer containing 150 mM NaCl, those fractions judged to be >99% pure after Coomassie staining were subjected to centrifugation in an Ultrafree®-4 Centrifugal Filter (Millipore).
2.4Electrophoretic mobility shift assay
Electrophoretic mobility shift assays were performed using the 77 bp MAE16-4 probe fragment spanning nucleotides −31–+40 of the bldD promoter as described previously . 0–40 pmol of His6-BldD and/or GST-BldD and 1–2 ng (20–40 fmol) of end-labeled MAE16-4 probe were added to each 20 μl reaction, and after incubation at 30°C the bound and free DNA products were separated on a 1.5% glycerol-containing, 8% polyacrylamide gel. Gels were dried and exposed to a phosphor screen before analysis using a phosphorimager (Molecular Dynamics model 445 SI).
3Results and discussion
3.1Constructed bldD null mutants have the same phenotype as the bldD point mutant
3.2BldD exists mainly as a dimer in solution and as a dimer when bound to its own promoter
Since we had previously predicted that BldD binds to an inverted repeat sequence in its own promoter/operator region , it seemed reasonable to suggest that it might bind DNA in a dimeric form with one monomer interacting with each half site. To address this possibility, the oligomeric state of BldD was examined both in solution and when bound to a fragment of DNA encompassing the known BldD binding site  in its own promoter/operator region.
In order to determine the oligomeric state in solution, BldD was overexpressed as an amino-terminal His6 fusion protein  and purified as described in Section 2. BldD judged to be >99% pure was then subjected to size exclusion chromatography and its elution profile was compared to the profile of protein standards run under the same conditions (Fig. 3). BldD eluted in three peaks which, by comparison to the elution profiles of the standards, were believed to correspond to tetrameric, dimeric and monomeric forms of the protein. In two separate gel filtration experiments, most of the BldD protein was in the dimer form, as has been found for many transcription factors (reviewed in ), and estimation of the areas under the peaks allowed us to determine that the ratio of tetramer:dimer:monomer was approximately 2:7:1. Very recently, gel filtration chromatography also suggested that BldB, a small transcription factor that appears to regulate its own synthesis , dimerizes in solution .
In order to determine the oligomeric state of BldD when bound to DNA, an electrophoretic mobility shift assay was conducted in which both the His6-BldD (described above), as well as GST-BldD (see Section 2), were employed. Electrophoretic mobility shift assays were performed using the 77 bp MAE16-4 probe . As had been seen previously for the His6-BldD , two shifted bands were observed both in the presence of His6-BldD and GST-BldD (Fig. 4). Since the GST-BldD was significantly larger than the His6-BldD, the GST-BldD protein-bound DNA was less mobile in the gel. When equal amounts of the two fusion proteins were mixed together and incubated with the end-labeled probe, the two bands corresponding to the GST-BldD-bound DNA, and the two bands corresponding to the His6-BldD-bound DNA were seen. In addition, a protein-bound fragment of intermediate mobility was also observed. Since no intermediate fragment would be expected if BldD bound as a monomer, and more than one intermediate fragment would be expected if BldD bound as a tetramer (for example, intermediates would include [His6-BldD][GST-BldD]3; [His6-BldD]2[GST-BldD]2; [His6-BldD]3[GST-BldD]), the intermediate fragment most likely represents binding by a [His6-BldD][GST-BldD] heterodimer.
Although our findings suggest that BldD exists primarily as a dimer in solution and that it is also in the dimeric form once bound to DNA, we cannot conclude with certainty that it is the dimer form of BldD that makes initial contact with its target DNA. The presence of a small fraction of the monomer in solution leaves open the possibility that BldD binding in vivo occurs via the monomer pathway  in which two monomers could bind sequentially to each of the half sites and then assemble their dimerization interface when bound to the DNA. Although use of the monomer pathway has been shown to increase the specificity of protein:DNA complex formation , it seems unlikely that BldD follows the monomer pathway for binding to its target promoters since no binding was observed when the left half site fragment of the bldD promoter/operator was separated from the right half site and used as a probe in gel mobility shift experiments .
This work was supported by the Alberta Heritage Foundation for Medical Research and the Natural Sciences and Engineering Research Council of Canada. We are grateful to Mark Glover, University of Alberta, for helpful discussions about the electrophoretic mobility shift assays, to Plant Bioscience Ltd. for the gift of the REDIRECT constructs and strains, and to Justin Nodwell for the pSETΩ.