Multi‐layered inhibition of Streptomyces development: BldO is a dedicated repressor of whiB

Summary BldD‐(c‐di‐GMP) sits on top of the regulatory network that controls differentiation in Streptomyces, repressing a large regulon of developmental genes when the bacteria are growing vegetatively. In this way, BldD functions as an inhibitor that blocks the initiation of sporulation. Here, we report the identification and characterisation of BldO, an additional developmental repressor that acts to sustain vegetative growth and prevent entry into sporulation. However, unlike the pleiotropic regulator BldD, we show that BldO functions as the dedicated repressor of a single key target gene, whiB, and that deletion of bldO or constitutive expression of whiB is sufficient to induce precocious hypersporulation.

Movie S1 Time-lapse microscopy of the wild type strain carrying the FtsZ-YPet fusion. DIC (A) and YFPchannel (B) movies are at 5 frames per second. The time following the first image is indicated at the bottom left. Images were taken every 1 hour (DIC 150 ms; YFP 350 ms). Movies were assembled in the Fiji software package (http://fiji.sc/Fiji).
Movie S2 Time-lapse microscopy of the bldO mutant strain carrying the FtsZ-YPet fusion. DIC (A) and YFPchannel (B) movies are at 5 frames per second. The time following the first image is indicated at the top left. Images were taken every 20 mins (DIC 150 ms; YFP 350 ms). Movies were assembled in the Fiji software package (http://fiji.sc/Fiji).
Movie S3 Time-lapse microscopy of the complemented bldO mutant strain carrying the FtsZ-YPet fusion. DIC (A) and YFP-channel (B) movies are at 5 frames per second. The time following the first image is indicated at the bottom left. Images were taken every 1 hour for first 8 hours and every 20 mins subsequently (DIC 150 ms; YFP 350 ms). Movies were assembled in the Fiji software package (http://fiji.sc/Fiji).

Text S1 -Supplementary Experimental Procedures
Chromatin immunoprecipitation, library construction, sequencing and ChIP-seq data analysis. ChIP was conducted using M2 gel suspension (Sigma-Aldrich A2220) as described previously (Bush et al., 2013) for the strains S. venezuelae ATCC 10712 and SV52-pIJ10613 (∆bldO::apr attBΦBT1::bldO-3xFLAG - Table   S1) except that cultures were grown in DNB liquid medium. Library construction and sequencing were performed as described previously (Bush et al., 2013) by The Earlham Institute, Norwich Research Park Norwich, UK. ChIP-seq data analysis was conducted as described previously (Bush et al., 2013), except that here for both the "diff" and "apv" values the corresponding value for the wild-type (WT) negative control was subtracted from the initial value to arrive at a final value. (Qiagen), followed by an additional DNase I treatment (Turbo DNA-free, Ambion) until they were free of DNA contamination (determined by PCR amplification of hrdB). RNA was quantified and equal amounts (500 ng) of total RNA from each sample was converted to cDNA using SuperScript II reverse transcriptase and random primers (Invitrogen). cDNA was then used as template in qRT-PCR performed using the SensiFAST SYBR No-ROX kit (Bioline). Three technical replicates were used for each gene. Specific qPCR primers (Table S1, final concentration of 250 nM) were used to amplify the whiB target gene (whiBqRT_F and whiBqRT_R) and the hrdB reference gene (hrdBqRT_F and hrdBqRT_R). To normalize for differing primer efficiency, a standard curve was constructed using chromosomal DNA. Melting curve analysis was used to confirm the production of a specific single product from each primer pair. qRT-PCR was performed using a CFX96 Touch instrument using hardshell white PCR plates (BioRad), sealed with thermostable film covers (Thermo). PCR products were detected with SYBR green fluorescent dye and amplified according to the following protocol: 95°C, 3 min, then 45 cycles at 95°C 5 sec, 60°C 10 sec and 72°C 7 sec. Melting curves were generated at 65 to 95°C with 0.5°C increments. The experiments (including RNA extraction) was repeated once independently. The BioRad CFX manager software was used to calculate starting quantity (SQ) values for whiB at each time point. These values were divided by the mean SQ value derived from the hrdB reference at the corresponding time points, generating a value for relative expression. The resulting values were normalised against the mean relative expression of the wild type at 10 hours, which was set to 1. The resulting normalised relative expression is reported in Figure 8.

DNase I Footprinting.
DNase I footprinting experiments to study BldO binding to the whiB promoter were carried out essentially as described previously (Bush et al., 2103) and according to the description supplied with the Sure Track footprinting kit (Amersham Pharmacia Biotech). DNA probes containing the whiB2p promoter were prepared by PCR using the primers whiB2p_F and whiB2p_R (Table S1). To study binding of BldO to the forward strand, whiB2p_F was first radiolabelled and to study binding to the reverse strand, whiB2p_R was first radiolabelled.
Western Blotting. Samples of frozen mycelium, originating from 5 ml liquid DNB cultures, were resuspended in 0.4 ml ice-cold sonication buffer [20 mM Tris pH 8.0, 5 mM EDTA, 1 x EDTA-free protease inhibitors (Roche)] and sonicated (5x 15 sec on/15 sec off) at 4.5 micron amplitude. Lysates were then centrifuged at 16,000 xg for 15 min at 4˚C to remove cell debris. Total protein concentration was determined using the Bradford assay (Biorad). 2.5 µg of total protein from each time point was loaded in triplicate into a microplate (proteinsimple #043-165) and anti-FLAG antibody (Sigma F4725) diluted 1:100. BldO-3xFLAG levels, originating from the SV52-pIJ10616 strain and the wild type negative control (Table S1) were then assayed using the automated Western blotting machine Wes (ProteinSimple, San Jose, CA), according to the manufacturer's guidelines.
Scanning electron microscopy. Colonies were mounted on the surface of an aluminum stub with optimal cutting temperature compound (Agar Scientific Ltd, Essex, UK), plunged into liquid nitrogen slush at approximately -210°C to cryopreserve the material, and transferred to the cryostage of an Alto 2500 cryotransfer system (Gatan, Oxford, England) attached to a Zeiss Supra 55 VP field emission gun scanning electron microscope (Carl Zeiss Ltd, Germany). The surface frost was sublimated at -95°C for 3 min before the sample was sputter coated with platinum for 2 min at 10 mA at below -110°C. Finally, the sample was moved onto the cryostage in the main chamber of the microscope, held at approximately -130°C, and viewed at 1.2 to 5.0 kV.