Illumina HiSeq 2000 sequencing; Mutation of the cysteine residues that coordinate the WhiB [4Fe-4S] cluster prevents in vivo DNA binding by both WhiB and WhiA
Mutation of the cysteine residues that coordinate the WhiB [4Fe-4S] cluster prevents in vivo DNA binding by both WhiB and WhiA
Streptomyces venezuelae strain NRRL B-65442 (NCBI Ref: NZ_CP018074.1) was used as the wild type strain. The WhiB and WhiA deletion strains were made using 'Redirect' PCR targeting (Gust et al., 2003; Gust et al., 2004), to replace the genes with a single apramycin resistance (apr) cassette in the wild type Streptomyces venezuelae strain. The Δwhi[AB]::apr locus was transduced back into wild type S. venezuelae using the transducing phage SV1 (Stuttard et al., 1982)to generate the deletion strains. Mutant alleles of whiB and 3xFLAG-[Gly4Ser]3-whiB were synthesized in which all four cysteine TGC codons were altered either to serine TGC codons (GenScript). These alleles were subcloned into pMS82 using the primer pair whiBFLAGnes_F/R to create the plasmids pIJ10607, pIJ10608, pIJ10609 and pIJ10610. Plasmids were introduced into the ΔwhiB::apr mutant SV7 by conjugation. The 3xFLAG-[Gly4Ser]3-whiB variant plasmid pIJ10610 was also introduced into the ΔwhiA ΔwhiB::apr double mutant SV51. Additionally, a plasmid (pIJ10611), based on pIJ10750 was constructed to encode 3xFLAG-[Gly4Ser]3-WhiA driven from its own promoter (using the primer pair whiAcomp_82MCS_F/R and pIJ10601 as a template) and variant WhiB driven by its own promoter (using the primer pair whiBcomp_82MCS_F/R and pIJ10608 as a template). This was then introduced into the ΔwhiA ΔwhiB::apr double mutant SV51. Formaldehyde was added to cultures at a final concentration of 1% (vol/vol), and incubation was continued for 30 min. Glycine was then added to a final concentration of 125 mM to stop the cross-linking. The samples were left at room temperature for 5 min and washed twice in phosphate-buffered saline (PBS) buffer (pH 7.4). The pellets were resuspended in 0.5 ml of lysis buffer (10 mM Tris-Cl, pH 8.0, 50 mM NaCl), containing 15 mg/ml of lysozyme and 1x protease inhibitor (Roche Applied Science) and incubated at 37 degree C for 25 min or until lysed. Subsequently, 0.5 ml immunoprecipitation (IP) buffer (50 mM Tris-Cl, pH 8.0, 250 mM NaCl, 0.8% [vol/vol] Triton X-100), containing 1x protease inhibitor, was added, and the samples were chilled on ice. The samples were sonicated for six cycles of 20 s each at 10 micrometer to shear chromosomal DNA into fragments ranging from 300 to 1,000 bp on average. The samples were centrifuged twice at 16,000g at 4 degree C for 10 min to clear the cell extracts. For total DNA extraction, 50 microlitres of the cell extracts were incubated with 0.2 mg/ml proteinase K (Roche) for 2 h at 55 degree C, then extracted twice with phenol-chloroform and once with chloroform and further purified using QiaQuick columns (Qiagen). DNA was eluted in 50 microlitres EB buffer (10 mM Tris-Cl, pH 8.5) and quantified using a NanoDrop spectrophotometer (Thermo Scientific).  Library construction and sequencing were performed by The Genome Analysis Centre (TGAC), Norwich Research Park, Norwich, United Kingdom. The TruSeq ChIP sample preparation kit from Illumina Inc. (part 15023092, revision A, August 2012) was used according to the recommended protocol. In brief, 5 to 10 ng ChIP DNA is blunt ended and phosphorylated, and a single A nucleotide is added to the 3 ends of the fragments in preparation for ligation to an adapter with a single-base T overhang. The ligation products are purified and accurately size selected by agarose gel electrophoresis. Size-selected DNA is then purified and PCR amplified to enrich for fragments that have adapters on both ends. The final purified product is then quantified using a combination of Bioanalyzer DNA HS Chip from Agilent Technology 2100 Bioanalyzer and Qubit 2.0 from Invitrogen. The libraries were normalized to 10 nM and pooled, and quantitative PCR (q-PCR) was performed prior to cluster generation.