ABSTRACTWhiB is the founding member of a family of proteins (theWhiB-like [Wbl] family) that carry a [4Fe-4S] iron-sulfur cluster and play key roles in diverse aspects of the biology of actinomycetes, including pathogenesis, antibiotic resistance, and the control of development. InStreptomyces, WhiB is essential for the process of developmentally controlled cell division that leads to sporulation. The biochemical function of Wbl proteins has been controversial; here, we set out to determine unambiguously if WhiB functions as a transcription factor using chromatin immunoprecipitation sequencing (ChIP-seq) inStreptomyces venezuelae. In the first demonstration ofin vivogenome-wide Wbl binding, we showed that WhiB regulates the expression of key genes required for sporulation by binding upstream of ~240 transcription units. Strikingly, the WhiB regulon is identical to the previously characterized WhiA regulon, providing an explanation for the identical phenotypes ofwhiAandwhiBmutants. Using ChIP-seq, we demonstrated thatin vivoDNA binding by WhiA depends on WhiB and vice versa, showing that WhiA and WhiB function cooperatively to control expression of a common set of WhiAB target genes. Finally, we show that mutation of the cysteine residues that coordinate the [4Fe-4S] cluster in WhiB prevents DNA binding by both WhiB and WhiAin vivo.IMPORTANCEDespite the central importance ofWhiB-like (Wbl) proteins in actinomycete biology, a conclusive demonstration of their biochemical function has been elusive, and they have been difficult to study, particularlyin vitro, largely because they carry an oxygen-sensitive [4Fe-4S] cluster. Here we used genome-wide ChIP-seq to investigate the function ofStreptomycesWhiB, the founding member of the Wbl family. The advantage of this approach is that the oxygen sensitivity of the [4Fe-4S] cluster becomes irrelevant once the protein has been cross-linked to DNAin vivo. Our data provide the most compellingin vivoevidence to date that WhiB, and, by extension, probably all Wbl proteins, function as transcription factors. Further, we show that WhiB does not act independently but rather coregulates its regulon of sporulation genes with a partner transcription factor, WhiA.
Genome-Wide Chromatin Immunoprecipitation Sequencing Analysis Shows that WhiB Is a Transcription Factor That Cocontrols Its Regulon with WhiA To Initiate Developmental Cell Division inStreptomyces
