Abstract
ABSTRACTWhiB is the founding member of a family of proteins (the WhiB-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. In Streptomyces, 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) in Streptomyces venezuelae. In the first demonstration of in vivo genome-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 of whiA and whiB mutants. Using ChIP-seq, we demonstrated that in vivo DNA 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 WhiA in vivo.
Highlights
The complex life cycle of streptomycetes involves two distinct filamentous cell forms, the growing or vegetative hyphae and the reproductive or aerial hyphae, which differentiate into long chains of spores
The study of Streptomyces development has been hampered because the classical model species, S. coelicolor, sporulates only on solid medium and ~95% of the colony is found within the agar as vegetative mycelium, with the aerial hyphae representing only the remaining ~5% of the total biomass
Scanning electron microscopy (SEM) and DNA staining by propidium iodide (PI) showed that the S. venezuelae whiB mutant forms long, extended aerial hyphae that fail to lay down sporulation septa or to segregate their chromosomes (Fig. 2)
Summary
The complex life cycle of streptomycetes involves two distinct filamentous cell forms, the growing or vegetative hyphae and the reproductive or aerial hyphae, which differentiate into long chains of spores. The reproductive hyphae cease tip growth and undergo a massive cell division event involving the synchronous formation of dozens of sporulation septa that divide the multigenomic tip cell into a long chain of unigenomic prespore compartments [5, 6] These cylindrical compartments differentiate into rounded, mature spores. The cells keep growing, producing long aerial hyphae that lack sporulation septa and contain uncondensed DNA [7, 8] These identical phenotypes have led to the suggestion that WhiA and WhiB might function together to control a distinct pathway within the sporulation regulatory network [3, 7, 8]. This makes the detection of subtle changes in the transcriptome asso-
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