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. 2016 Apr 19;7(2):e00523-16.
doi: 10.1128/mBio.00523-16.

Genome-Wide Chromatin Immunoprecipitation Sequencing Analysis Shows that WhiB Is a Transcription Factor That Cocontrols Its Regulon with WhiA To Initiate Developmental Cell Division in Streptomyces

Matthew J Bush  1 , Govind Chandra  2 , Maureen J Bibb  2 , Kim C Findlay  3 , Mark J Buttner  2
Affiliations

Genome-Wide Chromatin Immunoprecipitation Sequencing Analysis Shows that WhiB Is a Transcription Factor That Cocontrols Its Regulon with WhiA To Initiate Developmental Cell Division in Streptomyces

Matthew J Bush et al. mBio. .

Abstract

WhiB 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.

Importance: Despite the central importance of WhiB-like (Wbl) proteins in actinomycete biology, a conclusive demonstration of their biochemical function has been elusive, and they have been difficult to study, particularly in vitro, largely because they carry an oxygen-sensitive [4Fe-4S] cluster. Here we used genome-wide ChIP-seq to investigate the function of Streptomyces WhiB, 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 DNA in vivo. Our data provide the most compelling in vivo evidence 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.

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Figures

FIG 1
FIG 1
Deletion of whiB prevents sporulation. Shown are the phenotypes of wild-type S. venezuelae (WT), the constructed ΔwhiB::apr SV7 null mutant (ΔwhiB::apr), SV7 carrying the empty pMS82 vector (ΔwhiB::apr attBΦBT1::pMS82), and the complemented strain SV7/pIJ6761 (ΔwhiB::apr attBΦBT1::whiB). Strains were grown on MYM solid medium and photographed after 4 days.
FIG 2
FIG 2
whiA and whiB mutants have identical phenotypes. The data compare the phenotypes of wild-type S. venezuelae (WT), the ΔwhiA SV11 null mutant (ΔwhiA::apr) (12), the constructed ΔwhiB SV7 null mutant (ΔwhiB::apr), the ΔwhiA ΔwhiB SV51 double mutant (ΔwhiA ΔwhiB::apr), and the SV51/pIJ10604 complemented strain (ΔwhiA ΔwhiB::apr attBΦBT1::whiABcomp). Strains were examined by scanning electron microscopy (A), transmission electron microscopy (B), and fluorescence microscopy after staining DNA and the cell wall with 25 µg/ml propidium iodide (C) and 50 µg/ml wheat germ agglutinin (WGA) Alexa Fluor 488 (D), respectively. All of the hyphae shown are aerial hyphae. Strains were grown on MYM solid medium for 2 days before fluorescence microscopy and 4 days before electron microscopy. Scale bars are as indicated.
FIG 3
FIG 3
WhiA and WhiB have a shared regulon. The data compare anti-FLAG ChIP-seq results for WhiA and WhiB. ChIP traces are shown for 12 selected WhiA and WhiB target genes: ftsW, ftsZ, filP, whiG, sven1406, sven5313, sven3535, sven4724, sven1324, sven4756, sven6616, and sven3229. Color coding of the ChIP samples is as follows: 3×FLAG-[Gly4Ser]3-WhiB strain (WhiB-FLAG), red; corresponding S. venezuelae wild-type anti-FLAG negative control (WT), green; 3×FLAG-[Gly4Ser]3-WhiA strain (WhiA-FLAG), blue; and corresponding S. venezuelae wild-type anti-FLAG negative control (WT), purple. Plots span approximately 3 kb of DNA sequence. Genes running left to right are shown in green, and genes running right to left are shown in red. The black arrow indicates the gene subject to WhiA and WhiB regulation. The arrangement of the 12 panels mirrors that in Fig. 4.
FIG 4
FIG 4
WhiA and WhiB targets depend on both whiA and whiB for their expression. Data represent results of microarray transcriptional profiling for 12 selected WhiA and WhiB target genes (ftsW, ftsZ, filP, whiG, sven1406, sven5313, sven3535, sven4724, sven1324, sven4756, sven6616, and sven3229) during submerged sporulation in wild-type S. venezuelae (blue diamonds); the congenic whiA mutant, SV11 (red circles); and the congenic whiB mutant, SV7 (green triangles). In each panel, the x axis indicates the age of the culture in hours, and the y axis indicates the per-gene normalized transcript abundance (log2). For the wild type, 10 to 14 h corresponds to vegetative growth, 14 to 16 h corresponds to the onset of sporulation (fragmentation), and 16 h and beyond corresponds to sporulation. The arrangement of the 12 panels mirrors that in Fig. 3.
FIG 5
FIG 5
WhiB binding to its target promoters depends on WhiA and vice versa. ChIP-seq data for three representative WhiA and WhiB target genes, ftsW, ftsZ, and filP, are shown. (A) Anti-WhiA ChIP-seq in the presence and absence of WhiB. Color coding of the ChIP samples is as follows: S. venezuelae wild-type strain (WT), red; ΔwhiA negative control (ΔwhiA), blue; ΔwhiB strain (ΔwhiB), purple. (B) Anti-WhiB ChIP-seq in the presence and absence of WhiA. Color coding of the ChIP samples is as follows: S. venezuelae wild-type strain (WT), red; ΔwhiB negative control (ΔwhiB), blue; ΔwhiA strain (ΔwhiA), purple. Plots span approximately 3 kb of DNA sequence. Genes running right to left are shown in red. The black arrow indicates the gene subject to WhiA and WhiB regulation.
FIG 6
FIG 6
Mutation of the cysteine residues that coordinate the WhiB [4Fe-4S] cluster prevents DNA binding in vivo. Anti-FLAG ChIP-seq data for WhiB and WhiB (4C-S) are shown for three representative WhiA and WhiB target genes: ftsW, ftsZ, and filP. Color coding of the ChIP samples is as follows: 3×FLAG-[Gly4Ser]3-WhiB strain (WhiB-FLAG), red; 3×FLAG-[Gly4Ser]3-WhiB(4C-S) strain, green; corresponding S. venezuelae wild-type anti-FLAG negative control (WT), blue. Plots span approximately 3 kb of DNA sequence. Genes running right to left are shown in red. The black arrow indicates the gene subject to WhiA and WhiB regulation.
FIG 7
FIG 7
The regulatory network governing Streptomyces development. Flat-headed arrows indicate repression, and pointed arrows indicate activation. During vegetative growth, almost all of the genes of the core transcriptional regulatory cascade, including bldN, bldM, whiB, and whiG (upper red lines), are targets of BldD−(c-di-GMP)-mediated repression (3, 4, 47). BldD−(c-di-GMP) also represses genes encoding critical components of the cell division and chromosome segregation machineries required for sporulation septation, including FtsZ and SmeA-SffA (lower red lines). When the level of c-di-GMP perceived by BldD drops, BldD-mediated repression of almost the entire regulatory cascade is relieved. This allows σBldN to activate expression of BldM, which functions as a homodimer to activate expression of WhiB (33, 34). WhiAB proteins then coactivate key targets required for sporulation septation and chromosome segregation such as FtsZ, FtsW, and FtsK and the sporulation-specific sigma factor σWhiG that extends the regulatory cascade. σwhiG directs expression of WhiI and WhiH (58, 59). Finally, WhiI forms a heterodimer with BldM to activate the expression of genes required for spore maturation (34), including the smeA-sffA operon involved in chromosome segregation into spores (60) and the multigene whiE locus that specifies the synthesis of the spore pigment (61).
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