Response regulator heterodimer formation controls a key stage in Streptomyces development

Abstract

The orphan, atypical response regulators BldM and WhiI each play critical roles in Streptomyces differentiation. BldM is required for the formation of aerial hyphae, and WhiI is required for the differentiation of these reproductive structures into mature spores. To gain insight into BldM function, we defined the genome-wide BldM regulon using ChIP-Seq and transcriptional profiling. BldM target genes clustered into two groups based on their whi gene dependency. Expression of Group I genes depended on bldM but was independent of all the whi genes, and biochemical experiments showed that Group I promoters were controlled by a BldM homodimer. In contrast, Group II genes were expressed later than Group I genes and their expression depended not only on bldM but also on whiI and whiG (encoding the sigma factor that activates whiI). Additional ChIP-Seq analysis showed that BldM Group II genes were also direct targets of WhiI and that in vivo binding of WhiI to these promoters depended on BldM and vice versa. We go on to demonstrate that BldM and WhiI form a functional heterodimer that controls Group II promoters, serving to integrate signals from two distinct developmental pathways. The BldM-WhiI system thus exemplifies the potential of response regulator heterodimer formation as a mechanism to expand the signaling capabilities of bacterial cells.

Figure 1. Microarray transcription profiles of (A) Group-I and (B) Group-II genes in wild-type S. venezuelae during submerged sporulation and in congenic ΔbldM, ΔwhiA, ΔwhiB, ΔwhiD, ΔwhiG, ΔwhiH and ΔwhiI null mutants grown under identical conditions.

RNA samples were prepared at 2-hour intervals from 8 to 20 hours, by which time sporulation was nearing completion. For 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). Group-I genes (blue) depend on bldM but are independent of all the whi genes. Group-II genes (red) depend on bldM but also depend whiG and whiI. The average expression profile is indicated by the black line. Group I genes are activated at least two hours earlier than Group-II genes (see insets). Red bars indicate vegetative growth (V), fragmentation of mycelium (F), and spore formation (S).

Figure 2. BldM binds to the MEME-predicted Group-I consensus sequence.

A. The MEME-predicted palindromic Group-I consensus sequence. The height of the letters in the sequence logo, in bits, is proportional to the frequency of the A, C, T, or G nucleotides at each position of the motif. B. DNase I footprinting analysis of BldM on two Group-I promoters: sven1998 and sven4150. The BldM protein concentrations used were 0, 100, 250 and 500 nM for sven1998 and 0, 250 and 500 nM for sven4150. G+A indicates the Maxam and Gilbert sequence ladder. The protected regions are indicated by dotted lines and the positions of the MEME-predicted Group I binding motif are shown.

Figure 3. BldM and WhiI ChIP-Seq data for representative Group-I and Group-II promoters analysed in wild-type, ΔbldM and ΔwhiI backgrounds.

Only BldM binds at Group-I promoters and this occurs both in the wild type and in the ΔwhiI mutant. Both BldM and WhiI bind at Group-II promoters in the wild type, but BldM binding depends on WhiI and vice versa. The genes shown in red are the targets genes.

Figure 4. Bacterial two-hybrid analysis of BldM-WhiI interaction.

The listed pairs of constructs were transferred into the BACTH reporter strain E. coli BTH101 by transformation. The resulting transformants were selected on MacConkey-maltose agar containing 100 µg/ml carbenicillin and 50 µg/ml kanamycin and incubated at 30°C for two days before four independent clones were picked and subjected to β-galactosidase assays. Error bars show the standard error of the four replicates carried out for each pairwise combination.

Figure 5. Affinity purification of the BldM-WhiI complex.

N-terminally His₆-tagged BldM and either (A) untagged WhiI, or (B) N-terminally S_II-tagged WhiI, were co-expressed from the pETDuet-1 vector. In both cases, co-expression of BldM solubilized WhiI, which formed inclusion bodies when expressed alone. The His₆-BldMWhiI complex in (A) was purified over a HisTrap Ni affinity column, and the His₆-BldM-S_II-whiI complex in (B) was purified over consecutive HisTrap Ni and StrepTrap HP affinity columns.

Figure 6. BldM-WhiI binds to the MEME-predicted Group-II consensus sequence.

A. The MEME-predicted non-palindromic Group-II consensus sequence. The height of the letters in the sequence logo, in bits, is proportional to the frequency of the A, C, T, or G nucleotides at each position of the motif. B. DNase I footprinting analysis of BldM-WhiI, BldM and GST-WhiI binding to two Group-II promoters (sven1263 and murA2). The protein concentrations used were 100 and 200 nM. G+A indicates the Maxam and Gilbert sequence ladder. The protected regions are indicated by dotted lines and the positions of the MEME-predicted binding motifs are shown.

Figure 7. Schematic representation summarizing the regulatory network involved in controlling BldM and WhiI expression and the activation of Group-I and Group-II genes.

During vegetative growth, BldD represses bldC, bldN, whiG and bldM. At the onset of differentiation, this repression is relieved, σ^BldN activates bldM and σ^WhiG activates whiI. BldM homodimer activates early sporulation genes such as whiB and ssgR. Subsequently, BldM and WhiI form a heterodimer that activates late sporulation genes such as whiE and smeA-sffA. Thus heterodimer formation serves to integrate signals from two independent pathways (σ^WhiG-WhiI and σ^BldN-BldM). bldM expression is tightly controlled by at least four global regulators - BldD, BldC, σ^BldN and PhoP (a response regulator activated during phosphate depletion [59]).