Structural basis of dual activation of cell division by the actinobacterial transcription factors WhiA and WhiB

Significance

This work provides unique insight into the evolution and function of two highly unusual transcription factors, WhiA and WhiB, which control sporulation septation in the antibiotic-producing bacteria Streptomyces. Furthermore, in the closely related pathogen Mycobacterium tuberculosis ( Mtb), WhiA and WhiB are essential because they are required for cell division, and mutations in whiA and whiB are positively selected in clinical isolates of Mtb, implicating WhiA/B in virulence and/or resistance to drug treatment. Our work will thus provide functional insight into the molecular mechanism of a new class of transcription factors in an important bacterial clade that contains the major antibiotic producers while informing on the adaptive nature of WhiA/B mutations in virulence and antibiotic resistance in a deadly pathogen.

Abstract

Studies of transcriptional initiation in different bacterial clades reveal diverse molecular mechanisms regulating this first step in gene expression. The WhiA and WhiB factors are both required to express cell division genes in Actinobacteria and are essential in notable pathogens such as Mycobacterium tuberculosis. The WhiA/B regulons and binding sites have been elucidated in Streptomyces venezuelae ( Sven), where they coordinate to activate sporulation septation. However, how these factors cooperate at the molecular level is not understood. Here we present cryoelectron microscopy structures of Sven transcriptional regulatory complexes comprising RNA polymerase (RNAP) σ ^A-holoenzyme and WhiA and WhiB, in complex with the WhiA/B target promoter sepX. These structures reveal that WhiB binds to domain 4 of σ ^A (σ ^A ₄) of the σ ^A-holoenzyme, bridging an interaction with WhiA while making non-specific contacts with the DNA upstream of the −35 core promoter element. The N-terminal homing endonuclease-like domain of WhiA interacts with WhiB, while the WhiA C-terminal domain (WhiA-CTD) makes base-specific contacts with the conserved WhiA GACAC motif. Notably, the structure of the WhiA-CTD and its interactions with the WhiA motif are strikingly similar to those observed between σ ^A ₄ housekeeping σ-factors and the −35 promoter element, suggesting an evolutionary relationship. Structure-guided mutagenesis designed to disrupt these protein–DNA interactions reduces or abolishes developmental cell division in Sven, confirming their significance. Finally, we compare the architecture of the WhiA/B σ ^A-holoenzyme promoter complex with the unrelated but model CAP Class I and Class II complexes, showing that WhiA/WhiB represent a new mechanism in bacterial transcriptional activation.