WhiB6 Transduces Contact‐Dependent Signaling in Mycobacterium smegmati s and Coordinately Induces Both ESX‐1 and ESX‐4

ABSTRACT Bacteria have evolved complex conditional pathways that respond to environmental stresses and signals. We use conjugation in Mycobacterium smegmatis to identify contact‐recognition and response pathways that mediate interactions between donor and recipient cells. Contact with a compatible donor cell initiates a response in the recipient that requires the ESX‐1 secretion system and subsequently activates the dormant ESX‐4 secretion system. The links of this signal transduction pathway, the mechanism of coordination and dependency between ESX‐1 and ESX‐4 secretion systems, are unknown. Previous studies identified SigM as a cell‐contact responsive sigma factor dedicated to activating ESX‐4. WhiB proteins are iron–sulfur‐binding stress‐response transcription factors exclusively found in Actinobacteria. WhiB6 has been shown to regulate ESX‐1 associated gene expression in other mycobacteria. Here, we show that WhiB6 is required both for conjugation and for transducing cell‐contact dependent signaling in the recipient cell. Our RNA‐seq, ChIP‐seq, and proteomic profiling data define a WhiB6 regulon that supports conjugative cell–cell interaction. The WhiB6 regulon includes genes encoding ESX‐1, ESX‐4, SigM, as well as dispersed operons that likely support ESX secretion. Our data demonstrate that WhiB6 is epistatic to SigM and ESX‐4 in this signal transduction pathway. This work shows that WhiB6 functions as a signal transduction node in recipient cells: it coordinates the expression of two ESX systems and it also induces uncharacterized proteins that collectively constitute a complete secretion response to recipient contact with a donor cell.