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Significance Mycobacteria use ESX systems to transport protein substrates across the cytoplasmic membrane. The ESX-1 system is required for mycobacterial pathogenesis inMycobacterium tuberculosis (M. tb ), the cause of tuberculosis (TB). Differences in the expression of genes encoding ESX substrates directly impactsM. tb transmission and virulence. Deletion of genes encoding ESX exporters results in reduced levels of ESX substrates in mycobacteria. Here, we define a fundamental mechanism of regulation of ESX-1 substrates inM. marinum , a pathogenic mycobacterial species and a model forM. tb . We demonstrate that the transcriptional regulation of genes encoding ESX-1 substrates is linked to the presence or absence of the ESX-1 exporter. These findings provide insight into how substrate levels are intricately controlled in mycobacteria.

WhiB6 regulation of ESX-1 gene expression is controlled by a negative feedback loop in Mycobacterium marinum