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

ESX (ESAT-6 system) export systems play diverse roles across mycobacterial species. Interestingly, genetic disruption of ESX systems in different species does not result in an accumulation of protein substrates in the mycobacterial cell. However, the mechanisms underlying this observation are elusive. We hypothesized that the levels of ESX substrates were regulated by a feedback-control mechanism, linking the levels of substrates to the secretory status of ESX systems. To test this hypothesis, we used a combination of genetic, transcriptomic, and proteomic approaches to define export-dependent mechanisms regulating the levels of ESX-1 substrates in Mycobacterium marinum WhiB6 is a transcription factor that regulates expression of genes encoding ESX-1 substrates. We found that, in the absence of the genes encoding conserved membrane components of the ESX-1 system, the expression of the whiB6 gene and genes encoding ESX-1 substrates were reduced. Accordingly, the levels of ESX-1 substrates were decreased, and WhiB6 was not detected in M. marinum strains lacking genes encoding ESX-1 components. We demonstrated that, in the absence of EccCb₁, a conserved ESX-1 component, substrate gene expression was restored by constitutive, but not native, expression of the whiB6 gene. Finally, we found that the loss of WhiB6 resulted in a virulent M. marinum strain with reduced ESX-1 secretion. Together, our findings demonstrate that the levels of ESX-1 substrates in M. marinum are fine-tuned by negative feedback control, linking the expression of the whiB6 gene to the presence, not the functionality, of the ESX-1 membrane complex.