LAUSR

Significance The Mycobacterium tuberculosis (Mtb) ESX-3 type VII secretion system plays a critical role in iron acquisition. Infection of mice with highly attenuated Mtb deletion mutants lacking esxG or esxH, genes encoding key ESX-3 substrates, unexpectedly yielded suppressor mutants with restored capacity to grow in vivo and in vitro in the absence of iron supplementation. Whole-genome sequencing identified two mechanisms of suppression, the disruption of a transcriptional repressor that regulates expression of an ESX-3 paralogous region encoding EsxR and EsxS, and a massive 38- to 60-fold gene amplification of this same region. These data are significant because they reveal a previously unrecognized iron acquisition regulon and inform mechanisms of Mtb chromosome evolution. Mycobacterium tuberculosis (Mtb) possesses five type VII secretion systems (T7SS), virulence determinants that include the secretion apparatus and associated secretion substrates. Mtb strains deleted for the genes encoding substrates of the ESX-3 T7SS, esxG or esxH, require iron supplementation for in vitro growth and are highly attenuated in vivo. In a subset of infected mice, suppressor mutants of esxG or esxH deletions were isolated, which enabled growth to high titers or restored virulence. Suppression was conferred by mechanisms that cause overexpression of an ESX-3 paralogous region that lacks genes for the secretion apparatus but encodes EsxR and EsxS, apparent ESX-3 orphan substrates that functionally compensate for the lack of EsxG or EsxH. The mechanisms include the disruption of a transcriptional repressor and a massive 38- to 60-fold gene amplification. These data identify an iron acquisition regulon, provide insight into T7SS, and reveal a mechanism of Mtb chromosome evolution involving “accordion-type” amplification.