Molecular bases for the loss of type VI secretion system activity during enteroaggregative E. coli experimental evolution

The type VI secretion system (T6SS) is a nanoweapon deployed by Gram-negative to inject effectors into target cells and hence involved in pathogenesis and bacterial competition. While T6SS gene clusters are found in all recently isolated commensals or pathogenic Escherichia coli strains, they are absent from classical laboratory strains. These E. coli strains, which have been used since decades for bacterial genetics, were usually grown in pure cultures, suggesting that T6SS might have been lost during evolution in absence of competitors. Here, we conducted a 640-generation experimental evolution by passaging the enteroaggregative E. coli (EAEC) 17-2 strain under controlled competition conditions against susceptible or immune recipient cells. EAEC T6SS activity was almost abolished when grown in the presence of immune recipients, while no difference with the ancestral strain was observed for EAEC grown in the presence of susceptible cells. Whole genome sequencing of 18 clones identified adaptative mutations responsible for the loss of T6SS activity, including mutations of the T6SS promoter and within genes encoding T6SS subunits, and revealed a novel regulatory mechanism involving RfaH-dependent antitermination. We further found that the RfaH binding site ops element is present in T6SS gene clusters of several species, suggesting a conserved RfaH-dependent regulation of T6SSs across enterobacteria. This work exemplifies the power of experimental evolution to understand T6SS genetic adaptation and to unravel new players for its function.