Trade-offs constrain adaptive pathways to T6 survival

Many microbial communities are characterized by intense competition for nutrients and space. One way for an organism to gain control of these resources is by eliminating nearby competitors. The Type VI Secretion System (T6) is a nano-harpoon used by many bacteria to inject toxins into neighboring cells. While much is understood about mechanisms of T6-mediated toxicity, little is known about the ways that competitors can defend themselves against this attack, especially in the absence of their own T6. Here we use directed evolution to examine the evolution of T6 resistance, subjecting eight replicate populations of Escherichia coli to T6 attack by Vibrio cholerae. Over ~500 generations of competition, the E. coli evolved to survive T6 attack an average of 27-fold better than their ancestor. Whole genome sequencing reveals extensive parallel evolution. In fact, we found only two pathways to increased T6 survival: apaH was mutated in six of the eight replicate populations, while the other two populations each had mutations in both yejM and yjeP. Synthetic reconstruction of individual and combined mutations demonstrate that yejM and yjeP are synergistic, with yejM requiring the mutation in yejP to provide a benefit. However, the mutations we identified are pleiotropic, reducing cellular growth rates, and increasing susceptibility to antibiotics and elevated pH. These trade-offs underlie the effectiveness of T6 as a bacterial weapon, and help us understand how the T6 shapes the evolution of bacterial interactions. SignificanceBacteria are the most abundant organisms on Earth and often live in dense, diverse communities, where they interact with each other. One of the most common interactions is antagonism. While most research has focused on diffusible toxins (e.g., antibiotics), bacteria have also evolved a contact-dependent nano-harpoon, the Type VI Secretion System (T6), to kill neighboring cells and compete for resources. While the co-evolutionary dynamics of antibiotic exposure is well understood, no prior work has examined how targets of T6 evolve resistance. Here, we use experimental evolution to observe how an Escherichia coli target evolves resistance to T6 when it is repeatedly competing with a Vibrio cholerae killer. After 30 rounds of competition, we identified mutations in three genes that improve E. coli survival, but found that these mutations come at a cost to other key fitness components. Our findings provide new insight into how contact-dependent antagonistic interaction drives evolution in a polymicrobial community.