Fig. 1: T6SS1 effector identification methodology.

a Venn diagram displaying the premise for using comparative genomics to identify T6SS1-related proteins and candidate effectors. b The first step in the T6SS1 effector identification methodology. An overview of the expected results from analyzing V. parahaemolyticus BB22OP proteins against the V. parahaemolyticus genome dataset; proteins belonging to the V. parahaemolyticus core genome are expected to have highly similar homologs in both T6SS1⁺ and T6SS1⁻ genomes, whereas the closely related homologs of T6SS1 components and putative effectors should only be found in T6SS1⁺ genomes. c The second step in the T6SS1 effector identification methodology. Schematic representation of the V. parahaemolyticus surrogate T6SS1 platform. Candidate antibacterial T6SS1 effectors are cloned, together with a putative neighboring immunity, into an expression vector under Pbad regulation. Expression vectors are introduced into a strain with a constitutively active T6SS1 (T6SS1^CA; the surrogate platform), and into a derivative mutant in which the T6SS1 is inactive (T6SS1⁻). The ability of these attacking strains to kill a competing parental prey strain in a T6SS1-dependent manner is monitored. The parental prey strain contains the same endogenous effector/immunity (E/I) pairs as the attackers. Thus, it can antagonize their attack if they did not acquire a genuine E/I pair; however, if the expression plasmid in the attacker encodes a genuine E/I pair, then the parental prey will not be able to resist T6SS1-mediated intoxication.