(A) Viability of E. coli cells grown on solid media harboring inducible plasmids expressing the C-terminal toxin domains of the three identified SiB196 LXG proteins or an empty vector control. (B) SiB196 colonies recovered after transformation with equal concentrations of constitutive expression plasmids carrying genes encoding the indicated proteins. ss-TelCtox is targeted to the sec translocon through the addition of the secretion signal sequence from S. pneumoniae LysM (SP_0107). Error bars represent ± SD (n = 3). Asterisk indicates a statistically significant difference in Si transformation efficiency relative to TelCtox (p<0.05). (C) Viability of E. coli cells grown on solid media harboring inducible plasmids co-expressing the indicated proteins. Empty vector controls are indicated by a dash. Mean c.f.u. values ± SD (n = 3) are plotted. Asterisks indicate statistically significant differences in E. coli viability relative to vector control (p<0.05) (D) Intra-species growth competition experiments between the indicated bacterial strains. Competing strains were mixed and incubated in liquid medium or on solid medium for 30 hr and both initial and final populations of each strain were enumerated by plating on selective media. The competitive index was determined by comparing final and initial ratios of the two strains. Asterisks indicate outcomes statistically different between liquid and solid medium (n = 3, p<0.05). (E) Intra-species growth competition experiments performed as in (D) except for the presence of a filter that inhibits cell-cell contact. No contact, filter placed between indicated donor and susceptible recipient (∆telB ∆tipB) strains; Contact, donor and susceptible recipient strains mixed on same side of filter. Asterisks indicate statistically different outcomes (n = 3, p<0.05). Note that recipient cell populations have an Esx-independent fitness advantage in these experiments by virtue of their relative proximity to the growth substrate. (F) Inter-species growth competition experiments performed on solid or in liquid (E. faecalis) medium between Si wild-type and ∆essC donor strains and the indicated recipient organisms. Si23775 lacks tipA and tipB and is therefore potentially susceptible to TelA and TelB delivered by SiB196. Asterisks indicate outcomes where the competitive index of wild-type was significantly higher than an ∆essC donor strain (n = 3, p<0.05). Genetic complementation of the mutant phenotypes presented in this figure was confounded by inherent plasmid fitness costs irrespective of the inserted sequence. As an alternative, we performed whole genome sequencing on strains ∆essC, ∆telB, ∆telC, ∆telB ∆tipB, and ∆telC ∆tipC, which confirmed the respective desired mutation as the only genetic difference between these strains. Sequences of these strains have been deposited to the NCBI Sequence Read Archive (BioProject ID: PRJNA388094).