(A) Schematics of the type III-B (above; adapted from [Silas et al., 2016]) and type I-F (below) CRISPR-Cas systems in M. mediterranea MMB-1. The type III-B operon contains genes encoding the adaptation proteins RT-Cas1 and Cas2, a 6-gene cmr cassette encoding the type III-B effector complex, the 58-spacer CRISPR02 array, the 8-spacer CRISPR03 array, and two genes of unknown function (Marme_0670, 0671). The Cas10 HD nuclease domain, required for DNA interference in some type III-B systems (Elmore et al., 2016), is not evident in the M. mediterranea Cas10 (Cmr2). The entire operon is flanked by two ~200 bp direct repeats (green arrows). The type I-F system contains cas1, cas2-3, four csy genes and a 52 spacer CRISPR04 array. Marme_3330, 3329, and 3326 are ORFs of unknown function. The 37-spacer CRISPR05 array, located ~250 kb away from the type I-F system has a near-canonical type I-F repeat sequence but no neighboring cas genes. An alignment of predicted 5’-crRNA handles (last 8 nt) from type I-F and type III-B CRISPR repeats is shown, with the remaining repeat nucleotides aligned separately (grey) and the crRNA processing sites depicted by the yellow triangle. (B) Overview of the plasmid interference assay using native CRISPR03 spacers. Configurations that allow the protospacer RNA to be targeted by the corresponding endogenous crRNA-Cmr complex lead to plasmid elimination and cell death on selective medium (+Kan). Inactivation of the type III-B CRISPR-Cas system results in plasmid stability and a corresponding increase in transconjugants. Numbers shown correspond to colony forming units per microliter (cfu/μl) measurements from the conjugations depicted. (C) Transcription-dependent plasmid interference in various M. mediterranea mutants using native CRISPR03 spacers. Log-transformed cfu/ml measurements from conjugations of plasmids with different protospacer configurations (relative to Pamp). Random R1 and R2 (pink text) are two different randomized sequences with base composition identical to the type III-B CRISPR repeat (brown text; CRISPR03-R). Upside down text denotes the reverse-complement. The GFP-Cmr2 strain is a control for the process of mutant construction.