(A) Genomic arrangement of T6SS chaperone-effector-immunity genes for characterized effector-associated gene family members (eag; shown in purple), which encode DUF1795 domain-containing chaperones. (B) Schematic depicting Eag chaperone interactions with the transmembrane domain (TMD) regions of the model chaperone-effector pair EagT6-Tse6. (C) Protein architecture and sequence logo for the prePAAR motif found in the N-terminus of Tse6. An alignment of 2,054 sequences was generated using the 61 N-terminal residues of Tse6 as the search query. The relative frequency of each residue and information content in bits was calculated at every position of the sequence and then normalized by the sum of each position’s information bits. Transparency is used to indicate probability of a residue appearing at a specific position. Residues colored in pink correspond to the prePAAR motif: AARxxDxxxH. (D) Genomes from genera of Proteobacteria known to contain functional T6SSs (Burkholderia, Escherichia, Enterobacter, Pseudomonas, Salmonella, Serratia, Shigella, Yersinia) were screened for unique prePAAR effectors. Percentage of total genomes that contained 1 to 6 prePAAR motifs is indicated. (E) Phylogenetic distribution of 1,166 non-redundant prePAAR-containing effectors identified in B. TM prediction algorithms were used to quantify the number of TM regions in each effector. The two classes that emerged are labeled in green (class I; 1 TM region-containing effectors) and blue (class II; 2 TM region-containing effectors). Branch lengths indicates evolutionary distances. (F) Effector sequences within class I or class II were aligned, and a sequence logo was generated based on the relative frequency of each residue at each position to identify characteristic motifs of both classes. Four different regions (r1–r4) after the PAAR and TM regions were found to harbor conserved residues. Class I effectors contain YD repeat regions (r1-3) characteristic of Rhs proteins, whereas a GxxxxGxxLxGxxxD motif (r4) was identified in class II effectors. (G) Western blot analysis of five effector-chaperone pairs that were selected from the indicated genera, based on the analysis in B. Each pair was co-expressed in E. coli and co-purified using nickel affinity chromatography. The class and number of TM regions from each pair are indicated. Locus tags for each pair (e, effector; c, chaperone) are as follows: Enterobacter (e: ECL_01567, c: ECL_01566), Shigella (e: SF0266, c: SF3490), Salmonella (e: SL1344_0286, c: SL1344_0285), Serratia (e: Spro_3017, c: Spro_3016), Pseudomonas (e: PA0093, c: PA0094). Note that the Rhs component of the class I prePAAR effector SF0266 is encoded by the downstream open-reading frame SF0267 (see Figure 1—figure supplement 1C for details).