Extended Data Fig. 2: Sequence conservation and functional analysis of LetA, related to Fig. 2.

a, Cellular assay to assess the function of the LetA variant where N- and C-terminal modules are co-expressed as separate open reading frames (split-LetA construct). 10-fold serial dilutions of strains indicated were spotted on LB agar with or without LSB or cholate, as noted. All strains are constructed in a ΔpqiAB background. b, Western blot showing results of pull-down assay, used to assess the interaction between the N-terminal LetA module, the C-terminal LetA module, and LetB when co-expressed as separate open reading frames (split-LetA construct). His-LetA (ZnR^N+TMD^N) was used as the bait for pull-down, and interaction with LetA-Strep (ZnR^C+TMD^C) and untagged LetB was assessed using α-Strep and α-LetB antibodies. Gel source data are provided in Supplementary Fig. 1f. Three independent biological replicates were performed starting with three different colonies, on different days, with similar results. c, 20 sequences of LetA and PqiA proteins representing Alpha-, Beta-, and Gammaproteobacteria, aligned using MUSCLE⁶⁹; Uniprot IDs are provided. Positions are colored based on sequence identity (see color key). Regions of interest are indicated. d, Cartoon representation of LetA; inset shows residues that are part of the ³⁶¹GRWSM-Ψ-D-Ψ-F³⁶⁹ motif in colored sticks. e, Sequence logo of the ³⁶¹GRWSM-Ψ-D-Ψ-F³⁶⁹ motif generated using WebLogo 3¹¹¹. Residues are colored based on hydrophilic (blue), neutral (green), or hydrophobic (black) properties.