Extended Data Fig. 3. LytH pulled down ActH, a previously uncharacterized polytopic membrane protein.

a, A homology model of LytH highlights the predicted catalytic residues identified by sequence alignment of S. aureus LytH with E. coli AmiC. b, TMHMM topology prediction suggests that LytH is anchored in the cytoplasmic membrane. c, Schematic representation of the BDL-labeling assay to assess LytH hydrolytic activity in vitro. Short glycan strands were synthesized from Lipid II using S. aureus SgtB^Y181D. E. faecalis PBPX was used to exchange the terminal amino acid of stem peptides for biotin-D-Lys (BDL, yellow spheres), enabling visualization of glycan strands by western blotting with streptavidin. d, Glycan strands synthesized in vitro were treated with LytH. No difference in the distribution or intensity of polymers was observed between the three lanes. Blot is representative of two independent experiments. e, SDS-PAGE analysis of the co-immunoprecipitation experiment to identify proteins that bound to full-length LytH. Purple asterisks mark the protein bands corresponding to the baits. A red box is drawn around the ActH protein band. ActH is encoded by the gene saouhsc_01649. The fractions are: (FT) flowthrough, (W1–W2) washes, and (E1–E2) elutions. The schematics for LytH constructs are shown with the predicted transmembrane (TM), SH3, and amidase catalytic domains annotated. The predicted domain structure of ActH is also shown. f, A cladogram based on 16S rRNA alignment of bacterial species that encode ActH homologs with a similar domain structure was generated. The tree shows 196 bacterial species. For ease of visualization, only some species are labeled. Firmicutes are highlighted in purple and Deltaproteobacteria are highlighted in red.