Figure 4. Representatives of Families 3 and 4 are T6S amidase EI pairs.

(A) Tae3^TY and Tae4^TM are peptidoglycan amidases with specificity for the m-DAP-D-alanine DD-bond and the γ-D-glutamyl-L-m-DAP bond, respectively. Partial HPLC chromatograms of sodium borohydride-reduced soluble E. coli peptidoglycan products resulting from digestion with Tae3^TY or Tae4^TM and subsequent cleavage with cellosyl. The control sample was digested with cellosyl alone.

(B) Simplified representation of Gram-negative peptidoglycan showing cleavage sites of effector families 1–4 (F1-4) based on HPLC data. Cleavage specificity on peptidoglycan with tetrapeptide (left) and pentapeptide (right) stems are depicted. Tse1 activity against pentapeptide-rich peptidoglycan has not been tested, as indicated by yellow stars. Abbreviations: GlcNAc, N-acetyl-glucosamine, MurNAc, N-acetyl-muramic acid.

(C) Tae3^TY and Tae4^TM are toxic in the periplasm and this toxicity is rescued specifically by cognate immunity proteins, Tai3^TY and Tai4^TM, respectively. Growth of E. coli harboring one (top panels) or two (bottom panel) vectors expressing the indicated genes. A dash indicates the empty vector. From left to right are increasing serial ten-fold dilutions. Expression data for this experiment are shown in Figure S2.

(D) Tae3^PF is secreted in a T6SS-dependent manner. Western blot analysis of supernatant (Sup) and cell-associated (Cell) fractions of the indicated P. fluorescens strains expressing vesicular stomatitis virus glycoprotein (VSV-G) tagged Tae3^PF (Tae3^PF –V).