An inner membrane protein is covalently attached to peptidoglycan in the γ-proteobacterium Dickeya dadantii.

Gram-negative (diderm) bacteria possess a multilayered envelope comprising an inner membrane, a thin peptidoglycan (PG) layer and an outer membrane. In Escherichia coli and certain other γ-proteobacteria, including Dickeya dadantii, Braun lipoprotein, Lpp, covalently tethers the outer membrane to PG. Here, we show that in D. dadantii an inner membrane protein, OutB, is covalently attached to PG by the same catalytic mechanism as Lpp. Specifically, two L,D-transpeptidases, Ldt03 and Ldt84, catalyze protein attachment with a preference for muropeptide monomers and dimers, respectively. By altering the Lpp length, we show that the extended Lpp+21 enhances OutB attachment to PG, whereas the truncated LppΔ21 reduces it. Furthermore, we show that the PG-anchoring sequence of OutB tolerates substantial amino acid substitutions and allows PG-tethering of a periplasmic reporter protein, suggesting that other periplasmic and/or membrane proteins may also be tethered to PG in proteobacteria.