FIG 8.

ElsL is the missing-link PG recycling l,d-carboxypeptidase in A. baumannii. (A) Western blot quantification of ElsL_FLAG and LdcA_FLAG. ΔelsL bacteria with inducible expression of each enzyme by P(IPTG) were cultured with the indicated IPTG concentration. Data points show protein level (relative to ElsL_FLAG, IPTG 50 μM) determined from image analysis. Bars show the mean ± SD (n = 3 biological replicates). (B) E. coli LdcA reverses the ΔelsL sulbactam hypersusceptibility defect. Susceptibility was determined by CFE assay. Plates contained the indicated sulbactam and IPTG concentrations. Data points show the geometric mean ± SD (n = 3 biological replicates). Arrows denote that colonies formed by ΔelsL + vector on 0.125 μg/mL sulbactam medium were pinpoint-sized. (C) Western blot quantification of ElsL_FLAG in ΔelsL bacteria with the indicated P(IPTG)-inducible allele cultured in the presence of 500 μM IPTG. Data points show the protein level (relative to ElsL_FLAG, IPTG 50 μM) as in panel A, except n = 2. (D) Intrinsic sulbactam resistance conferred by ElsL depends on an active site cysteine. Susceptibility to sulbactam was determined by CFE as in panel B. (E and F) ElsL has LDC activity in vitro. Shown are UPLC chromatograms of the reaction products following incubation of substrate (M4, murotetrapeptide, top; or M5, muropentapeptide, bottom) with the indicated purified enzyme (E). Numbered peaks were identified based on retention time and confirmed by MS (F). Minor peaks, which were trace muropeptide contaminants arising during manual HPLC purification of M4 and M5 substrates from C. crescentus PG (Materials and Methods), were identified as M5^Gly (at minute ∼3.6) and M2 (at minute ∼4.1).