Characterization of Chlamydia MurC‐Ddl, a fusion protein exhibiting D‐alanyl‐D‐alanine ligase activity involved in peptidoglycan synthesis and D‐cycloserine sensitivity

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Abstract
Recent characterization of chlamydial genes encoding functional peptidoglycan (PG)‐synthesis proteins suggests that the Chlamydiaceae possess the ability to synthesize PG yet biochemical evidence for the synthesis of PG has yet to be demonstrated. The presence of D‐amino acids in PG is a hallmark of bacteria. Chlamydiaceae do not appear to encode amino acid racemases however, a D‐alanyl‐D‐alanine (D‐Ala‐D‐Ala) ligase homologue (Ddl) is encoded in the genome. Thus, we undertook a genetics‐based approach to demonstrate and characterize the D‐Ala‐D‐Ala ligase activity of chlamydial Ddl, a protein encoded as a fusion with MurC. The full‐length murC‐ddl fusion gene from Chlamydia trachomatis serovar L2 was cloned and placed under the control of the arabinose‐inducible ara promoter and transformed into a D‐Ala‐D‐Ala ligase auxotroph of Escherichia coli possessing deletions of both the ddlA and ddlB genes. Viability of the E. coliΔddlAΔddlB mutant in the absence of exogenous D‐Ala‐D‐Ala dipeptide became dependent on the expression of the chlamydial murC‐ddl thus demonstrating functional ligase activity. Domain mapping of the full‐length fusion protein and site‐directed mutagenesis of the MurC domain revealed that the structure of the full fusion protein but not MurC enzymatic activity was required for ligase activity in vivo. Recombinant MurC‐Ddl exhibited substrate specificity for D‐Ala. Chlamydia growth is inhibited by D‐cycloserine (DCS) and in vitro analysis provided evidence for the chlamydial MurC‐Ddl as the target for DCS sensitivity. In vivo sensitivity to DCS could be reversed by addition of exogenous D‐Ala and D‐Ala‐D‐Ala. Together, these findings further support our hypothesis that PG is synthesized by members of the Chlamydiaceae family and suggest that D‐amino acids, specifically D‐Ala, are present in chlamydial PG.