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- Title
Characterization ofChlamydiaMurC-Ddl, a fusion protein exhibiting D-alanyl-D-alanine ligase activity involved in peptidoglycan synthesis and D-cycloserine sensitivity.
- Authors
McCoy, Andrea J.; Maurelli, Anthony T.
- Abstract
Recent characterization of chlamydial genes encoding functional peptidoglycan (PG)-synthesis proteins suggests that theChlamydiaceaepossess 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.Chlamydiaceaedo 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-lengthmurC-ddlfusion gene fromChlamydia trachomatisserovar L2 was cloned and placed under the control of the arabinose-induciblearapromoter and transformed into a D-Ala-D-Ala ligase auxotroph ofEscherichia colipossessing deletions of both theddlAandddlBgenes. Viability of theE. coliΔ ddlAΔ ddlBmutant in the absence of exogenous D-Ala-D-Ala dipeptide became dependent on the expression of the chlamydialmurC-ddlthus 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 activityin vivo. Recombinant MurC-Ddl exhibited substrate specificity for D-Ala.Chlamydiagrowth is inhibited by D-cycloserine (DCS) andin vitroanalysis provided evidence for the chlamydial MurC-Ddl as the target for DCS sensitivity.In vivosensitivity 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 theChlamydiaceaefamily and suggest that D-amino acids, specifically D-Ala, are present in chlamydial PG.
- Subjects
CHLAMYDIACEAE; CHLAMYDIA; BACTERIAL proteins; MICROBIAL proteins; ALANINE; LIGASES; PEPTIDOGLYCANS; MICROBIAL polysaccharides
- Publication
Molecular Microbiology, 2005, Vol 57, Issue 1, p41
- ISSN
0950-382X
- Publication type
Article
- DOI
10.1111/j.1365-2958.2005.04661.x