Abstract
ABSTRACTPeptidoglycan is a sugar/amino acid polymer unique to bacteria and essential for division and cell shape maintenance. The d-amino acids that make up its cross-linked stem peptides are not abundant in nature and must be synthesized by bacteria de novo. d-Glutamate is present at the second position of the pentapeptide stem and is strictly conserved in all bacterial species. In Gram-negative bacteria, d-glutamate is generated via the racemization of l-glutamate by glutamate racemase (MurI). Chlamydia trachomatis is the leading cause of infectious blindness and sexually transmitted bacterial infections worldwide. While its genome encodes a majority of the enzymes involved in peptidoglycan synthesis, no murI homologue has ever been annotated. Recent studies have revealed the presence of peptidoglycan in C. trachomatis and confirmed that its pentapeptide includes d-glutamate. In this study, we show that C. trachomatis synthesizes d-glutamate by utilizing a novel, bifunctional homologue of diaminopimelate epimerase (DapF). DapF catalyzes the final step in the synthesis of meso-diaminopimelate, another amino acid unique to peptidoglycan. Genetic complementation of an Escherichia coli murI mutant demonstrated that Chlamydia DapF can generate d-glutamate. Biochemical analysis showed robust activity, but unlike canonical glutamate racemases, activity was dependent on the cofactor pyridoxal phosphate. Genetic complementation, enzymatic characterization, and bioinformatic analyses indicate that chlamydial DapF shares characteristics with other promiscuous/primordial enzymes, presenting a potential mechanism for d-glutamate synthesis not only in Chlamydia but also numerous other genera within the Planctomycetes-Verrucomicrobiae-Chlamydiae superphylum that lack recognized glutamate racemases.
Highlights
Peptidoglycan is a sugar/amino acid polymer unique to bacteria and essential for division and cell shape maintenance
All chlamydial genomes encode a UDP-N-acetylmuramoyl-L-alanine-D-glutamate ligase [13], which adds D-Glu to the pentapeptide chain in all of the bacteria examined to date. murI is an essential gene for the vast majority of bacterial pathogens [14], and while homologues are present in the genomes of various environmental chlamydiae [10], no murI homologue has been identified in any pathogenic Chlamydia species
To assess whether chlamydial DAP epimerase is capable of generating sufficient D-Glu to restore growth in an E. coli D-Glu auxotroph, we inserted a copy of the C. trachomatis dapF gene into expression plasmid pBAD18, placing it under the control of an arabinose-inducible promoter
Summary
Peptidoglycan is a sugar/amino acid polymer unique to bacteria and essential for division and cell shape maintenance. The members of the family Chlamydiaceae are obligate intracellular bacterial pathogens that cause ocular, sexually transmitted, and respiratory infections They exhibit a unique biphasic developmental cycle, alternating between metabolically inert elementary bodies (EBs) and metabolically active reticulate bodies (RBs) [1]. The genomes of pathogenic Chlamydia species lack homologues of known racemases and D-amino acid aminotransferases [6]. In most Gram-negative bacteria, D-Glu is generated via the glutamate racemase MurI [11] and is invariant in the second position of the peptidoglycan pentapeptide chain [12]. Recent studies indicate that both pathogenic and environmental Chlamydia species contain m-DAP, D-Ala, and D-Glu in the stem peptides of their peptidoglycan [8, 9], indicating the presence of a potentially novel D-Glu synthesis pathway
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