Competing Substrates for the Bifunctional Diaminopimelic Acid Epimerase/Glutamate Racemase Modulate Peptidoglycan Synthesis in Chlamydia trachomatis

The Chlamydia trachomatis genome encodes multiple bifunctional enzymes, such as DapF, which is capable of both diaminopimelic acid (DAP) epimerase and glutamate racemase activity. Our previous work demonstrated the bifunctional activity of chlamydial DapF in vitro and in a heterologous system ( Escherichia coli ). In the present study, we employed a substrate competition strategy to demonstrate DapF Ct function in vivo in C. trachomatis We reasoned that, because DapF Ct utilizes a shared substrate-binding site for both racemase and epimerase activities, only one activity can occur at a time. Therefore, an excess of one substrate relative to another must determine which activity is favored. We show that the addition of excess l-glutamate or meso -DAP ( m DAP) to C. trachomatis resulted in 90% reduction in bacterial titers, compared to untreated controls. Excess l-glutamate reduced in vivo synthesis of m DAP by C. trachomatis to undetectable levels, thus confirming that excess racemase substrate led to inhibition of DapF Ct DAP epimerase activity. We previously showed that expression of dapF Ct in a murI (racemase) Δ dapF (epimerase) double mutant of E. coli rescues the d-glutamate auxotrophic defect. Addition of excess m DAP inhibited growth of this strain, but overexpression of dapF Ct allowed the mutant to overcome growth inhibition. These results confirm that DapF Ct is the primary target of these m DAP and l-glutamate treatments. Our findings demonstrate that suppression of either the glutamate racemase or epimerase activity of DapF compromises the growth of C. trachomatis Thus, a substrate competition strategy can be a useful tool for in vivo validation of an essential bifunctional enzyme.