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 (mDAP) to C. trachomatis resulted in 90% reduction in bacterial titers, compared to untreated controls. Excess L-glutamate reduced in vivo synthesis of mDAP 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) ΔdapF (epimerase) double mutant of E. coli rescues the D-glutamate auxotrophic defect. Addition of excess mDAP 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 mDAP 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.