Functional and biochemical analysis of the Chlamydia trachomatis ligase MurE

Abstract

Chlamydiae are unusual obligately intracellular bacteria that do not synthesize detectable peptidoglycan. However, they possess genes that appear to encode products with peptidoglycan biosynthetic activity. Bioinformatic analysis predicts that chlamydial MurE possesses UDP-MurNAc-L-Ala-D-Glu:meso-diaminopimelic acid (UDP-MurNAc-L-Ala-D-Glu:meso-A(2)pm) ligase activity. Nevertheless, there are no experimental data to confirm this hypothesis. In this paper we demonstrate that the murE gene from Chlamydia trachomatis is capable of complementing a conditional Escherichia coli mutant impaired in UDP-MurNAc-L-Ala-D-Glu:meso-A(2)pm ligase activity. Recombinant MurE from C. trachomatis (MurE(Ct)) was overproduced in and purified from E. coli in order to investigate its kinetic parameters in vitro. By use of UDP-MurNAc-L-Ala-D-Glu as the nucleotide substrate, MurE(Ct) demonstrated ATP-dependent meso-A(2)pm ligase activity with pH and magnesium ion optima of 8.6 and 30 mM, respectively. Other amino acids (meso-lanthionine, the ll and dd isomers of A(2)pm, D-lysine) were also recognized by MurE(Ct.) However, the activities for these amino acid substrates were weaker than that for meso-A(2)pm. The specificity of MurE(Ct) for three possible C. trachomatis peptidoglycan nucleotide substrates was also determined in order to deduce which amino acid might be present at the first position of the UDP-MurNAc-pentapeptide. Relative k(cat)/K(m) ratios for UDP-MurNAc-L-Ala-D-Glu, UDP-MurNAc-L-Ser-D-Glu, and UDP-MurNAc-Gly-D-Glu were 100, 115, and 27, respectively. Our results are consistent with the synthesis in chlamydiae of a UDP-MurNAc-pentapeptide in which the third amino acid is meso-A(2)pm. However, due to the lack of specificity of MurE(Ct) for nucleotide substrates in vitro, it is not obvious which amino acid is present at the first position of the pentapeptide.

FIG. 1.

SDS-PAGE of purified C. trachomatis MurE. The MurE_Ct protein was overproduced in E. coli cells in the His₆-tagged form. Purification on Ni²⁺-NTA agarose was performed as described in the text. Lanes: MW, molecular mass standards; MurE_Ct, purified MurE_Ct. Staining was performed with Coomassie brilliant blue R250.

FIG. 2.

Growth of E. coli TKL-11 carrying either plasmid pTrc99A (circles), pTrc99A::murE_Ec (squares), or pTrc99A::murE_Ct (triangles). Cells were grown at 30°C, and at the time indicated by the arrow, the cultures were shifted to 42°C.

FIG. 3.

Graphic representation of the kinetic parameters of MurE_Ct for UDP-MurNAc-l-Ala-d-Glu (A), UDP-MurNAc-Gly-d-Glu (B), and UDP-MurNAc-l-Ser-d-Glu (C). MurE assays were performed as described in Materials and Methods, and the data were fitted to the equation v = V_maxS/(K_m + S + [S²]/K_i).