L,L-diaminopimelate aminotransferase, a trans-kingdom enzyme shared by Chlamydia and plants for synthesis of diaminopimelate/lysine

Abstract

The synthesis of meso-diaminopimelic acid (m-DAP) in bacteria is essential for both peptidoglycan and lysine biosynthesis. From genome sequencing data, it was unclear how bacteria of the Chlamydiales order would synthesize m-DAP in the absence of dapD, dapC, and dapE, which are missing from the genome. Here, we assessed the biochemical capacity of Chlamydia trachomatis serovar L2 to synthesize m-DAP. Expression of the chlamydial asd, dapB, and dapF genes in the respective Escherichia coli m-DAP auxotrophic mutants restored the mutants to DAP prototrophy. Screening of a C. trachomatis genomic library in an E. coli DeltadapD DAP auxotroph identified ct390 as encoding an enzyme that restored growth to the Escherichia coli mutant. ct390 also was able to complement an E. coli DeltadapD DeltadapE, but not a DeltadapD DeltadapF mutant, providing genetic evidence that it encodes an aminotransferase that may directly convert tetrahydrodipicolinate to L,L-diaminopimelic acid. This hypothesis was supported by in vitro kinetic analysis of the CT390 protein and the fact that similar properties were demonstrated for the Protochlamydia amoebophila homologue, PC0685. In vivo, the C. trachomatis m-DAP synthesis genes are expressed as early as 8 h after infection. An aminotransferase activity analogous to CT390 recently has been characterized in plants and cyanobacteria. This previously undescribed pathway for m-DAP synthesis supports an evolutionary relationship among the chlamydiae, cyanobacteria, and plants and strengthens the argument that chlamydiae synthesize a cell wall despite the inability of efforts to date to detect peptidoglycan in these organisms.

Fig. 1.

Variant pathways for the synthesis of m-DAP/lysine in plants and bacteria. m-DAP/lysine biosynthesis genes present in chlamydiae are boxed. The genes of the upper m-DAP biosynthesis pathway are linked together in the genome of all chlamydiae (Inset) whereas the lower m-DAP homologue, dapF, is unlinked to the upper m-DAP biosynthesis pathway genes.

Fig. 2.

Complementation of ΔdapD::tet ΔdapF::kan and ΔdapD::tet ΔdapE::kan double mutants by ct390. (A and B) ATM764 (ΔdapD::tet ΔdapF::kan; A) and ATM769 (ΔdapD::tet ΔdapE::kan; B) carrying pUC19 (circles) or pC1 (triangles) were grown with (Upper) or without (Lower) DAP as described. At various times, the bacteria were diluted and plated onto LB agar with DAP to determine the number of viable cells.

Fig. 3.

m-DAP synthesis gene expression patterns during Chlamydia development. Mouse L2 fibroblast cells were infected as described. RNA was isolated at 8, 24, and 46 h after infection. cDNA was amplified by using primers internal to hsp60, Ct390, dapB, and dapF. +, DNA control; −, −RT control.