Abstract
An accumulation of evidence suggests that peptidoglycan, consistent with a bacterial cell wall, is synthesised around the chloroplasts of many photosynthetic eukaryotes, from glaucophyte algae to land plants at least as evolved as pteridophyte ferns, but the biosynthetic pathway has not been demonstrated. We employed mass spectrometry and enzymology in a twofold approach to characterize the synthesis of peptidoglycan in chloroplasts of the moss Physcomitrium (Physcomitrella) patens. To drive the accumulation of peptidoglycan pathway intermediates, P.patens was cultured with the antibiotics phosphomycin, D-cycloserine and carbenicillin, which inhibit key peptidoglycan pathway proteins in bacteria. Mass spectrometry of the TCA-extracted moss metabolome revealed elevated levels of five of the predicted intermediates from UDP-GlcNAc through to the UDP-MurNAc-D,L-diaminopimelate (DAP)-pentapeptide.
Most Gram negative bacteria, including cyanobacteria, incorporate meso-diaminopimelate (D,L-DAP) into the third residue of the stem peptide of peptidoglycan, as opposed to L-lysine, typical of most Gram positive bacteria. To establish the specificity of D,L-DAP incorporation into the P.patens precursors, we analysed the recombinant protein, UDP-MurNAc-tripeptide ligase (MurE), from both P.patens and the cyanobacterium Anabaena sp. strain PCC 7120. Both ligases incorporated D,L-DAP in almost complete preference to L-Lys, consistent with the mass spectrophotometric data, with catalytic efficiencies similar to previously documented Gram negative bacterial MurE ligases. We discuss how these data accord with the conservation of active site residues common to DL-DAP-incorporating bacterial MurE ligases and of the probability of a horizontal gene transfer event within the plant peptidoglycan pathway.
Footnotes
1 This work was supported by a Pump Priming Initiative from the University of Warwick
↵3 Senior author
A.J.D generated the heterologous expression vectors, prepared and assayed proteins, purified peptidoglycan intermediates from P. patens and wrote the manuscript; A.J.L. conceived use of TCA extraction, performed the mass spectrophotometric analysis and analysed the data; A.C.C. assisted with P. patens handling; D.I.R, and L.F initiated the enzymatic analysis and Pump Prime funding, C.G.D assisted with funding and A.J.L., D.I.R. and C.G.D. assisted with experimental discussion and critiqued the manuscript.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plcell/pages/General-Instructions) is: A.J.Dowson (a.j.dowson{at}warwick.ac.uk).