Plant peptidoglycan precursor biosynthesis: Conservation between moss chloroplasts and Gram-negative bacteria

Abstract

Accumulating evidence suggests that peptidoglycan, consistent with a bacterial cell wall, is synthesised around the chloroplasts of many photosynthetic eukaryotes, from glaucophyte algae to early-diverging land plants including pteridophyte ferns, but the biosynthetic pathway has not been demonstrated. Here, 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 fosfomycin, 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 the UDP-MurNAc-D,L-diaminopimelate (DAP)-pentapeptide. Most Gram negative bacteria, including cyanobacteria, incorporate meso-diaminopimelic acid (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 analyzed the recombinant protein UDP-N-acetylmuramoyl-L-alanyl-D-glutamate–2,6-diaminopimelate ligase (MurE) from both P. patens and the cyanobacterium Anabaena sp. (Nostoc 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.

The Chloroplast Envelope of Angiosperms Contains a Peptidoglycan Layer

“…Our findings regarding MurE function are in conflict with these studies but may be explained by different substrate specificities. In a recent report 28 it was shown that MurE from Anabaena and Physcomitrella specifically incorporated meso-Diaminopimelate (DAP) into muropeptides instead of L-Lysine, which we detected in higher plant PGN in our experiments (Fig. 3d and h).…”

“…The molecular structure of angiosperm plant PGN was revealed to be similar to that of its bacterial isoform, because it was detected in our experiments with azides of canonical amino acids of bacterial PGN and with proteins detecting bacterial PGN. This conclusion needs to be confirmed in the future by the extraction, isolation, and structural resolution of plant PGN, which is a long way to go, like the recent approaches in Physcomitrella showed 32 .…”

“…It is noteworthy that cyanobacteria as plastidic ancestors usually integrate DAP at this position 29 , and that plant MurE, like other bacterial PGN synthesis homologs in plants, does not seem to be of cyanobacterial origin 30,31 . Additionally, a second, yet uncharacterized, MurE isoform in all cryptogamic plants with higher homology to gymnosperm and angiosperm homologs has been reported 32 . Future experiments will show if both MurE isoforms also differ in their amino acid specificity, and if this is the reason for the failed complementation of Physcomitrella mutants by higher plant MurE.…”

Incompatibility and interchangeability in molecular evolution

“…The hypothesis that functional replacement is more likely to occur for proteins with limited molecular interactions is also supported by examples such as the extensive HGT in the peptidoglycan biosynthesis pathway for endosymbiotic bacteria/organelles (Husnik, et al 2013;Sato and Takano 2017;Dowson, et al 2022). The enzymes in this pathway catalyze individual reactions in series and do not assemble into large multisubunit complexes (Lovering, et al 2012).…”

“…For example, peptidoglycan is one of the defining features of the bacterial cell wall, and peptidoglycan biosynthesis in some plastids and endosymbiotic bacteria is now controlled by nuclear genes. But phylogenetic analyses have traced these peptidoglycan biosynthesis genes to disparate bacterial lineages (Husnik, et al 2013;Sato and Takano 2017;Dowson, et al 2022), meaning that the native enzymes originally present in the endosymbionts have been functionally replaced by homologs from entirely different phyla. Such examples support the broader argument that establishment of endosymbiotic relationships may often involve a series of multiple relationships that leave genetic footprints (Larkum, et al 2007;Bennett and Moran 2015;Gray 2015).…”