Visualization of Plastid Peptidoglycan in the Charophyte Alga Klebsormidium nitens Using a Metabolic Labeling Method

It is widely accepted that a symbiosis involving a cyanobacterium with a peptidoglycan wall led to the origin of plastids of photosynthetic eukaryotes. Recently, we visualized plastid peptidoglycan in a moss species by epifluorescence microscopy using metabolic labeling with click chemistry. In the present study, we applied the same method to visualize plastid peptidoglycan in the filamentous charophyte alga Klebsormidium nitens, as its genome contains a set of genes capable of synthesizing peptidoglycan. To visualize peptidoglycan, the generation of D-alanyl-D-alanine (DA-DA) must be blocked to allow the incorporation of ethynyl-DA-DA (EDA-DA) into peptidoglycan. Because a gene-targeting technique has not been established for K. nitens, we used D-cycloserine, which is an inhibitor of D-Ala : D-Ala ligase. Treatment with 500 mu M D-cycloserine arrested cell division and inhibited chloroplast division. The addition of 1 mM EDA-DA restored the rate of cell division to that observed in medium without D-cycloserine, suggesting that EDA-DA can integrate plastid peptidoglycan instead of DA-DA, thus restarting chloroplast division and therefore cell division. Subsequently, cells were subjected to click chemistry to attach an azide-modified Alexa 488 fluorophore to the EDA-DA probe. Microscopic observation indicated that both sides of chloroplasts have strong Alexa 488 fluorescence. Under conditions of no cell growth, the chloroplast peptidoglycan that existed prior to treatment with D-cycloserine might remain in the cell. Therefore, the fluorescence must be indicative of the location of newly synthesized peptidoglycan. Our observations suggest that chloroplast peptidoglycan is built along the chloroplast division plane and serves as an essential system for chloroplast division in K. nitens.