To click or not to click for short pulse-labeling of the bacterial cell wall

A method of choice to study the spatio-temporal dynamics of bacterial cell growth and division is to analyze the localization of cell wall synthesis regions by fluorescence microscopy. For this, nascent cell wall biopolymers need to be labeled with fluorescent reporters, like fluorescent d-alanines (FDAs) that can be incorporated into the peptidoglycan. To achieve high spatial and temporal resolution, dense, high-intensity fluorescence labeling must be obtained in the shortest possible time. However, modifications carried by d-Ala can hinder their uptake by the enzymes that incorporate them into the peptidoglycan, such as the d,d-transpeptidases. Conversely, these modifications can impede the elimination of the incorporated d-Ala derivatives by d,d-carboxypeptidases, making the labeling more persistent. In this context, we synthesized clickable d-Alas and tested their incorporation into the peptidoglycan using different labeling approaches, prior or after their conjugation to clickable fluorescent dyes through SPAAC reaction. Our data allow ranking of the d-Ala derivatives in terms of their ease of incorporation and resistance to trimming during one-step, "one-pot" two-step or sequential two-step labeling strategies. We further show that a hybrid "one-step" approach, in which a FDA is used in combination with clickable choline and fluorescent dye, enables two-color co-labeling of peptidoglycan and teichoic acids. Finally, we identify a strategy compatible with the cell fixation required for super-resolution microscopy, by combining one-step labeling with FDA and sequential two-step labeling with clickable choline and fluorescent dye, allowing to obtain two-color high-resolution images of peptidoglycan and teichoic acid synthesis regions.