Studying bacterial cell wall growth
The incorporation of D-amino acid-based probes, such as FDAAs, is conducted by peptidoglycan (PG) transpeptidases. PG is the main content of the bacterial cell wall. Its rigid structure protects the cells from environmental stress and dictates morphology changes through the cells' life cycle. Transpeptidases are the primary synthases of PG. Therefore, the metabolic labeling of FDAA through transpeptidase activity indicates its growth and remodeling activity. Killing the bacterial cells or inhibiting PG synthesis activity, such as antibiotic treatment, results in failed FDAA labeling.
Visualizing new PG synthesis sites
There are several methods to visualize PG growth using FDAA labeling. The most common and easy-to-do approach is to perform a short-pulse FDAA labeling. Because FDAA incorporation occurs at the sites having active transpeptidases, the fluorescently labeled area indicates the newly synthesized or remodeled PG structures. This can be done by incubating the growing cells with 0.5 to 1 mM FDAA for 10% doubling time of the species (e.g. 2 minutes for E. coli), followed by fixing the cells to stop their growth. A simplified scheme (Figure 1, Panel A) is shown below.
Figure 1. Different ways to visualize new PG synthesis/remodeling activities. (A) Comparison of short-pulse and long-pulse labeling. (B) Pulse-and-chase labeling. (C) Sequential FDAA labeling (virtual time-lapse labeling). Polarly growing bacteria, S. venezuelae is used as an example here. [1]
In comparison to a long-pulse FDAA labeling, where the cells are incubated with FDAA for 300% cell doubling time, short-pulse FDAA labeling only reveals the new PG synthesis/remodeling sites, not the whole PG structure. For example, in a polarly growing bacterium, Streptomyces venezuelae, short-pulse labeling results in the labeling of cell poles, indicating that new PG structures are made and extended from the cell terminals. On the other hand, in Staphylococcus aureus and Streptococcus pneumoniae, short-pulse labeling gives a band-shaped fluorescent region at the middle of cells, saying that these two species undergo a mid-cell growth manner for PG synthesis.
S. venezuelae (polar growth) [2] S. pneumoniae (mid-cell growth) [2]
The second approach to visualize new PG formation is pulse-and-chase labeling (Figure 1, Panel B). The cells are first incubated with FDAA for 300% doubling time of the species (long-pulse labeling). Following that, the cells are washed to remove FDAA in the solution and transferred to the fresh culture medium without fixation. In this case, old PG is fluorescently labeled but newly synthesized PG is not. One can observe new PG formation by recognizing unlabeled PG structures. The advantage of this pulse-and-chase approach over the short-pulse labeling is that PG growth can be monitored continuously in live cells. It provides more information about where PG growth proceeds and how fast it is. On the other hand, this approach suffers from lower sensitivity because we are now looking at fluorescence signal reduction (the new PG) from strongly fluorescent background (the old PG).
The third approach, and also the most advanced one, is sequential labeling using different colored FDAAs (Figure 1, Panel C). This method is also known as virtual time-lapse labeling, where multiple short-pulse labeling with different colored FDAAs is performed sequentially with washing steps in between. This results in a color pattern that reflects the ways that cells build PG structures. One can investigate when and where new PG is made in the cells by identifying the color and location of FDAA signals. An example of five-colored FDAA labeling in S. venezuelae is shown below.
Sequential FDAA labeling in a polarly growing species, S. venezuelae.The cells were incubated with green (Atto488ADA), orange (Cy3bADA), cyan (AF350DL) and then red FDAA (Atto610ADA) sequentially. [3]
References
[1] Radkov et al. Imaging Bacterial Cell Wall Biosynthesis. Annu. Rev. Biochem., 2018, 87:22.1-22.4.
[2] Kuru et al. In Situ Probing of Newly Synthesized Peptidoglycan in Live Bacteria with Fluorescent D-Amino Acids. Angew. Chem. 2012, 124, 12687-12691.
[3] Hsu et al. Full color palette of fluorescent D-amino acids for in situ labeling of bacterial cell walls Chem. Sci. 2017, 8, 6313-6321.