Quantifying bacterial cell wall growth activity
The incorporation of D-amino acid (DAA)-based probes, such as FDAAs, into bacterial peptidoglycan is conducted by the activities of peptidoglycan synthesis enzymes. With a given labeling time, the higher the peptidoglycan growth activity is, the stronger the labeling intensity is (more probe incorporation). Thus, DAA-probe labeling enables quantification of peptidoglycan growth activity. This can be done by performing an FDAA labeling for 10% to 100% doubling time of the species (as shown below). An incubation longer than 100% doubling time might result in saturation of FDAA incorporation; while ultra-short-pulse labeling less than 10% doubling time might cause FDAA signal too faint to be detected. After the fixation and washing steps, the fluorescence images of the labeled cells are collected. Their fluorescence intensity can be further quantified using ImageJ and its plugin, MicrobeJ.
One example of studying peptidoglycan growth activity using FDAA labeling comes from our recent FtsZ study. [1] FtsZ is a cytoskeletal protein that recruits the synthesis enzymes for septal peptidoglycan formation during cell division. FtsZ forms protein complexes and move dynamically at the cell division sites during the process. However, there is a long-standing question in this field: whether the rate of the FtsZ complex movement is implicated with peptidoglycan growth activity? To answer this question, we quantified FDAA intensity at the cell division sites while the FtsZ complex moving rate was manipulated, either increased or decreased. We reported that, speeding up FtsZ complex movement results in higher FDAA intensity, namely, higher cell wall growth activity; while decreasing FtsZ movement rate caused reduced FDAA intensity. This result suggests that the rate of FtsZ complex movement is implicated with the activity of the cell wall (peptidoglycan) formation.
The fluorescence intensity of FDAA-labeled peptidoglycan at the cell division sites was quantified and presented as violin diagram. In addition to the intensity, the spatial coverage of the FDAA signal along the cell division ring (along the Z-axis) was measured. [1]
References
[1] Bisson et al. Treadmilling by FtsZ filaments drives peptidoglycan synthesis and bacterial cell division. Science. 2017, 355,739-743.