Screening for a suitable cell membrane anchoring tag for Pseudomonas aeruginosa and applying it in cell membrane real-time tracking to investigate membrane aging

Membrane proteins that have been widely used in drug delivery and cell labeling can localize onto the cell membrane by interacting with lipid bilayers. A membrane-binding tag fused with a fluorescent protein can enable tracking of the cell outline. However, numerous known membrane proteins have species preferences, and thus, a suitable membrane-binding tag for Pseudomonas aeruginosa has not been reported. In this study, we examined the membrane-binding effects of a series of endogenous and exogenous proteins (peptides) in P. aeruginosa; the proteins included LacY, WspA, tsr and its truncated mutant (tsrMut), exotoxin A signal peptide (ESP), and TAT. Among them, tsrMut exhibited a faster and steadier membrane positioning ability than others, and it also did not interfere with bacteria growth. In addition, tsrMut could be further applied for identifying and tracking cell membrane aging areas in real-time. By linking it with a tandem fluorescent timer (EGFP-Tdimer2), the aging areas of the cell membrane could easily be displayed and observed under the microscope. These findings suggest that tsrMut is a highly favorable binding tag for P. aeruginosa and integrating the tag with an aging timer may be a promising approach for studying bacterial membrane senescence at the single-cell level. •Membrane aging in bacteria was difficult to observe.•The tsr mutant is the optimal membrane-binding tag for P.aeruginosa.•EGFP-Tdimer2 was linked to monitor the cell membrane aging in real-time.•We developed this method to study bacteria at the single-cell level.