Cell division cycle fluctuation of Pal concentration in Escherichia coli

The Tol-Pal proteins stabilise the outer membrane during cell division in many gram-negative bacteria, including Escherichia coli. Pal is an outer membrane lipoprotein that can bind peptidoglycan. It accumulates at the septum during division by a mobilisation-and-capture mechanism. This work further substantiates and extends knowledge of Pal's localisation in E. coli using immunolabeling, this method enables the detection of endogenous proteins. The midcell localisation of Pal and TolB, as seen with fluorescent protein fusions, during cell division, was confirmed. The retention of Pal in newly formed cell poles seemed to persist longer than observed with fluorescent Pal fusions. The concentration of endogenous Pal during the cell division cycle fluctuated: it decreased initially (to half the fluorescence concentration (32.1 au.µm-3) of the maximum (64.1 au.µm-3) reached during the cell cycle) and then increased during the second half of the cell division cycle. We probed for possible regulators and proposed two new putative regulators of Pal. Deleting the periplasmic protease, Prc decreased the total Pal abundance (to approximately 65% of the fluorescence concentration in wildtype cells) and affected its concentration fluctuation during the cell cycle. This suggests that Prc controls a cell division stage-specific regulator of Pal. Immunolabeling also supported the prediction that the small RNA MicA suppresses Pal expression (the fluorescence concentration of Pal in cells without MicA is double that of Pal in wildtype cells). However, the regulation by MicA occurred in a cell-cycle-independent manner. All these findings urge further research on the tight regulation of the dividing cell envelope stability.