Journal: mBio

Article Title: Two new enzymes that liberate undecaprenyl-phosphate to replenish the carrier lipid pool during envelope stress

doi: 10.1128/mbio.03710-24

Figure Lengend Snippet: UshA and UpsH maintain the free pool of UndP. Representative fluorescence and phase-contrast images of strains stained with fluorescently labeled MX2401 (MX2401-FL) to assess the free pools of UndP. Expression of the blue fluorescent protein highlights the cell cytoplasm and phase-contrast merged with propdium iodide (PI) staining highlighting cells with membrane permeability defects. ( A ) Expression of CsbB in the absence of YfhO traps UndP-GlcNAc and in cells lacking UshA the carrier lipid pool is dramatically reduced. The reduction in UndP impairs cell wall synthesis resulting in aberrant morphologies. Overexpression of UshA increases the levels of UndP and restores normal cell shape. ( B ) Depletion of the TUA flippase TuaB traps UndPP-TUA precursors and in the absence of UpsH depletes the carrier lipid pool. Overexpression of UpsH increases the levels of UndP and restores normal cell morphologies. Scale bar indicates 2 µm.

Article Snippet: MX2401 fluorescently labeled with CF488A (Biotium #92350) (MX2401-FL) (25 µM final) was added and incubated for 60 s. Cells were washed with 1× PBS, resuspended in 1/25 volume of 1× PBS, and spotted onto 1.5% agarose pads containing growth medium.

Techniques: Fluorescence, Staining, Labeling, Expressing, Membrane, Permeability, Over Expression