title: Localization of anionic phospholipids in Escherichia coli cells.
abstract: Cardiolipin (CL) is an anionic phospholipid with a characteristically large curvature and is of growing interest for two primary reasons: (i) it binds to and regulates many peripheral membrane proteins in bacteria and mitochondria, and (ii) it is distributed asymmetrically in rod-shaped cells and is concentrated at the poles and division septum. Despite the growing number of studies of CL, its function in bacteria remains unknown. 10-N-Nonyl acridine orange (NAO) is widely used to image CL in bacteria and mitochondria, as its interaction with CL is reported to produce a characteristic red-shifted fluorescence emission. Using a suite of biophysical techniques, we quantitatively studied the interaction of NAO with anionic phospholipids under physiologically relevant conditions. We found that NAO is promiscuous in its binding and has photophysical properties that are largely insensitive to the structure of diverse anionic phospholipids to which it binds. Being unable to rely solely on NAO to characterize the localization of CL in Escherichia coli cells, we instead used quantitative fluorescence microscopy, mass spectrometry, and mutants deficient in specific classes of anionic phospholipids. We found CL and phosphatidylglycerol (PG) concentrated in the polar regions of E. coli cell membranes; depletion of CL by genetic approaches increased the concentration of PG at the poles. Previous studies suggested that some CL-binding proteins also have a high affinity for PG and display a pattern of cellular localization that is not influenced by depletion of CL. Framed within the context of these previous experiments, our results suggest that PG may play an essential role in bacterial physiology by maintaining the anionic character of polar membranes.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Piercen M
authors.initials: PM
authors.lastname: Oliver
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: John A
authors.initials: JA
authors.lastname: Crooks
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Mathias
authors.initials: M
authors.lastname: Leidl
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Earl J
authors.initials: EJ
authors.lastname: Yoon
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Alan
authors.initials: A
authors.lastname: Saghatelian
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USA weibel@biochem.wisc.edu.
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Weibel
mesh_terms: D001692:Biological Transport; D002308:Cardiolipins / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D013194:Staining and Labeling
chemical_list: D002308:Cardiolipins