title: Studying biomolecule localization by engineering bacterial cell wall curvature.
abstract: In this article we describe two techniques for exploring the relationship between bacterial cell shape and the intracellular organization of proteins. First, we created microchannels in a layer of agarose to reshape live bacterial cells and predictably control their mean cell wall curvature, and quantified the influence of curvature on the localization and distribution of proteins in vivo. Second, we used agarose microchambers to reshape bacteria whose cell wall had been chemically and enzymatically removed. By combining microstructures with different geometries and fluorescence microscopy, we determined the relationship between bacterial shape and the localization for two different membrane-associated proteins: i) the cell-shape related protein MreB of Escherichia coli, which is positioned along the long axis of the rod-shaped cell; and ii) the negative curvature-sensing cell division protein DivIVA of Bacillus subtilis, which is positioned primarily at cell division sites. Our studies of intracellular organization in live cells of E. coli and B. subtilis demonstrate that MreB is largely excluded from areas of high negative curvature, whereas DivIVA localizes preferentially to regions of high negative curvature. These studies highlight a unique approach for studying the relationship between cell shape and intracellular organization in intact, live bacteria.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America ; Technical University Dresden and the Max-Bergmann-Centre for Biomaterials, Dresden, Germany.
authors.forename: Lars D
authors.initials: LD
authors.lastname: Renner
authors.affiliation: National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Prahathees
authors.initials: P
authors.lastname: Eswaramoorthy
authors.affiliation: National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Kumaran S
authors.initials: KS
authors.lastname: Ramamurthi
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America ; Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Weibel
mesh_terms: D001412:Bacillus subtilis / Q000166:cytology* / Q000378:metabolism; D057005:Bioengineering / Q000379:methods*; D002473:Cell Wall / Q000737:chemistry*; D004926:Escherichia coli / Q000166:cytology* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted; D008856:Microscopy, Fluorescence; D012685:Sepharose / Q000737:chemistry; D013104:Spheroplasts / Q000737:chemistry*
chemical_list: D029968:Escherichia coli Proteins; C078473:MreB protein, E coli; D012685:Sepharose