Molecular Microbiology (2004) 53 (6), 1771–1783 doi:10.1111/j.1365-2958.2004.04242.x© 2004 Blackwell Publishing Ltd Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Publishing Ltd, 2004 ? 2004 53 617711783 Original Article Complex spatial distribution and dynamics of LamBK. A. Gibbs et al. Accepted 1 June, 2004. *For correspondence. [email protected]; Tel. ( + 1) 650 725 7968; Fax ( + 1) 650 7236783. † Present address: Miami Valley Laboratories, The Proctor andGamble Company, Cincinnati, OH 45252, USA. Complex spatial distribution and dynamics of an abundant Escherichia coli outer membrane protein, LamB Karine A. Gibbs, 1 Daniel D. Isaac, 2 Jun Xu, 3† Roger W. Hendrix, 3 Thomas J. Silhavy 2 and Julie A. Theriot 1,4 * 1 Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA. 2 Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA. 3 Pittsburgh Bacteriophage Institute and Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA. 4 Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA. SummaryAdvanced techniques for observing protein localiza-tion in live bacteria show that the distributions aredynamic. For technical reasons, most such tech-niques have not been applied to outer membrane pro-teins in Gram-negative bacteria. We have developedtwo novel live-cell imaging techniques to observe thesurface distribution of LamB, an abundant integralouter membrane protein in Escherichia coli res-ponsible for maltose uptake and for attachment ofbacteriophage lambda. Using fluorescently labelledbacteriophage lambda tails, we quantitatively des-cribed the spatial distribution and dynamic movementof LamB in the outer membrane. LamB accumulatedin spiral patterns. The distribution depended on celllength and changed rapidly. The majority of the pro-tein diffused along spirals extending across the cellbody. Tracking single particles, we found that thereare two populations of LamB – one shows veryrestricted diffusion and the other shows greatermobility. The presence of two populations recalls thepartitioning of eukaryotic membrane proteinsbetween ‘mobile’ and ‘immobile’ populations. In thisstudy, we have demonstrated that LamB moves alongthe bacterial surface and that these movements arerestricted by an underlying dynamic spiral pattern.Introduction Over the past decade, advances in techniques for mea-suring protein localization in bacteria have revealed thatproteins involved in cell division and cell shape (e.g. FtsZand MreB), virulence proteins (e.g. IcsA), chemoreceptorsand flagella all have specific cellular distributions (Gold-berg et al ., 1993; Maddock and Shapiro, 1993; Steinhauer et al ., 1999; Lybarger and Maddock, 2000; 2001; Fu et al .,2001; Robbins et al ., 2001; Shapiro et al ., 2002). Notrestricted to one bacterial phylum, non-uniform subcellularlocalization reflects the complexity of the bacterial cell(Lybarger and Maddock, 2001). With an emphasis oncytoplasmic and inner membrane components, recentlive-cell imaging using green fluorescent protein (GFP)and its derivatives (Feilmeier et al ., 2000; Southward andSurette, 2002) has shown that these subcellular proteinpatterns can vary from indistinct compact accumulationsto elegant helical structures and that they can changerapidly. With the advancement of deconvolution micros-copy, the resolution of subcellular distributions hasincreased, revealing that a number of the ‘indistinct accu-mulations’, such as MinCDE in Escherichia coli and Bacil-lus subtilis , are in fact helices (Marston and Errington,1999; Shih et al ., 2003). Most prominent of these helicesin Gram-negative bacteria are structures containing MreB,SetB, and the complement of Min proteins, MinCDE(Espeli et al ., 2003; Shih et al ., 2003), all of which arecrucial for cell shape and division (Jones et al ., 2001; Shih et al ., 2003). GFP, however, does not fold properly forfluorescence when exported from the cytoplasm via thegeneral secretory pathway, therefore prohibiting its use forlocalizing outer membrane components (Feilmeier et al .,2000).While the field of bacterial cell biology is beginning toreveal the dynamic architecture inside bacteria, we stillhave little insight into the dynamics of the Gram-negativebacterial outer membrane surface in live cells. The overallarchitecture of the outer membrane, proteins interspersedwith phospholipids and lipopolysaccharides (LPS), hasbeen biochemically detailed with respect to compositionratios, fluidity and asymmetry of the inner versus outerleaflet (Jaffe and D’Ari, 1985; Souzu, 1986; Rodriguez-Torres et al ., 1993). However, the spatial distribution ofmembrane components, specifically proteins, along the1772 K. A. Gibbs et al. © 2004 Blackwell Publishing Ltd, Molecular Microbiology , 53 , 1771–1783 surface has been elusive. A few macromolecular com-plexes, such as flagella and pili, are readily observedanchored peritrichously along the bacterial surface in E.coli , or exclusively at one pole in other Gram-negativeorganisms (e.g. Pseudomonas aeruginosa and Vibriocholerae ) (McCarter, 2001; Mattick, 2002; Shapiro et al .,2002). These organelles differ from other outer membranestructures in that they are anchored through the cell bodyto both periplasmic and plasma membrane components,similar to the type II and type III secretion systems (Tha-nassi and Hultgren, 2000).IcsA (VirG) in Shigella flexneri is one of the few outermembrane proteins in Gram-negative Enterobacteriaceaewhose spatial localization has been determined. A viru-lence protein that promotes actin polymerization, IcsA issecreted at one pole of S. flexneri and diffuses laterallyacross the cell body (Goldberg et al ., 1993; Goldberg andTheriot, 1995; Steinhauer et al ., 1999; Charles et al .,2001; Robbins et al ., 2001). In predivisional cells, IcsAlocalizes asymmetrically to both cell poles, but is not foundin the middle of the cell (Goldberg et al ., 1993; Robbins et al ., 2001). Unlike the flagella and pili components, IcsAis believed to have no periplasmic or intracellular bindingpartners and is integrated fully into the outer membraneas a beta-barrel. Localization of IcsA raises the questionof whether other outer membrane proteins on the surfaceof Gram-negative bacteria might be localized to the poles.To
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