title: Genetic reporter system for positioning of proteins at the bacterial pole.
abstract: Spatial organization within bacteria is fundamental to many cellular processes, although the basic mechanisms underlying localization of proteins to specific sites within bacteria are poorly understood. The study of protein positioning has been limited by a paucity of methods that allow rapid large-scale screening for mutants in which protein positioning is altered. We developed a genetic reporter system for protein localization to the pole within the bacterial cytoplasm that allows saturation screening for mutants in Escherichia coli in which protein localization is altered. Utilizing this system, we identify proteins required for proper positioning of the Shigella autotransporter IcsA. Autotransporters, widely distributed bacterial virulence proteins, are secreted at the bacterial pole. We show that the conserved cell division protein FtsQ is required for localization of IcsA and other autotransporters to the pole. We demonstrate further that this system can be applied to the study of proteins other than autotransporters that display polar positioning within bacterial cells.
  
  
  OBJECTIVE
  Many proteins localize to specific sites within bacterial cells, and localization to these sites is frequently critical to proper protein function. The mechanisms that underlie protein localization are incompletely understood, in part because of the paucity of methods that allow saturation screening for mutants in which protein localization is altered. We developed a genetic reporter assay that enables screening of bacterial populations for changes in localization of proteins to the bacterial pole, and we demonstrate the utility of the system in identifying factors required for proper localization of the polar Shigella autotransporter protein IcsA. Using this method, we identify the conserved cell division protein FtsQ as being required for positioning of IcsA to the bacterial pole. We demonstrate further that the requirement for FtsQ for polar positioning applies to other autotransporters and that the method can be applied to polar proteins other than autotransporters.
authors.affiliation: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Kathryn R
authors.initials: KR
authors.lastname: Fixen
authors.forename: Anuradha
authors.initials: A
authors.lastname: Janakiraman
authors.forename: Sean
authors.initials: S
authors.lastname: Garrity
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Slade
authors.forename: Andrew N
authors.initials: AN
authors.lastname: Gray
authors.forename: Nilay
authors.initials: N
authors.lastname: Karahan
authors.forename: Ann
authors.initials: A
authors.lastname: Hochschild
authors.forename: Marcia B
authors.initials: MB
authors.lastname: Goldberg
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D005827:Genetics, Microbial / Q000379:methods*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D021381:Protein Transport; D012760:Shigella / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C068023:FtsQ protein, E coli; D008565:Membrane Proteins; D014157:Transcription Factors; C058483:virG protein, Shigella flexneri