title: Single molecule super-resolution imaging of proteins in living Salmonella enterica using self-labelling enzymes.
abstract: The investigation of the subcellular localization, dynamics and interaction of proteins and protein complexes in prokaryotes is complicated by the small size of the cells. Super-resolution microscopy (SRM) comprise various new techniques that allow light microscopy with a resolution that can be up to ten-fold higher than conventional light microscopy. Application of SRM techniques to living prokaryotes demands the introduction of suitable fluorescent probes, usually by fusion of proteins of interest to fluorescent proteins with properties compatible to SRM. Here we describe an approach that is based on the genetically encoded self-labelling enzymes HaloTag and SNAP-tag. Proteins of interest are fused to HaloTag or SNAP-tag and cell permeable substrates can be labelled with various SRM-compatible fluorochromes. Fusions of the enzyme tags to subunits of a type I secretion system (T1SS), a T3SS, the flagellar rotor and a transcription factor were generated and analysed in living Salmonella enterica. The new approach is versatile in tagging proteins of interest in bacterial cells and allows to determine the number, relative subcellular localization and dynamics of protein complexes in living cells.
authors.affiliation: Abt. Mikrobiologie, Universität Osnabrück, Osnabrück, Germany.
authors.forename: Britta
authors.initials: B
authors.lastname: Barlag
authors.affiliation: Abt. Biophysik, Universität Osnabrück, Osnabrück, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Beutel
authors.affiliation: Abt. Neurobiologie, Universität Osnabrück, Osnabrück, Germany.
authors.forename: Dennis
authors.initials: D
authors.lastname: Janning
authors.affiliation: Abt. Mikrobiologie, Universität Osnabrück, Osnabrück, Germany.
authors.forename: Frederik
authors.initials: F
authors.lastname: Czarniak
authors.affiliation: Abt. Biophysik, Universität Osnabrück, Osnabrück, Germany.
authors.forename: Christian P
authors.initials: CP
authors.lastname: Richter
authors.affiliation: Abt. Mikrobiologie, Universität Osnabrück, Osnabrück, Germany.
authors.forename: Carina
authors.initials: C
authors.lastname: Kommnick
authors.affiliation: Abt. Mikrobiologie, Universität Osnabrück, Osnabrück, Germany.
authors.forename: Vera
authors.initials: V
authors.lastname: Göser
authors.affiliation: CALMOS, Universität Osnabrück, Osnabrück, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Kurre
authors.affiliation: Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Fabiani
authors.affiliation: Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Marc
authors.initials: M
authors.lastname: Erhardt
authors.affiliation: Abt. Biophysik, Universität Osnabrück, Osnabrück, Germany.
authors.forename: Jacob
authors.initials: J
authors.lastname: Piehler
authors.affiliation: Abt. Mikrobiologie, Universität Osnabrück, Osnabrück, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Hensel
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D058947:Bacterial Secretion Systems / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism* / Q000494:pharmacology; D057054:Molecular Imaging / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D019779:Salmonella enterica / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins