title: Lighting Up Clostridium Difficile: Reporting Gene Expression Using Fluorescent Lov Domains.
abstract: The uses of fluorescent reporters derived from green fluorescent protein have proved invaluable for the visualisation of biological processes in bacteria grown under aerobic conditions. However, their requirement for oxygen has limited their application in obligate anaerobes such as Clostridium difficile. Fluorescent proteins derived from Light, Oxygen or Voltage sensing (LOV) domains have been shown to bridge this limitation, but their utility as translational fusions to monitor protein expression and localisation in a strict anaerobic bacterium has not been reported. Here we demonstrate the utility of phiLOV in three species of Clostridium and its application as a marker of real-time protein translation and dynamics through genetic fusion with the cell division protein, FtsZ. Time lapse microscopy of dividing cells suggests that Z ring assembly arises through the extension of the FtsZ arc starting from one point on the circumference. Furthermore, through incorporation of phiLOV into the flagella subunit, FliC, we show the potential of bacterial LOV-based fusion proteins to be successfully exported to the extracellular environment.
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary &Life Sciences, University of Glasgow, Glasgow G12 8TA U.K.
authors.forename: Anthony M
authors.initials: AM
authors.lastname: Buckley
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary &Life Sciences, University of Glasgow, Glasgow G12 8TA U.K.
authors.forename: Caitlin
authors.initials: C
authors.lastname: Jukes
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary &Life Sciences, University of Glasgow, Glasgow G12 8TA U.K.
authors.forename: Denise
authors.initials: D
authors.lastname: Candlish
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary &Life Sciences, University of Glasgow, Glasgow G12 8TA U.K.
authors.forename: June J
authors.initials: JJ
authors.lastname: Irvine
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary &Life Sciences, University of Glasgow, Glasgow G12 8TA U.K.
authors.forename: Janice
authors.initials: J
authors.lastname: Spencer
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN U.K.
authors.forename: Robert P
authors.initials: RP
authors.lastname: Fagan
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary &Life Sciences, University of Glasgow, Glasgow G12 8TA U.K.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Roe
authors.affiliation: Institute of Molecular Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA U.K.
authors.forename: John M
authors.initials: JM
authors.lastname: Christie
authors.affiliation: Division of Cell and Molecular Biology, Centre for Molecular Microbiology and Infection, Imperial College London, London SW7 2AZ U.K.
authors.forename: Neil F
authors.initials: NF
authors.lastname: Fairweather
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary &Life Sciences, University of Glasgow, Glasgow G12 8TA U.K.
authors.forename: Gillian R
authors.initials: GR
authors.lastname: Douce
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis; D015374:Biosensing Techniques / Q000379:methods*; D016360:Clostridioides difficile / Q000235:genetics* / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000096:biosynthesis; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D057054:Molecular Imaging / Q000379:methods*; D061848:Optical Imaging / Q000379:methods*; D014176:Protein Biosynthesis; D021381:Protein Transport
chemical_list: D001426:Bacterial Proteins; D003598:Cytoskeletal Proteins; D005456:Fluorescent Dyes; C071525:FtsZ protein, Bacteria; D049452:Green Fluorescent Proteins