title: Visualisation of the mechanosensitive channel of large conductance in bacteria using confocal microscopy.
abstract: The mechanosensitive channel of large conductance (MscL) plays an important role in the survival of bacterial cells to hypo-osmotic shock. This channel has been extensively studied and its sequence, structure and electrophysiological characteristics are well known. Here we present a method to visualise MscL in living bacteria using confocal microscopy. By creating a gene fusion between mscl and the gene encoding the green fluorescent protein (GFP) we were able to express the fusion protein MscL-GFP in bacteria. We show that MscL-GFP is present in the cytoplasmic membrane and forms functional channels. These channels have the same characteristics as wild-type MscL, except that they require more pressure to open. This method could prove an interesting, non-invasive, tool to study the localisation and the regulation of expression of MscL in bacteria.
authors.affiliation: School of Medicine and Pharmacology, QEII Medical Centre, The University of Western Australia, Crawley, WA 6009, Australia. christel@cyllene.uwa.edu.au
authors.forename: Christel
authors.initials: C
authors.lastname: Norman
authors.forename: Zhen-Wei
authors.initials: ZW
authors.lastname: Liu
authors.forename: Paul
authors.initials: P
authors.lastname: Rigby
authors.forename: Albert
authors.initials: A
authors.lastname: Raso
authors.forename: Yevgeniy
authors.initials: Y
authors.lastname: Petrov
authors.forename: Boris
authors.initials: B
authors.lastname: Martinac
mesh_terms: D018407:Bacterial Physiological Phenomena; D001426:Bacterial Proteins / Q000737:chemistry; D001703:Biophysics / Q000295:instrumentation / Q000379:methods*; D002462:Cell Membrane / Q000378:metabolism; D049109:Cell Proliferation; D003593:Cytoplasm / Q000378:metabolism; D004594:Electrophysiology; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D007473:Ion Channels / Q000737:chemistry*; D018613:Microscopy, Confocal / Q000379:methods*; D010957:Plasmids / Q000378:metabolism; D011312:Pressure; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013104:Spheroplasts / Q000378:metabolism; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D007473:Ion Channels; C086596:MscL protein, E coli; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins