Visualisation of the mechanosensitive channel of large conductance in bacteria using confocal microscopy.
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.
Norman
Christel
C
School of Medicine and Pharmacology, QEII Medical Centre, The University of Western Australia, Crawley, WA 6009, Australia. christel@cyllene.uwa.edu.au
Liu
Zhen-Wei
ZW
Rigby
Paul
P
Raso
Albert
A
Petrov
Yevgeniy
Y
Martinac
Boris
B
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
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D007473:Ion Channels; C086596:MscL protein, E coli; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins