Isolation and validation of an endogenous fluorescent nucleoid reporter in Salmonella Typhimurium.
In this study we adapted a Mud-based delivery system to construct a random yfp reporter gene (encoding the yellow fluorescent protein) insertion library in the genome of Salmonella Typhimurium LT2, and used fluorescence activated cell sorting and fluorescence microscopy to screen for translational fusions that were able to clearly and specifically label the bacterial nucleoid. Two such fusions were obtained, corresponding to a translational yfp insertion in iscR and iolR, respectively. Both fusions were further validated, and the IscR::YFP fluorescent nucleoid reporter together with time-lapse fluorescence microscopy was subsequently used to monitor nucleoid dynamics in response to the filamentation imposed by growth of LT2 at high hydrostatic pressure (40-45 MPa). As such, we were able to reveal that upon decompression the apparently entangled LT2 chromosomes in filamentous cells rapidly and efficiently segregate, after which septation of the filament occurs. In the course of the latter process, however, cells with a "trilobed" nucleoid were regularly observed, indicative for an imbalance between septum formation and chromosome segregation.
Laboratory of Food Micobiology, Department of Microbial and Molecular Systems (M2S), Faculty of Bioscience Engineering, KU Leuven, University of Leuven, Belgium.
Ioannis
I
Passaris
Laboratory of Food Micobiology, Department of Microbial and Molecular Systems (M2S), Faculty of Bioscience Engineering, KU Leuven, University of Leuven, Belgium.
Anirban
A
Ghosh
Laboratory of Food Micobiology, Department of Microbial and Molecular Systems (M2S), Faculty of Bioscience Engineering, KU Leuven, University of Leuven, Belgium.
William
W
Cenens
Laboratory of Food Micobiology, Department of Microbial and Molecular Systems (M2S), Faculty of Bioscience Engineering, KU Leuven, University of Leuven, Belgium.
Chris W
CW
Michiels
BIOSYST-MeBios, Faculty of Bioscience Engineering, KU Leuven, University of Leuven, Belgium.
Jeroen
J
Lammertyn
Laboratory of Food Micobiology, Department of Microbial and Molecular Systems (M2S), Faculty of Bioscience Engineering, KU Leuven, University of Leuven, Belgium.
Abram
A
Aertsen
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D012486:Salmonella typhimurium / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D049452:Green Fluorescent Proteins