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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 f...

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Autores principales: Passaris, Ioannis, Ghosh, Anirban, Cenens, William, Michiels, Chris W., Lammertyn, Jeroen, Aertsen, Abram
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3973593/
https://www.ncbi.nlm.nih.gov/pubmed/24695782
http://dx.doi.org/10.1371/journal.pone.0093785
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author Passaris, Ioannis
Ghosh, Anirban
Cenens, William
Michiels, Chris W.
Lammertyn, Jeroen
Aertsen, Abram
author_facet Passaris, Ioannis
Ghosh, Anirban
Cenens, William
Michiels, Chris W.
Lammertyn, Jeroen
Aertsen, Abram
author_sort Passaris, Ioannis
collection PubMed
description 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.
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spelling pubmed-39735932014-04-04 Isolation and Validation of an Endogenous Fluorescent Nucleoid Reporter in Salmonella Typhimurium Passaris, Ioannis Ghosh, Anirban Cenens, William Michiels, Chris W. Lammertyn, Jeroen Aertsen, Abram PLoS One Research Article 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. Public Library of Science 2014-04-02 /pmc/articles/PMC3973593/ /pubmed/24695782 http://dx.doi.org/10.1371/journal.pone.0093785 Text en © 2014 Passaris et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Passaris, Ioannis
Ghosh, Anirban
Cenens, William
Michiels, Chris W.
Lammertyn, Jeroen
Aertsen, Abram
Isolation and Validation of an Endogenous Fluorescent Nucleoid Reporter in Salmonella Typhimurium
title Isolation and Validation of an Endogenous Fluorescent Nucleoid Reporter in Salmonella Typhimurium
title_full Isolation and Validation of an Endogenous Fluorescent Nucleoid Reporter in Salmonella Typhimurium
title_fullStr Isolation and Validation of an Endogenous Fluorescent Nucleoid Reporter in Salmonella Typhimurium
title_full_unstemmed Isolation and Validation of an Endogenous Fluorescent Nucleoid Reporter in Salmonella Typhimurium
title_short Isolation and Validation of an Endogenous Fluorescent Nucleoid Reporter in Salmonella Typhimurium
title_sort isolation and validation of an endogenous fluorescent nucleoid reporter in salmonella typhimurium
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3973593/
https://www.ncbi.nlm.nih.gov/pubmed/24695782
http://dx.doi.org/10.1371/journal.pone.0093785
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