Abstract
A number of methods for visualization of specific mRNAs in bacterial cells have been developed recently, but not all of them are suitable for environmental species when examined under harsh growth conditions (e.g., exposure to hydrocarbons or chemical pollutants). One technique of choice is fluorescent in situ mRNA hybridization (FISH) in single fixed cells. With this method, the result of hybridization with fluorophore-conjugated probes responding to target gene permits to detect endogenous mRNA in subcellular regions. We describe here a FISH protocol using a Stellaris probe set targeted toward the xylX mRNA, transcribed in the presence of m-xylene from the TOL plasmid pWW0 of Pseudomonas putida mt-2.
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References
Montero Llopis P, Jackson AF, Sliusarenko O, Surovtsev I, Heinritz J, Emonet T, Jacobs-Wagner C (2010) Spatial organization of the flow of genetic information in bacteria. Nature 466:77–81
Nevo-Dinur K, Nussbaum-Shochat A, Ben-Yehuda S, Amster-Choder O (2011) Translation-independent localization of mRNA in E. coli. Science 331:1081–1084
Russell JH, Keiler KC (2009) Subcellular localization of a bacterial regulatory RNA. Proc Natl Acad Sci U S A 106:16405–16409
Broude NE (2011) Analysis of RNA localization and metabolism in single live bacterial cells: achievements and challenges. Mol Microbiol 80:1137–1147
Golding I, Paulsson J, Zawilski SM, Cox EC (2005) Real-time kinetics of gene activity in individual bacteria. Cell 123:1025–1036
Russell JH, Keiler KC (2012) RNA visualization in bacteria by fluorescence in situ hybridization. Methods Mol Biol 905:87–95
Skinner SO, Sepulveda LA, Xu H, Golding I (2013) Measuring mRNA copy number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization. Nat Protoc 8:1100–1113
Toran P, Smolina I, Driscoll H, Ding F, Sun Y, Cantor CR, Broude NE (2014) Labeling native bacterial RNA in live cells. Cell Res 24:894–897
Ramos JL, Marques S, Timmis KN (1997) Transcriptional control of the Pseudomonas TOL plasmid catabolic operons is achieved through an interplay of host factors and plasmid-encoded regulators. Annu Rev Microbiol 51:341–373
Acknowledgments
This work was supported by the BIO Program of the Spanish Ministry of Economy and Competitiveness; the ST-FLOW, ARISYS, and EVOPROG Contracts of the EU; the ERANET-IB, and the PROMT Project of the CAM.
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Kim, J., de Lorenzo, V. (2015). Detection of Specific mRNA Molecules in Hydrocarbon-Degrading Bacteria. In: McGenity, T.J., Timmis, K.N., Nogales, B. (eds) Hydrocarbon and Lipid Microbiology Protocols. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/8623_2015_100
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DOI: https://doi.org/10.1007/8623_2015_100
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