Following transforming DNA in Helicobacter pylori from uptake to expression.
Natural transformation is a potent driver for genetic diversification in bacterial populations. It involves exogenous DNA binding, uptake, transport and internalization into the cytoplasm, where DNA can be processed and integrated into the host chromosome. Direct visualisation of transforming DNA (tDNA) has been limited to its binding to the surface or, in the case of Gram-negative species, to its entrance into the periplasm. We present here for the first time the direct visualisation of tDNA entering the bacterial cytoplasm. We used as a model the Gram-negative pathogen Helicobacter pylori, characterised by a large intraspecies variability that results from high mutation rates and efficient horizontal gene transfer. Using fluorescently labelled DNA, we followed for up to 3 h the fate of tDNA foci formed in the periplasm and eventually internalised into the cytoplasm. By tracking at the single cell level the expression of a fluorescent protein coded by the tDNA, we show that up to 50% of the cells express the transforming phenotype. The overall transformation process in H. pylori, from tDNA uptake to expression of the recombinant gene, can take place in less than 1 h, without requiring a growth arrest, and prior to the replication of the chromosome.
CEA, Institute of Molecular and Cellular Radiobiology, F-92265 Fontenay aux Roses, France.|INSERM, U967, F-92265 Fontenay-aux-Roses, France.|Universités Paris Diderot et Paris Sud, UMR967, F-92265 Fontenay-aux-Roses, France.
Christopher
C
Corbinais
CEA, Institute of Molecular and Cellular Radiobiology, F-92265 Fontenay aux Roses, France.
Aurélie
A
Mathieu
CEA, Institute of Molecular and Cellular Radiobiology, F-92265 Fontenay aux Roses, France.|INSERM, U967, F-92265 Fontenay-aux-Roses, France.|Universités Paris Diderot et Paris Sud, UMR967, F-92265 Fontenay-aux-Roses, France.
Thierry
T
Kortulewski
CEA, Institute of Molecular and Cellular Radiobiology, F-92265 Fontenay aux Roses, France. pablo.radicella@cea.fr.|INSERM, U967, F-92265 Fontenay-aux-Roses, France. pablo.radicella@cea.fr.|Universités Paris Diderot et Paris Sud, UMR967, F-92265 Fontenay-aux-Roses, France. pablo.radicella@cea.fr.
J Pablo
JP
Radicella
CEA, Institute of Molecular and Cellular Radiobiology, F-92265 Fontenay aux Roses, France. stephanie.marsin@i2bc.paris-saclay.fr.|INSERM, U967, F-92265 Fontenay-aux-Roses, France. stephanie.marsin@i2bc.paris-saclay.fr.|Universités Paris Diderot et Paris Sud, UMR967, F-92265 Fontenay-aux-Roses, France. stephanie.marsin@i2bc.paris-saclay.fr.
Stéphanie
S
Marsin
D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D022761:Gene Transfer, Horizontal; D016480:Helicobacter pylori / Q000235:genetics* / Q000378:metabolism; D014169:Transformation, Bacterial / Q000235:genetics*
D004269:DNA, Bacterial; C036483:T-DNA