Following transforming DNA in Helicobacter pylori from uptake to expression

Abstract

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.

Horizontal gene transfer by natural transformation promotes both genetic and epigenetic inheritance of traits

“…Thus, it is likely that the mechanism for epigenetic inheritance described here is broadly applicable to this mode of horizontal gene transfer. In support of this, recent studies have shown that the expression of integrated DNA prior to cell division likely also occurs in naturally transformable Helicobacter pylori and Bacillus subtilis (Boonstra et al, 2018; Corbinais et al, 2016).…”

“…Fluorescent fusions to RecA do not fulfill this need because RecA interacts with ssDNA immediately upon uptake (Kidane and Graumann, 2005; Seitz and Blokesch, 2014); thus, making it difficult to distinguish between uptake of ssDNA into the cytoplasm from active integration into the chromosome. Also, recent work highlights the use of fluorescently labeled DNA to track the progress of DNA uptake and integration during NT (Boonstra et al, 2018; Corbinais et al, 2016); however, this approach suffers from the same shortfall. Recent work from our group has demonstrated that ComM is a hexameric helicase required for branch migration during NT in V. cholerae (Nero et al, 2018).…”

An efficient heat-shock protocol for transformation of Agrobacterium rhizogenes without spontaneous generation to antibiotic resistance

“…These phenomena have been reported for Salmonella enterica, Escherichia coli, and Pseudomonas aeruginosa and may be associated with alterations in the permeability of the outer cell membrane (Butler et al, 2010;Irazoki et al, 2017). The use of the high genetic adaptability strategy has been reported for Helicobacter pylori under laboratory conditions (Stingl et al, 2010;Corbinais et al, 2016) and in vivo for internal infections in humans (Corbinais et al, 2017), resulting in changes in membrane permeability, redox potential, and transporter proteins (Hu et al, 2016). Changes in the metabolic profiles may be induced by the composition and consistency of the culture medium, the addition of new metabolites or by cultivation in the presence of sublethal concentrations of antibiotics (Goh et al, 2002).…”

A peptide of a type I toxin-antitoxin system induces Helicobacter pylori morphological transformation from spiral-shape to coccoids

“…Confocal analysis of live cells was performed as previously described (61). Twenty µL of exponentially grown bacteria (10 cells) suspended in BHI with or without 1mM IPTG were deposited on 35mm glass bottom Petri dishes.…”