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Open AccessArticle

Direct Visualization of Horizontal Gene Transfer by Transformation in Live Pneumococcal Cells Using Microfluidics

Laboratoire de Microbiologie et Génétique Moléculaires, Centre de Biologie Intégrative (CBI), Centre National de la Recherche Scientifique (CNRS), Université de Toulouse, UPS, F-31000 Toulouse, France
Author to whom correspondence should be addressed.
Genes 2020, 11(6), 675;
Received: 19 May 2020 / Revised: 13 June 2020 / Accepted: 16 June 2020 / Published: 20 June 2020
(This article belongs to the Special Issue Horizontal Gene Transfer in Bacteria)
Natural genetic transformation is a programmed mechanism of horizontal gene transfer in bacteria. It requires the development of competence, a specialized physiological state during which proteins involved in DNA uptake and chromosomal integration are produced. In Streptococcus pneumoniae, competence is transient. It is controlled by a secreted peptide pheromone, the competence-stimulating peptide (CSP) that triggers the sequential transcription of two sets of genes termed early and late competence genes, respectively. Here, we used a microfluidic system with fluorescence microscopy to monitor pneumococcal competence development and transformation, in live cells at the single cell level. We present the conditions to grow this microaerophilic bacterium under continuous flow, with a similar doubling time as in batch liquid culture. We show that perfusion of CSP in the microfluidic chamber results in the same reduction of the growth rate of individual cells as observed in competent pneumococcal cultures. We also describe newly designed fluorescent reporters to distinguish the expression of competence genes with temporally distinct expression profiles. Finally, we exploit the microfluidic technology to inject both CSP and transforming DNA in the microfluidic channels and perform near real time-tracking of transformation in live cells. We show that this approach is well suited to investigating the onset of pneumococcal competence together with the appearance and the fate of transformants in individual cells. View Full-Text
Keywords: Streptococcus pneumoniae; competence; transformation; fluorescence microscopy; microfluidic Streptococcus pneumoniae; competence; transformation; fluorescence microscopy; microfluidic
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Mortier-Barrière, I.; Polard, P.; Campo, N. Direct Visualization of Horizontal Gene Transfer by Transformation in Live Pneumococcal Cells Using Microfluidics. Genes 2020, 11, 675.

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