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Applied and Environmental Microbiology, July 2006, p. 4923-4930, Vol. 72, No. 7
0099-2240/06/$08.00+0     doi:10.1128/AEM.02605-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Flow Cytometric Testing of Green Fluorescent Protein-Tagged Lactobacillus rhamnosus GG for Response to Defensins

Sigrid C. J. De Keersmaecker,1 Kristien Braeken,1 Tine L. A. Verhoeven,1 Mónica Perea Vélez,1 Sarah Lebeer,1 Jos Vanderleyden,1* and Pascal Hols2

Centre of Microbial and Plant Genetics, K.U. Leuven, Kasteelpark Arenberg 20, 3001 Leuven, Belgium,1 Unité de Génétique, Institut des Sciences de la Vie (ISV), Université catholique de Louvain, Louvain-la-Neuve, Belgium2

Received 4 November 2005/ Accepted 15 April 2006

Lactobacillus rhamnosus GG is of general interest as a probiotic. Although L. rhamnosus GG is often used in clinical trials, there are few genetic tools to further determine its mode of action or to develop it as a vehicle for heterologous gene expression in therapy. Therefore, we developed a reproducible, efficient electroporation procedure for L. rhamnosus GG. The best transformation efficiency obtained was 104 transformants per µg of DNA. We validated this protocol by tagging L. rhamnosus GG with green fluorescent protein (GFP) using the nisin-controlled expression (NICE) system. Parameters for overexpression were optimized, which allowed expression of gfp in L. rhamnosus GG upon induction with nisin. The GFP+ strain can be used to monitor the survival and behavior of L. rhamnosus GG in vivo. Moreover, implementation of the NICE system as a gene expression switch in L. rhamnosus GG opens up possibilities for improving and expanding the performance of this strain. The GFP-labeled strain was used to demonstrate that L. rhamnosus GG is sensitive to human beta-defensin-2 but not to human beta-defensin-1.

* Corresponding author. Mailing address: Centre of Microbial and Plant Genetics, K.U. Leuven, Kasteelpark Arenberg 20, 3001 Leuven, Belgium. Phone: 32 16 321631. Fax: 32 16 321966. E-mail: jozef.vanderleyden{at}biw.kuleuven.be.

Applied and Environmental Microbiology, July 2006, p. 4923-4930, Vol. 72, No. 7
0099-2240/06/$08.00+0     doi:10.1128/AEM.02605-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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Copyright © 2006 by the American Society for Microbiology. All rights reserved.