title: Flow cytometric testing of green fluorescent protein-tagged Lactobacillus rhamnosus GG for response to defensins.
abstract: 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 10(4) transformants per microg 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.
authors.affiliation: Centre of Microbial and Plant Genetics, K.U. Leuven, Kasteelpark Arenberg 20, 3001 Leuven, Belgium. jozef.vanderleyden@biw.kuleuven.be
authors.forename: Sigrid C J
authors.initials: SC
authors.lastname: De Keersmaecker
authors.forename: Kristien
authors.initials: K
authors.lastname: Braeken
authors.forename: Tine L A
authors.initials: TL
authors.lastname: Verhoeven
authors.forename: Mónica
authors.initials: M
authors.lastname: Perea Vélez
authors.forename: Sarah
authors.initials: S
authors.lastname: Lebeer
authors.forename: Jos
authors.initials: J
authors.lastname: Vanderleyden
authors.forename: Pascal
authors.initials: P
authors.lastname: Hols
mesh_terms: D000890:Anti-Infective Agents / Q000494:pharmacology*; D018274:Electroporation; D005434:Flow Cytometry / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D052201:Lacticaseibacillus rhamnosus / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D009561:Nisin / Q000494:pharmacology*; D014169:Transformation, Bacterial; D023083:beta-Defensins / Q000494:pharmacology*
chemical_list: D000890:Anti-Infective Agents; C406407:DEFB1 protein, human; C106641:DEFB4A protein, human; D023083:beta-Defensins; D009561:Nisin; D049452:Green Fluorescent Proteins