The molecular response of Lactobacillus plantarum to intestinal passage and conditions

Lactobacillus plantarumis a lactic acid bacterium (LAB) that is encountered in many environmental niches, including dairy, meat and a variety of vegetable fermentations. Next to the occurrence of L. plantarum in our diets, this microbe is frequently encountered as a natural inhabitant of thegastrointestinal tract(GI-tract).This thesis describes the identification and functional analysis of L. plantarumpromoters andgenes that play a role during intestinal passage of this LAB. During these studies the mouse GI-tract and in vitro studies were utilized as models for the human situation. Three main strategies that allow the identification of conditionally active genes have been implemented in L. plantarum , namely an in vitro screen to trap bile-inducible promoters, DNA micro-array technology to identify mRNAs that are up-regulated in the presence of bile, and a resolvase-based in vivo expression technology (R-IVET) screen to select promoters that are induced in the GI-tract of a mouse model system.Matching of the genes identified with these three strategies revealed two genes, encoding an integral membrane protein and anargininosuccinate synthase, which appeared to be induced by bile in vitro as well as in vivo in the mouse GI-tract.As the duodenum is the intestinal site of bile release, expression of these two genes at this specific location in the host's GI-tract was investigatedusingquantitative reverse transcription PCR. The results demonstrated that the expression levels of these two genes were dramatically increased in L. plantarum cells isolated from the mouse duodenum relative to cells grown on standard laboratory medium.Furthermore, gene disruption mutants were constructed in 12 of the in vivo induced ( ivi ) genes identified by the R-IVET screen. The effect of the mutations on the survival and persistence of L. plantarum were investigated using quantitative PCRto monitor the relative population dynamics of the group of mutants in faecal samples after passage through the GI-tract of mice.These data indicate that the products of three of the disrupted genes (an orphanIIC transport component of a cellobiose PTS system, an extracellular protein that contains an LPQTNE motif involved in anchoring of this protein to the bacterial cell wall, and acopper transporting ATPase) play a key-role in the survival and/or persistence of L. plantarum during passage of the GI-tract. Overall, the results described in this thesis represent a first step towards molecular elucidation of the behavior of L. plantarum in a highly complex and inaccessible niche like the GI-tract. These results contribute to the development of eventual molecular models that describe GI-tract behavior of LAB, which are fundamental for the explanation of probiotic effects associated with these microbes and related species at the molecular level.