Genetic characterization of the bile salt response in Lactobacillus plantarum and analysis of responsive promoters in vitro and in situ in the gastrointestinal tract

Abstract

In this paper we describe the growth, morphological, and genetic responses of Lactobacillus plantarum WCFS1 to bile. Growth experiments revealed that a stepwise increase in the porcine bile concentration led to a gradual decrease in the maximal growth rate. Moreover, the final density reached by an L. plantarum culture growing in MRS containing 0.1% bile was approximately threefold lower than that in MRS lacking bile. The morphology of the cells grown in MRS containing 0.1% bile was investigated by scanning electron microscopy, which revealed that cells clumped together and had rough surfaces and that some of the cells had a shrunken and empty appearance, which clearly contrasted with the characteristic rod-shaped, smooth-surface morphology of L. plantarum cells grown in MRS without bile. An alr complementation-based genome-wide promoter screening analysis was performed with L. plantarum, which led to identification of 31 genes whose expression was potentially induced by 0.1% porcine bile. Remarkably, 11 membrane- and cell wall-associated functions appeared to be induced by bile, as were five functions involved in redox reactions and five regulatory factors. Moreover, the lp_0237 and lp_0775 genes, identified here as genes that are inducible by bile in vitro, were previously identified in our laboratory as important for L. plantarum in vivo during passage in the mouse gastrointestinal tract (P. A. Bron, C. Grangette, A. Mercenier, W. M. de Vos, and M. Kleerebezem, J. Bacteriol. 186:5721-5729, 2004). A quantitative reverse transcription-PCR approach focusing on these two genes confirmed that the expression level of lp_0237 and lp_0775 was significantly higher in cells grown in the presence of bile and cells isolated from the mouse duodenum than in cells grown on laboratory medium without bile.

FIG. 1.

Maximal growth rateS of L. plantarum in the presence of increasing concentrations of bile salts. A full-grown culture was diluted 50-fold in MRS containing no porcine bile (□), 0.01% porcine bile (▵), 0.05% porcine bile (○), 0.10% porcine bile (▪), or 0.15% porcine bile (▴), and growth was monitored by measuring the OD₆₀₀. The resulting data were used to calculate the maximal logarithmic growth rateS (0.56, 0.54, 0.52, 0.44, and 0.14 h⁻¹, respectively). The arrow indicates the time at which the morphology of the cells was investigated (Fig. 2).

FIG. 2.

Morphological changes in L. plantarum during bile salt stress. After 4 h of exposure to no bile salts (A), 0.05% bile salts (B), 0.10% bile salts (C), or 0.15% bile salts (D) cells were investigated by scanning electron microscopy.