Genomic and genetic characterization of the bile stress response of probiotic Lactobacillus reuteri ATCC 55730

Abstract

Probiotic bacteria encounter various stresses after ingestion by the host, including exposure to the low pH in the stomach and bile in the small intestine. The probiotic microorganism Lactobacillus reuteri ATCC 55730 has previously been shown to survive in the human small intestine. To address how L. reuteri can resist bile stress, we performed microarray experiments to determine gene expression changes that occur when the organism is exposed to physiological concentrations of bile. A wide variety of genes that displayed differential expression in the presence of bile indicated that the cells were dealing with several types of stress, including cell envelope stress, protein denaturation, and DNA damage. Mutations in three genes were found to decrease the strain's ability to survive bile exposure: lr1864, a Clp chaperone; lr0085, a gene of unknown function; and lr1516, a putative esterase. Mutations in two genes that form an operon, lr1584 (a multidrug resistance transporter in the major facilitator superfamily) and lr1582 (unknown function), were found to impair the strain's ability to restart growth in the presence of bile. This study provides insight into the possible mechanisms that L. reuteri ATCC 55730 may use to survive and grow in the presence of bile in the small intestine.

FIG. 1.

Representative growth curve of L. reuteri ATCC 55730 used for microarray experiments. Arrows represent time points where samples were taken for RNA isolation. Open arrows represent samples for bile shock experiments; filled arrows represent samples for bile adaptation experiments. Oxgall (0.5%) was added at 250 min.

FIG. 2.

Comparison of survival rates after 30 min of exposure to 0.3% oxgall for the L. reuteri ATCC 55730 wild type and lr1864 (ClpL), lr1516 (putative esterase), lr0085 (unknown), lr1706 (Dps), lr1265 (multidrug resistance protein in the ABC transporter family), and lr1584 (multidrug resistance protein in the major facilitator superfamily) mutant strains. Cultures were plated onto MRS plates after bile exposure to determine the number of viable cells. Error bars represent standard deviation. *, P < 0.001 compared with wild type.