| Lactobacillus acidophilus NCFM has been widely used as a probiotic strain in yogurt and dietary supplements. Currently, there is interest in using this strain to deliver vaccines and other biotherapeutics to the gastrointestinal tract. Bile tolerance is crucial for this strain's ability to survive and persist in this environment. A functional genomics approach was used to identify and characterize mechanisms of bile tolerance in L. acidophilus.;Whole genome microarrays were employed to examine the effects of bile on gene transcription in this strain. These experiments showed that after bile exposure, genes involved in carbohydrate metabolism were generally induced, while genes involved in other aspects of cellular growth were mostly repressed. These experiments also identified an 8 gene operon, encoding a two-component regulatory system (2CRS), a transporter, an oxidoreductase, and four hypothetical proteins, for which transcription was significantly induced in the presence of bile. The role of six of these genes was examined by creating NCFM strains with deletion mutations in each of these genes. Mutations in the transporter, the histidine protein kinase (HPK), the response regulator (RR), and a hypothetical protein each resulted in loss of tolerance to bile. Mutations in genes encoding another hypothetical protein and a putative oxidoreductase resulted in significant increases in bile tolerance. Transcriptional analysis of the 2CRS mutant strains showed that deletion of the HPK had no effect on the induction of the operon, whereas mutating the RR strain increased operon induction when cells were exposed to bile. These results indicate that the 2CRS plays a role in bile tolerance, and the operon it resides in is negatively controlled by the response regulator. Genes found in this operon were shown to contribute to both bile tolerance and bile sensitivity.;The role of transporter genes in bile tolerance was examined. NCFM derivatives containing deletion mutations in the bile-induced transporter genes LBA1429, LBA1446, and LBA1679 were examined, along with a strain containing a deletion mutant in LBA0552, a transporter that was equally expressed in the presence and absence of bile. These mutants showed unique patterns of sensitivity to a variety of bile salts, antibiotics, and detergents. Analysis of ciprofloxacin and taurocholate accumulation in these mutants indicated that LBA0552 accumulated more ciprofloxacin than other strains, and that all transporter mutant strains accumulated more taurocholate than NCFM. These results suggest that the transporters play an important role in bile salt export and bile tolerance in L. acidophilus NCFM, as well as mediating tolerance to other inhibitory compounds.;The global transcriptional effects of a bile-induced 2CRS were examined using microarrays. Transcription was compared between the HPK mutant, the RR mutant, and NCFM in the presence and absence of 0.5% oxgall when strains were grown with media containing lactose. When exposed to bile, numerous genes involved in carbohydrate metabolism were expressed more highly in the 2CRS mutants. When cells were grown in glucose or fructose and exposed to bile, however, the induction of carbohydrate metabolism genes was similar in all strains. These results indicate that the 2CRS plays a repressive role on carbohydrate metabolism gene expression in the presence of lactose and bile. When added to intestinal epithelial cells in vitro, the adherence of all strains was decreased when glucose-grown cells were exposed to oxgall, however the adherence of lactose-grown cells was unaffected by bile exposure. The results demonstrate a regulatory network operating between carbohydrate metabolism and bile exposure, and describe the effects of bile on gene transcription and adherence phenotypes of L. acidophilus. |
