Bile-inducible efflux transporter from Bifidobacterium longum NCC2705, conferring bile resistance

Abstract

Bifidobacteria are normal inhabitants of the human gut. Some strains of this genus are considered health promoting or probiotic, being included in numerous food products. In order to exert their health benefits, these bacteria must overcome biological barriers, including bile salts, to colonize and survive in specific parts of the intestinal tract. The role of multidrug resistance (MDR) transporters in bile resistance of probiotic bacteria and the effect of bile on probiotic gene expression are not fully understood. In the present study, the effect of subinhibitory concentrations of bile on the expression levels of predicted MDR genes from three different bifidobacterial strains, belonging to Bifidobacterium longum subsp. longum, Bifidobacterium breve, and Bifidobacterium animalis subsp. lactis, was tested. In this way, two putative MDR genes whose expression was induced by bile, BL0920 from B. longum and its homolog, Bbr0838, from B. breve, were identified. The expression of the BL0920 gene in Escherichia coli was shown to confer resistance to bile, likely to be mediated by active efflux from the cells. To the best of our knowledge, this represents the first identified bifidobacterial bile efflux pump whose expression is induced by bile.

FIG. 1.

Effect of subinhibitory concentrations of bile (0.1, 0.08, and 0.05%, respectively) on the transcription of putative MDR-encoding genes in B. longum subsp. longum NCC2705 (a), B. breve UCC2003 (b), and B. animalis subsp. lactis BB-12 (c) relative to the transcription of such genes in the absence of bile. The horizontal axis is placed at 1, which is the ratio reflecting the change in the expression level.

FIG. 2.

Phylogenetic relationship analysis of the permease domains of BL0920, Bbr0838, and other highly similar putative MDR proteins. The tree was constructed using the fast minimum evolution method and the Grishin distance model (12). Database accession numbers are given in parentheses.

FIG. 3.

Bile-mediated effects on the culture viability following 30, 90, and 180 min of exposure to medium containing 3% bile. Results were calculated as change in viability relative to culture viability immediately prior to the addition of bile.

FIG. 4.

Fluorescence levels in cells and supernatants of tDE3 cells carrying the empty pETblue-1 plasmid or the same plasmid containing the BL0920 gene (plasmid pETBL0920), after cells were loaded with fluorescent UDCA followed by 5 min of incubation (37°C) in the presence of glucose. Dark gray bars represent the fluorescence in the supernatants, and light gray bars represent the fluorescence in the cells. Values are expressed as percent change relative to the fluorescence values at time zero.

FIG. 5.

Organization of the B. longum subsp. longum NCC2705 genomic region containing the BL0920 gene and its flanking sequences and the corresponding sequences in B. breve UCC2003, B. adolescentis ATCC 15703, and B. longum subsp. infantis ATCC 15697. The pin-like symbol indicates a Rho-independent transcription terminator. The arrows indicate the relative positions and direction of transcription of the ORFs. White arrows indicate ORFs without significant homology. Arrows with the same filling pattern are considered to encode homologous proteins. Gray-shaded areas indicate DNA homologous sequences. HP, hypothetical protein; MFS, major facilitator superfamily; ssDNA, single-stranded DNA; CoA, coenzyme A.