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A bile-inducible membrane protein mediates bifidobacterial bile resistance.

Ruiz L, O'Connell-Motherway M, Zomer A, de los Reyes-Gavilán CG, Margolles A, van Sinderen D - Microb Biotechnol (2012)

Bottom Line: The high level of similarity to bile efflux pumps from other bifidobacteria suggests a significant and general role for Bbr_0838 in bile tolerance.Insertional inactivation of Bbr_0838 in B. breve UCC2003 resulted in a strain, UCC2003:838(800) , which exhibited reduced survival upon cholate exposure as compared with the parent strain, a phenotype that was reversed when a functional, plasmid-encoded Bbr_0838 gene was introduced into UCC2003:838(800) .This study represents the first in-depth analysis of a bile-inducible locus in bifidobacteria, identifying a key gene relevant for bifidobacterial bile tolerance.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiología y Bioquímica de Productos Lácteos, Instituto de Productos Lácteos de Asturias, Villaviciosa, Asturias, Spain.

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Schematic representation of relevant regions of Bbr_0838 gene in B. breve UCC2003 and B. breve UCC2003::838 chromosomes. A. Chromosomal DNA is represented by a thin line, Bbr_0838 by a black arrow, Bbr_0838 fragment employed for homologous recombination by a grey bar and pORI19 by a dashed line. Representative EcoRI sites for Southern hybridization are indicated. B. Southern hybridization of EcoRI digested chromosomal DNA from B. breve UCC2003 (lane 1) and 8 representative insertional mutants (lanes 2 to 4 correspond to UCC2003::838800 clones and lanes 5 to 9 correspond to UCC2003::8381000clones) are shown in B. Sizes of hybridized fragments are shown at the left of the panel. pORI19‐8381000‐tetW was used as a probe and positive control of hybridization was performed on lane 10.
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f2: Schematic representation of relevant regions of Bbr_0838 gene in B. breve UCC2003 and B. breve UCC2003::838 chromosomes. A. Chromosomal DNA is represented by a thin line, Bbr_0838 by a black arrow, Bbr_0838 fragment employed for homologous recombination by a grey bar and pORI19 by a dashed line. Representative EcoRI sites for Southern hybridization are indicated. B. Southern hybridization of EcoRI digested chromosomal DNA from B. breve UCC2003 (lane 1) and 8 representative insertional mutants (lanes 2 to 4 correspond to UCC2003::838800 clones and lanes 5 to 9 correspond to UCC2003::8381000clones) are shown in B. Sizes of hybridized fragments are shown at the left of the panel. pORI19‐8381000‐tetW was used as a probe and positive control of hybridization was performed on lane 10.

Mentions: The bile‐inducibility of Bbr_0838 (Fig. 1C) coupled with its homology to reported bile efflux systems in other bifidobacteria (> 98% identity to betA from B. longum NCC2705) suggests a role for Bbr_0838 in supporting B. breve UCC2003 bile tolerance. In support of this theory, our previous work demonstrated the ability of Bbr_0838 to enhance bile survival of recombinant Lactococcus lactis clones expressing the Bbr_0838 (Ruiz et al., 2011). In order to further investigate the physiological role of Bbr_0838 in B. breve, two Bbr_0838 insertion mutant strains designated UCC2003::838800 and UCC2003::8381000, were constructed (see Experimental procedures). Southern hybridization using EcoRI‐digested genomic DNA and pORI19‐8381000‐tetW as a probe unequivocally confirmed that the disruptions of Bbr_0838 were the result of site‐specific homologous recombination events (Fig. 2). Moreover, the stability of the integrated plasmid in UCC2003::838800 and UCC2003::8381000 strains was analysed following 50 generations in the absence of the selective antibiotic, showing that at least 96% of the cells retained the insertion.


A bile-inducible membrane protein mediates bifidobacterial bile resistance.

Ruiz L, O'Connell-Motherway M, Zomer A, de los Reyes-Gavilán CG, Margolles A, van Sinderen D - Microb Biotechnol (2012)

Schematic representation of relevant regions of Bbr_0838 gene in B. breve UCC2003 and B. breve UCC2003::838 chromosomes. A. Chromosomal DNA is represented by a thin line, Bbr_0838 by a black arrow, Bbr_0838 fragment employed for homologous recombination by a grey bar and pORI19 by a dashed line. Representative EcoRI sites for Southern hybridization are indicated. B. Southern hybridization of EcoRI digested chromosomal DNA from B. breve UCC2003 (lane 1) and 8 representative insertional mutants (lanes 2 to 4 correspond to UCC2003::838800 clones and lanes 5 to 9 correspond to UCC2003::8381000clones) are shown in B. Sizes of hybridized fragments are shown at the left of the panel. pORI19‐8381000‐tetW was used as a probe and positive control of hybridization was performed on lane 10.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815329&req=5

f2: Schematic representation of relevant regions of Bbr_0838 gene in B. breve UCC2003 and B. breve UCC2003::838 chromosomes. A. Chromosomal DNA is represented by a thin line, Bbr_0838 by a black arrow, Bbr_0838 fragment employed for homologous recombination by a grey bar and pORI19 by a dashed line. Representative EcoRI sites for Southern hybridization are indicated. B. Southern hybridization of EcoRI digested chromosomal DNA from B. breve UCC2003 (lane 1) and 8 representative insertional mutants (lanes 2 to 4 correspond to UCC2003::838800 clones and lanes 5 to 9 correspond to UCC2003::8381000clones) are shown in B. Sizes of hybridized fragments are shown at the left of the panel. pORI19‐8381000‐tetW was used as a probe and positive control of hybridization was performed on lane 10.
Mentions: The bile‐inducibility of Bbr_0838 (Fig. 1C) coupled with its homology to reported bile efflux systems in other bifidobacteria (> 98% identity to betA from B. longum NCC2705) suggests a role for Bbr_0838 in supporting B. breve UCC2003 bile tolerance. In support of this theory, our previous work demonstrated the ability of Bbr_0838 to enhance bile survival of recombinant Lactococcus lactis clones expressing the Bbr_0838 (Ruiz et al., 2011). In order to further investigate the physiological role of Bbr_0838 in B. breve, two Bbr_0838 insertion mutant strains designated UCC2003::838800 and UCC2003::8381000, were constructed (see Experimental procedures). Southern hybridization using EcoRI‐digested genomic DNA and pORI19‐8381000‐tetW as a probe unequivocally confirmed that the disruptions of Bbr_0838 were the result of site‐specific homologous recombination events (Fig. 2). Moreover, the stability of the integrated plasmid in UCC2003::838800 and UCC2003::8381000 strains was analysed following 50 generations in the absence of the selective antibiotic, showing that at least 96% of the cells retained the insertion.

Bottom Line: The high level of similarity to bile efflux pumps from other bifidobacteria suggests a significant and general role for Bbr_0838 in bile tolerance.Insertional inactivation of Bbr_0838 in B. breve UCC2003 resulted in a strain, UCC2003:838(800) , which exhibited reduced survival upon cholate exposure as compared with the parent strain, a phenotype that was reversed when a functional, plasmid-encoded Bbr_0838 gene was introduced into UCC2003:838(800) .This study represents the first in-depth analysis of a bile-inducible locus in bifidobacteria, identifying a key gene relevant for bifidobacterial bile tolerance.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiología y Bioquímica de Productos Lácteos, Instituto de Productos Lácteos de Asturias, Villaviciosa, Asturias, Spain.

Show MeSH
Related in: MedlinePlus