The Bifidobacterium longum NCIMB 702259T ctr Gene Codes for a Novel Cholate Transporter

Preexposure of Bifidobacterium longum NCIMB 702259^T to cholatecaused increased resistance to cholate, chloramphenicol, anderythromycin. The B. longum ctr gene, encoding a cholate effluxtransporter, was transformed into the efflux-negative mutantEscherichia coli KAM3, conferring resistance to bile salts andother antimicrobial compounds and causing the efflux of [¹⁴C]cholate.

INTRODUCTION

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Introduction

Bifidobacteria are major components of the human intestinalmicroflora (13) and are widely used as probiotics in food supplements.Probiotic survival depends on resistance to antibiotics andto inhibitory host-produced substances, such as bile salts (9).Bifidobacteria are resistant to a range of antibiotic compounds(5), which could allow them to withstand concurrent antibioticadministration. This study aimed to identify and prove the functionalityof a possible efflux system encoded by the ctr gene in Bifidobacteriumlongum which may contribute to bile and antibiotic resistance.

Adaptation to sodium glycocholate and antibiotics.

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Adaptation to sodium...

To determine the intrinsic MICs for B. longum NCIMB 702259^T(NCIMB, United Kingdom) of antimicrobial agents, 10 µlof a standard cell suspension (optical density at 600 nm, 0.5)of a culture grown anaerobically on BYG agar (14) was spottedonto BYG plates containing a twofold dilution range of sodiumglycocholate, ampicillin, chloramphenicol, erythromycin, ortetracycline. Adaptation to the antibiotics was tested usinga method modified from the work of Carsenti-Etesse et al. (4).Mid-exponential-phase B. longum cells grown in BYG broth werestreaked for four passages onto sodium glycocholate gradientplates, and the MICs for these cells were tested as describedabove. Adapted B. longum showed an increase in resistance tosodium glycocholate, chloramphenicol, and erythromycin but notto ampicillin and tetracycline (Table 1). This indicated thatB. longum may possess multidrug transporters, since these areoften regulated by the compounds that they transport but mayconfer resistance to structurally unrelated antimicrobial agents(3).

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TABLE 1. MICs of antimicrobial agents tested against B. longum NCIMB 702259^a

Cloning and antimicrobial characterization of the ctr gene.

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Cloning and Antimicrobial...

Open reading frame BL1102 (B. longum NCC 2705, GenBank accessionnumber AE014295) was identified as a possible sodium-dependentbile acid transporter. The BL1102 orthologue was isolated fromB. longum NCIMB 702259^T genomic DNA (14), using standard PCRprotocols and the primers ctrans-F (5'-AGCTGAATTCGCGCAACAGG-3')and ctrans-R (5'-ACGCCCGGTACCTCAATCG-3'). EcoRI and KpnI restrictionenzyme sites (underlined) were introduced to ctrans-F and ctrans-R,respectively, to assist subcloning into pBluescriptSK. The nucleotidesequence of the insert in the recombinant plasmid pCtr was determined(14), and nucleotide and amino acid homology searches were performedusing the BLAST algorithm and NCBI databases (1). The deducedamino acid sequence of Ctr was 100% identical to that of BL1102.Plasmid pCtr was transformed into competent (2) Escherichiacoli KAM3 (11), a K-12 derivative lacking the multidrug transporterAcrAB. The MICs of acriflavine, sodium dodecyl sulfate (Merck),chloramphenicol, erythromycin, ethidium bromide, tetracycline(Sigma), and sodium glycocholate (Difco) were determined usingthe broth dilution method (8). Plasmid pCtr conferred cholateresistance on E. coli KAM3, increasing the MIC of sodium glycocholateby 16-fold (Table 2). Resistance to the antimicrobial agentswas increased by two- to fourfold.

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TABLE 2. MICs for E. coli KAM3 harboring pCtr and the control vector pBluescriptSK as determined by the broth dilution method^a

Efflux of [¹⁴C]cholate.

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Efflux of [14c]cholate.

To determine whether pCtr conferred resistance to bile throughthe active efflux of the compound, de-energized washed cellsuspensions of E. coli KAM3 (pCtr or pBluescriptSK) that hadbeen grown to mid-exponential phase in Luria-Bertani broth (14)were preloaded with [carboxyl-¹⁴C]cholic acid (New England NuclearCorp.). The amount of cell-associated radioactivity was monitoredwith and without the addition of glucose as an energy source.The method of Yokota et al. (17) was used with the followingmodifications. Washed cells were resuspended to an optical densityat 600 nm of 4, aliquots of 1.94 ml were used in the experiment,and all incubation was at 37°C. To preload the cells withcholate, 40 µl of 5.8 mM [¹⁴C]cholate (16 mCi/mmol) wasadded (final cholate concentration, 0.116 mM). The amount ofradioactivity associated with each aliquot was used to calculatethe counts per minute per mmol of cholate. The results wereexpressed as nmol cholate/µg protein, determined usinga DC protein assay kit (Bio-Rad). In the absence of glucose,the external and internal cellular cholate concentrations reachedequilibrium within 35 min (Fig. 1A and B). Upon the additionof glucose, cholate was transiently accumulated in the presenceand absence of pCtr. In the absence of pCtr, an equilibriumwas again reached after 40 min (Fig. 1B). When pCtr was present,however, there was a decrease in the level of cell-associatedcholic acid (Fig. 1A), indicating an active efflux of cholate.Glucose metabolism results in a slow generation of a pH gradient,which is likely to drive the initial accumulation of cholate.The subsequent activity of the Na⁺/H⁺ antiporter results inthe generation of a sodium gradient necessary to drive the cholatetransporter. This is evidence that the ctr gene of B. longumencodes a cholate efflux transport system that is functionalin E. coli.

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FIG. 1. Energy-dependent extrusion of [¹⁴C]cholate in E. coli KAM3 harboring (A) pCtr or (B) pBluescriptSK. Cells were preloaded with cholate, and the amount of cell-associated cholate was subsequently measured over time with the addition of glucose (dotted lines) or without glucose (solid lines) Glucose was added to a final concentration of 10 mM at 35 min as indicated by the arrow. During each experiment, samples were taken in triplicate, and the entire experiment was also performed in triplicate. Error bars indicate the deviation from the mean.

Bioinformatic and phylogenetic analysis of the Ctr protein.

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Bioinformatic and Phylogenetic...

Ctr belongs to the sodium/bile acid family (SBF) of transporters(10), showing the signature motif of this family (Fig. 2). Analysisof the predicted membrane topology revealed the presence ofnine transmembrane segments as well as a highly conserved prolineresidue, corresponding to P²⁹⁰ in the human bile transporter(Fig. 2), which is an essential residue for bile acid transport(15). The phylogenetic relationship of various SBF proteinsfrom different taxa was determined using the neighbor-joiningmethod of ClustalW (Fig. 3). The Ctr protein is closely relatedto a number of sodium bile acid cotransporter proteins frombacteria, including two Streptococcus species and Leuconostocmesenteroides. Prior to this study, the members of the SBF familywith proved function were all in eukaryotes, and in mammals,these transmembrane proteins are responsible for the cotransportof sodium and bile acids across the plasma membrane in the liverand ileum (6, 7). SBF transporters from plants, namely, Arabidopsisthaliana and Oryza sativa (12), form a separate distinct cluster.These are inducible during growth, but neither their effluxfunction nor their substrates have been established. The ACR3protein from Saccharomyces cerevisiae is an efflux transmembraneprotein involved in resistance to arsenic compounds (16).

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FIG. 2. Multiple sequence alignment of the significant regions of the putative B. longum bile transporter Ctr (GenBank accession no. DQ017587) with closely related bacterial sodium/bile acid transporters. Sequences included are from the following organisms: B. longum (Ctr) (NP_696274), Streptococcus thermophilus (S.the) (YP_141686), Streptococcus mutans (S.mut) (NP_721034), Leuconostoc mesenteroides (L.mes) (ZP_00063561), Oceanobacillus iheyensis (O.ihe) (NP_691915), Bacillus subtilis (B.sub) (CAB13827.1), Pseudomonas fluorescens (P.flu) (ZP_00262238.1), and Neisseria meningitides (N.men) (NP_273747). Amino acids conserved in all sequences are shaded in dark gray, and amino acids conserved in over 75% of the sequences are shaded in light gray. The conserved proline residue is indicated by an asterisk, and the SBF signature motif is underlined.

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FIG. 3. Phylogenetic relationship analysis of bacterial sodium/bile acid transporters, namely, Ctr from B. longum, Streptococcus thermophilus, Streptococcus mutans, Leuconostoc mesenteroides, Oceanobacillus iheyensis, Bacillus subtilis, Pseudomonas fluorescens, and Neisseria meningitidis; the plant sodium/bile acid symporter-like proteins AtSbf1 (Arabidopsis thaliana) and OsSbf1 (Oryza sativa); mammalian ileal sodium/bile acid transporters (IBAT) from Homo sapiens, Mus musculus, Oryctolagus cuniculus, and Rattus norwegicus; mammalian hepatic sodium/bile acid transporters (HBAT) from M. musculus and O. cuniculus; and the arsenate resistance protein (ACR3) from Saccharomyces cerevisiae. Database accession numbers are given in parentheses. The tree was constructed using the neighbor-joining method, and bootstrap values (250 replicates) are given at the branch points.

In this study we have confirmed that the ctr gene of B. longumencodes a cholate transporter which is responsible for the effluxof cholate from E. coli and confers resistance to a number ofstructurally unrelated antimicrobial compounds. This is thefirst characterization of a bile acid transporter of the SBFfamily in bacteria and the first multidrug transporter to becharacterized from a Bifidobacterium species.

Nucleotide sequence accession number.

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Nucleotide sequence accession...

The GenBank accession number for the putative B. longum biletransporter Ctr is DQ017587.

ACKNOWLEDGMENTS

We thank the National Research Foundation (NRF) South Africafor financial support of this project. C. E. Price acknowledgesan NRF bursary and a University of Cape Town travel scholarship.

FOOTNOTES

* Corresponding author. Mailing address: Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, 7701 Cape Town, South Africa. Phone: 27 21 6502183. Fax: 27 21 6897573. E-mail: val{at}science.uct.ac.za

REFERENCES

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