Bifidobacterium Longum Ncc2705 Fructose Abc Transporter System

Bifidobacteria are one of the predominant groups of the commensal microflora in the gastrointestinal tract (GIT).They exert various beneficial effects on the health of the human host like nutrition amelioration, immunity enhancement, anti-anaphylaxis, anti-neoplasms, anti-infection and intestines functions regulation. In addition, Bifidobacteria also are researched as vectors of neoplasms-targeted therapy and gene vaccine.In our study, a novel ABC transporter system for fructose uptake in Bifidobacterium longum NCC2705 was detected and validated.B. longum NCC2705 was able to utilize 6 monosaccharides including fructose, galactose, ribose, xylose, glucose and mannose. The molar ratio of acetic acid to lactic acid varied from 1.27 to 2.61, higher levels of lactic acid were produced during growth on sugars other than glucose, while acetic acid was produced at higher levels during growth on glucose and mannose. Interestingly, growth curves demonstrated that B. longum NCC2705 had a higher growth rate on fructose, ribose, xylose, and galactose than glucose and mannose as a carbon and energy source, pointing towards a preferential utilization of sugars.To identify the different catabolic pathways of monosaccharide fermentation, the proteomic profiles of the B. longum strain NCC2705 in stationary phase grown on fructose, galactose, mannose, ribose, xylose, or glucose were compared using multiple overlapping narrow pH range(4-7). Assessing the relative differences of protein expression by image analysis revealed a total of 136 spots representing 68 proteins that exhibited a change of 3-fold or greater in cultures grown on fructose, galactose, mannose, ribose or xylose compared to glucose. All of the 68 proteins were identified by MALDI-TOF/TOF MS/MS and/or ESI-MS/MS mass spectrometry and included: 1) components of the ABC transport system; 2) metabolic enzymes involved in breakdown of different monosaccharides; 3) key stress proteins; 4) proteins related to translation; 5) transcriptional regulators and 6) hypothetical proteins. The cellular localizations of the 68 identified proteins were predicted using PSORT version 2.0 including cytoplasmic, cytoplasmic membrane, cell wall and extracellular. Interestingly, 14 proteins including 8 ABC transporters family proteins had unknown cellular localization. Moreover four proteins were predicted to have a signal peptide.These results indicate that changes in carbon sources may have a more global regulatory effect in B. longum NCC2705. In the present experiment, we found that posttranslational protein modifications are common in B. longum NCC2705. To determine whether the changes in protein mobility reflect protein phosphorylation, Pro-Q Diamond phosphoprotein staining was performed on excised sections of 2-D gels. THis revealed that different spots of GroEL, Eno and Tal as well as Pgm stained positive with Pro-Q Diamond indicating different phosphorylation patterns of these proteins. To further determined the phosphorylation sites, Western Blot analysis was performed using antibodies against phosphor-serine, -threonine, and -tyrosine (P-Ser, P-Thr, and P-Tyr, respectively). None of the proteins stained positive with the P-Thr-specific antibody but all four proteins contained phosphorylated serine residues. Additionally, GroEL and Eno were phosphorylated at tyrosine residues.The most intriguing finding from our observations was that BL0033, a putative sugar-binding protein of an ABC transporter system exhibited specifically high levels of expression in the cells grown on fructose, ribose and xylose. As highlighted on the partial map, the spot intensities of BL0033 under these conditions are increased more than 50-fold compared to bacteria grown on glucose.There were more than five isoforms of BL0033 differing in charge identified by MALDI-TOF/TOF MS/MS and ESI-MS/MS. Subsequent Pro-Q Diamond and Western Blot analysis revealed that BL0033 was phosphorylated on both serine and tyrosine residues with the major spot carrying a phosphorylation at a serine residue and four spots carrying tyrosine phosphorylation.Result of bioinformatics,BL0033 belongs to a sugar ABC transport system that is composed by BL0033\BL0034\BL0035\BL0036. BL0033 is predicted as a sugar binding protein of a ABC transport system; BL0034 is predicted as a ATP protein of a ABC transport system; BL0035 and BL0036 are predicted as transporters in a ABC transport system. Due to the results of comparation of both at the protein and transcriptional levels of different time pionts and concentrations in cells grown on fructose, ribose, xylose or glucose, we found that BL0033 could be introduced by ribose and xylose,especially by fructose. Further more,BL0033\BL0035\BL0036 were demonstrated their ability of sugar binding by capillary electrophoresis (CE) while BL0034 was demonstrated as a ATP binding protein by ATP-binding test.Purified BL0033\BL0034\BL0035\BL0036 protein with GST or His tag were prepared, then with GST pull-down and Western Blot, the interactions with any of other 3 of these 4 proteins were validated. To further map the BL0033 binding sites with BL0034, five truncated versions of BL0033 and BL0034 (BL0033/1-23, BL0033/36-314, BL0034/8-244, BL0034/33-220 and BL0034/289-481 spanning different motifs of these proteins) were constructed and expressed. These truncated proteins were used in further GST pull-down assays which showed that BL0033/1-23 spanning the first 23 amino acids interacted strongly with BL0034/8-244 and BL0034/33-220. THis region of BL0033 contains a DNA recombination-mediator protein A motif which interacts with the ATP-binding cassette motif in BL0034 located between amino acid 33 to 220. Also, it had been known that BL0033 was phosphorylated on both serine and tyrosine residues as described above. Further analysis using Pro-Q Diamond staining and Western Blot revealed that the truncated mutants BL0033/36-314 (containing periplasmic binding proteins) were phosphorylated. BL0033/36-314aa is the binding region for fructose, ribose and xylose, suggesting that the substrate such as fructose, ribose or xylose can triggered the phosphorylation of BL0033 during the substrate binds to BL0033 and is transported from extracellular into the intracellular.In conclusion, BL0033\BL0034\BL0035\BL0036 composed a classic ABC transport system including a secreted receptor protein, a membrane-integral component, and an ATPase subunit.So We propose to name BL0033-BL0036 as Fru-ABC Transport System in B. longum NCC2705.