title: Mechanistic Study of Utilization of Water-Insoluble Saccharomyces cerevisiae Glucans by Bifidobacterium breve Strain JCM1192.
abstract: Bifidobacteria exert beneficial effects on hosts and are extensively used as probiotics. However, due to the genetic inaccessibility of these bacteria, little is known about their mechanisms of carbohydrate utilization and regulation.Bifidobacterium brevestrain JCM1192 can grow on water-insoluble yeast (Saccharomyces cerevisiae) cell wall glucans (YCWG), which were recently considered as potential prebiotics. According to the results of1H nuclear magnetic resonance (NMR) spectrometry, the YCWG were composed of highly branched (1→3,1→6)-β-glucans and (1→4,1→6)-α-glucans. Although the YCWG were composed of 78.3% β-glucans and 21.7% α-glucans, only α-glucans were consumed by theB. brevestrain. The ABC transporter (malEFG1) and pullulanase (aapA) genes were transcriptionally upregulated in the metabolism of insoluble yeast glucans, suggesting their potential involvement in the process. A nonsense mutation identified in the gene encoding an ABC transporter ATP-binding protein (MalK) led to growth failure of an ethyl methanesulfonate-generated mutant with yeast glucans. Coculture of the wild-type strain and the mutant showed that this protein was responsible for the import of yeast glucans or their breakdown products, rather than the export of α-glucan-catabolizing enzymes. Further characterization of the carbohydrate utilization of the mutant and three of its revertants indicated that this mutation was pleiotropic: the mutant could not grow with maltose, glycogen, dextrin, raffinose, cellobiose, melibiose, or turanose. We propose that insoluble yeast α-glucans are hydrolyzed by extracellular pullulanase into maltose and/or maltooligosaccharides, which are then transported into the cell by the ABC transport system composed of MalEFG1 and MalK. The mechanism elucidated here will facilitate the development ofB. breveand water-insoluble yeast glucans as novel synbiotics.IMPORTANCEIn general,Bifidobacteriumstrains are genetically intractable. Coupling classic forward genetics with next-generation sequencing, here we identified an ABC transporter ATP-binding protein (MalK) responsible for the import of insoluble yeast glucan breakdown products byB. breveJCM1192. We demonstrated the pleiotropic effects of the ABC transporter ATP-binding protein in maltose/maltooligosaccharide, raffinose, cellobiose, melibiose, and turanose transport. With the addition of transcriptional analysis, we propose that insoluble yeast glucans are broken down by extracellular pullulanase into maltose and/or maltooligosaccharides, which are then transported into the cell by the ABC transport system composed of MalEFG1 and MalK. The mechanism elucidated here will facilitate the development ofB. breveand water-insoluble yeast glucans as novel synbiotics.
authors.affiliation: School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong SAR.
authors.forename: Hoi Yee
authors.initials: HY
authors.lastname: Keung
authors.affiliation: School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong SAR.
authors.forename: Tsz Kai
authors.initials: TK
authors.lastname: Li
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Lok To
authors.initials: LT
authors.lastname: Sham
authors.affiliation: School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong SAR.
authors.forename: Man Kit
authors.initials: MK
authors.lastname: Cheung
authors.affiliation: School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong SAR.
authors.forename: Peter Chi Keung
authors.initials: PCK
authors.lastname: Cheung
authors.affiliation: School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong SAR hoishankwan@cuhk.edu.hk.
authors.forename: Hoi Shan
authors.initials: HS
authors.lastname: Kwan
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D000069984:Bifidobacterium breve / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism; D003912:Dextrins / Q000494:pharmacology; D005936:Glucans / Q000378:metabolism*; D006003:Glycogen / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D008320:Maltose / Q000378:metabolism / Q000494:pharmacology; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D012995:Solubility; D058616:Synbiotics; D014867:Water; D047071:beta-Glucans / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D003912:Dextrins; D005936:Glucans; C068021:MalK protein, Bacteria; D047071:beta-Glucans; D014867:Water; D008320:Maltose; D006003:Glycogen; D006026:Glycoside Hydrolases; C100181:pullulanase
keywords: ABC transporters; Bifidobacterium breve; genome resequencing; mutants; yeast glucans