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. 2016 Sep 22;11(9):e0162983.
doi: 10.1371/journal.pone.0162983. eCollection 2016.

Genome Analysis and Characterisation of the Exopolysaccharide Produced by Bifidobacterium longum subsp. longum 35624™

Friedrich Altmann  1 , Paul Kosma  1 , Amy O'Callaghan  2 , Sinead Leahy  2 , Francesca Bottacini  2 , Evelyn Molloy  2 , Stephan Plattner  3 , Elisa Schiavi  4   5 , Marita Gleinser  2 , David Groeger  5 , Ray Grant  5 , Noelia Rodriguez Perez  4 , Selena Healy  3 , Elisabeth Svehla  1 , Markus Windwarder  1 , Andreas Hofinger  1 , Mary O'Connell Motherway  2 , Cezmi A Akdis  4 , Jun Xu  6 , Jennifer Roper  3 , Douwe van Sinderen  2 , Liam O'Mahony  4
Affiliations

Genome Analysis and Characterisation of the Exopolysaccharide Produced by Bifidobacterium longum subsp. longum 35624™

Friedrich Altmann et al. PLoS One. .

Abstract

The Bifibobacterium longum subsp. longum 35624™ strain (formerly named Bifidobacterium longum subsp. infantis) is a well described probiotic with clinical efficacy in Irritable Bowel Syndrome clinical trials and induces immunoregulatory effects in mice and in humans. This paper presents (a) the genome sequence of the organism allowing the assignment to its correct subspeciation longum; (b) a comparative genome assessment with other B. longum strains and (c) the molecular structure of the 35624 exopolysaccharide (EPS624). Comparative genome analysis of the 35624 strain with other B. longum strains determined that the sub-speciation of the strain is longum and revealed the presence of a 35624-specific gene cluster, predicted to encode the biosynthetic machinery for EPS624. Following isolation and acid treatment of the EPS, its chemical structure was determined using gas and liquid chromatography for sugar constituent and linkage analysis, electrospray and matrix assisted laser desorption ionization mass spectrometry for sequencing and NMR. The EPS consists of a branched hexasaccharide repeating unit containing two galactose and two glucose moieties, galacturonic acid and the unusual sugar 6-deoxy-L-talose. These data demonstrate that the B. longum 35624 strain has specific genetic features, one of which leads to the generation of a characteristic exopolysaccharide.

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Conflict of interest statement

I have read the journal's policy and the authors of this manuscript have the following competing interests: David Groeger, Elisa Schiavi and Ray Grant are employees of Alimentary Health Pharma Davos AG, Jun Xu is an employee of Procter & Gamble, while Stephan Plattner, Selena Healy and Jennifer Roper are employees of Alimentary Health Ltd. Liam O’Mahony has received research funding from GSK and is a consultant to Alimentary Health Ltd. Cezmi Akdis has received research support from Novartis and Stallergenes and consulted for Actellion, Aventis and Allergopharma. Friedrich Altmann, Paul Kosma, Elisabeth Svehla, Markus Windwarder, Andreas Hofinger, Amy O’Callaghan, Sinead Leahy, Francesca Bottacini, Evelyn Molloy, Noelia Rodriguez Perez, Elisa Schiavi, Marita Gleinser, Mary O’Connell Motherway and Douwe van Sinderen have no competing interests. While competing interests are declared for some of the authors, the content of this article was neither influenced nor constrained by this fact and this does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Phylogenetic tree based on the B. longum core-genome.
(A) The B. longum subsp. longum phylogenetic group. (B) The B. longum subsp. infantis phylogenetic group. B. longum subsp. longum and B. longum subsp. infantis type strains are indicated in blue text. Lactobacillus salivarius was included as an outlier.
Fig 2
Fig 2. EPS gene cluster.
Illustration of the EPS cluster located in the B. longum 35624 genome and comparison to similar clusters located in B. longum 105-A, B. longum subsp. longum JCM1217 and B. longum subsp. longum NCC2705. Each gene is colour-coded according to function which is indicated in the legend located at the end of the page. Percentages represent the percent of sequence similarity at the protein level with corresponding genes in the B. longum 35624 genome. The locus tags of the first and last genes located in the EPS clusters of B. longum 105-A, B. longum subsp. longum JCM1217 and B. longum subsp. longum NCC2705 are also indicated in the illustration.
Fig 3
Fig 3. B. longum 35624 electron microscopy.
(A) A layer of extracellular polysaccharide is clearly visible by electronic microscopy of the 35624 strain. (B) The isolated and purified EPS is illustrated.
Fig 4
Fig 4. B. longum 35624 EPS characterization.
(A) Comparison of the EPS (solid line) with a dextran standards dotted line) demonstrated that EPS had an average molecular mass much higher than 1 MDa (Mw). (B) HPLC analysis of anthranilic acid-labeled monosaccharides of EPS revealed the presence of glucose (Glc), galactose (Gal), some galacturonic acid (GalA) and two additional peaks with masses corresponding to an aldobiuronic acid and a deoxy-hexose later identified as 6-deoxy-talose. The upper trace in (B) is the standard mixture and the lower trace shows the results of the EPS sample. Numbers in the lower trace give the masses of the compounds as determined by off-line ESI-MS.
Fig 5
Fig 5. Mild acid hydrolysis of EPS.
(A) Separation of EPS fragments by PGC HPLC with MS/MS detection. The extracted ion chromatogram for mass 1008.39 Da shows four peaks. Their reducing end sugar was clearly revealed by ESI-MS/MS. Their assignment as either Gal or Glc and the interpretation in terms of fragment structures was done a posteriori based on MALDI-TOF data and on knowledge of the EPS structure. (B) Example of a MALDI-TOF/TOF fragment spectrum showing b-ions from the non-reducing and y- and y´ (= 1,5x) -ions from the reducing end.
Fig 6
Fig 6. B. longum 35624 EPS characterization.
(A) The 600 MHz 1H NMR proton spectrum of the acid-treated 35624 EPS (D2O, 338 K) is illustrated. A part of the high-field region is displayed in the insert. (B) Expansion plot of the 150 MHz 13C NMR spectrum of the acid-treated 35624 exopolysaccharide. The anomeric signals on the left confirmed the presence of a hexasaccharide repeat unit.
Fig 7
Fig 7. B. longum 35624 EPS proton and carbon signals.
(A) A selected region of the multiplicity-edited, gradient enhanced 1H, 13C-HSQC NMR spectrum of the exopolysaccharide. Letters denote the residues as given in the structural formula and arabic numerals denote the respective pyranose position. Resonances from anomeric carbons/protons, glycosylation sites and resolved signals are annotated. (B) Selected region of the 1H, 13C-HSQC-TOCSY NMR spectrum (600 MHz) of the acid-treated 35624 EPS. Arabic numerals before and after oblique stroke denote carbons and protons, respectively.
Fig 8
Fig 8. B. longum 35624 EPS composition and structure.
The structure is annotated as the chemical formula and in condensed form. Capital letters denote the residues as in Figs 6 and 7.
See this image and copyright information in PMC

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