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Cited by 134Polysaccharides Composition and ApplicationsProbiotics and Fermented FoodsEscherichia coli research studies

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Structure and Biosynthesis of Two Exopolysaccharides Produced by Lactobacillus johnsonii FI9785

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Dertli, Enes, Colquhoun, Ian J., Gunning, A. Patrick, Bongaerts, Roy J., Le Gall, Gwénaëlle, Bonev, Boyan B., Mayer, Melinda J., Narbad, Arjan·2013·Journal of Biological Chemistry
DOI 10.1074/jbc.m113.507418PMC PMC3814790
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Abstract

Exopolysaccharides were isolated and purified from Lactobacillus johnsonii FI9785, which has previously been shown to act as a competitive exclusion agent to control Clostridium perfringens in poultry. Structural analysis by NMR spectroscopy revealed that L. johnsonii FI9785 can produce two types of exopolysaccharide: EPS-1 is a branched dextran with the unusual feature that every backbone residue is substituted with a 2-linked glucose unit, and EPS-2 was shown to have a repeating unit with the following structure: -6)-α-Glc p -(1–3)-β-Glc p -(1–5)-β-Gal f -(1–6)-α-Glc p -(1–4)-β-Gal p -(1–4)-β-Glc p -(1-. Sites on both polysaccharides were partially occupied by substituent groups: 1-phosphoglycerol and O -acetyl groups in EPS-1 and a single O -acetyl group in EPS-2. Analysis of a deletion mutant (Δ epsE ) lacking the putative priming glycosyltransferase gene located within a predicted eps gene cluster revealed that the mutant could produce EPS-1 but not EPS-2, indicating that epsE is essential for the biosynthesis of EPS-2. Atomic force microscopy confirmed the localization of galactose residues on the exterior of wild type cells and their absence in the Δ epsE mutant. EPS2 was found to adopt a random coil structural conformation. Deletion of the entire 14-kb eps cluster resulted in an acapsular mutant phenotype that was not able to produce either EPS-2 or EPS-1. Alterations in the cell surface properties of the EPS-specific mutants were demonstrated by differences in binding of an anti-wild type L. johnsonii antibody. These findings provide insights into the biosynthesis and structures of novel exopolysaccharides produced by L. johnsonii FI9785, which are likely to play an important role in biofilm formation, protection against harsh environment of the gut, and colonization of the host. Background: Bacterial cell surface polysaccharides are important in pathogenesis, cell adhesion, and protection against harsh environments. Results: Two novel exopolysaccharide (EPS) structures were identified in Lactobacillus johnsonii . Conclusion: The eps cluster is essential for production of both EPS, but epsE is required only for the heteropolymer. Significance: This study will guide functional analysis of EPS in survival and colonization of gut commensals.

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Figures

FIGURE 1. Molecular organization of the eps cluster of L. johnsonii FI9785. The cluster has 14 genes that are predicted to encode a transcriptional regulator (epsA), a polymerization and chain length determination protein (epsB), a tyrosine-protein kinase (epsC), a protein-tyrosine phosphate phosphohydrolase (epsD), the priming glycosyltransferase UDP-phosphate galactosephosphotransferase (epsE) and five glycosyltransferases (1178–1174), an oligosaccharide repeat unit polymerase (1173), a mutase (glf), an oligosaccharide translocase (epsU), and an EPS biosynthesis protein (1170) (9).
FIGURE 1. Molecular organization of the eps cluster of L. johnsonii FI9785. The cluster has 14 genes that are predicted to encode a transcriptional regulator (epsA), a polymerization and chain length determination protein (epsB), a tyrosine-protein kinase (epsC), a protein-tyrosine phosphate phosphohydrolase (epsD), the priming glycosyltransferase UDP-phosphate galactosephosphotransferase (epsE) and five glycosyltransferases (1178–1174), an oligosaccharide repeat unit polymerase (1173), a mutase (glf), an oligosaccharide translocase (epsU), and an EPS biosynthesis protein (1170) (9).CC BY · PubMed Central
FIGURE 2. NMR analysis shows two novel exopolysaccharides. A, 600-MHz 1H NMR spectra (anomeric region, 338 K, D2O) of exopolysaccharides produced by L. johnsonii FI9785 and two mutant strains. Sugar units b and c are from EPS-1, and units a and d–h are from EPS-2. Peaks labeled m are from the growth medium, those labeled S are from the supernatant fraction, and those labeled P are from the pellet fraction. B, 150-MHz 13C NMR spectra (anomeric region, 338 K, D2O) of exopolysaccharides produced by L. johnsonii FI9785 and a mutant strain. Sugar units b and c are from EPS-1, and units a and d–h are from EPS-2. Peaks labeled m are from the growth medium. C, 600-MHz two-dimensional NMR spectra (338 K, D2O) of exopolysaccharides from L. johnsonii epsCD88N (S). Left, TOCSY spectrum showing coupling networks associated with each anomeric signal; right, ROESY spectrum. Labels indicate hydrogens brought into proximity across glycosidic linkages (a1–f3, c1–b2, etc.).
FIGURE 2. NMR analysis shows two novel exopolysaccharides. A, 600-MHz 1H NMR spectra (anomeric region, 338 K, D2O) of exopolysaccharides produced by L. johnsonii FI9785 and two mutant strains. Sugar units b and c are from EPS-1, and units a and d–h are from EPS-2. Peaks labeled m are from the growth medium, those labeled S are from the supernatant fraction, and those labeled P are from the pellet fraction. B, 150-MHz 13C NMR spectra (anomeric region, 338 K, D2O) of exopolysaccharides produced by L. johnsonii FI9785 and a mutant strain. Sugar units b and c are from EPS-1, and units a and d–h are from EPS-2. Peaks labeled m are from the growth medium. C, 600-MHz two-dimensional NMR spectra (338 K, D2O) of exopolysaccharides from L. johnsonii epsCD88N (S). Left, TOCSY spectrum showing coupling networks associated with each anomeric signal; right, ROESY spectrum. Labels indicate hydrogens brought into proximity across glycosidic linkages (a1–f3, c1–b2, etc.).CC BY · PubMed Central
FIGURE 3. Structure of exopolysaccharides EPS-1 and EPS-2. The sugar rings in EPS-1 and EPS-2 are labeled A–H, and these letters correspond with the (lowercase) labeling of the NMR signals in Fig. 2.
FIGURE 3. Structure of exopolysaccharides EPS-1 and EPS-2. The sugar rings in EPS-1 and EPS-2 are labeled A–H, and these letters correspond with the (lowercase) labeling of the NMR signals in Fig. 2.CC BY · PubMed Central
FIGURE 4. Solid state 31P MAS NMR spectra of EPS from wild type L. johnsonii and from the ΔepsE mutant. Peak numbering corresponds to listing in Table 5.
FIGURE 4. Solid state 31P MAS NMR spectra of EPS from wild type L. johnsonii and from the ΔepsE mutant. Peak numbering corresponds to listing in Table 5.CC BY · PubMed Central
FIGURE 5. Accumulation of exopolysaccharides on the cell surface of L. johnsonii FI9785 and derivative strains. TEM analysis of L. johnsonii FI9785 and mutant strains in MRS medium showing the variation in the EPS layer. Bar, 100 nm.
FIGURE 5. Accumulation of exopolysaccharides on the cell surface of L. johnsonii FI9785 and derivative strains. TEM analysis of L. johnsonii FI9785 and mutant strains in MRS medium showing the variation in the EPS layer. Bar, 100 nm.CC BY · PubMed Central
FIGURE 6. Anti-wild type antibody responses to the wild type and derivative strains measured by flow cytometry. Results are the mean of duplicate experiments ± S.D. (error bars) Significant differences were determined by an independent t test compared with the wild type. *, p < 0.05; **, p < 0.005.
FIGURE 6. Anti-wild type antibody responses to the wild type and derivative strains measured by flow cytometry. Results are the mean of duplicate experiments ± S.D. (error bars) Significant differences were determined by an independent t test compared with the wild type. *, p < 0.05; **, p < 0.005.CC BY · PubMed Central
FIGURE 7. Force-volume images obtained with a PA1-functionalized AFM tip. Shown are L. johnsonii (wild type) topography (A) and adhesion (B) as well as L. johnsonii (ΔepsE mutant) topography (C) and adhesion (D).
FIGURE 7. Force-volume images obtained with a PA1-functionalized AFM tip. Shown are L. johnsonii (wild type) topography (A) and adhesion (B) as well as L. johnsonii (ΔepsE mutant) topography (C) and adhesion (D).CC BY · PubMed Central
FIGURE 8. Adhesion data from force-volume data. A, distribution of rupture force magnitudes using data from Fig. 7 depicted as histograms. Inset, expanded view of data >70 pN. B, distribution of rupture distances. C, distribution of rupture forces obtained from a PA1-functionalized AFM tip probing EPS extracts covalently attached to a glass slide. Red, wild type; green, ΔepsE mutant; blue, wild type in galactose solution.
FIGURE 8. Adhesion data from force-volume data. A, distribution of rupture force magnitudes using data from Fig. 7 depicted as histograms. Inset, expanded view of data >70 pN. B, distribution of rupture distances. C, distribution of rupture forces obtained from a PA1-functionalized AFM tip probing EPS extracts covalently attached to a glass slide. Red, wild type; green, ΔepsE mutant; blue, wild type in galactose solution.CC BY · PubMed Central
FIGURE 9. In situ characterization of the physical properties of EPS-2. Example force spectra (A–C) from the L. johnsonii wild type were fitted to a wormlike chain model (brown line). Lc, derived contour length; Lp, derived persistence length. Arrow, the rupture point between the lectin on the AFM tip and the extracellular polysaccharide. Red line, approach; blue line, retract.
FIGURE 9. In situ characterization of the physical properties of EPS-2. Example force spectra (A–C) from the L. johnsonii wild type were fitted to a wormlike chain model (brown line). Lc, derived contour length; Lp, derived persistence length. Arrow, the rupture point between the lectin on the AFM tip and the extracellular polysaccharide. Red line, approach; blue line, retract.CC BY · PubMed Central

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Metadata

Authors
Dertli, E., Colquhoun, I. J., Gunning, A. P., Bongaerts, R. J., Le Gall, G., Bonev, B. B., Mayer, M. J., & Narbad, A.
Affiliations
  • Quadram Institute (GB)
  • University of Nottingham (GB)
Corresponding author
Mayer, Melinda J.
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2013-11
Volume
288
Issue
44
Pages
31938-31951
ISSN
0021-9258 (ISSN-L)
DOI
10.1074/jbc.m113.507418
PMC
PMC3814790
OpenAlex
W1964087334
Cited by
134
References
58
Citations by year
2026: 13 · 2025: 13 · 2024: 4 · 2023: 13 · 2022: 11 · 2021: 10 · 2020: 14 · 2019: 13 · 2018: 10 · 2017: 11 · 2016: 4 · 2015: 14 · 2014: 4
FWCI
10.85
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DSpace@Bayburt (submittedVersion, cc-by-nc-nd)
  • UEA Digital Repository (University of East Anglia) (submittedVersion)
Funders
Biotechnology and Biological Sciences Research Council ((BB/J004529/1, BBS/E/F/00044453, BB/J004545/1)
Topics
  • Life Sciences › Agricultural and Biological Sciences › Food Science › Polysaccharides Composition and Applications
  • Life Sciences › Agricultural and Biological Sciences › Food Science › Probiotics and Fermented Foods
  • Life Sciences › Immunology and Microbiology › Microbiology › Escherichia coli research studies
Keywords
exopolysaccharides, exopolysaccharide biosynthesis, heteropolymers, galactose substitution, O-acetylation, cell surface polysaccharides, biofilm formation
MeSH terms
Bacterial Proteins, Carbohydrate Conformation, Genes, Bacterial, Multigene Family, Glucosyltransferases, Lactobacillus, Mutation, Polysaccharides, Bacterial

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