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Structure and biosynthesis of two exopolysaccharides produced by Lactobacillus johnsonii FI9785.

Dertli E, Colquhoun IJ, Gunning AP, Bongaerts RJ, Le Gall G, Bonev BB, Mayer MJ, Narbad A - J. Biol. Chem. (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: From the Gut Health and Food Safety Programme, Institute of Food Research, Colney, Norwich NR4 7UA, United Kingdom.

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-Glcp-(1-3)-β-Glcp-(1-5)-β-Galf-(1-6)-α-Glcp-(1-4)-β-Galp-(1-4)-β-Glcp-(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 (ΔepsE) 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 ΔepsE 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.

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Related in: MedlinePlus

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.
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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.

Mentions: The structure of EPS-1 was determined mainly from experiments on the wild type (WT-bacterial pellet) sample. Rings b and c were found to be both α-Glcp; b1 and c1 had 3J12 = 3.5 Hz, consistent with α configuration. In both rings, H1 was linked to H5 through all intermediate protons in the TOCSY experiment, and the shapes of the cross-peaks indicated substantial couplings throughout, as expected for Glcp. The HSQC-TOCSY experiment linked H1 for each ring to all carbons of the same ring, including C6. In particular, b1 and c1 were linked to C6 signals at 68.66 and 63.42 ppm, respectively. Chemical shifts of EPS-1 are reported in Table 3. The connectivities (Table 2) showed that EPS-1 consists of a chain of α-(1,6)-linked Glcp residues (ring b), all of which are additionally substituted at position 2 with a single α-Glcp (ring c), as shown in Fig. 3. The chemical shifts of rings b and c are close to those reported for (1,2,6)α-Glcp and t-α-Glcp in a dextran isolated from Leuconostoc citreum E497 (19); however, EPS-1 contained none of the unbranched (1,6)α-Glcp residues that were the major constituents of the L. citreum E497 dextran backbone.


Structure and biosynthesis of two exopolysaccharides produced by Lactobacillus johnsonii FI9785.

Dertli E, Colquhoun IJ, Gunning AP, Bongaerts RJ, Le Gall G, Bonev BB, Mayer MJ, Narbad A - J. Biol. Chem. (2013)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814790&req=5

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.
Mentions: The structure of EPS-1 was determined mainly from experiments on the wild type (WT-bacterial pellet) sample. Rings b and c were found to be both α-Glcp; b1 and c1 had 3J12 = 3.5 Hz, consistent with α configuration. In both rings, H1 was linked to H5 through all intermediate protons in the TOCSY experiment, and the shapes of the cross-peaks indicated substantial couplings throughout, as expected for Glcp. The HSQC-TOCSY experiment linked H1 for each ring to all carbons of the same ring, including C6. In particular, b1 and c1 were linked to C6 signals at 68.66 and 63.42 ppm, respectively. Chemical shifts of EPS-1 are reported in Table 3. The connectivities (Table 2) showed that EPS-1 consists of a chain of α-(1,6)-linked Glcp residues (ring b), all of which are additionally substituted at position 2 with a single α-Glcp (ring c), as shown in Fig. 3. The chemical shifts of rings b and c are close to those reported for (1,2,6)α-Glcp and t-α-Glcp in a dextran isolated from Leuconostoc citreum E497 (19); however, EPS-1 contained none of the unbranched (1,6)α-Glcp residues that were the major constituents of the L. citreum E497 dextran backbone.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: From the Gut Health and Food Safety Programme, Institute of Food Research, Colney, Norwich NR4 7UA, United Kingdom.

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-Glcp-(1-3)-β-Glcp-(1-5)-β-Galf-(1-6)-α-Glcp-(1-4)-β-Galp-(1-4)-β-Glcp-(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 (ΔepsE) 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 ΔepsE 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.

Show MeSH
Related in: MedlinePlus