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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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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.
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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.

Mentions: TEM showed the accumulation of the EPS to the cell surface, where they formed a capsule as an outer cell surface layer in L. johnsonii FI9785 (Fig. 5). An EPS layer still accumulated at the cell surface of the ΔepsE mutant, consisting solely of EPS-1, whereas the EPS layer was absent from the Δeps_cluster mutant (Fig. 5). We analyzed all strains using TEM, but the observed differences in the thickness of the EPS layer did not match the yields of EPS measured in previous work, suggesting that the preparation procedure resulted in the loss of some EPS from the cell surface (Fig. 5) into the culture medium. Washing with buffers that have no EPS cross-linking potential has been reported to remove capsular EPS (21); in particular, the epsCD88N mutant shown previously to have an increased accumulation of EPS (9) appeared to have a similar or slightly reduced capsule thickness compared with the wild type strain, and this may have implications for the nature of the interactions of the EPS within the capsule and with the cell wall.


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)

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

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

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.
Mentions: TEM showed the accumulation of the EPS to the cell surface, where they formed a capsule as an outer cell surface layer in L. johnsonii FI9785 (Fig. 5). An EPS layer still accumulated at the cell surface of the ΔepsE mutant, consisting solely of EPS-1, whereas the EPS layer was absent from the Δeps_cluster mutant (Fig. 5). We analyzed all strains using TEM, but the observed differences in the thickness of the EPS layer did not match the yields of EPS measured in previous work, suggesting that the preparation procedure resulted in the loss of some EPS from the cell surface (Fig. 5) into the culture medium. Washing with buffers that have no EPS cross-linking potential has been reported to remove capsular EPS (21); in particular, the epsCD88N mutant shown previously to have an increased accumulation of EPS (9) appeared to have a similar or slightly reduced capsule thickness compared with the wild type strain, and this may have implications for the nature of the interactions of the EPS within the capsule and with the cell wall.

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