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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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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.).
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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.).

Mentions: An initial screening of all pellet and supernatant EPS samples by 1H NMR at 300 K showed that two anomeric signals at 5.17 and 5.11 ppm were a major feature of all cell surface-associated (pellet) EPS preparations. These signals were also present in the supernatant series, although in most cases, they were no longer the major ones in the anomeric region. The polysaccharide sugar rings were partially acetylated because a cluster of at least six methyl singlet signals was observed between 1.98 and 2.08 ppm plus, in some samples, an isolated singlet at 2.16 ppm. Representative samples were selected for detailed NMR studies, and for these, the temperature was increased to 338 K as a significant sharpening of 1H signals was obtained (Fig. 2A) (e.g. the apparent singlets at 5.17 (labeled b1) and 5.11 ppm (c1) were revealed as doublets); also, the residual HDO signal (4.41 ppm) did not interfere with any other peaks at this temperature.


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)

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

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

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.).
Mentions: An initial screening of all pellet and supernatant EPS samples by 1H NMR at 300 K showed that two anomeric signals at 5.17 and 5.11 ppm were a major feature of all cell surface-associated (pellet) EPS preparations. These signals were also present in the supernatant series, although in most cases, they were no longer the major ones in the anomeric region. The polysaccharide sugar rings were partially acetylated because a cluster of at least six methyl singlet signals was observed between 1.98 and 2.08 ppm plus, in some samples, an isolated singlet at 2.16 ppm. Representative samples were selected for detailed NMR studies, and for these, the temperature was increased to 338 K as a significant sharpening of 1H signals was obtained (Fig. 2A) (e.g. the apparent singlets at 5.17 (labeled b1) and 5.11 ppm (c1) were revealed as doublets); also, the residual HDO signal (4.41 ppm) did not interfere with any other peaks at this temperature.

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