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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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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.
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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.

Mentions: The lower base-line adhesion values surrounding the mode in both sets may well be due to nonspecific adhesion between the AFM tip and the cell surfaces. This can arise from several sources; one is electrostatic interaction between the tip and cell, although in the current experiment, this should be minimal due to the screening action of the buffer solution used. Another possible source can be penetration of the AFM tip apex into the bacterial cell wall during the approach phase of the measurement. This causes capillary adhesion as the tip is pulled away from the cell surface. In order to minimize this, the maximum loading force was kept to a moderately low value (300 pN), but some penetration or deformation of the cell surface is inevitable when one considers the sharpness of AFM tips (typical radius of curvature, 5–30 nm), although cells have been shown to tolerate such puncturing (23). Both of these nonspecific sources of adhesion tend to occur at (or relatively close to) the tip-sample detachment point (defined as 0 nm in the force-distance curves), whereas specific adhesion between the lectin on the AFM tip and the EPS will occur at distances well beyond the tip-sample detachment point, allowing discrimination of the origins of adhesive peaks in the force spectra. The reason for the shift in position of specific adhesion is due to two factors; the probe molecule (PA1 lectin) is tethered to the AFM tip via a flexible PEG linker, which is ∼12 nm in length, and the EPS targeted will extend under the load exerted by the retracting AFM tip-cantilever assembly before the ligand and receptor are torn from each other (i.e. the rupture point; arrow in Fig. 9). This provides a useful means for discrimination of the adhesive forces observed for each sample, comparison of the range of distances at which rupture occurs. Fig. 8B displays the adhesion data categorized by the distance at which they occurred and shows that the modal values in this case are different for each sample (140 nm for the wild type sample and 35 nm for the ΔepsE mutant). This suggests that the adhesion of the functionalized tip to the wild type sample represents specific interactions with the galactose residues of EPS-2. Validation of the lectin-functionalized tip binding to extracted EPS from the wild type and the ΔepsE deletion mutant (both covalently attached to glass slides) confirmed that the PA1 lectin bound only to EPS from the wild type. The frequency of binding was reduced in the presence of free galactose, confirming that it was due to lectin-carbohydrate association (Fig. 8C).


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)

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

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

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.
Mentions: The lower base-line adhesion values surrounding the mode in both sets may well be due to nonspecific adhesion between the AFM tip and the cell surfaces. This can arise from several sources; one is electrostatic interaction between the tip and cell, although in the current experiment, this should be minimal due to the screening action of the buffer solution used. Another possible source can be penetration of the AFM tip apex into the bacterial cell wall during the approach phase of the measurement. This causes capillary adhesion as the tip is pulled away from the cell surface. In order to minimize this, the maximum loading force was kept to a moderately low value (300 pN), but some penetration or deformation of the cell surface is inevitable when one considers the sharpness of AFM tips (typical radius of curvature, 5–30 nm), although cells have been shown to tolerate such puncturing (23). Both of these nonspecific sources of adhesion tend to occur at (or relatively close to) the tip-sample detachment point (defined as 0 nm in the force-distance curves), whereas specific adhesion between the lectin on the AFM tip and the EPS will occur at distances well beyond the tip-sample detachment point, allowing discrimination of the origins of adhesive peaks in the force spectra. The reason for the shift in position of specific adhesion is due to two factors; the probe molecule (PA1 lectin) is tethered to the AFM tip via a flexible PEG linker, which is ∼12 nm in length, and the EPS targeted will extend under the load exerted by the retracting AFM tip-cantilever assembly before the ligand and receptor are torn from each other (i.e. the rupture point; arrow in Fig. 9). This provides a useful means for discrimination of the adhesive forces observed for each sample, comparison of the range of distances at which rupture occurs. Fig. 8B displays the adhesion data categorized by the distance at which they occurred and shows that the modal values in this case are different for each sample (140 nm for the wild type sample and 35 nm for the ΔepsE mutant). This suggests that the adhesion of the functionalized tip to the wild type sample represents specific interactions with the galactose residues of EPS-2. Validation of the lectin-functionalized tip binding to extracted EPS from the wild type and the ΔepsE deletion mutant (both covalently attached to glass slides) confirmed that the PA1 lectin bound only to EPS from the wild type. The frequency of binding was reduced in the presence of free galactose, confirming that it was due to lectin-carbohydrate association (Fig. 8C).

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