Size-distribution of levans used in the study.

<p>Size-distribution of levans used in the study.</p

A Highly Active Endo-Levanase BT1760 of a Dominant Mammalian Gut Commensal <i>Bacteroides thetaiotaomicron</i> Cleaves Not Only Various Bacterial Levans, but Also Levan of Timothy Grass

<div><p><i>Bacteroides thetaiotaomicron</i>, an abundant commensal of the human gut, degrades numerous complex carbohydrates. Recently, it was reported to grow on a β-2,6-linked polyfructan levan produced by <i>Zymomonas mobilis</i> degrading the polymer into fructooligosaccharides (FOS) with a cell surface bound endo-levanase BT1760. The FOS are consumed by <i>B</i>. <i>thetaiotaomicron</i>, but also by other gut bacteria, including health-promoting bifidobacteria and lactobacilli. Here we characterize biochemical properties of BT1760, including the activity of BT1760 on six bacterial levans synthesized by the levansucrase Lsc3 of <i>Pseudomonas syringae</i> pv. tomato, its mutant Asp300Asn, levansucrases of <i>Zymomonas mobilis</i>, <i>Erwinia herbicola</i>, <i>Halomonas smyrnensis</i> as well as on levan isolated from timothy grass. For the first time a plant levan is shown as a perfect substrate for an endo-fructanase of a human gut bacterium. BT1760 degraded levans to FOS with degree of polymerization from 2 to 13. At optimal reaction conditions up to 1 g of FOS were produced per 1 mg of BT1760 protein. Low molecular weight (<60 kDa) levans, including timothy grass levan and levan synthesized from sucrose by the Lsc3Asp300Asn, were degraded most rapidly whilst levan produced by Lsc3 from raffinose least rapidly. BT1760 catalyzed finely at human body temperature (37°C) and in moderately acidic environment (pH 5–6) that is typical for the gut lumen. According to differential scanning fluorimetry, the T_m of the endo-levanase was 51.5°C. All tested levans were sufficiently stable in acidic conditions (pH 2.0) simulating the gastric environment. Therefore, levans of both bacterial and plant origin may serve as a prebiotic fiber for <i>B</i>. <i>thetaiotaomicron</i> and contribute to short-chain fatty acids synthesis by gut microbiota. In the genome of <i>Bacteroides xylanisolvens</i> of human origin a putative levan degradation locus was disclosed.</p></div

Initial velocities of reducing sugar release by the endo-levanase BT1760 from various levans added at 5 g/L.

<p>* Synthesized by <i>Pseudomonas syringae</i> pv. tomato levansucrase Lsc3 or its mutant Asp300Asn (D300N) from sucrose or raffinose (Raf). Mean values and standard deviation were calculated from at least three independent experiments. For additional information on levans, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169989#pone.0169989.t001" target="_blank">Table 1</a>.</p

Parameters of FOS production by endo-levanase BT1760 from six different levans at optimal reaction duration.

<p>Parameters of FOS production by endo-levanase BT1760 from six different levans at optimal reaction duration.</p

Clustal Omega alignment of the <i>Bacteroides thetaiotaomicron</i> endo-levanase BT1760 with putative levanase (D4IW69) of <i>Butyrivibrio fibrisolvens</i> 16/4 (BF 16/4).

<p>Identical residues between the sequences are shown on black background and marked with an asterisk. Similar residues are designated below the alignment with dots. Predicted N-terminal signal peptides are shown on grey background. Predicted nucleophiles, proton donors and the ‘RDP’ motifs are shown on red background. Cc–Clustal consensus.</p

The effect of temperature on catalytic activity of the endo-levanase BT1760.

<p>(A) Temperature optimum of BT1760. Release of reducing sugars from levan by BT1760 was recorded at varied temperatures and relative activities were calculated. 100% of activity corresponds to 123.6 ± 6.4 U/mg, measured at 37°C. (B) Thermostability of BT1760. The endo-levanase BT1760 was incubated at indicated temperatures for 30 min, and residual endo-levanase activity was then measured at 37°C by recording reducing sugar release from levan. 100% of activity corresponds to 124.0 ± 1.8 U/mg. The average and standard deviation values are calculated form at least two independent measurements with two parallel samples analysed. Detailed description of the methods is presented in Materials and Methods.</p

A Highly Active Endo-Levanase BT1760 of a Dominant Mammalian Gut Commensal <i>Bacteroides thetaiotaomicron</i> Cleaves Not Only Various Bacterial Levans, but Also Levan of Timothy Grass - Fig 8

<p><b>Time course of degradation of six different levans by the BT1760 into products of varied DP (A-F).</b> Levans synthesized by Lsc3 or its mutant Asp300Asn (D300N) are designated by an asterisk. Reaction products were analysed using HPLC. Up to four parallel samples were analysed to calculate the average values and standard deviations. Details of the reaction conditions and used methods are presented in Materials and Methods section.</p

Comparison of fructan PUL of <i>Bacteroides thetaiotaomicron</i> (<i>Bt</i>) VPI-5482 [7] with genomic locus of <i>Bacteroides xylanisolvens</i> (<i>Bx</i>) CL03T12C04 harboring a close homologue of the <i>B</i>. <i>thetaiotaomicron</i> endo-levanase BT1760.

<p>Colour code is used to designate homologous proteins of the two loci. ORFs of levan-hydrolyzing proteins of <i>B</i>. <i>thetaiotaomicron</i> and their homologues in <i>B</i>. <i>xylanisolvens</i> are in blue. Intervening unrelated genes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169989#pone.0169989.ref007" target="_blank">7</a>] are in white.</p

Acid-resistance of levans and dahlia inulin.

<p>Fructans (10 g/L) were incubated in 0.01 M hydrochloric acid (pH 2.0) at 37°C for 24 h and sampled over this period for reducing sugar and thin layer chromatography (TLC) analysis. The bars indicate the extent of hydrolysis of the fructan calculated as percentage of released reducing sugars from the total amount of reducing sugars in completely hydrolysed sample. Standard deviation values shown on bars are calculated from at least two independent experiments with at least two parallel samples. TLC shows fructan hydrolysis products at 7 and 24 h of incubation. Samples spotted onto a TLC plate are numbered from 1 to 7 according to respective numbers on the bar diagramme. The chromatogram was developed in chloroform: acetic acid: water (60:70:10; v/v/v). Sugar markers (M) on TLC: 7 g/L levan (L), 8 mM nystose (N), 8 mM 1-kestose (K), 30 mM sucrose (S) and 30 mM fructose (F). Details of the methods are presented in Materials and Methods section.</p

Levan degradation by <i>Escherichia coli</i> expressing endo-levanase BT1760 of <i>Bacteroides thetaiotaomicron</i> from the pURI3-BT1760Cter plasmid.

<p>(A) 5 μL of the liquid culture of recombinant <i>E</i>. <i>coli</i> was spotted onto a LB plate containing Amp, IPTG and 5 g/L of Lsc3-produced HMW levan and grown overnight at room temperature. Agar discs were cut off from regions marked with black rings, melted by heating and 0.5 μL of the sample was applied to a thin layer chromatography (TLC) plate. (B) TLC analysis of levan degradation from regions 3–6 of the agar plate in panel A and on lanes 7 and 8 products from 5 g/L levan by 1 h and 4 days of reaction with the lysate from recombinant <i>E</i>. <i>coli</i>. The chromatogram was developed in chloroform: methanol: water (90:65:15, v/v/v). Reference sugars on the TLC plate: fructose (F) and 1-kestose (K) on lane 1; sucrose (S) and nystose (N) on lane 2. F1-F5; levan degradation products with respective degree of polymerization.</p