The Gram-positive bacterium Romboutsia ilealis harbors a polysaccharide synthase that can produce (1,3;1,4)-β-D-glucans

Abstract

(1,3;1,4)-β-D-Glucans are widely distributed in the cell walls of grasses (family Poaceae) and closely related families, as well as some other vascular plants. Additionally, they have been found in other organisms, including fungi, lichens, brown algae, charophycean green algae, and the bacterium Sinorhizobium meliloti. Only three members of the Cellulose Synthase-Like (CSL) genes in the families CSLF, CSLH, and CSLJ are implicated in (1,3;1,4)-β-D-glucan biosynthesis in grasses. Little is known about the enzymes responsible for synthesizing (1,3;1,4)-β-D-glucans outside the grasses. In the present study, we report the presence of (1,3;1,4)-β-D-glucans in the exopolysaccharides of the Gram-positive bacterium Romboutsia ilealis CRIB^T. We also report that RiGT2 is the candidate gene of R. ilealis that encodes (1,3;1,4)-β-D-glucan synthase. RiGT2 has conserved glycosyltransferase family 2 (GT2) motifs, including D, D, D, QXXRW, and a C-terminal PilZ domain that resembles the C-terminal domain of bacteria cellulose synthase, BcsA. Using a direct gain-of-function approach, we insert RiGT2 into Saccharomyces cerevisiae, and (1,3;1,4)-β-D-glucans are produced with structures similar to those of the (1,3;1,4)-β-D-glucans of the lichen Cetraria islandica. Phylogenetic analysis reveals that putative (1,3;1,4)-β-D-glucan synthase candidate genes in several other bacterial species support the finding of (1,3;1,4)-β-D-glucans in these species.

Fig. 1. Exploring the functions of the GT2 polysaccharide synthase gene through gain-of-function experiments.

The cDNA encoding GT2 polysaccharide synthase was cloned into the pDDGFP-2 vector through homologous recombination. The resulting construct was then transformed into the quadruple knockout yeast Saccharomyces cerevisiae LoGSA. Expression of the GT2 gene was carried out following the methodology described in Newstead et al. and Drew et al.. Post-transformation, positive expression clones were identified through in-gel fluorescence screening. The polysaccharide synthesized by the overexpressed GT2 proteins was subsequently digested by associated polysaccharide hydrolases. The resulting oligosaccharide profiles were analyzed by MALDI-TOF MS and HPAEC-PAD.

Fig. 2. Oligosaccharide profiling of R. ilealis CRIB^T EPS.

R. ilealis CRIB^T EPS was precipitated with ethanol and treated with the (1,3;1,4)-β-d-glucan-specific enzyme lichenase for 24 h at 50 °C. The HPAEC-PAD chromatogram of lichenase-treated EPS (EPS + lichenase) was compared with the control chromatogram without lichenase treatment (EPS control) and with chromatograms of pure standard oligosaccharides. The latter were cellotriose (C3), cellotetraose (C4), cellopentaose (C5) and cellohexaose (C6) derived from cellulose; laminaribiose (L2), laminaritriose (L3) and laminaritetraose (L4) derived from laminarin; and M3 (DP3) and M4 (DP4) derived from (1,3;1,4)-β-d-glucan. Numbers refer to degrees of polymerization (DP). The two peaks from lichenase-treated EPS (EPS + lichenase) have the same retention times as M3 and M4. EPS, exopolysaccharides.

Fig. 3. Oligosaccharide profiling of RiGT2 LoGSA by HPAEC-PAD and MALDI-TOF MS.

a HPAEC-PAD chromatograms of the pure oligosaccharide standards (STDs) DP3 and DP4 compared with chromatograms of the lichenase hydrolysates of RiGT2 LoGSA and the empty vector (EV) LoGSA. The lichenase hydrolysate of AIR prepared from RiGT2 LoGSA has an abundant peak with a retention time corresponding to the DP3 standard oligosaccharide and a minor peak corresponding to the DP4 standard oligosaccharide, giving a DP3/DP4 ratio of ~15:1. b MALDI-TOF spectrum of the RiGT2 LoGSA lichenase hydrolysate. The signal at m/z 527 corresponds to DP3, whereas the signal from DP4 is undetectable. EV empty vector, AIRs alcohol insoluble residues, STDs oligosaccharide standards, DP degrees of polymerization.

Fig. 4. Linkage analysis of the DP3 (1,3;1,4)-β-d-glucan oligosaccharide from RiGT2 LoGSA.

a HPAEC-PAD chromatogram of the purified DP3 oligosaccharide derived from a lichenase digest of RiGT2 LoGSA. b MALDI-TOF spectrum shows a single ion with an m/z 527 that corresponds to the molecular weight of DP3 (G4G3G). c The oligosaccharide was reduced by NaBD₄, followed by GC–MS analysis of the partially methylated alditol acetate (PMAA) derivatives prepared from the sample. The total ion current (TIC) chromatogram shows the presence of 3-linked glucitol (3-glucitol) from the reduced reducing end, terminal glucopyranose (t-Glcp) from the non-reducing end, and 4-linked glucopyranose (4-Glcp) as the residue in the middle of the trisaccharide chain.

Fig. 5. Confocal microscopy of RiGT2-GFP and indirect immunofluorescence microscopy of (1,3;1,4)-β-d-glucans.

Images of induced (a–f) and non-induced (g–l) RiGT2 LoGSA were obtained using an inverted confocal microscope. The RiGT2 protein expressed in RiGT2 transgenic yeast containing C-terminal GFP fluoresced green, but no fluorescence was shown in non-induced cells (RiGT2-GFP). The location of the (1,3;1,4)-β-d-glucans was determined by indirect immunofluorescence using a mouse monoclonal antibody against (1,3;1,4)-β-d-glucan (BS400-3) and goat anti-mouse IgG (H + L) conjugated to Alexa Fluor^TM 555 showing a red fluorescence, which overlapped GFP fluorescence (Merge2), indicating (1,3;1,4)-β-d-glucans were produced only in yeast with RiGT2-GFP expression. Brightfield and merged fields are also shown for comparison. Representative of three independent experiments. a, g DNA staining with 1 µg/mL DAPI (blue). b, h RiGT2-GFP fusion protein (green). c, i Indirect immunofluorescence detection of (1,3;1,4)-β-d-glucan. Merged 1. Merged images of DAPI, RiGT2-GFP, and Anti-(1,3;1,4)-β-d-glucan. Merged 2. Merged images of RiGT2-GFP and Anti-(1,3;1,4)-β-d-glucan. BF brightfield images of yeast cells. All images have been exposed to the same laser intensity when visualizing the fluorescence. BF brightfield. a–f Induced RiGT2-GFP transformed yeast. g–l Non-induced RiGT2-GFP transformed yeast. White arrows in panels Anti-(1,3;1,4)-β-d-glucan and Merged 1 and Merged 2 indicate red fluorescence of Alexa Fluor 555.

Fig. 6. The specific activity of the RiGT2-containing microsomal fraction by radiometry assay.

Microsomal fractions (MF) from both RiGT2 and empty vector (EV) LoGSA were incubated with MgCl₂, the substrate UDP-Glc and ¹⁴C-labeled UDP-Glc as a radiotracer. The reactions were terminated by 66% EtOH and the alcohol-insoluble fractions were filtered and dried for scintillation counting. a The reactions were performed without UDP-Glucose (no UDP-Glc), without MFs (no RiGT2), with MFs heated to 95 °C for 10 min (heat inactivated), without lichenase (−Lichenase) and with lichenase (+Lichenase). The activity of RiGT2-containing fractions was normalized by the activity from EV MF and relative to the reaction in the absence of lichenase (−lichenase). b Additional c-di-GMP at different concentrations (30, 60, and 90 µM) was added to the reaction mixtures. Data are presented as mean values ± SD. Each data point represents an independent experiment. The error bars indicate the standard deviation (SD) calculated from three replicates (n = 3).

Fig. 7. Comparison of CsBcsA and the AlphaFold2 model of RiGT2.

a Ribbon representation of the crystal structure of the C. sphaeroides cellulose synthase complex consisting of BcsA and BcsB (PDB 4HG6 [https://www.rcsb.org/structure/4HG6]), and b the predicted AlphaFold2 model of RiGT2. The domains in the C. sphaeroides BcsA-BcsB complex are colored as follows: CsBcsA GT domain (GTd), orange; TM domain (TMd), blue; interface helices (IFH), green; PilZ domain (PilZd), pink; CsBcsB, gray; cellulose chain, yellow; and the gating loop connecting interface helix 3 and the PilZ domain is colored yellow. The cellulose chain runs from the center of the GT domain in BcsA and exits the channel at the BcsA-BcsB interface at the periplasmic face. The membrane bilayer has been delineated by a blue dotted line. The same colors were used for RiGT2. c Overlay of the active site in CsBcsA (green; PDB 4HG6) and the RiGT2_A model (pink). The view is rotated 180° with respect to panel (b). The two terminal glucosyl units Glc-17 and Glc-18 (yellow) and the UDP molecule (light blue with orange phosphate groups) in the CsBcsA/BcsB complex are shown. The residues that are part of the [D,D,D,Q(Q/R)XRW] signatures are shown, see text for details. Residue names are shown as pairs where the first residue corresponds to that in CsBcsA and the second to the equivalent residue in RiGT2. (Schrödinger, L. & DeLano, W., 2020. PyMOL, Available at: http://www.pymol.org/pymol).

Fig. 8. The phylogenetic analysis showing the possible homologs in other species of Gram-positive bacteria.

Neighbor joining-likelihood phylogenetic analysis of (1,3;1,4)-β-d-glucan synthase candidates by MEGA version X^, . The midpoint-rooted phylogenetic tree shows two sub-clades. Sequence entries of all candidates are listed in Supplementary Table 4. R. ilealis CRIB^T (1,3;1,4)-β-d-glucan synthase is marked with a blue dot. Species selected for subsequent (1,3;1,4)-β-d-glucan analysis are marked with red dots. Bar scale indicates distance.

Fig. 9. (1,3;1,4)-β-d-Glucan oligosaccharide profiles of selected Gram-positive bacteria.

EPSs from C. ventriculi, C. bornimence, R. peoriensis, and C. tyrobutyrisum were digested by lichenase and the hydrolysates analyzed by HPAEC-PAD and compared with a similar experiment with the EPS from R. ilealis. A mixture of standard pure DP3 and DP4 oligosaccharides was also analyzed under the same conditions. Peaks with the same retention time as the standard DP3 are present in the hydrolysates of all species and peaks with the same retention time as the standard DP4 are present in the hydrolysates of all species except C. bornimence. The chromatogram with control groups aligned is shown in Supplementary Fig. 17. STDs, oligosaccharide standards. DP degrees of polymerization.