Identification of WxL and S-Layer Proteins from Lactobacillus brevis with the Ability to Bind Cellulose and Xylan

Abstract

Xylanase releases xylo-oligosaccharides from dietary xylan, which stimulate the growth of the gut bacteria lactobacilli. Many lactobacilli adhere to dietary fibers, which may facilitate the assimilation of xylo-oligosaccharides and help them gain competence in the gut, but the underlying mechanisms remain elusive. Herein we report, from the highly abundant transcripts of Lactobacillus brevis cultured in wheat arabinoxylan supplemented with a xylanase, the identification of genes encoding four putative cell-surface WxL proteins (Lb630, Lb631, Lb632, and Lb635) and one S-layer protein (Lb1325) with either cellulose- or xylan-binding ability. The repetitively occurring WxL proteins were encoded by a gene cluster, among which Lb630 was chosen for further mutational studies. The analysis revealed three aromatic residues (F30, W61, and W156) that might be involved in the interaction of the protein with cellulose. A homology search in the genome of Enterococcus faecium identified three WxL proteins with conserved counterparts of these three aromatic residues, and they were also found to be able to bind cellulose and xylan. The findings suggested a role of the cell-surface WxL and S-layer proteins in assisting the cellular adhesion of L. brevis to plant cell wall polysaccharides.

Keywords: Lactobacillus brevis; S-layer protein; WxL protein; cell adhesion; gut bacteria; xylanase.

Figure 1

Growth of L. brevis in MRS supplemented with different carbon sources. WAX: wheat arabinoxylan; WAX+Xyn: wheat arabinoxylan plus xylanase; Control: no carbon source.

Figure 2

Scanning electron microscopy (SEM) analysis indicated that L. brevis cells were directly attached to the crystalline cellulose. The bars indicate 10.0 μm.

Figure 3

Binding of Lb630, Lb631, Lb632, Lb635, and Lb1325 to crystalline cellulose (a) and insoluble WAX (b).

Figure 4

Amino-acid sequence alignment of Lb630 and its homologs in E. faecalis. Solid arrows represent strictly conserved aromatic amino acids, empty arrows are similar residues, and diamonds indicate non-conserved residues. Rectangle boxes with shadow indicate strictly conserved amino acids. Rectangular boxes are similar residues. The amino acids in bold letters represent identical and similar ones.

Figure 5

Binding of the Lb630 homologs in E. faecalis to cellulose and xylan. Binding of Ef1216, Ef1840, and Ef2403 to Avicel crystalline cellulose (a) and insoluble wheat arabinoxylan (b).

Figure 6

A model depicting how the WxL and S-layer proteins may help L. brevis to utilize xylooligosaccharides. For simplification, wheat arabinoxylan was shown to represent dietary xylan. The WxL and S-layer proteins were presented as modeled structures using AlphaFold2 or RoseTTAFold.