In vivo Functional Characterization of Hydrophilic X2 Modules in the Cellulosomal Scaffolding Protein

Abstract

As part of free cellulases or scaffolding proteins in cellulosomes, the hydrophilic non-catalytic X2 module is widely distributed in cellulolytic Clostridia or other Firmicutes bacteria. Previous biochemical studies suggest that X2 modules might increase the solubility and substrate binding affinity of X2-bearing proteins. However, their in vivo biological functions remain elusive. Here we employed CRISPR-Cas9 editing to genetically modify X2 modules by deleting the conserved motif (NGNT) from the CipC scaffoldin. Both single and double X2 mutants (X2-N: near the N terminus of CipC; X2-C: near the C terminus of CipC) presented similar stoichiometric compositions in isolated cellulosomes as the wildtype strain (WT). These X2 mutants had an elongated adaptation stage during growth on cellulose compared to cellobiose. Compared to WT, the double mutant ΔX2-NC reduced cellulose degradation by 15% and the amount of released soluble sugars by 63%. Since single X2 mutants did not present such obvious physiological changes as ΔX2-NC, there seems to be a functional redundancy between X2 modules in CipC. The in vivo adhesion assay revealed that ΔX2-NC decreased cell attachment to cellulose by 70% but a weaker effect was also overserved in single X2 mutants. These results highlight the in vivo biological role of X2 in increasing cellulose degradation efficiency by enhancing the binding affinity between cells and cellulose, which provides new perspectives for microbial engineering.

Keywords: Clostridium cellulolyticum; X2 module; cellulose degradation; cellulosome; motif deletion.

FIGURE 1

Precise deletion of the conserved motif from the X2 modules to maintain the structural integrity of the CipC protein. (A) An overview of the strategy for constructing the dual X2 module mutant by the Cas9 nickase-based genome editing. Both plasmids pCas9n-X2-C-delete-donor and pCas9n-X2-N-delete-donor were used for the ΔX2-NC mutant construction. LH, left homologous; RH, right homologous; P4, P4 promoter (Xu et al., 2015); Fd, ferredoxin promoter (Xu et al., 2015); CBM, carbohydrate binding module; CO, Cohesin. (B) DNA sequence showing the deletion of conserved motif from X2 modules in the cipC gene. (C) SDS-PAGE analysis of cellulosomes extracted from WT and all mutant strains (15 μg protein/lane).

FIGURE 2

Deletion of the conserved motif (NGNT) may lead to a conformational change of the X2 module. (A) The structure of the X2-N module protein; (B) the structure of ΔX2-N module in which the conserved NGNT residues were deleted. (C) Structures overlapping between X2-N and ΔX2-N modules. (D) The structure of the X2-C module protein; (E) the structure of ΔX2-C module in which the conserved NGNT residues were deleted; (F) structures overlapping between X2-C and ΔX2-C modules.

FIGURE 3

Disruption of X2 modules increased the lag phase and decreased the cellulose degradation efficiency when mutants were grown on cellulose. (A) Growth profiles of WT, ΔX2-N, ΔX2-C, and ΔX2-NC grown on cellobiose. (B) Growth profiles of WT, ΔX2-N, ΔX2-C, and ΔX2-NC grown on cellulose. (C) Cellulose degradation profiles of WT, ΔX2-N, ΔX2-C, and ΔX2-NC. (D) Released total soluble sugars in supernatant of medium at final time point for each strain when grown on cellulose. Data are presented as the mean of three biological replicates and error bars represent standard deviation (SD).

FIGURE 4

The in vivo function of the X2 module was related to the binding affinity between cells and cellulose. Panels (A,B), the relative cell adhesion capability between cells and cellulose for each strain in early exponential (A) and late-exponential phase (B). Data are presented as the mean of three biological replicates and error bars represent SD. (C) The binding of X2-C, ΔX2-C, and CBM3a proteins to the cell surfaces of E. coli, C. thermocellum, or C. cellulolyticum respectively, determined by western blot. CBM3a was detected in all three strains (as blue arrow indicated), indicating it could bind to the cell surface for both Gram-negative and Gram-positive bacteria. The X2-C could not be detected for any of them, indicating it can not directly bind to the cell surface. The weak band of ΔX2-C was detected in C. thermocellum and C. cellulolyticum, indicating it had a weak binding affinity with the cell surface of the Gram-positive bacteria. (D) Binding of X2-C, CBM3a, and BSA protein to crystalline cellulose, determined by SDS-PAGE. The CBM3a protein and the BSA protein were used as the positive and negative control respectively. The CBM3a was detected in the cellulose pellet (as blue arrow indicated) and X2-C was only detected in the supernatant fraction as same as the BSA negative control (as brown arrow indicated), indicating that X2-C can not directly bind to the cellulose. Lane 1, BSA + Cellulose in supernatant fraction; Lane 2, CBM + Cellulose in supernatant fraction; Lane 3, X2-C + Cellulose in supernatant fraction; Lane 4, blank; Lane 5, BSA + Cellulose in cellulose-containing pellet; Lane 6, CBM + Cellulose in cellulose-containing pellet; Lane 7, X2-C + Cellulose in cellulose-containing pellet.