Proteomic Analysis Reveals Enzymes for β-D-Glucan Formation and Degradation in Levilactobacillus brevis TMW 1.2112

Abstract

Bacterial exopolysaccharide (EPS) formation is crucial for biofilm formation, for protection against environmental factors, or as storage compounds. EPSs produced by lactic acid bacteria (LAB) are appropriate for applications in food fermentation or the pharmaceutical industry, yet the dynamics of formation and degradation thereof are poorly described. This study focuses on carbohydrate active enzymes, including glycosyl transferases (GT) and glycoside hydrolases (GH), and their roles in the formation and potential degradation of O2-substituted (1,3)-β-D-glucan of Levilactobacillus (L.) brevis TMW 1.2112. The fermentation broth of L. brevis TMW 1.2112 was analyzed for changes in viscosity, β-glucan, and D-glucose concentrations during the exponential, stationary, and early death phases. While the viscosity reached its maximum during the stationary phase and subsequently decreased, the β-glucan concentration only increased to a plateau. Results were correlated with secretome and proteome data to identify involved enzymes and pathways. The suggested pathway for β-glucan biosynthesis involved a β-1,3 glucan synthase (GT2) and enzymes from maltose phosphorylase (MP) operons. The decreased viscosity appeared to be associated with cell lysis as the β-glucan concentration did not decrease, most likely due to missing extracellular carbohydrate active enzymes. In addition, an operon was discovered containing known moonlighting genes, all of which were detected in both proteome and secretome samples.

Keywords: Levilactobacillus brevis TMW 1.2112; exopolysaccharide; glycosyl hydrolase; glycosyltransferase; moonlighting proteins; proteome; secretome; β-glucan.

Figure 1

Growth characteristics of the fermentation broth: (A) cell count in cfu/mL, OD, and pH, (B) changes in the viscosity, and (C) β-glucan and D-glucose concentration in culture supernatants. Values are mean values of four-fold biological replicates including standard deviations.

Figure 2

Secretome analysis by COG classification. The in silico analyzed secretome was compared with samples from the beginning (8 h) and end (7 days) of fermentation of at least three out of four replicates. On the right, the total numbers of identified gene locus IDs are stated.

Figure 3

Proteomics analysis of secreted proteins. Volcano plot for the differential abundance analysis of 8 h vs. 7 d secretomes: GHs and GTs (encircled), proteins with higher abundance at 8 h (blue dots), proteins with higher abundance at 7 days (light green dots), and potential moonlighting proteins (red dots).

Figure 4

Proteomics analysis of cellular proteins over fermentation. (A) Principal component analysis (PCA) of the five fermentation time points. (B) Heat map using Pearson correlation displaying the protein abundance at five different time points (8 h, 24 h, 2 days, 3 days, and 7 days) after the hierarchical clustering of 1641 proteins. Three- to four-fold biological replicates were used for the analysis. The changes in enzyme expression are depicted by color intensity, as indicated below the figure.

Figure 5

Cellular proteome analysis by COG classification. The in silico proteome was compared at five time points (8 h, 24 h, 2 days, 3 days, and 7 days) in at least three out of four or two out of three replicates.

Figure 6

Expression of GHs and GTs in the cell lysate. (A) Heat map of the expressed enzymes over time. The three clusters of proteins are indicated by the color bar on the right (orange, purple, and red). Relative fold change of protein expression compared to the mean depicted by color intensity, as stated below the figure (a range between −1.7-fold and 1.7-fold). Correlation coefficient values with viscosity, β-glucan, and D-glucose are listed next to the specific GHs and GTs. (B) The correlation of the expression of α-glucosidase AZI09_04165, β-1,3-glucosidase (bglB) AZI09_02170, and the MP AZI09_04670 with viscosity, including correlation coefficient values (r) and p-values (p).

Figure 7

Gene set overrepresentation analysis of proteins correlating with (A) viscosity, (B) β-glucan content, and (C) D-glucose content. Top bar charts indicated the positively and bottom bar chart the negatively correlating proteins with corresponding GO terms (y-axis). The x-axis represents the gene counts. GO terms comprising proteins of interest are indicated by (a) GHs and GTs; (b) β-glucan-biosynthesis-associated proteins (other than GHs and GTs), and (c) moonlighting-associated protein enolase (AZI09_08765).

Figure 8

β-glucan biosynthesis of L. brevis TMW 1.2112. (A) Metabolic pathway of the β-glucan biosynthesis, as suggested from genomic data and proteomic analysis. (B) Suggested maltose operons: malR = transcriptional regulator, MP = maltose phosphorylase, malT = MFS maltose transporter and β-PGM = β-phosphoglucomutase.

Figure 9

Operon of putative moonlighting proteins. GADPH (glyceraldehyde-3-phosphate dehydrogenase), PGK (phophoglycerate kinase), TPI (triose-phosphat isomerase), and enolase.