Identification of Genes Required for Glucan Exopolysaccharide Production in Lactobacillus johnsonii Suggests a Novel Biosynthesis Mechanism

Abstract

Lactobacillus johnsonii FI9785 makes two capsular exopolysaccharides-a heteropolysaccharide (EPS2) encoded by the eps operon and a branched glucan homopolysaccharide (EPS1). The homopolysaccharide is synthesized in the absence of sucrose, and there are no typical glucansucrase genes in the genome. Quantitative proteomics was used to compare the wild type to a mutant where EPS production was reduced to attempt to identify proteins associated with EPS1 biosynthesis. A putative bactoprenol glycosyltransferase, FI9785_242 (242), was less abundant in the Δeps_cluster mutant strain than in the wild type. Nuclear magnetic resonance (NMR) analysis of isolated EPS showed that deletion of the FI9785_242 gene (242) prevented the accumulation of EPS1, without affecting EPS2 synthesis, while plasmid complementation restored EPS1 production. The deletion of 242 also produced a slow-growth phenotype, which could be rescued by complementation. 242 shows amino acid homology to bactoprenol glycosyltransferase GtrB, involved in O-antigen glycosylation, while in silico analysis of the neighboring gene 241 suggested that it encodes a putative flippase with homology to the GtrA superfamily. Deletion of 241 also prevented production of EPS1 and again caused a slow-growth phenotype, while plasmid complementation reinstated EPS1 synthesis. Both genes are highly conserved in L. johnsonii strains isolated from different environments. These results suggest that there may be a novel mechanism for homopolysaccharide synthesis in the Gram-positive L. johnsonii. IMPORTANCE Exopolysaccharides are key components of the surfaces of their bacterial producers, contributing to protection, microbial and host interactions, and even virulence. They also have significant applications in industry, and understanding their biosynthetic mechanisms may allow improved production of novel and valuable polymers. Four categories of bacterial exopolysaccharide biosynthesis have been described in detail, but novel enzymes and glycosylation mechanisms are still being described. Our findings that a putative bactoprenol glycosyltransferase and flippase are essential to homopolysaccharide biosynthesis in Lactobacillus johnsonii FI9785 indicate that there may be an alternative mechanism of glucan biosynthesis to the glucansucrase pathway. Disturbance of this synthesis leads to a slowgrowth phenotype. Further elucidation of this biosynthesis may give insight into exopolysaccharide production and its impact on the bacterial cell.

Metabolism Characteristics of Lactic Acid Bacteria and the Expanding Applications in Food Industry

“…Lactobacillus johnsonii (Mayer et al, 2020) Glucan will extend to the crumb porosity of bread Improvement of bread texture Limosilactobacillus reuteri (Leemhuis et al, 2014) (Continued) Levilactobacillus brevis (Lyu et al, 2019) Levilactobacillus brevis D17 (Gong et al, 2019) GadC transports L-glutamate into the cell Lactococcus lactis (Small and Waterman, 1998) Glutamate decarboxylase and pyridoxal-5 -phosphate participate in the decarboxylation reaction of L-glutamate Lactococcus lactis (Cui et al, 2020) The cell immobilization technology increase GABA production Levilactobacillus brevis RK03 (Hsueh et al, 2017) Levilactobacillus brevis (Shi et al, 2017) Flavor substances SHMT gene encodes a serine hydroxymethyltransferase with threonine aldolase activity Produce flavor substances (2,3-butanedione and 2,3-pentanedione, etc.) in wine, vinegar, bread, sourdough and cheese…”

Characterization of inositol lipid metabolism in gut-associated Bacteroidetes

“…3). The BT_1524 gene is predicted to encode an integral membrane protein with a Gtr-A motif (Pfam); other Gtr-A family proteins are involved in cell surface polysaccharide or exopolysaccharide synthesis [25][26][27] .…”

Effects of polysaccharides-riched Prunus mume fruit juice concentrate on uric acid excretion and gut microbiota in mice with adenine-induced chronic kidney disease

“…Since PFC may have positive effect on the excretion of UA, it is therefore encouraged to further study whether this is associated with changes in the abundance of Lactobacillus in the gut. The species Lactobacillus_johnsonii_FI9785 and Lactobacillus_animalis are known to have probiotic effects in animals ( Karunasena et al, 2013 ; Mayer et al, 2020 ), and we found that the abundances of these particular species were significantly greater in CKD-P group, which implies that PFC may promote the multiplication of probiotic Lactobacillus species.…”