Scientific Publications
Identification of genes required for glucan exopolysaccharide production in Lactobacillus johnsonii suggests a novel mechanism of biosynthesis.
Applied and environmental microbiology
Lactobacillus johnsonii FI9785 makes two capsular exopolysaccharides -a heteropolysaccharide (EPS2) encoded by the eps operon, and a branched glucan homopolysaccharide (EPS1). The homopolysaccharide is synthesised 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, 242, was less abundant in the ?eps_cluster mutant than in the wild type. NMR analysis of isolated EPS showed that deletion of the 242 gene 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 neighbouring gene 241 suggested 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 there may be a novel mechanism for homopolysaccharide synthesis in the Gram-positive L. johnsoniiImportanceExopolysaccharides 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 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 glucansynthase pathway. Disturbance of this synthesis leads to a slow growth phenotype. Further elucidation of this biosynthesis may give insight into exopolysaccharide production and its impact on the bacterial cell.
Applied and environmental microbiology
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