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

Melinda J Mayer,Alfonsina D’Amato,Gwénaëlle Le Gall,Arjan Narbad,Ian J Colquhoun

doi:10.1128/aem.02808-19

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. johnsoniiIMPORTANCE 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 slow-growth phenotype. Further elucidation of this biosynthesis may give insight into exopolysaccharide production and its impact on the bacterial cell.

Highlights

Lactobacillus johnsonii FI9785 makes two capsular exopolysaccharides—a heteropolysaccharide (EPS2) encoded by the eps operon and a branched glucan homopolysaccharide (EPS1)
In order to identify proteins involved in EPS biosynthesis, the proteome of the wild type was compared to that of a mutant with a reduced EPS capsule to highlight proteins which were missing or downregulated in the mutant
Apart from variations in proteins associated with ribosome structure, translation, and protein synthesis, very few biological processes seemed strongly affected in the soluble protein content by the loss of EPS2 synthesis in the Δeps_cluster mutant strain

Summary

Introduction

Lactobacillus johnsonii FI9785 makes two capsular exopolysaccharides—a heteropolysaccharide (EPS2) encoded by the eps operon and a branched glucan homopolysaccharide (EPS1). Deletion of 241 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. Lactobacillus johnsonii FI9785 is a poultry isolate which has shown promise as a competitive exclusion agent against Clostridium perfringens [18] and Campylobacter jejuni [19] This strain makes 2 capsular exopolysaccharides—EPS2, a heteropolysaccharide containing glucose and galactose encoded by a 14-gene eps operon of the Wzx/Wzy type, and EPS1, a branched dextran homopolysaccharide with an ␣-(1¡6) backbone and ␣-(1¡2) branches which are present on every unit of the backbone and consist of a single glucose (Glc) residue [20, 21]. We compared the proteome of the wild-type L. johnsonii FI9785 EPS producer with the Δeps_cluster mutant strain to attempt to identify proteins involved in homopolysaccharide biosynthesis

Methods

Results

Conclusion