Biochemistry and functional analysis of exopolysaccharide production in Lactobacillus johnsonii

Lactic Acid Bacteria (LAB) produce unique exopolysaccharides (EPS) that are important in food industry but they also play critical role in bacterial interactions during colonisation of the gastrointestinal tract. The role of this layer in the virulence of pathogenic bacteria has been well described but the biological importance of the EPS layer of probiotic bacteria have not been studied to the same extent. The aim of this thesis is to investigate the structure, biosynthesis mechanism and the biological role of the EPS produced by a probiotic strain of Lactobacillus johnsonii FI9785 that is able to competitively exclude Clostridium perfringens from gastrointestinal tract. L. johnsonii harbours an eps gene cluster and a spontaneous mutation in the phosphoregulatory system of this cluster resulted in a colony switch from a rough morphology to a smooth one; similarly deletion of the epsE gene, that encodes the putative priming glycosyltransferase of the EPS biosynthesis, caused a huge increase in aggregation of L. johnsonii, which were shown to be related with EPS production levels. Structural analysis of the purified EPS showed that L. johnsonii could produce two types of EPS: EPS-1 and EPS-2. EPS-1 is a branched dextran with the unusual feature that every backbone residue is substituted with a 2-linked glucose unit and EPS-2 is composed of four glucose and two galactose units with a novel structure. Several mutants were generated with deletion of individual genes in eps cluster or the entire cluster to study the EPS biosynthesis mechanism. The ΔepsE mutant produced only EPS-1 but not EPS-2 whilst the deletion of the putative transcriptional regulator, epsA, and the entire eps cluster resulted in an acapsular phenotypes. These alterations in the cell surface of EPS specific mutants were demonstrated by differences in binding of an anti-wild type L. johnsonii antibody. The loss of the EPS layer increased the adhesion and autoaggregation properties of L. johnsonii in vitro but EPS layer was found to be protective against several antimicrobials and environmental stress conditions. Additionally EPS layer was shown to be important on physicochemical properties and biofilm formation of L. johnsonii. Furthermore the in vivo persistence properties of acapsular mutant and wild type were assessed in a mouse model and no difference was detected in their persistence. Finally, several glycosyltransferases within the eps cluster were expressed, activity assays were performed and a potential glycosyltransferase was biochemically characterised