Investigation of the biosynthesis of exopolysaccharides within the biofilm matrix of Pseudomonas aeruginosa and Pseudomonas syringae pv. actinidiea : a thesis presented in partial fulfilment of the requirements for degree of Doctor of Philosophy in Microbiology and Genetics at Massey University, Manawatu, New Zealand

Abstract

Polysaccharides are highly abundant natural biopolymers, which have biologically significant structural functions in living organisms. Various polysaccharides, with specific physicochemical properties, contribute to biofilm formation; defined as cell aggregations surrounded by extracellular polymeric substances. They are also important in the context of bacterial pathogenesis, while some have been harnessed for industrial and biomedical applications due to their unique chemical compositions and properties. In present study, we aimed at studying biofilm formation by Pseudomonas aeruginosa and P. syringae pv. actinidiae, respectively known as human and plant pathogens. In this context we focused on the production of exopolysaccharides, which predominantly constitute the biofilm matrix of these pathogenic bacteria. Here, we uncovered that the polysaccharide isolated from P. syringae pv. actinidiae biofilm mainly consists of rhamnose, fucose and glucose and it was cautiously introduced as a novel polysaccharide. In the context of disease control, and developing a management program, we provided some evidences for the effectiveness of chlorine dioxide and kasugamycin in the control of the bacteria living in both biofilm and planktonic modes. Furthermore, we investigated alginate biosynthesis as major polysaccharide contributing to mucoid biofilm formation by P. aeruginosa. We generated various mutants producing a variety of alginates with different chemical compositions. Also, this enabled us to analyse functional relationships of protein subunits involved in multiple steps of alginate biosynthesis including alginate polymerization, modification and secretion. We present evidence that while alginate unravelled that while alginate is polymerised and translocated across the membrane by a multiprotein complex, acetylation and epimerisation events positively and negatively correlated with the polymerization of the alginate or molecular mass, respectively. Analysis of the biofilms showed that biofilm architecture and cell-to-cell interactions were differently impacted by various compositions of the alginates. Also, this study provided insights into the c-di-GMP mediated activation of alginate polymerization upon binding to c-di-GMP as well as assigning functional roles to Alg8 and Alg44 including their subcellular localization and distribution. Here, we also used current knowledge of the alginate biosynthesis pathway to assess the production of alginate from biotechnologically accepted heterologous hosts including Escherichia coli and Bacillus megaterium strains. Primarily, we evaluated the production and functionality of the minimal protein requirements in nonpathogenic heterologous hosts, required for producing alginate precursor, and proceeding into polymerization and secretion steps. Overall, we concluded that polysaccharides play a major role in the formation of bacterial biofilms while chemical composition is a key determinant for biofilm architecture and development. This contribution to understanding the biosynthesis of bacterial polysaccharides and their properties could provide the necessary knowledge not only for developing novel therapeutics, but also for harnessing such biopolymers for various industrial applications and production via biotechnological procedures