Molecular mechanism of export of alginate in Pseudomonas aeruginosa : a thesis presented in partial fulfilment of the requirements for [the] degree of Doctor of Philosophy in Microbiology at Massey University, New Zealand

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen; infecting insects, plants and humans. It is of particular relevance to cystic fibrosis (CF) patients where it causes pulmonary infection and the leading cause of morbidity and mortality. The CF lung environment selects for a variant of P. aeruginosa characterised by the overproduction of an exopolysaccharide called alginate. It has been hypothesized that outer membrane protein AlgE forms a channel through which alginate is secreted into the extracellular environment. Furthermore, studies have suggested that proteins involved in the polymerisation, modification and export of alginate form a multiprotein complex that span the bacterial envelope. The aim of this thesis was to investigate the role of AlgE in polymerisation and secretion of alginate. For this purpose algE knockout mutant was created in PDO300. Results showed that AlgE does not have a role in alginate polymerisation however it has a role in secretion of alginate and stability of the alginate biosynthesis machinery. By performing FLAG epitope insertion mutagenesis the topology of AlgE was verified and site-directed mutagenesis further showed that the positive electrostatic field inside the AlgE lumen is required for efficient secretion of negatively charged alginate. By employing mutual stability analysis, evidence was provided for the existence of trans-envelope multiprotein complex required for alginate biosynthesis. Co-immuniprecipitaion assay suggest that AlgE interacts with periplasmic located AlgK and, most probably, this interaction is mediated by the peripasmic turn 4 of AlgE. Pull-down assays further showed that AlgK interacts with another periplasmic protein AlgX which in turn interacts with the inner membrane protein Alg44. Based on mutual stability analysis it was proposed that Alg44 interacts with Alg8 which might interacts with AlgG as well. Our results also support the existence of internal promoters for AlgE and AlgG.