The role of AlgK in alginate biosynthesis by Pseudomonas aeruginosa : a thesis presented in partial fulfilment of the requirements of the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand

Abstract

Alginate is a polysaccharide produced by brown seaweeds and two bacterial genera Azotobacter and Pseudomonas. While seaweed alginate finds numerous industrial and medical applications, alginate produced by Azotobacter and Pseudomonas spp., is important for cyst and biofim formation, respectively. A member of Pseudomonas, Pseudomonas aeruginosa, is the leading cause of death in Cystic Fibrosis (CF) patients. This pathogen over-produces alginate upon infection of the CF lung, protecting it from host immune responses and antibiotics while clogging up the patients’ airways leading to poor prognosis. Alginate biosynthesis occurs in four stages: (1) precursor synthesis in the cytoplasm (AlgA, D and C), (2) polymerisation at the inner membrane (Alg8 and Alg44), (3) periplasmic translocation and modification (AlgK, X, L, G, I, J and F), and (4) secretion (AlgE) across the outer membrane. The latter three stages are facilitated by a putative multi-protein complex spanning the entire envelope fraction. Currently, it is unknown how this complex is assembled and the roles certain components of the complex play in alginate biosynthesis are not clear. The periplasmic protein AlgK is a key component of this complex. This protein has multiple protein-protein interaction domains, suggesting that it could be critical for assembling functional alginate biosynthesis machinery. In the present study, an algK mutant was generated and used to determine the impact of AlgK’s absence on (i) alginate yield and size, and (ii) the stability of other components of the alginate biosynthesis machinery. This study demonstrates that AlgK is essential for polymerisation and is required for the stability of components involved in polymerisation (Alg44), translocation (AlgX), and secretion (AlgE). We also show that AlgK interacts with periplasmic AlgX but not with inner membrane Alg44 or outer membrane AlgE. Overall, this study sheds light on the role of AlgK in alginate production and the assembly of the alginate biosynthesis machinery.