Browsing by Author "Wang, Yajie"
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- ItemThe 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(Massey University, 2013) Wang, YajieAlginate 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.
- ItemUnderstanding aspects of alginate biosynthesis and regulation by Pseudomonas aeruginosa : a thesis presented in partial fulfilment of the requirements of the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand(Massey University, 2017) Wang, YajieAlginate is a medically and industrially important polymer produced by seaweeds and certain bacteria. The bacterium Pseudomonas aeruginosa over-produces alginate during cystic fibrosis lung infections, forming biofilms, making the infection difficult to treat. Bacteria make alginate using membrane spanning multi-protein complexes. Although alginate biosynthesis and regulation have been studied in detail, there are still major gaps in knowledge. In particular, the requirement of AlgL (a periplasmic alginate degrading enzyme) and role played by MucR (an inner membrane c-di-GMP modulator) are not well understood. Here I show that AlgL and MucR are not essential for alginate production during biofilm growth. My findings suggest that while catalytically active AlgL negatively affects alginate production, expressing catalytically inactive AlgL enhances alginate yields. Furthermore, preliminary data show AlgL is not required for the stability or functionality of the alginate biosynthesis complex, suggesting that it is a free periplasmic protein dispensable for alginate production. These findings support the prediction that the primary function of AlgL is to degrade misguided alginate from the periplasm. For MucR, I show for the first time that its sensor domain mediates nitrate-induced suppression of alginate biosynthesis. This appears to occur at multiple levels in a manner only partially dependent on c-di-GMP signaling. These results indicate that MucR is associated with the negative effect of nitrate (and possibly denitrification) on alginate production. On the basis of these results, I propose a combination of nitrate (or denitrification intermediates), exogenous lyases and antimicrobial agents could be used to eliminate established chronic biofilm infections. Furthermore, catalytically inactive AlgL and/or homologs of MucR with disabled sensor motifs could be harnessed in non-pathogenic bacteria for producing tailor-made alginates.