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
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Date
2017
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Massey University
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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.
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Keywords
Microbial exopolysaccharides, Pseudomonas aeruginosa, Pseudomonas syringae, Biofilms, Research Subject Categories::NATURAL SCIENCES::Biology::Organism biology::Microbiology