Spatial and temporal localisation of exopolysaccharide gene expression in mucoid and non-mucoid Pseudomonas aeruginosa biofilms : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology, Massey University, Manawatu, New Zealand
The biofilm, or surface-associated microbial community, is the preferred method of growth for most bacteria. Pseudomonas aeruginosa is an ubiquitous, opportunistic pathogen capable of biofilm formation in a wide range of natural and clinical environments. In particular, biofilms formed by P. aeruginosa in the lungs of people with cystic fibrosis (CF) are responsible for a significant decline in the health and prognosis of these patients. Once established, P. aeruginosa biofilms begin to excrete an exopolysaccharide (EPS) called alginate which protects the bacterial microcolonies from antimicrobial molecules and confers a mucoid phenotype. Once this phenotypic switch has occurred, the biofilm becomes impossible to eradicate and ultimately leads to the death of the patient. Here, fluorescent signalling systems and confocal laser scanning microscopy (CLSM) have been used to spatially and temporally resolve the expression of three EPSs produced by P. aeruginosa; the pellicle-forming EPS (Pel), the EPS encoded by the polysaccharide synthesis locus (Psl) and alginate. In order to observe the effect (if any) of EPS production on spatial localisation of the cells within the biofilm, the biofilm-associated characteristics of three P. aeruginosa double-knockout mutants, each able to produce only one EPS has been observed. In analysing these biofilm structures, it was found that Pel has a role in facilitating an increased surface area of the biofilm, while Psl-producing mutants form a biofilm structure with a significantly increased biomass. By visualising fluorescent signals throughout a biofilm consisting of a mixture of the three mutants, the spatial localisation of EPS-producing bacterial populations has been observed. Here, Pel-producing mutants tended to aggregate at the attachment surface, suggesting a role in adhesion of the biofilm structure. Spatial and temporal localisation of EPS promoter activity was achieved by transforming the prototypic P. aeruginosa PAO1 strain with one of three plasmids encoding unstable gfp expression under the control of each EPS’s promoter sequence. Overall, this study has demonstrated the applications and limitations of fluorescence-based localisation of bacterial gene expression throughout P. aeruginosa biofilm development. Collectively, this information can help to guide future investigations into the expression and regulation of the genes associated with a biofilm phenotype, with the aim of identifying a target for effective therapy against this important pathogen.