Pseudomonas aeruginosa is a ubiquitous environmental microorganism that opportunistically colonizes immune-compromised hosts. P. aeruginosa is capable of establishing complex. matrix-encased biofilms during colonization of both environmental and living host surfaces. Biofilms formed by P. aeruginosa are physiologically very different from free-living P. aeruginosa cells, and exhibit increased resistance to environmental stresses, including antibiotic treatment. While the development and establishment of P aeruginosa biofilms has been extensively studied in vitro, several new behaviours of P. aeruginosa in biofilms have recently been observed that may greatly impact on the spread, recolonization and function of biofilms. These processes include bacteriophage mediated lysis and dispersal of P. aeruginosa biofilms, and the generation of phenotypic and genetic variation among bacterial cells that disperse from the biofilm. In this project, the role of bacteriophage activity and phenotypic variation in the development of P. aeruginosa biofilms has been investigated. Induction of a Pf1-like prophage of P aeruginosa (here named Pf4), during biofilm formation was characterized and was shown to increase over the progression of biofilm development. It was observed in this study that the activity of Pf4 caused the emergence of small colony variant (SCY) phenotypes in the effluent run-off from P. aeruginosa biofilms. Computational analysis of the genome ofPf4 resulted in the identification of a novel Toxin-Antitoxin (TA) gene pair, not previously identified within the genome of P. aeruginosa, of which the putative toxin gene product was determined here to play a role in growth-inhibition and the small colony phenotype of P. aeruginosa. TA gene pairs are proposed to induce stress responses in host cells and therefore play a role in survival during periods of environmental stresses such as oxidative or starvation stress. To study the effects of the Pf4 toxin and its possible role in the stress response of P aeruginosa, the Pf4 toxin gene was cloned and placed under the regulation of an inducible arabinose promoter. The proteome expression and biofilm formation as a result of toxin over expression were compared. The proteomic studies performed here indicated that P. acrllginosu biofilms do respond to expression of the toxin component of this putative TA element by increased expression of stress related proteins. Many stress-related groups of proteins were found to be over expressed during induction of the toxin indicating a possible role in stress survival of P. acrllginosa. Homology studies of the Pf4 toxin indicated a strong structural sequence relationship with the toxin ParE of the ParDE TA system. The mode of action of ParE toxin had previously been determined and showed the ParE toxin to be a strong gyrase inhibitor. The Pf4 antitoxin was, however, found to have homology to the Phd antitoxin of the Phd-Doe system of bacteriophage PI. The mode of action of the Doc protein remains to be clearly determined. To better understand the interaction between the Pf4 antitoxin and its cognate toxin protein interaction studies were performed. Peptide fragments of the Pf4 antitoxin were generated for an SPR binding assay and this study identified putative peptide sequences that are responsible for binding of the Pf4 antitoxin to its cognate toxin. Further investigation of a selected strong binding peptide showed that there were 3 key amino acids that were important in binding to the Pf4 toxin, namely His65, Ser67 and A 69 sp. Overall, this study has identified a key role for bacteriophage Pf4 in biofilm development and phenotypic variation in P. aeruginosu, and has provided initial insight into the molecular mechanisms by which this bacteriophage influences growth and gene expression in this organism.
| Identifer | oai:union.ndltd.org:ADTP/258291 |
| Date | January 2007 |
| Creators | Lau, Mathew Thye Ngak, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW |
| Publisher | Publisher:University of New South Wales. Biotechnology & Biomolecular Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://unsworks.unsw.edu.au/copyright, http://unsworks.unsw.edu.au/copyright |
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