The release of cells from a biofilm to the surrounding environment is poorly understood and the importance of this stage of biofilm development has only recently been realized. A key part of this process is the generation of phenotypic variants in the biofilm dispersal population. This thesis reports on the characterization of biofilm development of Serratia marcescens MG1, the analysis of the biofilm dispersal population, and the identification of the conditions that trigger phenotypic diversification. Furthermore, it provides an insight into the molecular understanding of how phenotypic variation is being generated, and demonstrates the clinical and environmental implications of phenotypic diversification during bacterial pathogenesis and bacterial persistence. Characterization of the microcolony biofilm development of S. marcescens revealed that the S. marcescens biofilm develops through a process involving microcolony formation, hollowing of mature microcolonies, and a sudden biofilm expansion within a very short period (< 24h) resulting in an increase in biofilm biomass with a radiation of biofilm structures at days 3 to 4. The biofilm expansion phase consistently correlated to an increase in the number of dispersal variant morphotypes. Studies of variant induction in planktonic cultures and biofilm flow cells demonstrated that phenotypic diversification in S. marcescens is not only a biofilm-specific phenomenon, but also involves biofilm-specific morphotypes. These morphological variants can only be isolated from the microcolony biofilm morphotype and not from the filamentous biofilms, leading to the hypothesis that there is a strong diversifying selection that is specific to the microcolony biofilms. To further explore how these variants were generated, molecular analyses revealed that exopolysaccharides and lipopolysaccharides are important moieties that are involved in phenotypic variation in S. marcescens biofilms. The etk gene, encoding a tyrosine protein kinase within the exopolysaccharide biosynthesis operon, was found to contain single nucleotide polymorphisms (SNPs) that were present in the 'sticky' variants but not in the 'non-sticky' wild-type or the 'non sticky' small colony variants. Furthermore, infrequent-restriction-site PCR (IRS-PCR), BIOLOG metabolic profiling, and gene sequence analyses, suggest that phenotypic diversification in S. marcescens is likely to involve mutational hotspots in specific genes. The biofilm-derived morphotypic variants differed extensively in cell ultrastructure properties, and exhibited specialized colonization and virulence traits, such as attachment, biofilm formation, swimming and swarming motilities, protease production, and hemolysin production. It was also demonstrated that phenotypic diversification contributed to a varying degree of resistance to protozoan predation, and bacterial pathogenecity in Caenorhabditis elegans, highlighting the complexity of the dispersal populations from S. marcescens biofilms. Furthermore, mixed-culture experiments involving multiple variant isolates (with or without the parental wild-type) showed that the persistence and virulence potential of S. marcescens can be synergistically enhanced in the Acanthamoeba castellanii grazing model and in the C. elegans infection model, respectively. This indicates that the different bacterial morphotypes work in concert to provide S. marcescens with enhanced protection against environmental perturbations and a competitive edge during the infection process. It was proposed that phenotypic diversification is not only an integral part of S. marcescens biofilm life-cycle, but also represents an important strategy for bacteria to greatly enhance its survival and persistence in different environments, ranging from aquatic and soil ecosystems, to those of the infected hosts.
| Identifer | oai:union.ndltd.org:ADTP/257648 |
| Date | January 2007 |
| Creators | Koh, Kai-Shyang, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW |
| 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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