The eukaryote-associated Pseudoalteromonas tunicata produces two pigments and several other bioactive compounds that are able to inhibit a range of marine organisms including bacteria, protozoa, fungi, algal spores and invertebrate larvae. Early studies suggested that the production of bioactive compounds is correlated with pigmentation in P. tunicata. In one of these studies, a transposon mutagenesis library identified a white mutant, wmpD-, which had been disrupted in a gene encoding a component of the type 11 secretion (T2S) machinery. The T2S system is involved in the transport of different extracellular enzymes in many bacteria. In some cases, the T2S pathway also exports proteins that remain attached to the cells. This thesis aimed to investigate the role of the T2S pathway in the production of the pigments and bioactive compounds in P. tunicata. In order to gain insight into this relationship, two proteomics approaches (2D-PAGE and iTRAQ) were applied to investigate the profile of the secreted proteins (or secretome) in P. tunicata wild-type and the white mutant wmpD-. Proteomic analysis using 2D-PAGE revealed that 23 proteins were differentially expressed between P. tunicata Wt and the mutant wmpD-. The identities of some of these proteins could be correlated with the function of the T2S system in P. tunicata. The role of one of the proteins identified using 2D-PAGE was further investigated through the construction of a gene knockout mutant (hiik mutant). The supernatant activity of the hiik mutant was compared to that of P. tunicata Wt, and it was found that the HiiA protease is required to block the activity of antimicrobial peptides, such as cecropins, produced by eukaryotic hosts in the environment. The second proteomics approach (iTRAQ) used in this thesis, enabled the relative quantitation of a number of proteins in the supernatant of P. tunicata Wt and the white mutant wmpD-. Some proteins with no function to date (hypothetical) were absent in the extracellular fraction of the wmpD- mutant, indicating they may be transported to the extracellular environment via the T2S pathway in P. tunicata. The comparative analysis of the secretome also revealed that TonS-related proteins, involved in iron acquisition, were up-regulated in the wmpD- mutant, possibly to compensate for the lack of TonS-dependant receptors in the outer membrane. Assays for iron binding activity showed that P. tunicata Wt seems to release iron binding compounds (or siderophores) constitutively into the supernatant, in contrast to the white mutant wmpD-, which responds to iron limitation by increasing the production of siderophores. Further outer membrane fractionation studies, indicated that the P. tunicata T2S system is likely to be involved in the transport of TonB-dependant receptors to the outer membrane. The overall results discussed in this thesis indicate that the T2S system has an essential role in the general physiology of P. tunicata, as for iron metabolism, as well as in the in the relationship between this bacterium and eukaryotic hosts in the environment.
| Identifer | oai:union.ndltd.org:ADTP/258279 |
| Date | January 2007 |
| Creators | Evans, Flavia F., Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW |
| Publisher | Awarded by:University of New South Wales. Biotechnology & Biomolecular Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright Evans Flavia F.., http://unsworks.unsw.edu.au/copyright |
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