Title page for ETD etd-08182008-085625

Document Type Master's Dissertation
Author Du Plessis, David Johannes Francois
URN etd-08182008-085625
Document Title Regulation of rhamnolipid biosynthesis in the Pseudomonas aeruginosa PAOI biofilm population
Degree MSc
Department Microbiology and Plant Pathology
Advisor Name Title
Prof V S Brozel Co-Supervisor
Prof J Theron Supervisor
  • rhamnolipid biosynthesis
  • Pseudomonas aeruginosa
Date 2006-05-02
Availability unrestricted
Pseudomonas aeruginosa, a ubiquitous environmental bacterium and an opportunistic human pathogen, forms biofilms through a series of interactions between the cells and adherence to surfaces. Not only does rhamnolipid contribute to the pathogenic potential of P. aeruginosa, but it has also been reported that the bacterium utilises rhamnolipid to actively maintain the void spaces surrounding microcolonies, thus contributing to the architecture of P. aeruginosa biofilms. The P. aeruginosa rhlAB operon encodes the enzyme rhamnosyltransferase I, which produces mono-rhamnolipid, and the induction of rhlAB is dependent on the quorum sensing transcription activator RhIR complexed with the auto inducer N-butyryl-homoserine lactone. In this study, several aspects related to rhamnolipid biosynthesis and regulation in P. aeruginosa PAO1 were investigated.

As a first step, a biochemical assay was developed and optimised whereby the concentration of rhamnolipid could be accurately quantified following its extraction from small sample volumes. Although the optimised rhamnolipid assay is not able to distinguish between different rhamnolipids or between different homologs of a specific rhamnolipid, it is, however, simple to perform, cost¬effective and does not rely on the use of specialised equipment. Subsequently an rhlAB-deficient mutant strain of P. aeruginosa PAOI strain was generated. For this purpose, three allelic exchange strategies, i.e. plasmid incompatibility, the use of a SacB counter-selectable marker and a combination of these approaches, were investigated by making use of newly constructed allelic exchange vector systems. The results that were obtained indicated that, of the three approaches, the latter was most efficient in generating the desired P. aeruginosa mutant strain, and 90% of the derived strains were found to be double reciprocal mutants. Reporter gene technology, using the genes encoding for stable and unstable variants of the green fluorescent protein (GFP), was finally used to investigate the transcriptional activity of the rhlA promoter in P. aeruginosa biofilms under conditions of continuous flow using glass as substratum. For this purpose, mini-CTX-GFP reporter vectors, containing stable and unstable variants of the gfp reporter gene, were constructed that allow for integration of a single copy of the transcriptional fusion in a defined, non-essential region onto the P. aeruginosa genome. Several global regulators have been reported to playa role in regulating quorum sensing and/or rhamnolipid biosynthesis in P. aeruginosa, amongst other, the sigma factors RpoS and RpoN. Therefore, rhlA promoter activity was also investigated in biofilms of P. aeruginosa strains lacking either RpoN or RpoS. Although structural differences between the biofilms formed by the P. aeruginosa wild-type PAD 1 and respective mutant strains were noted, transcription of rhlA appeared to be constitutive from 24 h onwards and did not appear to be localised to specific areas within the microcolonies or biofilms. These results, combined with those obtained by batch analysis, indicated that RpoS positively regulates rhlA transcription, whilst RpoN did not appear to influence rhlA promoter activity under the conditions used in this study.

© 2005 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria.

Please cite as follows:

Du Plessis, DJF 2005, Regulation of rhamnolipid biosynthesis in the Pseudomonas aeruginosa PAOI biofilm population, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-08182008-085625/ >


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