The Presence and Role of Secondary Metabolites in Biofilm Forming Bacteria Isolated from Activated Sludge
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The Presence and Role of Secondary Metabolites in Biofilm Forming Bacteria Isolated from Activated Sludge by Önder Kimyon A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy School of Biotechnology and Biomolecular Sciences Faculty of Science The University of New South Wales Sydney, Australia January 2017 1 PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Kimyon First name: Önder Other name/s: Abbreviation for degree as given in the University calendar: PhD School: Shool of Biotechnology and Biomolecular Sciences Faculty: Science Title: Mr. Abstract 350 words maximum: (PLEASE TYPE) Biofilms are known as highly-structured microbial aggregates encased in a self-produced matrix composed of extracellular polymeric substances. Bacteria commonly grow in surface-associated (inert or organic) or suspended biofilms (bioflocs) in their natural environment. The role of secondary metabolites in regulation of bacterial biofilm formation has been frequently studied, however, there is still much to be learned regarding the role of secondary metabolites in biofilm communities given the diversity of secondary metabolites produced by bacteria. The thesis presented here explores the presence and role of secondary metabolites in biofilm forming bacteria, including an interplay between N-acetylglucosamine (NAG) and acylated homoserine lactone mediated gene expression. This thesis investigated the presence of quorum sensing (QS) and chitinase activities in chitin colonising bacteria isolated from activated sludge. The culture collection was dominated by Gammaproteobacteria, and QS, quorum quenching and chitinase activities are shown to be common among isolated bacteria. Further, a novel AHL-detection method was developed through modification of currently used AHL screening techniques. The novel method improved the AHL detection activity up to 1,000 fold in comparison to published reports. This thesis also investigated the impact of NAG on QS activities of LuxR, LasR and CviR regulated gene expression by examining the effect of NAG on QS regulated green fluorescent protein (GFP), violacein and extracellular chitinase expression. It was discovered that NAG inhibits AHL dependent gene transcription in AHL reporter strains within the range of 50% to 80% reduction at low millimolar concentrations (0.25 mM to 5 mM). Evidence is presented supporting a role for both competitive inhibition at the AHL binding site of LuxR type transcriptional regulators and catabolite repression. Further, this thesis explored the role of Serratia secondary metabolite prodigiosin on biofilm formation of P. aeruginosa. In this study, the link between prodigiosin and biofilm inhibition through the production of redox active metabolites is presented. Overall, the results presented here described how secondary metabolites are prevalent in bacteria colonising on chitin surface, and the impact of NAG on bacterial secondary metabolism (quorum sensing). This study also described the inhibitory effect of prodigiosin on biofilm formation of P. aeruginosa. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). 20.01.2017 …………………………………………………………… ……………………………………..……………… ……….……………………...…….… Signature Witness Signature Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. FOR OFFICE USE ONLY Date of completion of requirements for Award: 2 ORIGINALITY STATEMENT ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed …………………………............. Date ……………20.01.2017….............. 3 COPYRIGHT STATEMENT ‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' Signed ………………………….......................... Date ……………20.01.2017…........................... AUTHENTICITY STATEMENT ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Signed ………………………….......................... Date ……………20.01.2017…........................... 4 Table of Contents ACKNOWLEDGMENTS .............................................................................................. 9 ABSTRACT ................................................................................................................... 10 LIST OF PUBLICATIONS .......................................................................................... 12 LIST OF ABBREVIATIONS ...................................................................................... 13 LIST OF FIGURES ...................................................................................................... 15 LIST OF TABLES ........................................................................................................ 17 CHAPTER 1 GENERAL INTRODUCTION ............................................................. 18 1.1. BACTERIAL BIOFILMS AND BIOFLOCS ...................................................... 18 1.1.1. The industrial implications of biofilms .......................................................... 20 1.1.1.2. The activated sludge process ................................................................... 20 1.1.1.2.1. Microbial ecology of activated sludge ................................................... 22 1.1.2. The medical implications of biofilms ............................................................ 23 1.1.3. Regulation of attachment by surface properties ............................................ 24 1.1.3.1. Importance of chitin ................................................................................ 25 1.1.3.1.1. Attachment to chitin ......................................................................... 26 1.1.3.1.2. Chitin biodegradation ....................................................................... 27 1.1.3.1.3. Applications of N-acetylglucosamine (NAG) .................................. 27 1.1.4. Extracellular polymeric substance (EPS) matrices ........................................ 28 1.1.4.1. Role of extracellular DNA (eDNA) in biofilms ...................................... 29 1.1.5. Secondary metabolite chemistry in biofilms ................................................. 30 1.1.5.1. Extracellular electron transfer in biofilms............................................... 30 1.1.5.2. Chemical warfare in biofilms .................................................................. 31 1.1.5.3. Quorum Sensing ...................................................................................... 33 1.1.5.3.1. LuxI/LuxR like quorum sensing systems ......................................... 35 1.1.5.3.2. The role of quorum sensing in biofilm formation and bioflocculation .......................................................................................................................... 37 1.1.5.3.3. AHL-mediated quorum sensing in sludge systems .......................... 38 1.1.5.3.4. Inhibition of quorum sensing ..........................................................