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Phd Thesis Aida An investigation of the phenotypic and genotypic changes in Pseudomonas putida CP1 following substrate-dependent autoaggregation A thesis submitted to Dublin City University in fulfilment of the requirements for the award of the degree of Doctor of Philosophy by Wan Syaidatul Aqma Wan Mohd Noor B.Sc. School of Biotechnology and National Institute for Cellular Biotechnology, Dublin City University, Dublin 9, Ireland. Research Supervisor: Dr. Bríd Quilty August 2013 I hereby certify that this material, which I now submit for assessment on the programme of study leading to the award of Ph.D. is entirely my own work, that I have exercised reasonable care to ensure that the work is original, and does not to the best of my knowledge breach any law of copyright, and has not been taken from the work of others save and to the extent that such work has been cited and acknowledged within the text of my work. Signed: _______________________________ I.D. Number: _______________________________ Date: _______________________________ Acknowledgement I bow my head with great reverence to Him for giving me strength, inspiration and the cause behind every effort. I want to thank Dr. Bríd Quilty for the tremendous support for over the past four years. I thank her especially for always being available and teaching me the most fundamental principles as a teacher, a mentor and a researcher. She will always be my role-model. I am grateful to NICB team, especially Dr. Padraig Doolan for his feedbacks and help with the microarray study. A special thanks to Dr. Finbarr O’Sullivan and Dr. Niall Barron who helped me during the last phase of my research. It has been a real pleasure and an honor to work with them all. I would like to thank all staff in School of Biotechnology, Dublin City University. I wish to thank my girls - Markella, Ana and Joanna for making me laugh and enjoy lab work even during the most intense periods. Also, to all good friends that in the last four years helped Dublin feel like home especially Saidah, Aqida, Izwan, Raihana, Ika, Suliana, Shima, Naz, Tuty, Shuib, Muhad and Hooi Ling. A last but important word of appreciation goes to my family for the support and prayer. To my beautiful daughter Aisyah, you have turned me into a better and happier person. Above all, to my husband the love of my life and my best friend, Abdul Hadi, who has helped me tremendously by believing in me and making me believe in myself, telling me I’m the best and for having spent so much time and energy taking care of Aisyah in the last year of my Ph.D. Nothing of what I have accomplished would be possible without his incredible emotional support, love and friendship. Finally, I would like to acknowledge my sponsors Ministry of Higher Education of Malaysia (MOHE) and Universiti Kebangsaan Malaysia (UKM). Table of contents Abstract …………………………………………………………………………...i List of Abbreviations …………………………………………………………….ii List of Tables …………………………………………………………………….iv List of Figures …………………………………………………………………...vii 1 Introduction................................................................................................... 2 1.1 Pseudomonads............................................................................................. 2 1.1.1 Pseudomonas putida ................................................................................. 5 1.1.2 Gram-negative bacterial cell structure .................................................... 10 1.1.3 Metabolism in Pseudomonas putida ....................................................... 13 1.1.4 Chlorophenol degradation....................................................................... 17 Metabolism of mono-chlorophenols ................................................... 18 Microorganisms reported to degrade chlorophenols........................... 22 1.2 Autoaggregation in bacteria...................................................................... 24 1.2.1 Extracellular polymeric substances (EPS) .............................................. 26 1.2.2 Interactions involved in the formation of aggregates.............................. 29 1.2.3 Genotypic changes in autoaggregation ................................................... 33 1.3 Microbial stress responses ........................................................................ 36 1.3.1 Mechanisms of gene regulation under stress .......................................... 41 Two-component signalling systems.................................................... 41 Sigma factors....................................................................................... 42 C-di-GMP............................................................................................ 44 Chemotaxis and Flagellae ................................................................... 45 1.3.2 Molecular approaches used in understanding stress responses............... 48 1.4 Aims and objectives of the project............................................................ 53 2 Materials and methods ............................................................................... 55 2.1 Bacterial cultures....................................................................................... 55 2.2 Chemicals.................................................................................................. 55 2.3 Buffers and solutions ................................................................................ 56 2.4 Media and gels .......................................................................................... 58 2.5 pH measurement........................................................................................ 60 2.6 Dry weight measurement .......................................................................... 60 2.7 Cell characterisation.................................................................................. 60 2.8 Culture conditions and biodegradation studies ......................................... 63 2.9 Microscopic studies................................................................................... 65 2.10 Aggregate studies...................................................................................... 67 2.11 Extracellular polymeric substances (EPS) extraction ............................... 68 2.12 Lipopolysaccharide (LPS) extraction........................................................ 71 2.13 RNA preparation....................................................................................... 74 2.14 Microarray studies..................................................................................... 78 2.15 Quantitative real time RT-PCR (qRT-PCR)............................................. 86 2.16 Quantitative real time RT-PCR (qRT-PCR) assay ................................... 89 2.17 Data analysis ............................................................................................. 91 3 Results .......................................................................................................... 93 3.1 A comparative study of P. putida CP1 and P. putida KT2440................. 93 3.2 Phenotypic and genotypic responses of P. putida CP1 when grown on mono-chlorophenols........................................................................................ 105 3.2.1 Phenotypic responses of P. putida CP1 when grown on mono- chlorophenols .............................................................................................. 105 Growth of P. putida CP1 and P. putida KT2440 on mono- chlorophenols .................................................................................... 105 Analysis of the aggregates ................................................................ 110 Extracellular polymeric substances (EPS) ........................................ 112 3.2.2 Expression profiling analysis................................................................ 116 5 Comparative study for CP1 vs 100 ppm 2-chlorophenol, CP1 vs 100 ppm 3-chlorophenol, CP1 vs 200 ppm 4-chlorophenol and CP1 vs 50 ppm 4-chlorophenol .......................................................................... 116 Comparative study 50 ppm4CP vs 200 ppm4CP.............................. 119 Comparative analysis of CP1 vs 200 ppm 4CP-100 ppm 2CP-100 ppm 3CP.................................................................................................... 124 qRT-PCR........................................................................................... 131 3.3 Phenotypic and genotypic responses of P. putida CP1 when grown on 0.5% (w/v) fructose......................................................................................... 132 3.3.1 Phenotypic responses of P. putida CP1 when grown on 0.5% (w/v) fructose........................................................................................................ 132 Growth of P. putida CP1 and P. putida KT2440 on 0.5% (w/v) fructose and 0.5% (w/v) glucose....................................................... 132 Analysis of the aggregates ................................................................ 134 Extracellular polymeric substances (EPS) ........................................ 136 3.3.2 Expression profiling analysis................................................................ 139 Comparative study CP1vsFructose ................................................... 139 Comparative study CP1 vs. 200 ppm 4CP-100 ppm 2CP-100 ppm 3CP and CP1vsFructose............................................................................ 154 qRT-PCR..........................................................................................
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