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Identification of Potential Common Cyclic‑Di‑GMP Regulated Biomarkers Among Three Gram Negative Bacteria Via Transcriptomic and Metabolomic Profiling

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Identification of Potential Common Cyclic‑Di‑GMP Regulated Biomarkers Among Three Gram Negative Bacteria Via Transcriptomic and Metabolomic Profiling This document is downloaded from DR‑NTU (https://dr.ntu.edu.sg) Nanyang Technological University, Singapore. Identification of potential common cyclic‑di‑GMP regulated biomarkers among three gram negative bacteria via transcriptomic and metabolomic profiling Cai, Zhao 2017 Cai, Z. (2017). Identification of potential common cyclic‑di‑GMP regulated biomarkers among three gram negative bacteria via transcriptomic and metabolomic profiling. Doctoral thesis, Nanyang Technological University, Singapore. http://hdl.handle.net/10356/69468 https://doi.org/10.32657/10356/69468 Downloaded on 06 Oct 2021 21:03:43 SGT IDENTIFICATION OF POTENTIAL COMMON CYCLIC-DI-GMP REGULATED BIOMARKERS AMONG THREE GRAM NEGATIVE BACTERIA VIA TRANSCRIPTOMIC AND METABOLOMIC PROFILING CAI ZHAO Interdisciplinary Graduate School Singapore Centre for Environmental Life Science Engineering 2016 IDENTIFICATION OF POTENTIAL COMMON CYCLIC-DI-GMP REGULATED BIOMARKERS AMONG THREE GRAM NEGATIVE BACTERIA VIA TRANSCRIPTOMIC AND METABOLOMIC PROFILING CAI ZHAO Interdisciplinary Graduate School Singapore Centre for Environmental Life Science Engineering A thesis submitted to the Nanyang Technological University in partial fulfillment of the requirement for the degree of Doctor of Philosophy 2016 Acknowledgement I would like to express my sincere gratitude towards Professor Michael Givskov, Professor Staffan Kjelleberg, and Assistant Professor Yang Liang for giving me the opportunity to work in SCELSE and their guidance throughout my Ph.D study. I would like to thank Professor Sanjay Swarup, Dr. Victor Nesati and Dr. Peter Benke from Metabolite Biology Lab, SCELSE NUS for collaboration on my Ph.D project and providing me technical support and raw data on Liquid Chromatography Mass Spectrometry analysis. All LC-MS raw data were receiving from Dr. Victor Nesati and Dr. Peter Benke with courtesy. I would also like to thank Dr. Liu Yang for her help and guidance in transcriptomics works and sequencing analysis; Dr. Chua Songlin and Ms. Chen Yicai for helping me in lab skills and data analysis; Ms. Li Yingying and Mr. Ding Yichen for their guidance in PCR works and data analysis; all other group members for their assistance and help throughout the years. I am very grateful that my family members are always by my side all these years giving me their support, especially my mum, Mdm. Ma Guiling, for her encouragement and consolation at all the difficult times. Last but but not least, I would like to thank IGS and SCELSE for providing me financial, administrative and technical support to accomplish my Ph.D study. I Table of Contents Acknowledgement .................................................................................................................. I List of Figures ......................................................................................................................IV List of Tables ...................................................................................................................... VII List of Abbreviations and Symbols ......................................................................................IX List of Publications ................................................................................................................ X Summary ..............................................................................................................................XI Chapter 1. Introduction ....................................................................................................... 1 1.1. Biofilms .................................................................................................................... 1 1.2. Bis-(3’-5’)-cyclic dimeric GMP ............................................................................... 2 1.3. Quorum sensing ........................................................................................................ 4 1.4. Knowledge gap ......................................................................................................... 5 1.5. Hypothesis and objectives ........................................................................................ 6 1.6.1. Biosynthesis and breakdown of c-di-GMP ........................................................... 9 1.6.2. Regulation by c-di-GMP ..................................................................................... 13 1.6.2.1. C-di-GMP regulation on biosynthesis of exopolysaccharides ........................ 14 1.6.2.2. Regulation of motility by c-di-GMP .............................................................. 21 1.6.2.3. Quorum sensing ............................................................................................... 26 1.6.2.4. C-di-GMP regulation on biofilm dispersion ................................................... 36 1.6.2.5. C-di-GMP and virulence ................................................................................. 37 Chapter 2. Identification of signature genes regulated by c-di-GMP through transcriptomics analysis ....................................................................................................... 40 2.1. Materials and methods ............................................................................................ 41 2.2. Results .................................................................................................................... 45 2.3. Discussion .............................................................................................................. 67 2.3.1. Transcriptomics analysis of P. aeruginosa mutants ........................................... 67 2.3.2. Transcriptomics analysis of B. cenocepacia mutants ......................................... 78 II 2.3.3. Transcriptomics analysis of K. pneumoniae mutants ......................................... 83 Chapter 3. Identification of signature metabolites regulated by c-di-GMP through metabolomics profiling ........................................................................................................ 87 3.1. Materials and methods ............................................................................................ 88 3.2. Results .................................................................................................................... 91 3.3. Discussion ............................................................................................................ 125 3.3.1. Metabolomics analysis of P. aeruginosa .......................................................... 126 3.3.2. Metabolomics analysis of B. cenocepacia ........................................................ 130 3.3.3. Metabolomics analysis of K. pneumoniae ........................................................ 134 Chapter 4. RpoN (σ54) controls virulence factors by modulating PqsR in pqs quorum sensing of P. aeruginosa ....................................................................................... 139 4.1. Materials and methods .......................................................................................... 140 4.2. Results .................................................................................................................. 146 4.3. Discussion ............................................................................................................ 157 Chapter 5. Conclusion and Future Plan .......................................................................... 160 5.1. Conclusion ............................................................................................................... 160 5.2. Future Plan ............................................................................................................... 163 Chapter 6. References ..................................................................................................... 165 Chapter 7. Appendix ....................................................................................................... 183 7.1. Transcriptomics data ................................................................................................ 183 7.2. Metabolomics data ................................................................................................... 194 7.3. Gene expression of ∆rpoN mutant ........................................................................... 224 III List of Figures Figure 1.1 Stages of biofilm development. Figure 1.2 Modulation of c-di-GMP and its cellular functions. Figure 1.3 Enzyme regulators and receptors of c-di-GMP. Figure 1.4 Regulation of c-di-GMP regulated by WspR and Roc/Sad system. Figure 1.5 Pel,Psl and alginate gene operons. Figure 1.6 Biodynthesis of Pel polysaccharide. Figure 1.7 Biosynthesis of Psl polysaccharide. Figure 1.8 Hierarchical relationship among three quorum sensing systems. Figure 1.9 Genetic regulation and formation of PQS molecule. Figure 1.10 Cep and Cci quorum sensing in Burkholderia cenocepacia. Figure 2.1 Construction of c-di-GMP mutants of all three pathogens. Figure 2.2 C-d-GMP concentrations in mutants of three pathogens. Figure 2.3 Box plot of genes regulated in P. aeruginosa/pYedQ2 and P. aeruginosa/pY -hjH. Figure 2.4 PCA plot of genes in P. aeruginosa/pYedQ2 and P. aeruginosa/pYhjH mut- ants. Figure 2.5 Heatmap of genes regulated in P. aeruginosa/pYedQ2 and P. aeruginosa/p- YhjH. Figure 2.6 Box plot of genes regulated in B.cenocepacia/pYedQ2 and B.cenocepacia/ pYhjH. Figure 2.7 PCA plot of genes in B.cenocepacia/pYedQ2 and B.cenocepacia/pYhjH repl -icates. Figure 2.8 Heatmap of genes regulated in B. cenocepacia/pYedQ2
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