This document is downloaded from DR‑NTU (https://dr.ntu.edu.sg) Nanyang Technological University, Singapore. Metabolomics analysis of bacterial interactions with the environment : perspective from human gut and heavy metal stress Jain, Abhishek 2018 Jain, A. (2018). Metabolomics analysis of bacterial interactions with the environment : perspective from human gut and heavy metal stress. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/88007 https://doi.org/10.32657/10220/46936 Downloaded on 07 Oct 2021 20:45:35 SGT METABOLOMICS ANALYSIS OF BACTERIAL INTERACTIONS WITH THE ENVIRONMENT: PERSPECTIVE FROM HUMAN GUT AND HEAVY METAL STRESS ABHISHEK JAIN INTERDISCIPLINARY GRADUATE SCHOOL NANYANG ENVIRONMENT & WATER RESEARCH INSTITUTE 2018 INTERDISCIPLINARY GRADUATE SCHOOL METABOLOMICS ANALYSIS OF BACTERIAL NANYANG ENVIRONMENT & WATER RESEARCH INSTITUTE INTERACTIONS WITH THE ENVIRONMENT: PERSPETIVE 2018 FROM HUMAN GUT AND HEAVY METAL STRESS ABHISHEK JAIN Interdisciplinary Graduate School NANYANG ENVIRONMENT & WATER RESEARCH INSTITUTE A thesis submitted to the Nanyang Technological University in partial fulfillment of the requirement for the degree of Doctor of Philosophy 2018 ACKNOWLEDGEMENT This thesis would not have been possible without the support of many individuals whom I met during my PhD candidature. First and foremost, I would like to express my sincere gratitude and appreciation to my supervisor, Professor Chen Wei Ning, William, who has been supportive and motivational throughout the course of my PhD. Thank you for your invaluable advice to make my research project a better one and the numerous opportunities you have afforded me. My gratitude extends to my co-supervisor Associate Professor Koh Cheng Gee and my mentor Assistant Professor Yang Liang for the continuous support and guidance to reach the successful outcome of the study. I appreciate all the support offered by the administration and academia at the Advanced Environmental Biotechnology Centre (AEBC), Nanyang Environment and Water Research Institute (NEWRI) and Interdisciplinary Graduate School (IGS). My sincere thanks extend to all the volunteers who participated in this study. I would like to acknowledge the financial support from Nanyang Technological University, Interdisciplinary Graduate School and Nanyang Environment and Water Research Institute (NEWRI). My heartfelt appreciation goes to my co-workers and friends for lifting me up when the times were hard. I would like to thank my family in the lab, for all the wonderful memories. I would also to thank Lee Jie Lin Jaslyn for her guidance on the experiment and instrument. I also express my deepest gratitude to my family members and friends for all the undying support and encouragements Lastly, my heartfelt thanks and gratitude to my beloved wife, Ragini Jain, whose unwavering love and support has been the greatest motivation and driving force during my PhD candidature. i Table of Contents Acknowledgement……………………………………………………………………………………… i Abstract………………………………………………………………………………………………... vi List of figures…………………………………………………………………………………………. ix List of tables…………………………………………………………………………………………… xi Chapter 1. Introduction .......................................................................................................................... 1 1.1 Aims and objectives ........................................................................................................................... 2 Chapter 2. Literature Review ................................................................................................................. 5 Part I Metabolomics as a monitoring tool ............................................................................................. 5 2. 1 Metabolomics .................................................................................................................................... 5 2.1.1 Targeted and untargeted metabolomics approaches .................................................................. 6 2.2 Analytical tools in Metabolomics...................................................................................................... 7 2.2.1 Gas chromatography-mass spectrometry (GC-MS) based metabolomics ................................. 7 2.2.2 Liquid chromatography-mass spectrometry (LC-MS) based metabolomics ............................ 8 2.3 Applications of metabolomics ........................................................................................................... 9 2.3.1 Environment metabolomics ....................................................................................................... 9 2.3.2 Metabolomics and gut microbiome ......................................................................................... 10 Part II Role of gut microbiome in human nutrition and metabolism ............................................... 12 2.4 Human gut microbiome ................................................................................................................. 12 2.5 Diet is the most influential factor in determining gut microbiome composition………………16 2.5.1 Long term vs short term dietary patterns ................................................................................. 16 2.5.2 Vegetarian diet and Western diet ............................................................................................. 17 2.5.3 Food Constituents .................................................................................................................... 19 2.6 Metabolic functions associated with gut microbiome ................................................................. 20 Part III Heavy metals toxicity and microbial life…………………………………………………...23 2.7 Food as a source of toxic metals…………………………………………………………………..23 2.8 Other sources of heavy metals in the environment……………………………………………...24 2.9 Microbial interactions with the heavy metals in the environment……………………………..25 2.10 Caulobacter crescentus : a model organism to study cell cycle ................................................. 27 2.11 Cell Cycle of Caulobacter crescentus…………………………………………………………….28 ii 2.11.1 The core engine driving cell cycle progression in C. crescentus……………………………29 2.11.2 DnaA/GcrA/CtrA regulatory cascade is critical for cell cycle progression………………....31 2.11.3 Mechanism of interaction between essential proteins, MipZ FtsZ and ParB, during cell division…………………………………………………………………………………………………..32 Chapter 3. Similarities and differences in gut microbiome composition correlate with dietary patterns of Indian and Chinese adults………………………………………………………………..34 Abstract………………………………………………………………………………………………...34 3.1 Introduction………………………………………………………………………………………...35 3.2 Materials and Methods…………………………………………………………………………….36 3.2.1 Recruitment of volunteers……………………………………………………………………36 3.2.2 Sample collection…………………………………………………………………………….38 3.2.3 DNA extraction………………………………………………………………………………38 3.2.4 Library preparation and sequencing………………………………………………………….39 3.2.5 Data analysis…………………………………………………………………………………39 3.2.6 Statistical analysis……………………………………………………………………………40 3.3 Results………………………………………………………………………………………………41 3.3.1 Microbiota from Indian adults is as diverse as microbiota from Chinese adults……………46 3.3.2 Gut microbiota composition and relative abundance differ between healthy Indian and Chinese adults…………………………………………………………………………………………...47 3.3.3 LEfse analysis identified gut bacterial biomarkers of diet…………………………………..50 3.4 Discussion…………………………………………………………………………………………...51 3.4.1 Dominance of Bacteroidetes, Prevotella, and Lactobacillus in gut bacteria of Indians is consistent with their dietary habits………………………………………………………………………53 3.5 Conclusions…………………………………………………………………………………………55 Chapter 4. Fecal and urine metabolites association with diet or ethnicity and gut microbiome of Indian and Chinese adults: an untargeted metabolomics analysis…………………………………56 Abstract…………………………………………………………………………………………………56 4.1 Introduction………………………………………………………………………………………...57 4.2 Materials and methods……………………………………………………………………………..59 4.2.1 Chemicals…………………………………………………………………………………...59 4.2.2 Recruitment of volunteers…………………………………………………………………..59 iii 4.2.3 Sample collection…………………………………………………………………………...60 4.3 GC-MS sample preparation and metabolite extraction…………………………………………60 4.3.1 GC-MS analysis and metabolites detection…………………………………………………61 4.4 LC-MS metabolomics analysis and metabolites identification…………………………………62 4.5 Statistical analysis…………………………………………………………………………………63 4.6 Results and discussion…………………………………………………………………………….64 4.6.1 Fecal and urine metabolomics revealed differences between Indian and Chinese adults…………………………………………………………………………………………………….74 4.6.2 Metabolites that differentiate between Indian and Chinese adults could be associated with their diet…………………………………………………………………………………………………77 4.6.3 Gut microbiome is correlated with fecal and urine metabolites………………………………82 4.7 Conclusions………………………………………………………………………………………...85 Chapter 5. Involvement of organic acids and amino acids in ameliorating Ni(ii) toxicity induced cell cycle dysregulation in Caulobacter crescentus: a metabolomics analysis………………………86 Abstract…………………………………………………………………………………………………86 5.1 Introduction………………………………………………………………………………………...88 5.2 Materials and methods…………………………………………………………………………….90 5.2.1 Bacterial strains, media, and growth…………………………………………………………90 5.2.2 Toxic metals effect on growth and survival………………………………………………….91 5.2.3 RNA Extraction………………………………………………………………………………92