UNIVERSITY OF CALIFORNIA, SAN DIEGO An integrated workflow for the multi-omic characterization of microorganisms A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Bioengineering by Haythem Latif Committee in charge: Bernhard Ø. Palsson, Chair Douglas Bartlett Michael Heller Milton H. Saier, Jr. Karsten Zengler Kun Zhang 2015 Copyright Haythem Latif, 2015 All rights reserved. The dissertation of Haythem Latif is approved, and it is acceptable in quality and form for publication on micro- film and electronically: Chair University of California, San Diego 2015 iii DEDICATION To my mother and father for all that you have sacrificed, all that you have provided, and all that you have taught me. iv EPIGRAPH "It is the characteristic of the magnanimous man to ask no favor but to be ready to do kindness to others." -Aristotle "What I see in Nature is a magnificent structure that we can comprehend only very imperfectly, and that must fill a thinking person with a feeling of humility. This is a genuinely religious feeling that has nothing to do with mysticism." -Einstein v TABLE OF CONTENTS Signature Page.................................. iii Dedication..................................... iv Epigraph.....................................v Table of Contents................................. vi List of Figures..................................x List of Tables................................... xii Acknowledgements................................ xiii Vita........................................ xvii Abstract of the Dissertation........................... xix Chapter 1 Introduction...........................1 1.1 The genome that launched 1,000 genomes.........1 1.2 The complexity of microbial genomes revealed by multi-omic characterization........................2 1.3 Systems microbiology approach to omics data integration.3 1.4 Previous implementations of the systems approach to multi- omic data integration....................4 1.5 Updating and expanding the previous workflow......6 1.6 Bibliography.........................8 Chapter 2 Genome assembly improvements using next-generation sequenc- ing technology........................... 13 2.1 Abstract........................... 13 2.2 Introduction......................... 14 2.3 Results and Discussion................... 15 2.3.1 Thermotoga maritima ATCC genomovar sequence and gene annotation for a missed ≈9 kb region.. 15 2.3.2 Escherichia coli serotype O157:H7 EDL933 assem- bly........................... 17 2.4 Conclusion.......................... 20 2.5 Acknowledgements..................... 20 2.6 Bibliography......................... 22 vi Chapter 3 The genome organization of Thermotoga maritima reflects its lifestyle.............................. 27 3.1 Abstract........................... 27 3.2 Author Summary...................... 28 3.3 Introduction......................... 28 3.4 Results............................ 31 3.4.1 An integrative, multi-omic approach for the annota- tion of the genome organization.......... 31 3.4.2 Identification of promoters and RBSs followed by quantitative intra- and interspecies analysis of bind- ing free energies................... 35 3.4.3 T. maritima promoter-containing intergenic regions reveal a unique distribution of 50UTRs and spatial limitations on regulation.............. 42 3.4.4 T. maritima has an actively transcribed genome that is tightly correlated to protein abundances.... 45 3.5 Discussion.......................... 47 3.6 Materials and Methods................... 50 3.6.1 Culture conditions and physiology......... 50 3.6.2 Genome resequencing and annotation updates.. 50 3.6.3 Transcription start site determination....... 51 3.6.4 Transcriptome characterization and gene expression 51 3.6.5 Proteomics, peptide mapping, and protein abundance quantitation..................... 52 3.6.6 Promoter element motif analysis and position weight matrix (PWM) generation............. 53 3.6.7 Information content calculations.......... 54 3.6.8 Ribosome binding site energy calculations.... 55 3.6.9 Rho-independent terminator site determination. 56 3.6.10 Prediction of small RNAs............. 56 3.6.11 Transcription unit assembly............ 56 3.6.12 Transcription factor binding site mapping..... 57 3.6.13 Data deposition................... 57 3.7 Acknowledgments...................... 57 3.8 Bibliography......................... 58 Chapter 4 Adaptive evolution of Thermotoga maritima reveals plasticity of the ABC transporter network.................. 67 4.1 Abstract........................... 67 4.2 Introduction......................... 68 4.3 Materials and Methods................... 70 4.3.1 Culture conditions and physiology......... 70 4.3.2 Genomic DNA sequencing and variant analysis.. 71 vii 4.3.3 RNA-seq and transcript abundance estimation... 71 4.3.4 Gene expression analysis............... 72 4.3.5 Data Deposition................... 72 4.4 Results............................ 73 4.4.1 Glucose evolution and evolved phenotypic proper- ties.......................... 73 4.4.2 Genetic variants in evolved cultures on glucose.. 75 4.4.3 Gene expression analysis of eTMglc mutant cultures. 77 4.5 Discussion.......................... 80 4.6 Acknowledgments...................... 84 4.7 Bibliography......................... 84 Chapter 5 Integrated analysis of molecular and systems level function of Crp using ChIP-exo.......................... 90 5.1 Abstract........................... 90 5.2 Introduction......................... 91 5.3 Results............................ 93 5.3.1 ChIP-exo data provides genome-scale, in vivo mech- anistic insights into bacterial transcription activa- tion.......................... 93 5.3.2 ChIP-exonuclease coupled with gene expression de- lineates the full Crp regulon............ 107 5.4 Discussion.......................... 117 5.5 Materials and Methods................... 121 5.5.1 Strains and Culturing Conditions......... 121 5.5.2 ChIP Experiments................. 122 5.5.3 Gene Expression.................. 123 5.6 Author Contributions.................... 124 5.7 Acknowledgements..................... 124 5.8 Bibliography......................... 124 Chapter 6 A streamlined ribosome profiling protocol for the characterization of microorganisms........................ 133 6.1 Abstract........................... 133 6.2 Introduction......................... 134 6.3 Results and Discussion................... 135 6.4 Conclusion.......................... 137 6.5 Materials and Methods................... 139 6.5.1 Reagents....................... 139 6.5.2 Procedure...................... 140 6.5.3 Recipes....................... 147 6.5.4 Troubleshooting................... 150 6.5.5 Equipment...................... 151 viii 6.6 Acknowledgements..................... 152 6.7 Author Contributions.................... 152 6.8 Bibliography......................... 152 Chapter 7 Trash to treasure: Production of biofuels and commodity chemi- cals via syngas fermenting microorganisms........... 154 7.1 Abstract........................... 154 7.2 Graphical Abstract..................... 155 7.3 Introduction......................... 155 7.4 Syngas fermenting microorganisms............. 157 7.5 The Wood-Ljungdahl pathway............... 157 7.6 Energy conservation in acetogens............. 162 7.7 Advances in genetic manipulation tools.......... 163 7.8 Strain engineering to obtain desired production phenotypes 165 7.9 Rational strain design and process optimization through a systems-level approach................... 166 7.10 Summary and opportunities................ 168 7.11 Acknowledgements..................... 169 7.12 Bibliography......................... 169 Chapter 8 Conclusion............................. 178 8.1 Model organisms and their knowledgebases......... 178 8.2 Closing the knowledge gap using an integrated, multi-omic characterization workflow.................. 179 8.3 The benefits of a consolidated data generation platform.. 179 8.4 Applications of microbial knowledgebases......... 182 8.5 Perspective on the future of multi-omic data integration. 183 8.6 Bibliography......................... 186 ix LIST OF FIGURES Figure 1.1: The four principle steps of integrating multi-omic datasets....5 Figure 2.1: T. maritima genome gap revealed................. 18 Figure 2.2: Updated E. coli serotype O157:H7 EDL933 assembly....... 19 Figure 3.1: Generation of multiple genome-scale datasets integrated with bioin- formatics predictions reveals the genome organization...... 32 Figure 3.2: Identification and quantitative comparison of genetic elements for transcription and translation initiation.............. 36 Figure 3.3: Arrangement of genomic features contained within promoter-containing intergenic regions (PIRs)...................... 43 Figure 3.4: Global analysis of mRNA and protein expression levels..... 46 Figure 4.1: Glucose evolution time course................... 74 Figure 4.2: Mutations to the gluEFK and bglEFGKL ABC transporter oper- ons.................................. 76 Figure 4.3: Gene expression analysis of eTMglc cultures........... 79 Figure 4.4: Gene expression analysis of the different functional categories of proteins found in the ABC2 importer families for carbohydrate uptake (3.A.1.1 & 3.A.1.2) and the peptide/opine/nickel uptake family (3.A.1.5) for cultures grown on glucose........... 81 Figure 5.1: TSS aligned and oriented σ70 ChIP-exo data reveals DNA footprint patterns consistent with stable transcription initiation intermedi- ates.................................. 94 Figure