ANALYSIS OF THE AMMONIUM ASSIMILATION PATHWAYS OF THE HUMAN COLONIC BACTERIUM, BACTEROIDES THETAIOTAOMICRON BY MICHAEL IAKIVIAK DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Animal Sciences in the Graduate College of the University of Illinois at Urbana-Champaign, 2017 Urbana, Illinois Doctoral Committee Professor Roderick I. Mackie, Chair Professor Isaac K.O. Cann Assistant Professor Jason Ridlon Assistant Professor Patrick Degnan Abstract In ruminants, efficient rumen function and proper host metabolism is dependent on the nitrogen supply in the feed. Assimilated ammonium accounts for up to 70% of the microbial protein production, which satisfies up to 85% of the host protein requirements. Similar numbers for the human colon have not been determined. However, colonic bacteria are responsible for the production of ammonium, derived from host-secreted urea and endogenous and dietary proteins, that provides the preferred nitrogen source for microbial growth. Bacteroides thetaiotaomicron, a model organism for human gut Bacteroidetes, encodes genes for the capture of ammonium through the two primary pathways, the glutamate dehydrogenase (GDH) pathway and the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway. To gain insight into the genomic features underlying ammonium uptake and assimilation in this bacterium, comparative transcriptomic analysis using RNA-Seq was employed on cultures growing under excess or limiting ammonium concentrations. A single genomic locus, encoding for the GS/GOGAT pathway, was identified with highly increased transcription when the organism grows under limiting ammonium concentration. The relative contribution of each gene to ammonium assimilation was assessed through construction of genomic deletion strains for each of the three GS, one GOGAT, and two GDH genes. The deletion of two genes, the NADPH-dependent glutamate dehydrogenase (gdhA) and the glutamine synthetase type 3 (glnN2) significantly impeded growth of the organisms under both nitrogen conditions. Taken together, the results demonstrate the importance of the GDH pathway for constitutive ii ammonium assimilation, and GlnN2 for glutamine biosynthesis. However, when the organism grows under nitrogen limitation, the GS/GOGAT pathway, including glnN1, is highly induced. To extrapolate the significance of the findings, a comparative bioinformatic analysis, using all of the available sequenced Bacteroides genomes, revealed high conservation of the critical genomic loci in gut species. Understanding of nitrogen metabolism in gut microbes is essential for a complete depiction of their ecological implications on the host´s metabolism in health and disease. iii Table of Contents List of Figures ................................................................................................................. vii List of Tables .................................................................................................................. xii Chapter 1. Literature review ............................................................................................ 1 1.1 Introduction ........................................................................................................ 1 1.2 Gut microbiota .................................................................................................... 6 1.3 Bacterial nitrogen metabolism ............................................................................ 8 1.4 Enzymatic pathways of ammonium assimilation .............................................. 11 1.5 Ammonium assimilation in gut Bacteroidetes ................................................... 22 1.6 Proposition for research ................................................................................... 29 Chapter 2. Comparative genomic analysis for phylogenetic conservation of ammonium assimilation genes within Bacteroides and Prevotella ................................................... 31 2.1 Introduction ...................................................................................................... 31 2.2 Materials and Methods ..................................................................................... 35 2.3 Results and Discussion .................................................................................... 36 Chapter 3. Nitrogen utilization patterns of B. thetaiotaomicron and genome wide transcriptional response to extracellular ammonium ..................................................... 48 3.1 Introduction ...................................................................................................... 48 iv 3.2 Materials and Methods ..................................................................................... 52 3.3 Results ............................................................................................................. 56 3.4 Discussion ........................................................................................................ 72 Chapter 4. Probing the genetic machinery of ammonium assimilation through phenotypic and competitive fitness analyses ................................................................ 80 4.1 Introduction ...................................................................................................... 80 4.2 Materials and Methods ..................................................................................... 84 4.3 Results ............................................................................................................. 91 4.4 Discussion ...................................................................................................... 103 Chapter 5. Discussion and Future Perspectives ......................................................... 108 5.1 Introduction .................................................................................................... 108 5.2 Major finding................................................................................................... 110 5.3. Implication to the Enteric Paradigm ................................................................ 115 5.4 Future Prospective ......................................................................................... 128 5.5 Conclusion ..................................................................................................... 131 References .................................................................................................................. 134 v Appendix A. Media Composition ................................................................................. 167 Appendix B. Two New Xylanases with Different Substrate Specificities from the Human Gut Bacterium Bacteroides intestinalis DSM 17393 .................................................... 169 Appendix C. Functional and modular analyses of diverse endoglucanases from Ruminococcus albus 8, a specialist plant cell wall degrading bacterium ..................... 206 vi List of Figures Figure 1. Schematic diagram of microbial nitrogen cycling in the rumen and monogastrics animals .................................................................................................. 4 Figure 2. Enteric paradigm for ammonium assimilation based on E. coli .............. 13 Figure 3. Catalytic mechanisms of ammonium assimilation ................................... 15 Figure 4. Diagram of four genomic loci containing ammonium assimilation genes ...................................................................................................................................... 27 Figure 5. Phylogenetic conservation of loci encoding high affinity genes in Bacteroides species .................................................................................................... 39 Figure 6. Phylogenetic conservation of the locus encoding low affinity genes in Bacteroides sp. ........................................................................................................... 40 Figure 7. Conservation of genes encoded by the high affinity loci in Bacteroides fragilis strains .............................................................................................................. 42 Figure 8. Conservation of genes encoded by the low affinity locus in Bacteroides fragilis strains .............................................................................................................. 43 Figure 9. Conservation of ammonium assimilatory genes in Prevotella species .. 45 Figure 10. Growth of B. thetaiotaomicron VPI-5482 on non-limiting concentrations of various nitrogen sources ....................................................................................... 57 Figure 11. Ammonium concentration of B. thetaiotaomicron VPI-5482 growing on non-limiting concentrations of various nitrogen sources ....................................... 59 Figure 12. Growth of B. thetaiotaomicron VPI-5482 on limiting concentrations of individual and mixed nitrogen sources ..................................................................... 60 vii Figure 13. Growth of B. thetaiotaomicron VPI-5482 on limiting concentrations of mixed nitrogen sources .............................................................................................. 61 Figure 14. Ammonium concentration of B. thetaiotaomicron VPI-5482 on limiting concentrations of individual and mixed nitrogen sources ...................................... 62 Figure 15. Growth parameters of B. thetaiotaomicron VPI-5482 during growth in continuous