University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 12-2018 Investigating the Role of Carotenoids in Membrane Organization of Pantoea sp. YR343 Sushmitha Vijaya Kumar University of Tennessee, [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Recommended Citation Vijaya Kumar, Sushmitha, "Investigating the Role of Carotenoids in Membrane Organization of Pantoea sp. YR343. " PhD diss., University of Tennessee, 2018. https://trace.tennessee.edu/utk_graddiss/5304 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Sushmitha Vijaya Kumar entitled "Investigating the Role of Carotenoids in Membrane Organization of Pantoea sp. YR343." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Life Sciences. Jennifer Morrell-Falvey, Major Professor We have read this dissertation and recommend its acceptance: Gladys Alexandre, Francisco Barrera, Dale Pelletier, Robert Standaert Accepted for the Council: Dixie L. Thompson Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) Investigating the Role of Carotenoids in Membrane Organization of Pantoea sp. YR343 A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Sushmitha Vijaya Kumar December 2018 DEDICATION To my family for their unconditional love and support And To Siva Karthik, you are my rock ii ACKNOWLEDGEMENTS There have been many people who have walked alongside me for the past few years for whom I am grateful for. First and foremost, I would like to thank my advisor Dr. Jennifer Morrell-Falvey for her continued support, guidance and patience. I am grateful for the freedom she provided me, that helped me become a better researcher. All in all, I am lucky to have you as my mentor. I also would like to thank my committee members, Dr. Dale Pelletier, Dr. Gladys Alexandre, Dr. Robert Standaert and Dr. Francisco Barrera for your valuable suggestions and support. I am much obliged to Dr. Mitchel Doktycz, Dr. Graham Taylor and Dr. Dave Alison for their continued support in the lab. Dr. Amber Bible, thank you for being an awesome mentor and for all the scientific discussions. It has been so much fun working alongside you and I truly admire your work ethics. Many thanks to Cathy for being an awesome secretary. To all my lab members at ORNL, I have thoroughly enjoyed working alongside you. This work would not be completed without friends like you. I would like to thank Dr. Albrecht von Arnim for recruiting me and also for the valuable advice along the way. To Terrie Yeatts, for all the help. Many thanks to everyone in the GST program. To my doctors, for being very accommodating and helping me finish this journey strong. To my loving and supportive family, my parents, Latha and A. R. Vijaya Kumar, thanks for letting me chase my dreams and being so patient. Many thanks to my sister Samyuktha and brother-in law Srikant, for supporting me at every step of the way. To my handsome nephew Reyansh, who thinks greatly of his aunt. Finally, to my best friend Karthik, words cannot describe how instrumental you have been in this journey of mine. Your kindness, patience and continued love has helped enjoy this journey. I am so grateful for you. iii ABSTRACT Bacterial cell membranes are complex mixtures of lipids and proteins, the combination of which confers biophysical properties to the membrane and allows it to respond to environmental conditions. Pantoea sp. YR343 is a plant-associated gram-negative gamma-proteobacteria characterized by the presence of carotenoids in the membrane. Pantoea sp. YR343 mutants lacking phytoene synthase (encoded by crtB), which catalyzes the first step in carotenoid biosynthesis, failed to produce carotenoids and displayed enhanced sensitivity to reactive oxygen species. The crtB mutant also displayed unexpected defects in biofilm formation, secretion of indole-3-acetic acid, and root colonization compared to wildtype cells. We hypothesized that the phenotypic defects observed in the crtB mutant were due to the changes in both lipid and protein composition which modulate the biophysical properties of the membrane and influence signaling and transport. Mass spectrometry analysis of the lipid head and tails revealed a significant abundance of phosphotidylethanolamine and unsaturated fatty acids in the crtB mutant cells. Using both fluorescence anisotropy (FA) and atomic force microscopy on whole cells, we showed that the crtB mutant membranes were less fluid when compared to wildtype membranes. We further characterized the membranes using spheroplasts and natural extract vesicles, which showed that presence of the outer wall does not impact membrane fluidity, but the presence of membrane proteins and carotenoids do have a strong impact. Proteomic profiling of wildtype and crtB mutant membranes revealed a significant loss of several membrane protein classes in the crtB mutant. Among the different classes of proteins, signaling and transport, outer membrane biogenesis and cell motility protein classes were the most affected in the mutant. The loss of specific protein classes may explain, at least in part, the phenotypic defects associated with the crtB mutant. Our study highlights the importance of carotenoids beyond its anti-oxidant potential. Loss of bacterial carotenoids changes membrane organization and dynamics by affecting the composition and distribution of both lipids and membrane proteins. Importantly, carotenoids regulate membrane fluidity, which is an important parameter for bacterial adaptation and survival. iv TABLE OF CONTENTS CHAPTER 1 Background and Overview 1 The Lipid Bilayer 3 Distribution and role of sterols in cell membranes 4 Membrane phase transition 5 Membrane organization and lipid rafts 5 Membrane fluidity 7 Carotenoids- properties and functions 8 Role of carotenoids in membrane fluidity 9 Plant-microbe interaction 10 Pantoea sp. YR343 11 ΔcrtB, a carotenoid deficient strain of Pantoea sp. YR343 11 References 14 Appendix 22 CHAPTER 2 Deletion of carotenoids from membranes of Pantoea sp. YR343 results in altered lipid composition and changes in membrane biophysical properties 27 Abstract 28 Introduction 28 Results 30 Discussion 33 Materials and methods 35 References 40 Appendix 45 CHAPTER 3 Loss of carotenoids leads to changes in membrane protein classes in Pantoea sp. YR343 56 Abstract 57 Introduction 57 Results and Discussion 59 Materials and methods 68 References 73 Appendix 79 CHAPTER 4 Conclusions and Future Directions 101 References 105 APPENDIX 106 Appendix-A 107 References 112 Appendix-B 113 VITA 138 v LIST OF FIGURES Figure 1.1 Schematic representation of biological membranes with a heterogenous distribution of phospholipids and proteins 23 Figure 1.2. A simplified model of lipid rafts in eukaryotic membranes 24 Figure 1.3. Biosynthesis and functions of carotenoids 25 Figure 1.4 Phenotypic defects associated with ΔcrtB mutant 26 Figure 2.1 crtB deletion causes changes in membrane lipid head group composition 46 Figure 2.2 Significant decrease in % abundance of unsaturated fatty acids (C16:1 and C18:1) in the ΔcrtB mutant cells 48 Figure 2.3 Representative AFM deflection images of Pantoea sp. YR343 and ∆crtB cells at 28°C and 45°C 50 Figure 2.4 Atomic force microscopy (AFM) reveals less elastic ΔcrtB mutant cell 51 Figure 2.5 Fluorescence anisotropy with DPH indicates a rigid ∆crtB mutant membrane 52 Figure 2.6 Removal of outer membrane reveals no difference in fluorescence polarization between Pantoea sp. YR343 and ∆crtB mutant 53 spheroplast Figure 2.7 Properties of extruded vesicles of Pantoea sp. YR343 and ∆crtB mutant 54 Figure 2.8 Fluorescence anisotropy with DPH on natural extract vesicles indicates a fluid ∆crtB mutant membrane 55 Figure 3.1 Identification and analysis of the proteins identified in whole cell, membrane pellet, DRM and DSM fractions of Pantoea sp. YR343 80 and ΔcrtB mutant cells Figure 3.2 Histogram representing the % of proteins with predicted transmembrane helix domains (THMM) for each sample 81 Figure 3.3 Hierarchical clustering of all proteins identified in all four fractions of Pantoea sp. YR343 and ΔcrtB mutant 82 Figure 3.4 Volcano plot illustrating significantly differentially abundant 83 proteins Figure 3.5 Top orthologous groups for all significant proteins in whole cell, membrane pellet, DRM and DSM samples 88 Figure 3.6 Clustered heatmap of gene expression in Pantoea sp. YR343 and ΔcrtB mutant 89 Figure 3.7 Loss of carotenoids affects bacterial cell motility 93 vi LIST OF TABLES Table 2.1 Normalized µm concentration of all lipid head groups identified 47 by lipidomics Table 2.2 crtB deletion causes changes in membrane fatty acid 49 composition Table 3.1 Whole-genome gene ontology (GO) term annotation using 84 Blast2GO software Table 3.2 List of significantly upregulated or downregulated proteins 90 involved in cell wall/ membrane/ envelope biogenesis Table 3.3 List of significantly