The Role of PlsX in Fatty Acid Synthesis and Acid Adaptation in Streptococcus mutans by Benjamin W. Cross Submitted in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Supervised by Professor Robert G. Quivey, Jr. Department of Microbiology and Immunology School of Medicine and Dentistry University of Rochester Rochester, NY 2016 ii Dedication To John Colabrese and Kristen Butela, for teaching me how to be a scientist. To my parents, Michael and Gina Cross, for teaching me how to be. iii Biographical Sketch Benjamin Cross was born on January 18th, 1988 in Weirton, West Virginia. He graduated from Weir High School in 2006 as class Valedictorian. Benjamin attended the University of Pittsburgh from 2006 to 2010, where he was introduced to microbiology research in the lab of Dr. Jeffrey Lawrence. He graduated from the University of Pittsburgh with a Bachelor of Science degree in Microbiology, a minor in Chemistry, and a Certificate in the Conceptual Foundations of Medicine. In the Fall of 2010 Benjamin began graduate studies in Microbiology at the University of Rochester School of Medicine and Dentistry. Benjamin began his dissertation research under the direction of Dr. Robert G. Quivey, Jr. in the Spring of 2011. List of Publications Cross B, Garcia A, Faustoferri RC, Quivey RG, Jr. 2016. PlsX deletion impacts fatty acid synthesis and acid adaptation in Streptococcus mutans. Microbiology. Published ahead of print; DOI: 10.1099/mic.0.000252. Cross B, Faustoferri RC, Quivey RG Jr. 2016. What Are We Learning and What Can We Learn from the Human Oral Microbiome Project? Curr Oral Health Rep 3:56–63. Faustoferri RC, Santiago B, Baker J, Cross B, Xiao J, and Quivey RG, Jr. Acid- adaptive responses of S. mutans and mechanisms of integration with oxidative stress. de Bruijn, F. (ed), Stress and Environmental Control of Gene Expression in Bacteria, 1st Edition. Wiley-Blackwell Publishers. (in press) iv Acknowledgements My sincere thanks to the entire Quivey Lab, past and present, with special thanks to Brendaliz Santiago, Jon Baker, Andrew Buckley, Matt MacGilvray, Adam Derr, Kaisha Gonzalez, Ariana Garcia, Roberta Faustoferri, and Robert Quivey. I am grateful for the help and guidance of Mark Dumont, Steven Gill, Jose Lemos, and Marty Pavelka. I would like to thank my friends and family for their support, especially Alia Souissi, for her patience throughout the writing of this document. v Abstract Streptococcus mutans is one of the primary causative agents of dental caries in humans. S. mutans ferments dietary sugars in the mouth to produce organic acids. These acids lower local pH values resulting in demineralization of the tooth enamel, leading to caries. To survive acidic environments, S. mutans employs several adaptive mechanisms, including a shift from saturated to unsaturated fatty acids in membrane phospholipids. Evidence suggests that this shift requires de novo fatty acid and phospholipid synthesis; therefore, understanding these synthesis pathways is crucial for understanding how S. mutans adapts to low pH and causes caries. PlsX is an acyl- ACP:phosphate transacylase that links the fatty acid synthesis pathway to the phospholipid synthesis pathway, and is central to the movement of unsaturated fatty acids into the membrane. It has recently been discovered that plsX is not essential in S. mutans. This study explores how the loss of plsX affects the ability of S. mutans to alter its membrane fatty acid profile and survive at low pH. The plsX deletion mutant (∆plsX) is not a fatty acid or phospholipid auxotroph, indicating that some alternative pathway is capable of carrying out the first step of phospholipid synthesis. Gas chromatography of fatty acid methyl esters (GC-FAME) indicates that deletion of plsX impacts the regulation of fatty acid synthesis, altering the length and saturation of fatty acids. Surprisingly, ∆plsX survives significantly longer than the parent strain, UA159, when subjected to an acid challenge of pH 2.5. This enhanced survival may be due to the increased F-ATPase activity observed at low pH. This enhanced F-ATPase activity may be due to the altered fatty acid profile, or may be vi part of a response to membrane stress. Supplementing ∆plsX with exogenous unsaturated fatty acids does not restore any wild-type phenotypes; however, incorporation of exogenous fatty acids is 2-fold greater in ∆plsX, compared to UA159. Exogenous oleic acid was observed to decrease survival in acid challenge for both ∆plsX and UA159. These results clearly indicate that the loss of plsX affects both the fatty acid synthesis pathway and the acid-adaptive response of S. mutans. vii Contributors and Funding Sources This research was supported by the Training Program in Oral Sciences, T90 DE021985-05 (B. C.), as well as DE013683, DE017425. and DE017157 (R.G.Q.), all granted by the National Institute of Dental and Craniofacial Research. This work was supervised by a dissertation committee consisting of Professors Robert Quivey, Martin Pavelka, Jose Lemos, and Steven Gill of the Department of Microbiology and Immunology, and Professor Mark Dumont of the Department of Biochemistry and Biophysics. All of the experiments in this document were performed by the author except: Chapter 4, Figures 4.5, 4.7, and 4.8 were performed with assistance from Ariana Garcia. Chapter 5, Figures 5.4 and 5.5 were performed with assistance from Ariana Garcia. Chapter 5, Figures 5.2 and 5.3 were performed with assistance from Benjamin Metcalf. Chapter 5, Figure 5.6 was performed by Benjamin Metcalf and Catlyn Blanchard. viii Table of Contents Dedication ii   Biographical Sketch iii   Acknowledgements iv   Abstract v   Contributors and Funding Sources vii   List of Tables xi   List of Figures xii Chapter 1: Introduction 1   1.1 Streptococcus mutans and dental caries 2   1.2 The metabolic basis of Streptococcus mutans virulence 4   1.3 The role of fatty acid synthesis in acid adaptation 7   1.4 A possible connection between phospholipid synthesis and acid adaptation 10   1.5 Hypothesis and rationale 12 Chapter 2: Materials and Methods 16   2.1 Bacterial Strains 17   2.2 Growth conditions 18   2.3 Multiple sequence alignment of PlsX proteins 20   2.4 PCR confirmation of plsX deletion 20   2.5 Quantitative real-time PCR 20 ix 2.6 Bioscreen Growth Curves 21   2.7 Transformation Efficiency 22   2.8 Gas Chromatography of Fatty Acid Methyl Esters (GC-FAME) 23   2.9 Chloramphenicol acetyltransferase (CAT) assays 23   2.10 Growth media with exogenous fatty acids 25   2.11 Acid Challenge Experiments 25   2.12 Hydrogen Peroxide Challenge 26   2.13 ATPase Activity Assay 26   2.14 Proton Permeability 27   Chapter 3: PlsX is not Essential to S. mutans 32   3.1 Abstract 33   3.2 Introduction 34   3.3 Results 37   Confirming the deletion and complementation of plsX 37   Testing ΔplsX for auxotrophy 44   Preliminary investigation of the alternative acyl-phosphate pathway 49   3.4 Discussion 54   Chapter 4: PlsX and Fatty Acid Synthesis in S. mutans 57   4.1 Abstract 58   4.2 Introduction 59 x 4.3 Results 65   Fatty acid profile of the ΔplsX strain 65   Deletion of plsX causes no transcription changes in functionally-related genes 75   Elevated Fatty Acid Uptake in the ∆plsX strain 80   4.4 Discussion 92   Chapter 5: PlsX and Acid Adaptation in S. mutans 97   5.1 Abstract 98   5.2 Introduction 99   5.3 Results 102   Acid and peroxide challenge of ΔplsX 102   F F -ATPase activity is altered in ΔplsX 116   1 0 ∆plsX membrane integrity 119   5.4 Discussion 124 Chapter 6: Conclusions and Future Directions 131   6.1 PlsX is not essential to S. mutans 132   6.2 PlsX has a role in the regulation of fatty acid synthesis 134   6.3 PlsX has a complex effect on acid adaptation 137   6.4 Clinical Significance 140 References 144