Physiology and Evolution of Methylamine Metabolism across Methylobacterium extorquens strains The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Nayak, Dipti Dinkar. 2014. Physiology and Evolution of Methylamine Metabolism across Methylobacterium extorquens strains. Doctoral dissertation, Harvard University. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:13065009 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Physiology and evolution of methylamine metabolism across Methylobacterium extorquens strains A dissertation presented by Dipti Dinkar Nayak to The Department of Organismic and Evolutionary Biology in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Biology Harvard University Cambridge, Massachusetts August 2014 ©2014 - Dipti Dinkar Nayak All rights reserved. Dissertation Advisor: Professor Christopher J. Marx Dipti Dinkar Nayak Physiology and evolution of methylamine metabolism across Methylobacterium extorquens strains Abstract The interplay between physiology and evolution in microorganisms is extremely relevant from the stand-point of human health, the environment, and biotechnology; yet microbial physiology and microbial evolution largely continue to grow as disjoint fields of research. The goal of this dissertation was to use experimental evolution to study methylamine metabolism in Methylobacterium extorquens species. Methylotrophs like the M. extorquens species grow on reduced single carbon compounds and are the largest biological sink for methane. M. extorquens AM1, the model system for the study of aerobic methylotrophy, has an unstable genome and severe growth defects as a result of laboratory domestication. First, I describe the genomic, genetic, and phenotypic characterization of a new model system for the study of aerobic methylotrophy: M. extorquens PA1. This strain has a stable genome, was recently isolated from a known ecological niche, and is closely related to AM1. Whereas PA1 grew 10-50% faster than AM1on most substrates, it was five-fold slower on methylamine. The PA1 genome encodes a poorly characterized but ecologically relevant N-methylglutamate pathway whereas AM1 also encodes the well- characterized methylamine dehydrogenase for methylamine oxidation. I characterized the genetics of the N-methylglutamate pathway in PA1 to resolve a linear topology that requires the formation of two, unique amino acid intermediates during methylamine oxidation. I also showed that methylamine metabolism via the N-methylglutamate pathway routes carbon flux in a manner completely different from previous instances of methylotrophy. Next, I evolved replicate populations of PA1 on methylamine for 150 generations. Based on the empirical heuristic that the initial fitness is negatively correlated to the rate of adaptation, it was expected that the fitness gain would be rapid. However, methylamine fitness did not improve at all; adaptive constraints led to evolutionary recalcitrance despite low initial fitness. These adaptive constraints were alleviated by the horizontal gene transfer of an alternate, functionally degenerate metabolic module. Finally, I uncovered ecologically distinct roles for two functionally iii degenerate routes for methylamine oxidation pathways in the AM1 genome; the highly expressed, efficient route is primarily used for growth and the tightly regulated, energetically expensive route is used for assimilating nitrogen in methylamine-limiting environments. iv Table of Contents Acknowledgements……………………………………………………………………………...vi Chapter 1…………………………………………………………………………………………1 Introduction Chapter 2……………………………………………………………………………………..…17 Genetic and phenotypic characterization of methylotrophy in Methylobacterium extorquens strains PA1 and AM1 Chapter 3……………………………………………………………………………………..…42 Methylamine utilization via a linear N-methylglutamate pathway in Methylobacterium extorquens PA1 does not require the tetrahydrofolate dependent C1 transfer pathway Chapter 4………………………………………………………………………………………..73 Horizontal Gene Transfer overcomes the adaptive constraints posed by a sub-optimal pathway for methylamine utilization in Methylobacterium extorquens PA1 Chapter 5………………………………………………………………………………………..94 Experimental evolution reveals ecologically distinct roles for two functionally degenerate routes for methylamine oxidation in Methylobacterium extorquens AM1 Appendix 1……………………………………………………………………………………..126 Supplementary Material for Chapter 2 Appendix 2……………………………………………………………………………………..135 Supplementary Material for Chapter 3 Appendix 3……………………………………………………………………………………..142 Supplementary Material for Chapter 5 v Acknowledgements This dissertation, all the steps leading up to it and beyond, would not be possible without the support of friends, mentors, and family. First, I owe a debt of gratitude to Chris for welcoming me into his research group; for giving me the intellectual freedom to pursue a completely new research thread; and for the words of encouragement at opportune moments. Chris’ razor sharp intellect and scientific creativity combined with an addictive enthusiasm for research is awe-inspiring. He has been an outstanding mentor over the years and it was a joy to be a graduate student in the Marx lab. I would also like to specifically thank a few members of the Marx lab; Deepa Agashe and Will Harcombe for long and meandering yet extremely illuminating scientific discussions and Sean Carroll for being a sounding board on all matters C1 related and for working as a partner-in-crime on the betaine project. A big thank you to everyone else in the Marx lab for reading manuscripts, patiently sitting through and critiquing practice talks, sharing technical knowhow, and being awesome! Colleen Cavanaugh deserves a special mention. The first time I met her, we spent three hours animatedly discussing the wonderful, curious world of methylotrophy. I admire her passion for microbiology and really appreciate the regular doses of positive reinforcement from her end; those kind words have meant a lot. I am grateful to all the faculty members (Martin Polz, Graham Walker, Peter Girguis, and especially Michael Desai) who have taken the time to read this dissertation, my thesis proposal, and served as a part of my thesis committee over the last few years; their feedback, support and encouragement was invaluable. I’d also like to thank my collaborators, Yousif Shamoo and Milya Davileva at Rice University and Marty Ytreberg at University of Idaho for their time and energy; I’ve learnt a lot from these collaborations. A big thank you to Chris Preheim for running the OEB graduate program like a well-oiled machine, as well as to Nikki Hughes, Krista Carmichael, and Becky Chetham for administrative support through the years. vi Heather, Roxie, Kiana, Liz, Didem and Laura, having all of you around made grad school so much fun. Thank you all being such wonderfully quirky people and for the support and laughter through these past five years. Aditi and Nithya, thank you for your friendship through the years, and for travelling far and wide to spend time together. I cannot even fathom what I’d be doing now if wasn’t for the sage advice that the two of you have given me over the last fifteen years. I am grateful to have extremely kind and encouraging in-laws, and a wonderful, caring family. I will always be indebted to my maternal grandparents for being such an integral part of my childhood and for defying gender stereotypes; she is one of the most progressive, fearless, and independent women I’ve met and he is the most gentle, patient soul I know. I am so glad to have Mahesh as a sibling; I’ve always admired his quick witted humor and cheerful disposition and am extremely proud of his accomplishments. My husband Aditya is an inspiration. It has and will always be reassuring to have his dazzling intellect, pragmatic perspective, distinct sense of humor, and unconditional love at my side; I, too, look forward to our journey together. Finally, I am so fortunate to have two brilliant, forward-thinking individuals, Suman and Dinkar, as parents; they are the sole reason I have made it this far. vii Chapter 1 Introduction “Most basically, natural selection converts accident into apparent design, randomness into organized pattern”- Julian Huxley, Evolution in Action. The interplay between physiology and evolution is, arguably, the single-most important factor that governs the diverse metabolic and other physiological features observed in microorganisms (Falkowski et al. 2008; T. Ferenci 2007). More than a century of research in microbial physiology has resulted in sophisticated tools for the genetic manipulation of various bacteria (Holloway et al,. 1979; Simon et al. 1983; Sonenshein et al. 1993; Hopwood 1999; Marx and Lidstrom 2002) and archaea (Pritchett et al. 2004; Allers and Mevarech 2005; Wagner et al. 2009); and the discovery and characterization of thousands of enzymes and proteins involved in metabolism, transport, cell division, virulence, and many other traits (Tatusov et al. 1997; Marakova et al. 1999; Bateman et al. 2000; Haft et al. 2003). However, one-third of the genes in E. coli K12 - conceivably, the best characterized bacterial strain - remain functionally unannotated