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 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.
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/13065009 |
| Date | 01 January 2015 |
| Creators | Nayak, Dipti Dinkar |
| Contributors | Marx, Christopher J |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Rights | open |
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