How do new metabolic capacities arise and what environmental conditions favor ecological specialists vs. generalists? A key aspect in the origin and maintenance of diverse metabolisms is fitness tradeoffs. Tradeoffs are proposed to occur either due to some mutations providing a benefit under one set of conditions but leading to decreased fitness in other situations (antagonistic pleiotropy), or through passive loss of non-utilized functions due to mutation accumulation. The balance of specialization versus generalization rests upon both environmental factors and an organism’s underlying ‘genetic architecture’.
Even though the catabolism of various sugars by Escherichia coli only differs in a few initial steps upstream of glycolysis, experimental evolution on particular saccharides resulted in reduced fitness on some alternative sugars. The potential for tradeoffs should be even greater between fundamentally different metabolic modes.
Approximately 100 genes are specifically required for methylotrophy, and central metabolic fluxes are fundamentally different during C1 vs. multi-carbon growth. These characteristics suggest significant fitness tradeoffs should exist between C1 and multi-carbon growth. Consistent with this suggestion, methylotrophs typically have a restricted diet of one or more C1 compounds and few, if any, multicarbon compounds. This is in contrast to the typically broad substrate range exhibited by the nonmethylotrophic sister taxa to given methylotrophs. In the case of Methylobacterium, which can only utilize a limited number of C2 - C4 organic acids, closely related rhizobia species can chomp-up dozens, if not hundreds of different multicarbon compounds.
Four evolution regimes have been initiated (8 replicates for each, diluted 64-fold every 48 hours for 1000+ generations per year) to examine the fitness tradeoffs associated with specialized metabolism using the facultative methylotroph Methylobacterium:
- Specialization – Cultures are propagated on either methanol (C1), or succinate (C4), each of which provides an equivalent maximal growth rate (~4 hrs). Specialization is predicted to result in decreased fitness on alternative substrates, likely due to antagonistic pleiotropy.
- Adaptation to ecological complexity – Cultures will be either alternated between methanol and succinate (temporally variable) or propagated on a combination of methanol and succinate (mixed substrate). Theory predicts that the tradeoffs of the temporally variable environment will result in dominance of generalists with increased fitness under both conditions, but less fit in either than the single-resource specialists. In contrast, the mixed substrate treatment is predicted to result in increased ecological complexity, such as coexisting single-nutrient specialists.
Analysis of the initial phenotypic changes through time and across replicates is underway (they are at 900+ generations as of 11/04). Two types of data are being collected: fitness relative to the ancestor in multiple environments (determined by head-to-head competition) and key discernable physiological parameters (maximal growth rate, lag time, catabolic breadth, cell size, etc). The initial outcome of these efforts will be the ability to assess:
- The extent of optimization and degree of parallelism between replicates.
- Whether fitness tradeoffs occur and if they are consistent with antagonistic pleiotropy or mutation accumulation.
- The relationship between ecological complexity and within-population diversity.
These projects will be extended by exploring the physiological basis for the improved fitness, and moving toward identification of the underlying beneficial mutations. Identification of thebeneficial mutations enables further projects revolving around evolutionary questions:
- Do parallel phenotypic changes have the same genetic basis?
- Is epistasis observed amongst beneficial mutations, and is it antagonistic or synergistic?
- Do genome arrangements play a significant role in adaptation?
Additionally, pursuing the physiological basis of fitness improvements may lead to the discovery of novel information regarding methylotrophy itself. With E. coli, nearly all beneficial mutations that have been identified have occurred in previously known genes. Since much less is known for Methylobacterium, however, particularly regarding the regulation of methylotrophy, this approach may lead to a significant amount of novel information regarding methylotrophic metabolism.