Harvard University - Department of Molecular & Cellular Biology

HOW SWEET IT IS (TO BE STUCK BY YOU) [MURRAY LAB]

by John Koschwanez and Andrew Murray

April 2nd, 2013

John Koschwanez (l) and Andrew Murray

How do microbes evolve to utilize nutrients that they can't import? We used engineering and experimental evolution of budding yeast to study this question. Individual yeast cells grow very poorly in low concentrations of sucrose, the main sugar found in the grape juice that becomes wine. Sucrose is made of a molecule of glucose bound to a molecule of fructose and the bond between them must be broken to allow cells to metabolize the sugar. The sucrose is hydrolyzed at the cell wall, which surrounds the cell rather than being inside it, and nearly all of the resulting glucose and fructose diffuses away before being captured. We thought of three different ways yeast could grow in sucrose. First, yeast cells can grow in multicellular clumps, allowing each cell in the clump to capture glucose and fructose diffusing from its neighbors' cell walls. Second, a cell can increase its production of invertase, the enzyme that hydrolyzes sucrose. Finally, a cell can directly import sucrose and then hydrolyze it inside the cell, thus keeping all the glucose and fructose for itself. We engineered all three of these strategies into lab yeast and confirmed that each strategy allowed growth in sucrose.

We then asked, "What would evolution do?" and passaged unicellular, lab yeast in sucrose for about 300 generations. 11 of the 12 clones picked from 10 parallel populations formed multicellular clumps. This confirms our prediction from our earlier work (Koschwanez, Foster, and Murray, 2011) that the sharing of public goods (in this case the glucose and fructose that result from sucrose hydrolysis) would select for simple multicellularity. 10 of 12 clones increased invertase expression, none of the 12 imported sucrose, and 11 of the 12 increased the expression of glucose and fructose importers, a strategy we had not predicted. To find the mutations that had driven the improved growth on sucrose, we needed to separate the mutations that caused the phenotype from hitch-hiking, neutral mutations that had been dragged along for the ride. We backcrossed the evolved strains to the ancestor strain and found 80 putative causal mutations among the 12 clones. To show that these really were the mutations that had driven evolution we then genetically engineered them back into the starting strain. We did this for two of the strains, one with 5 mutations and one with 8 mutations, to confirm the causality of the mutations and ask how much each one affected growth in a variety of different environments. We speculate that microbes have evolved combinations of three strategies, multicellularity, increased enzyme expression, and increased simple nutrient transport, to utilize nutrients they can't import. We also speculate that the sharing of public goods was the initial selection for simple multicellularity in ancient organisms.

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