Our understanding of the origins of new metabolic functions is based upon anecdotal genetic and biochemical evidence. Some auxotrophies can be suppressed by overexpressing substrate-ambiguous enzymes (i.e., those that catalyze the same chemical transformation on different substrates). Other enzymes exhibit weak but detectable catalytic promiscuity in vitro (i.e., they catalyze different transformations on similar substrates). Cells adapt to novel environments through the evolution of these secondary activities, but neither their chemical natures nor their frequencies of occurrence have been characterized en bloc. Here, we systematically identified multifunctional genes within the Escherichia coli genome. We screened 104 single-gene knockout strains and discovered that many (20%) of these auxotrophs were rescued by the overexpression of at least one noncognate E. coli gene. The deleted gene and its suppressor were generally unrelated, suggesting that promiscuity is a product of contingency. This genome-wide survey demonstrates that multifunctional genes are common and illustrates the mechanistic diversity by which their products enhance metabolic robustness and evolvability.
Evolutionary innovations that enable organisms to colonize new ecological niches are rare compared to gradual evolutionary changes in existing traits. We discovered that key mutations in the gltA gene, which encodes citrate synthase (CS), occurred both before and after Escherichia coli gained the ability to grow aerobically on citrate (Cit+ phenotype) during the Lenski long-term evolution experiment. The first gltA mutation, which increases CS activity by disrupting NADH-inhibition of this enzyme, is beneficial for growth on the acetate and contributed to preserving the rudimentary Cit+ trait from extinction when it first evolved. However, after Cit+ was refined by further mutations, this potentiating gltA mutation became deleterious to fitness. A second wave of beneficial gltA mutations then evolved that reduced CS activity to below the ancestral level. Thus, dynamic reorganization of central metabolism made colonizing this new nutrient niche contingent on both co-opting and overcoming a history of prior adaptation.DOI:
http://dx.doi.org/10.7554/eLife.09696.001
Evolutionary innovations often arise from complex genetic and ecological interactions, which can make it challenging to understand retrospectively how a novel trait arose. In a long-term experiment, Escherichia coli gained the ability to use abundant citrate (Cit + ) in the growth medium after ∼31,500 generations of evolution. Exploiting this previously untapped resource was highly beneficial: later Cit + variants achieve a much higher population density in this environment. All Cit + individuals share a mutation that activates aerobic expression of the citT citrate transporter, but this mutation confers only an extremely weak Cit + phenotype on its own. To determine which of the other >70 mutations in early Cit + clones were needed to take full advantage of citrate, we developed a recursive genomewide recombination and sequencing method (REGRES) and performed genetic backcrosses to purge mutations not required for Cit + from an evolved strain. We discovered a mutation that increased expression of the dctA C 4 -dicarboxylate transporter greatly enhanced the Cit + phenotype after it evolved.
A general bacterial genome engineering framework, ‘Genome Editing via Targetrons and Recombinases' (GETR), is presented. GETR combines mobile group II introns (targetrons) and the Cre/lox system to allow genomic manipulations at a large scale.
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