ABSTRACTThe isolation of aerobic citrate-utilizingEscherichia coli(Cit+) in long-term evolution experiments (LTEE) has been termed a rare, innovative, presumptive speciation event. We hypothesized that direct selection would rapidly yield the same class ofE. coliCit+mutants and follow the same genetic trajectory: potentiation, actualization, and refinement. This hypothesis was tested with wild-typeE. colistrain B and with K-12 and three K-12 derivatives: anE. coliΔrpoS::kanmutant (impaired for stationary-phase survival), anE. coliΔcitT::kanmutant (deleted for the anaerobic citrate/succinate antiporter), and anE. coliΔdctA::kanmutant (deleted for the aerobic succinate transporter).E. coliunderwent adaptation to aerobic citrate metabolism that was readily and repeatedly achieved using minimal medium supplemented with citrate (M9C), M9C with 0.005% glycerol, or M9C with 0.0025% glucose. Forty-six independentE. coliCit+mutants were isolated from allE. coliderivatives except theE. coliΔcitT::kanmutant. Potentiation/actualization mutations occurred within as few as 12 generations, and refinement mutations occurred within 100 generations. Citrate utilization was confirmed using Simmons, Christensen, and LeMaster Richards citrate media and quantified by mass spectrometry.E. coliCit+mutants grew in clumps and in long incompletely divided chains, a phenotype that was reversible in rich media. Genomic DNA sequencing of fourE. coliCit+mutants revealed the required sequence of mutational events leading to a refined Cit+mutant. These events showed amplifiedcitTanddctAloci followed by DNA rearrangements consistent with promoter capture events forcitT. These mutations were equivalent to the amplification and promoter capture CitT-activating mutations identified in the LTEE.IMPORTANCEE. colicannot use citrate aerobically. Long-term evolution experiments (LTEE) performed by Blount et al. (Z. D. Blount, J. E. Barrick, C. J. Davidson, and R. E. Lenski, Nature 489:513–518, 2012,http://dx.doi.org/10.1038/nature11514) found a single aerobic, citrate-utilizingE. colistrain after 33,000 generations (15 years). This was interpreted as a speciation event. Here we show why it probably was not a speciation event. Using similar media, 46 independent citrate-utilizing mutants were isolated in as few as 12 to 100 generations. Genomic DNA sequencing revealed an amplification of thecitTanddctAloci and DNA rearrangements to capture a promoter to express CitT, aerobically. These are members of the same class of mutations identified by the LTEE. We conclude that the rarity of the LTEE mutant was an artifact of the experimental conditions and not a unique evolutionary event. No new genetic information (novel gene function) evolved.
Molecular cloning and comparative sequence analysis of fungal β-Xylosidases
