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MBE Advance Access originally published online on April 9, 2007
Molecular Biology and Evolution 2007 24(7):1480-1491; doi:10.1093/molbev/msm067
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© The Author 2007. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Research Articles

Stress-Driven In Vivo Selection of a Functional Mini-Gene from a Randomized DNA Library Expressing Combinatorial Peptides in Escherichia coli

Victor G. Stepanov and George E. Fox

Department of Biology and Biochemistry, University of Houston

E-mail: fox{at}uh.edu.

Accepted for publication March 26, 2007.

A plasmid-borne randomized mini-gene library expressing a population of combinatorial 20-mer peptides with no bias toward any biological function was used as an initial source of genetic variance during stress-driven evolution of Escherichia coli. The transformed bacteria were evolved under multiple rounds of selective pressure imposed by nearly lethal concentrations of NiCl2, AgNO3, or K2TeO3. At the final stage, clones conferring resistance to NiCl2 were found to carry identical functional mini-genes, which conferred significant nickel tolerance on the host cells. Expression of the mini-gene markedly improved growth parameters of the evolved clones at subinhibitory concentrations of NiCl2 while being slightly detrimental in the absence of the stress. This substantial increase in resistance, as compared with control cultures adapted in the absence of the mini-gene, is shown to be largely due to coadaptation with changes elsewhere in the E. coli genome. Clones resistant to AgNO3 and K2TeO3 harbored plasmid variants with an inactive mini-gene and with a deleted mini-gene operon, respectively. In those cases, an exploration of the mini-gene sequence space apparently was fruitless, and the developed toxicity tolerance was likely to be exclusively associated with acquired adaptive mutations. Overall, the results demonstrate a very natural outcome in which the mini-genes were expected to be either successfully integrated into the bacterial genetic network or rejected depending on their effect on host fitness. This approach is immediately useful as a laboratory model to study the dynamics of bacterial adaptive evolution at the molecular level and is especially relevant as a rapid method to study cellular response to recently acquired genetic material.

Key Words: adaptive evolution • mini-gene • in vivo selection • coadaptation • metal resistance

Dan Graur, Associate Editor


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