TY - JOUR
T1 - Expanded Genetic Codes Create New Mutational Routes to Rifampicin Resistance in Escherichia coli
AU - Hammerling, Michael J.
AU - Gollihar, Jimmy
AU - Mortensen, Catherine
AU - Alnahhas, Razan N.
AU - Ellington, Andrew D.
AU - Barrick, Jeffrey E.
N1 - Funding Information:
Acknowledgments This study was supported by the National Science Foundation BEACON Center for the Study of Evolution in Action (Grant number DBI-0939454); the Defense Advanced Research Projects Agency (Grant number HR0011-15-C0095); and the Welch Foundation (Grant number F-1780).We thank Jared Ellefson for the 5-hydroxytryptophan incorporation plasmid and members of the Barrick and Ellington labs for helpful suggestions and discussion.
Publisher Copyright:
© 2016 The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved.
PY - 2016/8/1
Y1 - 2016/8/1
N2 - Until recently, evolutionary questions surrounding the nature of the genetic code have been mostly limited to the realm of conjecture, modeling, and simulation due to the difficulty of altering this fundamental property of living organisms. Concerted genome and protein engineering efforts now make it possible to experimentally study the impact of alternative genetic codes on the evolution of biological systems. We explored how Escherichia coli strains that incorporate a 21st nonstandard amino acid (nsAA) at the recoded amber (TAG) stop codon evolve resistance to the antibiotic rifampicin. Resistance to rifampicin arises from chromosomal mutations in the β subunit of RNA polymerase (RpoB). We found that a variety of mutations that lead to substitutions of nsAAs in the essential RpoB protein confer robust rifampicin resistance. We interpret these results in a framework in which an expanded code can increase evolvability in two distinct ways: by adding a new letter with unique chemical properties to the protein alphabet and by altering the mutational connectivity of amber-adjacent codons by converting a lethal nonsense mutation into a missense mutation. Finally, we consider the implications of these results for the evolution of alternative genetic codes. In our experiments, reliance on a mutation to a reassigned codon for a vital trait is not required for the long-term maintenance of an expanded genetic code and may even destabilize incorporation of an nsAA, a result that is consistent with the codon capture model of genetic code evolution.
AB - Until recently, evolutionary questions surrounding the nature of the genetic code have been mostly limited to the realm of conjecture, modeling, and simulation due to the difficulty of altering this fundamental property of living organisms. Concerted genome and protein engineering efforts now make it possible to experimentally study the impact of alternative genetic codes on the evolution of biological systems. We explored how Escherichia coli strains that incorporate a 21st nonstandard amino acid (nsAA) at the recoded amber (TAG) stop codon evolve resistance to the antibiotic rifampicin. Resistance to rifampicin arises from chromosomal mutations in the β subunit of RNA polymerase (RpoB). We found that a variety of mutations that lead to substitutions of nsAAs in the essential RpoB protein confer robust rifampicin resistance. We interpret these results in a framework in which an expanded code can increase evolvability in two distinct ways: by adding a new letter with unique chemical properties to the protein alphabet and by altering the mutational connectivity of amber-adjacent codons by converting a lethal nonsense mutation into a missense mutation. Finally, we consider the implications of these results for the evolution of alternative genetic codes. In our experiments, reliance on a mutation to a reassigned codon for a vital trait is not required for the long-term maintenance of an expanded genetic code and may even destabilize incorporation of an nsAA, a result that is consistent with the codon capture model of genetic code evolution.
KW - antibiotic resistance
KW - codon capture
KW - evolvability
KW - sequence space
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U2 - 10.1093/molbev/msw094
DO - 10.1093/molbev/msw094
M3 - Article
C2 - 27189550
AN - SCOPUS:84983001795
SN - 0737-4038
VL - 33
SP - 2054
EP - 2063
JO - Molecular Biology and Evolution
JF - Molecular Biology and Evolution
IS - 8
ER -