title: Compensatory evolution in response to a novel RNA polymerase: orthologous replacement of a central network gene.
abstract: A bacteriophage genome was forced to evolve a new system of regulation by replacing its RNA polymerase (RNAP) gene, a central component of the phage developmental pathway, with that of a relative. The experiment used the obligate lytic phage T7 and the RNAP gene of phage T3. T7 RNAP uses 17 phage promoters, which are responsible for all middle and late gene expression, DNA replication, and progeny maturation, but the enzyme has known physical contacts with only 2 other phage proteins. T3 RNAP was supplied in trans by the bacterial host to a T7 genome lacking its own RNAP gene and the phage population was continually propagated on naive bacteria throughout the adaptation. Evolution of the T3 RNAP gene was thereby prevented, and selection was for the evolution of regulatory signals throughout the phage genome. T3 RNAP transcribes from T7 promoters only at low levels, but a single mutation in the promoter confers high expression, providing a ready mechanism for reevolution of gene expression in this system. When selected for rapid growth, fitness of the engineered phage evolved from a low of 5 doublings/h to 33 doublings/h, close to the expected maximum of 37 doublings/h. However, the experiment was terminated before it could be determined accurately that fitness had reached an obvious plateau, and it is not known whether further adaptation could have resulted in complete recovery of fitness. More than 30 mutations were observed in the evolved genome, but changes were found in only 9 of the 16 promoters, and several coding changes occurred in genes with no known contacts with the RNAP. Surprisingly, the T7 genome adapted to T3 RNAP also maintained high fitness when using T7 RNAP, suggesting that the extreme incompatibility of T7 elements with T3 RNAP is not an invariant property of divergence in these expression systems.
authors.affiliation: Section of Integrative Biology, University of Texas, USA. bull@bull.biosci.utexas.edu
authors.forename: J J
authors.initials: JJ
authors.lastname: Bull
authors.forename: R
authors.initials: R
authors.lastname: Springman
authors.forename: I J
authors.initials: IJ
authors.lastname: Molineux
mesh_terms: D017903:Bacteriophage T3 / Q000201:enzymology / Q000235:genetics; D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D019143:Evolution, Molecular; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics
chemical_list: D014764:Viral Proteins; C081591:bacteriophage T3 RNA polymerase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases