title: Evolution of phage with chemically ambiguous proteomes.
abstract: BACKGROUND
  The widespread introduction of amino acid substitutions into organismal proteomes has occurred during natural evolution, but has been difficult to achieve by directed evolution. The adaptation of the translation apparatus represents one barrier, but the multiple mutations that may be required throughout a proteome in order to accommodate an alternative amino acid or analogue is an even more daunting problem. The evolution of a small bacteriophage proteome to accommodate an unnatural amino acid analogue can provide insights into the number and type of substitutions that individual proteins will require to retain functionality.
  
  
  RESULTS
  The bacteriophage Qbeta initially grows poorly in the presence of the amino acid analogue 6-fluorotryptophan. After 25 serial passages, the fitness of the phage on the analogue was substantially increased; there was no loss of fitness when the evolved phage were passaged in the presence of tryptophan. Seven mutations were fixed throughout the phage in two independent lines of descent. None of the mutations changed a tryptophan residue.
  
  
  CONCLUSIONS
  A relatively small number of mutations allowed an unnatural amino acid to be functionally incorporated into a highly interdependent set of proteins. These results support the 'ambiguous intermediate' hypothesis for the emergence of divergent genetic codes, in which the adoption of a new genetic code is preceded by the evolution of proteins that can simultaneously accommodate more than one amino acid at a given codon. It may now be possible to direct the evolution of organisms with novel genetic codes using methods that promote ambiguous intermediates.
authors.affiliation: Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA. jbacher@scripps.edu
authors.forename: Jamie M
authors.initials: JM
authors.lastname: Bacher
authors.forename: James J
authors.initials: JJ
authors.lastname: Bull
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Ellington
mesh_terms: D017908:Allolevivirus / Q000235:genetics; D019943:Amino Acid Substitution; D001435:Bacteriophages / Q000235:genetics*; D019020:Directed Molecular Evolution / Q000379:methods*; D016679:Genome, Viral; D008969:Molecular Sequence Data; D009154:Mutation; D020543:Proteome / Q000737:chemistry / Q000235:genetics*; D014364:Tryptophan / Q000031:analogs & derivatives; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics*
chemical_list: D020543:Proteome; D014764:Viral Proteins; D014364:Tryptophan