title: Low-mutation-rate, reduced-genome Escherichia coli: an improved host for faithful maintenance of engineered genetic constructs.
abstract: BACKGROUND
  Molecular mechanisms generating genetic variation provide the basis for evolution and long-term survival of a population in a changing environment. In stable, laboratory conditions, the variation-generating mechanisms are dispensable, as there is limited need for the cell to adapt to adverse conditions. In fact, newly emerging, evolved features might be undesirable when working on highly refined, precise molecular and synthetic biological tasks.
  
  
  RESULTS
  By constructing low-mutation-rate variants, we reduced the evolutionary capacity of MDS42, a reduced-genome E. coli strain engineered to lack most genes irrelevant for laboratory/industrial applications. Elimination of diversity-generating, error-prone DNA polymerase enzymes involved in induced mutagenesis achieved a significant stabilization of the genome. The resulting strain, while retaining normal growth, showed a significant decrease in overall mutation rates, most notably under various stress conditions. Moreover, the error-prone polymerase-free host allowed relatively stable maintenance of a toxic methyltransferase-expressing clone. In contrast, the parental strain produced mutant clones, unable to produce functional methyltransferase, which quickly overgrew the culture to a high ratio (50% of clones in a 24-h induction period lacked functional methyltransferase activity). The surprisingly large stability-difference observed between the strains was due to the combined effects of high stress-induced mutagenesis in the parental strain, growth inhibition by expression of the toxic protein, and selection/outgrowth of mutants no longer producing an active, toxic enzyme.
  
  
  CONCLUSIONS
  By eliminating stress-inducible error-prone DNA-polymerases, the genome of the mobile genetic element-free E. coli strain MDS42 was further stabilized. The resulting strain represents an improved host in various synthetic and molecular biological applications, allowing more stable production of growth-inhibiting biomolecules.
authors.affiliation: Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, 62 Temesvári krt, H6726 Szeged, Hungary.
authors.forename: Bálint
authors.initials: B
authors.lastname: Csörgo
authors.forename: Tamás
authors.initials: T
authors.lastname: Fehér
authors.forename: Edit
authors.initials: E
authors.lastname: Tímár
authors.forename: Frederick R
authors.initials: FR
authors.lastname: Blattner
authors.forename: György
authors.initials: G
authors.lastname: Pósfai
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D005818:Genetic Engineering; D016680:Genome, Bacterial; D059645:Mutation Rate; D010957:Plasmids; D013014:SOS Response, Genetics; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D004259:DNA-Directed DNA Polymerase