The consequences of growth of a mutator strain of Escherichia coli as measured by loss of function among multiple gene targets and loss of fitness

We have examined the composition of members of mutator populations of Esche richia coli by employing an extensive set of phenotypic screens that allow us to monitor die function of >700 genes, constituting similar to 15% of th e genome. We looked at mismatch repair deficient cells after repeated cycle s of sing le colony isolation on rich medium to generate lineages that are forced through severe bottlenecks, and compared the results to those for wi ld-type strains. The mutator lineages continued to accumulate mutations rap idly with each increasing cycle of colony isolation. By die end of the 40th cycle, after similar to 1000 generations, most of the lineages had reduced colony size, 4% had died out, 55% had auxotrophic requirements (increasing to 80% after 60 cycles), and 70% had defects in at least one sugar or cata bolic pathway. In addition, 33% had a defect in cell motility, and 26% were either temperature-sensitive or cold-sensitive lethals. On the other hand, only 3% of die wild-type lineages had detectable mutations of any type aft er 40 cycles. By the 60th cycle, the typical mutator cell carried 4-5 inact ive genes among the 15% of the genome being monitored, indicating that the average cell carried at least 24-30 inactivated genes distributed throughou t the genome. Remarkably, 30% of the lineages had lost title ability to uti lize xylose as a carbon source. DNA sequencing revealed that most of the Xy l(-) mutants had a frameshift in a run of eight G's (GGGGGGGG) in the xylB gene, either adding or deleting one -G-. Further analysis indicated that re ndering E. coli deficient in mismatch repair unmasks hypermutable sites in certain genes or intergenic regions. Growth curves and competition tests on lineages that passed through 90 cycles of single colony isolation slowed t hat all lineages suffered reduced fitness. We discuss these results in term s of the value of mutators in cellular evolution.