Mechanisms causing rapid and parallel losses of ribose catabolism in evolving populations of Escherichia coli B

Abstract

Twelve populations of Escherichia coli B all lost D-ribose catabolic function during 2,000 generations of evolution in glucose minimal medium. We sought to identify the population genetic processes and molecular genetic events that caused these rapid and parallel losses. Seven independent Rbs(-) mutants were isolated, and their competitive fitnesses were measured relative to that of their Rbs(+) progenitor. These Rbs(-) mutants were all about 1 to 2% more fit than the progenitor. A fluctuation test revealed an unusually high rate, about 5 x 10(-5) per cell generation, of mutation from Rbs(+) to Rbs(-), which contributed to rapid fixation. At the molecular level, the loss of ribose catabolic function involved the deletion of part or all of the ribose operon (rbs genes). The physical extent of the deletion varied between mutants, but each deletion was associated with an IS150 element located immediately upstream of the rbs operon. The deletions apparently involved transposition into various locations within the rbs operon; recombination between the new IS150 copy and the one upstream of the rbs operon then led to the deletion of the intervening sequence. To confirm that the beneficial fitness effect was caused by deletion of the rbs operon (and not some undetected mutation elsewhere), we used P1 transduction to restore the functional rbs operon to two Rbs(-) mutants, and we constructed another Rbs(-) strain by gene replacement with a deletion not involving IS150. All three of these new constructs confirmed that Rbs(-) mutants have a competitive advantage relative to their Rbs(+) counterparts in glucose minimal medium. The rapid and parallel evolutionary losses of ribose catabolic function thus involved both (i) an unusually high mutation rate, such that Rbs(-) mutants appeared repeatedly in all populations, and (ii) a selective advantage in glucose minimal medium that drove these mutants to fixation.

FIG. 1

Frequency of Rbs⁻ cells over time in the 12 evolving populations. At generation 2000 all the populations contained between 97 and 100% Rbs⁻ cells.

FIG. 2

Fitnesses of seven spontaneous Rbs⁻ mutants relative to their progenitor, as measured by competition experiments. Error bars show 95% confidence intervals based on fivefold replication for each mutant.

FIG. 3

Losses of d-ribose catabolism in evolving populations caused by deletions in the rbs operon. (A) Map of the rbs operon in E. coli B ancestor, based on the genome sequence for E. coli K-12 (4). Arrows show the directions of gene transcription. An IS150 element is located upstream of the rbs operon. Thick lines indicate the left and right adjacent sequences used as probes. The primers used in PCR experiments (G5, G6, G76, and G77) and for constructing strain GBE127 (G266, G267, G268, and G269) are also shown, as are relevant HincII restriction sites. (B) The deletions in clones isolated from 11 of the 12 populations are shown as horizontal lines. Population Ara+2 is not depicted because an Rbs⁺ minority clone was sampled (see text). The right adjacent sequence of IS150 did not hybridize with any of the Rbs⁻ clones, whereas the left adjacent sequence hybridized in every case with a HincII fragment, whose size is shown in the column labeled Hyb. The sizes of the PCR products obtained from these clones using primers G76 and G77 are given in the column labeled PCR. The sizes, in the ancestral strain, of the hybridizing HincII fragment and PCR product are indicated at the bottom.