Whole-genome resequencing of Escherichia coli K-12 MG1655 undergoing short-term laboratory evolution in lactate minimal media reveals flexible selection of adaptive mutations

Abstract

Background: Short-term laboratory evolution of bacteria followed by genomic sequencing provides insight into the mechanism of adaptive evolution, such as the number of mutations needed for adaptation, genotype-phenotype relationships, and the reproducibility of adaptive outcomes.

Results: In the present study, we describe the genome sequencing of 11 endpoints of Escherichia coli that underwent 60-day laboratory adaptive evolution under growth rate selection pressure in lactate minimal media. Two to eight mutations were identified per endpoint. Generally, each endpoint acquired mutations to different genes. The most notable exception was an 82 base-pair deletion in the rph-pyrE operon that appeared in 7 of the 11 adapted strains. This mutation conferred an approximately 15% increase to the growth rate when experimentally introduced to the wild-type background and resulted in an approximately 30% increase to growth rate when introduced to a background already harboring two adaptive mutations. Additionally, most endpoints had a mutation in a regulatory gene (crp or relA, for example) or the RNA polymerase.

Conclusions: The 82 base-pair deletion found in the rph-pyrE operon of many endpoints may function to relieve a pyrimidine biosynthesis defect present in MG1655. In contrast, a variety of regulators acquire mutations in the different endpoints, suggesting flexibility in overcoming regulatory challenges in the adaptation.

Figure 1

Large genomic duplications. By viewing the coverage of mapped Solexa data graphically across all genomic coordinates, four large duplications were found in the lactate endpoints, two of which are present in two endpoints. The image shows the coverage of mapped Solexa reads from LactK in the region of a large duplication. In total, the following duplications were found: in LactB and LactK, a 4× and 3× duplication of approximately 40 kb from genomic coordinates 1253000 to 1294000; in LactF, a 3× duplication of approximately 12 kb from 1774000 to 1786000; in LactE, a 2× duplication of approximately 140 kb from 3620000 to 3760000; in LactA and LactE, a 2× duplication of approximately 87 kb from 3946000 to 4033000.

Figure 2

Frequency of mutations. The main graph shows the number of endpoint strains in which a specific gene was mutated out of the 11 adaptive endpoints. The smaller graph shows the number of endpoint strains that have acquired a mutation in at least one gene of a general category, such as metabolism or the cell envelope. The bar color of specific genes in the main graph corresponds to the gene's category classification in the smaller graph.

Figure 3

The rph-pyrE Δ82-bp mutation. An 82-bp deletion in the rph-pyrE operon was found in 7 of 11 lactate adapted strains. The mutation maps to the end of the rph gene, just before the pyrE attenuator loop, causing the translational stop codon (TAG, shown in bold) to move from some distance upstream of the attenuator to just downstream of the loop, likely relieving repression of pyrE by the attenuator. The sequence in and around the deleted region of the operon is shown. The sequence of the deleted region is shown as highlighted, while a 10-bp sequence that repeats after 82 bp is surrounded with a box. The repeating sequence may explain the frequent occurrence of the deletion as a result of DNA polymerase slippage during DNA replication [27].

Figure 4

Temporal order of acquired mutations. DNA extracted from frozen intermediate time points of the adaptive evolutions was Sanger sequenced at genomic locations corresponding to mutations in the endpoints. Time points that were sequenced for mutations are indicated by an arrowhead. The arrow is white if no mutations were identified that were not identified at a previous time point. The first day each mutation was observed is indicated with a dark arrow. Curves represent the growth rate trajectory during the period of adaptive evolution. (a) LactA, (b) LactC, (c) LactD, (d) LactE. The atoS, acpP, and yjbM genes are not represented in the figure because they were not identified as penetrating more than 50% of the population by day 60 of adaptive evolution.