Genetic Basis of Growth Adaptation of Escherichia coli after Deletion of pgi, a Major Metabolic Gene

Abstract

Bacterial survival requires adaptation to different environmental perturbations such as exposure to antibiotics, changes in temperature or oxygen levels, DNA damage, and alternative nutrient sources. During adaptation, bacteria often develop beneficial mutations that confer increased fitness in the new environment. Adaptation to the loss of a major non-essential gene product that cripples growth, however, has not been studied at the whole-genome level. We investigated the ability of Escherichia coli K-12 MG1655 to overcome the loss of phosphoglucose isomerase (pgi) by adaptively evolving ten replicates of E. coli lacking pgi for 50 days in glucose M9 minimal medium and by characterizing endpoint clones through whole-genome re-sequencing and phenotype profiling. We found that 1) the growth rates for all ten endpoint clones increased approximately 3-fold over the 50-day period; 2) two to five mutations arose during adaptation, most frequently in the NADH/NADPH transhydrogenases udhA and pntAB and in the stress-associated sigma factor rpoS; and 3) despite similar growth rates, at least three distinct endpoint phenotypes developed as defined by different rates of acetate and formate secretion. These results demonstrate that E. coli can adapt to the loss of a major metabolic gene product with only a handful of mutations and that adaptation can result in multiple, alternative phenotypes.

Metabolic impact of an NADH-producing glucose-6-phosphate dehydrogenase in Escherichia coli

“…The parental strains for the recombination procedure were: WT (MG1655 K12) and Dpgi (MG1655 K12 Dpgi; Charusanti et al, 2010).…”

A Model for Designing Adaptive Laboratory Evolution Experiments

“…ALEsim and the derived parameters (beneficial mutation rate and allowed increases in growth rate) were analyzed by using two previously performed ALE experiments on glucose (7, 18) and a legacy experiment on glycerol (14). The two glucose experiments yielded disparate final growth rates despite identical strains and media (E. coli K-12 MG1655 in M9 glucose minimal medium), i.e., 0.79 Ϯ 0.01 with 3 replicates and 1.00 Ϯ 0.02 with 6 replicates in the studies by Charusanti et al (18) and LaCroix et al (7), respectively. The only differences between the experiments were three experimental parameters, namely, batch culture volumes (250 ml versus 25 ml), ODs when passaged (variable OD versus OD at 600 nm [OD 600 ] of 1.2), and passage sizes (variable versus 800 l).…”

“…As an example, a previous study adapting Escherichia coli to glycerol in 250-ml batches started with a passage size of approximately 100 l and was using a passage size of less than 0.1 l by the end of the experiment (14). A more in-depth retrospective analysis revealed similar trends when passage amounts were significantly decreased (14)(15)(16)(17)(18). In those studies, the reduction in the population size, or bottleneck (i.e., passage size), became so significant that the calculated number of cells being passaged was on the order of 10 or even occasionally 1.…”

“…This can be seen in a comparison of two studies that evolved wild-type E. coli K-12 MG1655 on M9 glucose minimal medium (7,18). One study (7) used a consistent passage size of 800 l from 25-ml batches on an automated platform.…”

“…One study (7) used a consistent passage size of 800 l from 25-ml batches on an automated platform. The second study (18) was performed "by hand" and had widely varying passage sizes that were considerably smaller than those in the automated study. The outcomes of the ALE experiments were quite distinct.…”