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Diversity in lac Operon Regulation among Diverse Escherichia coli Isolates Depends on the Broader Genetic Background but Is Not Explained by Genetic Relatedness

Citation

Cooper, Tim et al. (2019), Diversity in lac Operon Regulation among Diverse Escherichia coli Isolates Depends on the Broader Genetic Background but Is Not Explained by Genetic Relatedness, Dryad, Dataset, https://doi.org/10.5061/dryad.8cz8w9gk9

Abstract

Transcription of bacterial genes is controlled by the coordinated action of cis- and trans-acting regulators. The activity and mode of action of these regulators can reflect different requirements for gene products in different environments. A well-studied example is the regulatory function that integrates the environmental availability of glucose and lactose to control the Escherichia coli lac operon. Most studies of lac operon regulation have focused on a few closely related strains. To determine the range of natural variation in lac regulatory function, we introduced a reporter construct into 23 diverse E. coli strains and measured expression with combinations of inducer concentrations. We found a wide range of regulatory functions. Several functions were similar to the one observed in a reference lab strain, whereas others depended weakly on the presence of cAMP. Some characteristics of the regulatory function were explained by the genetic relatedness of strains, indicating that differences varied on relatively short time scales. The regulatory characteristics explained by genetic relatedness were among those that best predicted the initial growth of strains following transition to a lactose environment, suggesting a role for selection. Finally, we transferred the lac operon, with the lacI regulatory gene, from five natural isolate strains into a reference lab strain. The regulatory function of these hybrid strains revealed the effect of local and global regulatory elements in controlling expression. Together, this work demonstrates that regulatory functions can be varied within a species and that there is variation within a species to best match a function to particular environments.

Methods

A gfp reporter driven by the lac promoter (Plac-GFP) was integrated into the attTn7 site of strains as indicated in file names. Regulatory input functions were characterized by measuring the expression of a Plac-GFP reporter at different combinations of cAMP and IPTG in DM supplemented with 2,000 g/ml glucose. This environment was used because glucose inhibits the production of cAMP, allowing measurement of the regulatory input function from as close to the basal level of Plac-GFP expression as possible. Strains containing the Plac-GFP reporter were preconditioned in DM medium supplemented with 2,000 g/ml (DM2000) glucose for 24 h and then transferred at a 1:1,000 dilution to the test environments containing combinations of DM2000 supplemented with cAMP and IPTG. cAMP was added at eight concentrations (0, 0.625, 1.25, 2.5, 5, 10, 20, and 40 mM), and IPTG was added at 10 or 6 concentrations (0, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 100, and 200 uM, or 0, 6.25, 12.5, 25, 50, and 100 uM). Strains were grown in these environments for 16 h to an optical density at 450 nm (OD450) of 0.1 to 0.2, which corresponded to mid-log growth phase, as determined by tracking changes in population OD using a VersaMax spectrophotometer.

 

Usage Notes

File names give the name of the measured strains followed by the replicate number (if any). Files contain a matrix of expression values determined using a flow cytometer to sample induced cell populations and reported as the median value over the measured reporter expression distribution for each inducer combination. cAMP concentrations increase through rows 1-8 and IPTG concentrations increase therough columns 1-6 or 1-10 as relevant to individual files. Concentrations are: cAMP 0, 0.625, 1.25, 2.5, 5, 10, 20, and 40 mM and IPTG 0, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 100, and 200 uM or 0, 6.25, 12.5, 25, 50, and 100 uM. 

Funding

National Science Foundation, Award: DEB-1253650

National Science Foundation, Award: IOS-071141