title: Stock culture heterogeneity rather than new mutational variation complicates short-term cell physiology studies of Escherichia coli K-12 MG1655 in continuous culture.
abstract: Nutrient-limited continuous cultures in chemostats have been used to study microbial cell physiology for over 60 years. Genome instability and genetic heterogeneity are possible uncontrolled factors in continuous cultivation experiments. We investigated these issues by using high-throughput (HT) DNA sequencing to characterize samples from different phases of a glucose-limited accelerostat (A-stat) experiment with Escherichia coli K-12 MG1655 and a duration regularly used in cell physiology studies (20 generations of continuous cultivation). Seven consensus mutations from the reference sequence and five subpopulations characterized by different mutations were detected in the HT-sequenced samples. This genetic heterogeneity was confirmed to result from the stock culture by Sanger sequencing. All the subpopulations in which allele frequencies increased (betA, cspG/cspH, glyA) during the experiment were also present at the end of replicate A-stats, indicating that no new subpopulations emerged during our experiments. The fact that ~31 % of the cells in our initial cultures obtained directly from a culture stock centre were mutants raises concerns that even if cultivations are started from single colonies, there is a significant chance of picking a mutant clone with an altered phenotype. Our results show that current HT DNA sequencing technology allows accurate subpopulation analysis and demonstrates that a glucose-limited E. coli K-12 MG1655 A-stat experiment with a duration of tens of generations is suitable for studying cell physiology and collecting quantitative data for metabolic modelling without interference from new mutations.
authors.affiliation: Competence Centre of Food and Fermentation Technologies, Akadeemia tee 15b, 12618 Tallinn, Estonia.|Tallinn University of Technology, Department of Chemistry, Akadeemia tee 15, 12618 Tallinn, Estonia.
authors.forename: Ranno
authors.initials: R
authors.lastname: Nahku
authors.affiliation: Competence Centre of Food and Fermentation Technologies, Akadeemia tee 15b, 12618 Tallinn, Estonia.|Tallinn University of Technology, Department of Chemistry, Akadeemia tee 15, 12618 Tallinn, Estonia.
authors.forename: Karl
authors.initials: K
authors.lastname: Peebo
authors.affiliation: Competence Centre of Food and Fermentation Technologies, Akadeemia tee 15b, 12618 Tallinn, Estonia.|Tallinn University of Technology, Department of Chemistry, Akadeemia tee 15, 12618 Tallinn, Estonia.
authors.forename: Kaspar
authors.initials: K
authors.lastname: Valgepea
authors.affiliation: The University of Texas at Austin, Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, Austin, TX 78712, USA.
authors.forename: Jeffrey E
authors.initials: JE
authors.lastname: Barrick
authors.affiliation: Tallinn University of Technology, Department of Food Processing, Ehitajate tee 5, 19086 Tallinn, Estonia.|Competence Centre of Food and Fermentation Technologies, Akadeemia tee 15b, 12618 Tallinn, Estonia.
authors.forename: Kaarel
authors.initials: K
authors.lastname: Adamberg
authors.affiliation: Competence Centre of Food and Fermentation Technologies, Akadeemia tee 15b, 12618 Tallinn, Estonia.|Tallinn University of Technology, Department of Chemistry, Akadeemia tee 15, 12618 Tallinn, Estonia.
authors.forename: Raivo
authors.initials: R
authors.lastname: Vilu
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D020137:Base Pair Mismatch; D048168:Escherichia coli K12 / Q000235:genetics* / Q000378:metabolism; D019143:Evolution, Molecular; D018740:Genetic Heterogeneity; D016680:Genome, Bacterial; D005947:Glucose / Q000378:metabolism; D009154:Mutation / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D005947:Glucose