Adaption to glucose limitation is modulated by the pleotropic regulator CcpA, independent of selection pressure strength

Abstract

BackgroundA central theme in (micro)biology is understanding the molecular basis of fitness i.e. which strategies are successful under which conditions; how do organisms implement such strategies at the molecular level; and which constraints shape the trade-offs between alternative strategies. Highly standardized microbial laboratory evolution experiments are ideally suited to approach these questions. For example, prolonged chemostats provide a constant environment in which the growth rate can be set, and the adaptive process of the organism to such environment can be subsequently characterized.ResultsWe performed parallel laboratory evolution of Lactococcus lactis in chemostats varying the quantitative value of the selective pressure by imposing two different growth rates. A mutation in one specific amino acid residue of the global transcriptional regulator of carbon metabolism, CcpA, was selected in all of the evolution experiments performed. We subsequently showed that this mutation confers predictable fitness improvements at other glucose-limited growth rates as well. In silico protein structural analysis of wild type and evolved CcpA, as well as biochemical and phenotypic assays, provided the underpinning molecular mechanisms that resulted in the specific reprogramming favored in constant environments.ConclusionThis study provides a comprehensive understanding of a case of microbial evolution and hints at the wide dynamic range that a single fitness-enhancing mutation may display. It demonstrates how the modulation of a pleiotropic regulator can be used by cells to improve one trait while simultaneously work around other limiting constraints, by fine-tuning the expression of a wide range of cellular processes.Electronic supplementary materialThe online version of this article (10.1186/s12862-018-1331-x) contains supplementary material, which is available to authorized users.

A Novel Method for Long-Term Analysis of Lactic Acid and Ammonium Production in Non-growing Lactococcus lactis Reveals Pre-culture and Strain Dependence

“…Lactococcus lactis subsp. cremoris NCDO712 ( Gasson, 1983 ), HB60 ( Bachmann et al, 2013 ), and MG1363 ( Wegmann et al, 2007 ) were grown on chemically defined medium for prolonged cultivation (CDMPC) as described previously ( Price et al, 2019 ). Medium was supplemented with either lactose 30 mM, galactose 55 mM, or glucose 55 mM depending on experimental design.…”

“…Medium was supplemented with either lactose 30 mM, galactose 55 mM, or glucose 55 mM depending on experimental design. All pre-cultures were standardized and started with a single use aliquot of glycerol stock which was 1,000-fold diluted in medium and cultured overnight (as described in Price et al, 2019 ). Overnight cultures were sub-cultured (40-fold dilution) in fresh medium and harvested during the early exponential phase (OD 0.1–0.2).…”

Searching for principles of microbial physiology

“…Mutations in key transcription factors or in promoter sequences are an example (Kussell 2013 ; Price et al . 2019 ).…”

“…We believe that this is the membrane compartment, also based on the outcome of a laboratory evolution experiment (Price et al . 2019 ). If the membrane compartment is filled with needed protein, and is the growth-rate limiting constraint, how can growth rate then still increase in the chenostat?…”

Transcriptional response of Lactococcus lactis during bacterial emulsification

“…An overnight culture (27 ml, OD 600 = 1.58) of L . lactis NCDO712 grown in chemically defined medium [65] with 1% lactose (LCDM) was diluted into 800 ml pre-warmed (30°C) fresh LCDM to an OD 600 of 0.1 and distributed in 25-ml aliquots over 16 tubes of 50 ml. The cultures were incubated at 30°C until an OD 600 of 0.43±0.03 was reached.…”