Standardized Assay Medium To Measure Lactococcus lactis Enzyme Activities while Mimicking Intracellular Conditions

Goel, Anisha; Santos, F.D.; Vos, Willem M. de; Teusink, Bas; Molenaar, Douwe

doi:10.1128/aem.05276-11

Cited by 53 publications

(49 citation statements)

References 51 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…lactis strains were grown overnight in a chemically defined medium (CDM) supplemented with glucose or cellobiose (41). Next, they were washed with CDM and diluted 20x in fresh CDM containing cellobiose, glucose, or a mixture of both; growth and fluorescence development were monitored by using a microtiter-plate reader (Tecan Group Ltd.) or FACS (BD Biosciences).…”

Section: Methodsmentioning

confidence: 99%

Bet-hedging during bacterial diauxic shift

Solopova

Gestel

Weissing

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

237

245

View full text Add to dashboard Cite

When bacteria grow in a medium with two sugars, they first use the preferred sugar and only then start metabolizing the second one. After the first exponential growth phase, a short lag phase of nongrowth is observed, a period called the diauxie lag phase. It is commonly seen as a phase in which the bacteria prepare themselves to use the second sugar. Here we reveal that, in contrast to the established concept of metabolic adaptation in the lag phase, two stable cell types with alternative metabolic strategies emerge and coexist in a culture of the bacterium Lactococcus lactis. Only one of them continues to grow. The fraction of each metabolic phenotype depends on the level of catabolite repression and the metabolic state-dependent induction of stringent response, as well as on epigenetic cues. Furthermore, we show that the production of alternative metabolic phenotypes potentially entails a bet-hedging strategy. This study sheds new light on phenotypic heterogeneity during various lag phases occurring in microbiology and biotechnology and adjusts the generally accepted explanation of enzymatic adaptation proposed by Monod and shared by scientists for more than half a century.phenotypic heterogeneity | Gram-positive bacteria | metabolic fitness I n nature, bacteria are confronted with a wide range of environmental conditions that change over time. These conditions often elicit specific metabolic responses that increase the division rate of a cell. For example, when bacterial cells are exposed to multiple sugars, they do not metabolize all sugars simultaneously, but rather use the sugar that allows the highest celldivision rate. Cells switch to the less-preferred sugar when the most-preferred one (in many cases glucose) is depleted. Jacques Monod coined this phenomenon "diauxie" (1). Diauxie is characterized by two growth cycles-the first one on the preferred sugar, followed by a second one on the less-preferred sugar. Both are separated by a short period during which the population apparently does not grow. This period is known as the diauxie lag phase. It is typically assumed that cells need time to make the necessary enzymatic adaptations to switch from one substrate to another (2). However, the behavior of individual cells during the lag phase has not been studied in detail. Here, we examine the diauxic shift at the single-cell level in Lactococcus lactis using time-lapse microscopy, in addition to the traditional approach of studying population growth characteristics. Surprisingly, the lag phase at the switch from glucose to cellobiose consumption by L. lactis largely results from the heterogeneous response of cells to the environmental change, rather than the time it takes for cells to make the necessary metabolic adaptations. This result challenges Monod's view of enzymatic adaptation and asks for a revision on the interpretation of population lag phases.

show abstract

Section: Methodsmentioning

confidence: 99%

Bet-hedging during bacterial diauxic shift

Solopova

Gestel

Weissing

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

237

245

View full text Add to dashboard Cite

show abstract

“…L. lactis MG1363 (43) and derivatives were used throughout this study. Experiments were carried out at 30°C in a chemically defined medium (CDM) (44), which was supplemented with 5 mM glucose (GCDM) with the exception of the competition experiment of NZ9000 and NZ9010 (proof-of-principle experiment) where 25 mM glucose was added. Bacterial plating was done on the rich medium M17 (Oxoid) supplemented with 25 mM glucose (GM17).…”

Section: Methodsmentioning

confidence: 99%

Availability of public goods shapes the evolution of competing metabolic strategies

Bachmann¹,

Fischlechner

Rabbers³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

176

220

View full text Add to dashboard Cite

Tradeoffs provide a rationale for the outcome of natural selection. A prominent example is the negative correlation between the growth rate and the biomass yield in unicellular organisms. This tradeoff leads to a dilemma, where the optimization of growth rate is advantageous for an individual, whereas the optimization of the biomass yield would be advantageous for a population. High-rate strategies are observed in a broad variety of organisms such as Escherichia coli, yeast, and cancer cells. Growth in suspension cultures favors fast-growing organisms, whereas spatial structure is of importance for the evolution of high-yield strategies. Despite this realization, experimental methods to directly select for increased yield are lacking. We here show that the serial propagation of a microbial population in a water-in-oil emulsion allows selection of strains with increased biomass yield. The propagation in emulsion creates a spatially structured environment where the growth-limiting substrate is privatized for populations founded by individual cells. Experimental evolution of several isogenic Lactococcus lactis strains demonstrated the existence of a tradeoff between growth rate and biomass yield as an apparent Pareto front. The underlying mutations altered glucose transport and led to major shifts between homofermentative and heterofermentative metabolism, accounting for the changes in metabolic efficiency. The results demonstrated the impact of privatizing a public good on the evolutionary outcome between competing metabolic strategies. The presented approach allows the investigation of fundamental questions in biology such as the evolution of cooperation, cell-cell interactions, and the relationships between environmental and metabolic constraints.A lthough the existence of tradeoffs in evolution seems to be undisputable, experimental evidence obtained under controlled conditions is scarce. Several examples failed to show tradeoffs (1-4), whereas others could find them (5, 6) or found general but not universal tradeoffs (7,8). A tradeoff between growth rate and growth yield in microbes (9-11) has direct implications for experiments carried out in liquid cultures. This is especially of importance during prolonged cultivations such as laboratory evolution experiments. In suspension, fast-growing variants outcompete slower growing ones at the cost of biomass yield (5). The yield versus rate optimization is governed by a dilemma where fast growth is advantageous from the perspective of an individual cell, whereas slow growth, and therefore high yield, is advantageous from the perspective of a population. This dilemma is consistent with a concept termed the tragedy of the commons (12). It is well described that spatial structure is essential for the selection of high-yield strategies (13-15). The yield/rate tradeoff of microbial growth has been linked to metabolic strategies (11) such as the switch between respiration and fermentation in yeast (9). It is suggested that the underlying cause of this tradeoff is based on...

show abstract

“…These strains were isolated from two veal calf farms, MRSA0026 and MRSA0027 from one and MRSA0028 and MRSA0029 from another. For the cultivation of the S. aureus strains, a chemically defined medium (CDMPC) (22) was used. The MRSA strains were assigned to sequence type 398 (ST398) and had the following characteristics (23): MRSA0026, mecA ϩ spa t011, methicillin MIC, 4 g/ml; MRSA0027, mecA-negative spa t011, methicillin MIC, 1 g/ml; MRSA0028, mecA ϩ spa t034, methicillin MIC, 64 g/ml; MRSA0029, mecA-negative spa t034, methicillin MIC, 1 g/ml.…”

Section: Methodsmentioning

confidence: 99%

Compensation of the Metabolic Costs of Antibiotic Resistance by Physiological Adaptation in Escherichia coli

Händel

Schuurmans

Brul

et al. 2013

Antimicrob Agents Chemother

View full text Add to dashboard Cite

b; Netherlands Food and Consumer Product Safety Authority, Office for Risk Assessment, Utrecht, The Netherlands c Antibiotic resistance is often associated with metabolic costs. To investigate the metabolic consequences of antibiotic resistance, the genomic and transcriptomic profiles of an amoxicillin-resistant Escherichia coli strain and the wild type it was derived from were compared. A total of 125 amino acid substitutions and 7 mutations that were located <1,000 bp upstream of differentially expressed genes were found in resistant cells. However, broad induction and suppression of genes were observed when comparing the expression profiles of resistant and wild-type cells. Expression of genes involved in cell wall maintenance, DNA metabolic processes, cellular stress response, and respiration was most affected in resistant cells regardless of the absence or presence of amoxicillin. The SOS response was downregulated in resistant cells. The physiological effect of the acquisition of amoxicillin resistance in cells grown in chemostat cultures consisted of an initial increase in glucose consumption that was followed by an adaptation process. Furthermore, no difference in maintenance energy was observed between resistant and sensitive cells. In accordance with the transcriptomic profile, exposure of resistant cells to amoxicillin resulted in reduced salt and pH tolerance. Taken together, the results demonstrate that the acquisition of antibiotic resistance in E. coli is accompanied by specifically reorganized metabolic networks in order to circumvent metabolic costs. The overall effect of the acquisition of resistance consists not so much of an extra energy requirement, but more a reduced ecological range.

show abstract

Standardized Assay Medium To Measure Lactococcus lactis Enzyme Activities while Mimicking Intracellular Conditions

Cited by 53 publications

References 51 publications

Bet-hedging during bacterial diauxic shift

Bet-hedging during bacterial diauxic shift

Availability of public goods shapes the evolution of competing metabolic strategies

Compensation of the Metabolic Costs of Antibiotic Resistance by Physiological Adaptation in Escherichia coli

Contact Info

Product

Resources

About