Fluctuating environment can negate cheater success due to speed-agility trade-off

Stability of microbial cooperation through common goods is susceptible to cheating. Evidence suggests that cheating plays a less prominent role in many natural systems than hitherto predicted by models of eco-evolutionary dynamics and evolutionary game theory. While several cheater negating factors such as spatial segregation have been identified, most consider single-nutrient regimes. Here we propose a cheater-suppressing mechanism based on previous experimental observations regarding the biochemical trade-off between growth speed and delay in switching to alternative nutrients. As changing the nutrient source requires redistribution of enzymatic resources to different metabolic pathways, the advantage in speed is offset by lower agility due to longer time required for resource re-allocation. Using an in silico model system of sucrose utilisation by Saccharomyces cerevisiae, we find that a trade-off between growth rate and diauxic lag duration can supress cheaters under fluctuating nutrient availability and thereby stabilise cooperation. The resulting temporal dynamics constrain cheaters despite their competitive benefit for the growth on the primary nutrient via avoided public goods synthesis costs. We further show that this speed-agility trade-off can function in synergy with spatial segregation to avoid the collapse of the community due to the cheaters. Taken together, the growth-agility trade-off may contribute to cheater suppression in microbial ecosystems experiencing fluctuating environments, such as plant root microbiota and gut microbiota.

Development of a semi-defined medium for high cell density cultivation of Escherichia coli in shake flasks: Part 2

The characteristics of the culture vessel determine, to a large extent, the type of growth medium suitable for use. For example, most growth media for high cell density cultivation are designed for expensive bioreactors operating either in continuous or fed-batch mode, where provision of additional nutrients and/or removal of metabolic waste products from the basal medium (comprising salts, buffer components and small amount of carbon and nitrogen sources) help increase biomass yield by maintaining culture conditions within the range conducive for growth. The inexpensive and ubiquitous shake flask, in contrast, is usually operated in batch mode and contains a comprehensive medium with all necessary nutrients for converting cells into biomass, and as a repository for secreted metabolites, some of which detrimental for cell growth. Thus, designing medium for high cell density cultivation in shake flask is an optimization process with the aim to increase biomass formation while reducing toxic metabolite secretion. This preprint reports improvements to a previously reported semi-defined medium for high cell density aerobic cultivation of Escherichia coli DH5α (ATCC 53868) in shake flasks. Specifically, by reducing the concentrations of glucose (from 6.0 to 4.0 g/L) and ammonium chloride (from 1.5 to 1.0 g/L), the following improvements were obtained: a shorter diauxic lag phase (3 versus 5 hours); a higher maximum optical density (12.0 versus 11.0) in a shorter total culture period (27 versus 48 hours), and smaller pH variation during cultivation (6.0 to 7.6 versus 5.5 to 7.8). Similar to the earlier study, glucose and yeast extract served as principal carbon sources in separate growth phases for E. coli in the improved formulated medium (FMimproved). Specifically, an OD600nm of 6.6 was attained after 9 hours of growth at 37 oC. After a lag phase of 3 hours, growth resumed on yeast extract and the OD600nm reached 12.0 after 27 hours. The broth’s pH decreased from 7.1 to 6.0 during the first growth phase, whereupon it gradually rose to 7.6 at the end of culture. A smaller pH decrease together with higher biomass yield in the first growth phase suggested that the lower glucose concentration in FMimproved might have prevented overflow metabolism (and associated negative effects on growth); thus, resulting in a shorter diauxic lag phase and total culture period. Collectively, increase in cell yield, as well as decrease in total culture time and a shorter diauxic lag phase arise from a small reduction in glucose concentration, which suggested that an optimum exist, beyond which occurrence of overflow metabolism would reduce cell yield and biomass formation. Part 1 of this work can be found at: https://peerj.com/preprints/115/

Development of a semi-defined medium for high cell density cultivation of Escherichia coli in shake flasks: Part 2

The characteristics of the culture vessel determine, to a large extent, the type of growth medium suitable for use. For example, most growth media for high cell density cultivation are designed for expensive bioreactors operating either in continuous or fed-batch mode, where provision of additional nutrients and/or removal of metabolic waste products from the basal medium (comprising salts, buffer components and small amount of carbon and nitrogen sources) help increase biomass yield by maintaining culture conditions within the range conducive for growth. The inexpensive and ubiquitous shake flask, in contrast, is usually operated in batch mode and contains a comprehensive medium with all necessary nutrients for converting cells into biomass, and as a repository for secreted metabolites, some of which detrimental for cell growth. Thus, designing medium for high cell density cultivation in shake flask is an optimization process with the aim to increase biomass formation while reducing toxic metabolite secretion. This preprint reports improvements to a previously reported semi-defined medium for high cell density aerobic cultivation of Escherichia coli DH5α (ATCC 53868) in shake flasks. Specifically, by reducing the concentrations of glucose (from 6.0 to 4.0 g/L) and ammonium chloride (from 1.5 to 1.0 g/L), the following improvements were obtained: a shorter diauxic lag phase (3 versus 5 hours); a higher maximum optical density (12.0 versus 11.0) in a shorter total culture period (27 versus 48 hours), and smaller pH variation during cultivation (6.0 to 7.6 versus 5.5 to 7.8). Similar to the earlier study, glucose and yeast extract served as principal carbon sources in separate growth phases for E. coli in the improved formulated medium (FMimproved). Specifically, an OD600nm of 6.6 was attained after 9 hours of growth at 37 oC. After a lag phase of 3 hours, growth resumed on yeast extract and the OD600nm reached 12.0 after 27 hours. The broth’s pH decreased from 7.1 to 6.0 during the first growth phase, whereupon it gradually rose to 7.6 at the end of culture. A smaller pH decrease together with higher biomass yield in the first growth phase suggested that the lower glucose concentration in FMimproved might have prevented overflow metabolism (and associated negative effects on growth); thus, resulting in a shorter diauxic lag phase and total culture period. Collectively, increase in cell yield, as well as decrease in total culture time and a shorter diauxic lag phase arise from a small reduction in glucose concentration, which suggested that an optimum exist, beyond which occurrence of overflow metabolism would reduce cell yield and biomass formation. Part 1 of this work can be found at: https://peerj.com/preprints/115/

Polymorphisms in the yeast galactose sensor underlie a natural continuum of nutrient-decision phenotypes

In nature, microbes often need to “decide” which of several available nutrients to utilize, a choice that depends on a cell’s inherent preference and external nutrient levels. While natural environments can have mixtures of different nutrients, phenotypic variation in microbes’ decisions of which nutrient to utilize is poorly studied. Here, we quantified differences in the concentration of glucose and galactose required to induce galactose-responsive (GAL) genes across 36 wildS. cerevisiaestrains. Using bulk segregant analysis, we found that a locus containing the galactose sensorGAL3was associated with differences in GAL signaling in eight different crosses. Using allele replacements, we confirmed thatGAL3is the major driver of GAL induction variation, and thatGAL3allelic variation alone can explain as much as 90% of the variation in GAL induction in a cross. TheGAL3variants we found modulate the diauxic lag, a selectable trait. These results suggest that ecological constraints on the galactose pathway may have led to variation in a single protein, allowing cells to quantitatively tune their response to nutrient changes in the environment.Author summaryIn nature, microbes often need to decide which of many potential nutrients to consume. This decision making process is complex, involving both intracellular constraints and the organism’s perception of the environment. To begin to mimic the complexity of natural environments, we grew cells in mixtures of two sugars, glucose and galactose. We find that in mixed environments, the sugar concentration at which cells decides to induce galactose-utilizing (GAL) genes is highly variable in natural isolates of yeast. By analyzing crosses of phenotypically different strains, we identified a locus containing the galactose sensor, a gene that in theory could allow cells to tune their perception of the environment. We confirmed that the galactose sensor can explain upwards of 90% of the variation in the decision to induce GAL genes. Finally, we show that the variation in the galactose sensor can modulate the time required for cells to switch from utilizing glucose to galactose. Our results suggest that signaling pathways can be highly variable across strains and thereby might allow for rapid adaption in fluctuating environments.

Development of a semi-defined medium for high cell density cultivation of Escherichia coli in shake flasks: Part 2

The characteristics of the culture vessel determine, to a large extent, the type of growth medium suitable for use. For example, most growth media for high cell density cultivation are designed for expensive bioreactors operating either in continuous or fed-batch mode, where provision of additional nutrients and/or removal of metabolic waste products from the basal medium (comprising salts, buffer components and small amount of carbon and nitrogen sources) help increase biomass yield by maintaining culture conditions within the range conducive for growth. The inexpensive and ubiquitous shake flask, in contrast, is usually operated in batch mode and contains, at the outset, a comprehensive medium with all necessary nutrients for conversion into biomass, and also serves as a repository for secreted metabolites - some of which are detrimental for cell growth. Thus, designing medium for high cell density cultivation in shake flask is an optimization process with the aim to increase biomass formation while reducing toxic metabolite secretion. This preprint reports improvements to a previously reported semi-defined medium for high cell density aerobic cultivation of Escherichia coli DH5α (ATCC 53868) in shake flasks. Specifically, by reducing the concentrations of glucose (from 6.0 to 4.0 g/L) and ammonium chloride (from 1.5 to 1.0 g/L), the following improvements were obtained: a shorter diauxic lag phase (3 versus 5 hours); a higher maximum optical density (12.0 versus 11.0) in a shorter total culture period (27 versus 48 hours), and smaller pH variation during cultivation (6.0 to 7.6 versus 5.5 to 7.8). Similar to the earlier study, glucose and yeast extract served as principal carbon sources in separate growth phases for E. coli in the improved formulated medium (FMimproved). Specifically, an OD600nm of 6.6 was attained after 9 hours of growth on glucose at 37 oC. After a lag phase of 3 hours, growth resumed on yeast extract and the OD600nm reached 12.0 after 27 hours. The broth’s pH decreased from 7.1 to 6.0 during the first growth phase, whereupon it gradually rose to 7.6 at the end of culture. A smaller pH decrease along with higher biomass yield in the first growth phase suggested that the lower glucose concentration in FMimproved might have prevented overflow metabolism (and associated negative effects on growth); thus, resulting in a shorter diauxic lag phase and total culture period. Collectively, increase in cell yield, as well as decrease in total culture time and a shorter diauxic lag phase arise from a small reduction in glucose concentration - which suggested that an optimum exist, beyond which occurrence of overflow metabolism would reduce cell yield and biomass formation.

Development of a semi-defined medium for high cell density cultivation of Escherichia coli in shake flasks: Part 2

The characteristics of the culture vessel determine, to a large extent, the type of growth medium suitable for use. For example, most growth media for high cell density cultivation are designed for expensive bioreactors operating either in continuous or fed-batch mode, where provision of additional nutrients and/or removal of metabolic waste products from the basal medium – comprising salts, buffer components and small amount of carbon and nitrogen sources - help increase biomass yield by maintaining culture conditions within the range conducive for growth. The inexpensive and ubiquitous shake flask, in contrast, is usually operated in batch mode and contains, at the outset, a comprehensive medium with all necessary nutrients for conversion into biomass, and also serves as a repository for secreted metabolites - some of which are detrimental for cell growth. Thus, designing medium for high cell density cultivation in shake flask is an optimization process with the aim to increase biomass formation while reducing toxic metabolite secretion. This preprint reports improvements to a previously reported semi-defined medium for high cell density aerobic cultivation of Escherichia coli DH5α (ATCC 53868) in shake flasks. Specifically, by reducing the concentrations of glucose (from 6.0 to 4.0 g/L) and ammonium chloride (from 1.5 to 1.0 g/L), the following improvements were obtained: a shorter diauxic lag phase (3 versus 5 hours); a higher maximal optical density (12.0 versus 11.0) in a shorter total culture period (27 versus 48 hours), and smaller pH variation during cultivation (6.0 to 7.6 versus 5.5 to 7.8). Similar to the earlier study, glucose and yeast extract served as principal carbon sources in separate growth phases for E. coli in the improved formulated medium (FMimproved). Specifically, an OD600nm of 6.6 was attained after 9 hours of growth on glucose at 37 oC. Following a lag phase of 3 hours, growth resumed on yeast extract and the OD600nm reached 12.0 after 27 hours. The broth’s pH decreased from 7.1 to 6.0 during the first growth phase, whereupon it gradually rose to 7.6 at the end of culture. A smaller pH decrease along with higher biomass yield in the first growth phase suggested that the lower glucose concentration in FMimproved might have prevented overflow metabolism - and associated negative effects on growth - thus, resulting in a shorter diauxic lag phase and total culture period. Collectively, increase in cell yield, as well as decrease in total culture time and a shorter diauxic lag phase arise from a small reduction in glucose concentration - which suggested that an optimum exist, beyond which occurrence of overflow metabolism would reduce cell yield and biomass formation.

Development of a semi-defined medium for high cell density cultivation of Escherichia coli in shake flask culture system: Part 2

The characteristics of the culture vessel determine, to a large extent, the type of growth medium suitable for use. For example, most growth media for high cell density cultivation are designed for expensive bioreactors operating either in continuous or fed-batch mode, where provision of additional nutrients and/or removal of metabolic waste products from the basal medium – comprising salts, buffer components and small amount of carbon and nitrogen sources - help increase biomass yield by maintaining culture conditions within the range conducive for growth. The inexpensive and ubiquitous shake flask, in contrast, is usually operated in batch mode and contains, at the outset, a comprehensive medium with all necessary nutrients for conversion into biomass and, also serves as a repository for secreted metabolites - some of which are detrimental for cell growth. Thus, designing medium for high cell density cultivation in shake-flask is an optimization process aiming to increase biomass formation while reducing toxic metabolite secretion. This preprint reports improvements to a previously reported semi-defined medium for high cell density aerobic cultivation of Escherichia coli DH5α (ATCC 53868) in shake flask. Specifically, by reducing the concentrations of glucose (from 6.0 to 4.0 g/L) and ammonium chloride (from 1.5 to 1.0 g/L), the following improvements were obtained: a shorter diauxic lag phase (3 versus 5 hours); a higher maximal optical density (12.0 versus 11.0) in a shorter total culture period (27 versus 48 hours), and smaller pH variation during cultivation (6.0 to 7.6 versus 5.5 to 7.8). Similar to the earlier study, glucose and yeast extract served as principal carbon sources in separate growth phases for E. coli in the improved formulated medium (FMimproved). Specifically, an OD600nm of 6.6 was attained after 9 hours of growth on glucose at 37 oC. Following a lag phase of 3 hours, growth resumed on yeast extract and the OD600nm reached 12.0 after 27 hours. The broth’s pH decreased from 7.1 to 6.0 during the first growth phase, whereupon it gradually rose to 7.6 at the end of culture. A smaller pH decrease along with higher biomass yield in the first growth phase suggested that the lower glucose concentration in FMimproved might have prevented overflow metabolism - and associated negative effects on growth - thus, resulting in a shorter diauxic lag phase and total culture period. Collectively, increase in cell yield, as well as decrease in total culture time and a shorter diauxic lag phase arise from a small reduction in glucose concentration - which suggested that an optimum exist, beyond which, occurrence of overflow metabolism would reduce cell yield and biomass formation.

The Escherichia coli yjfP Gene Encodes a Carboxylesterase Involved in Sugar Utilization during Diauxie

<b><i>Background:</i></b> Acetylation and efflux of carbohydrates during cellular metabolism is a well-described phenomenon associated with a detoxification process to prevent metabolic congestion. It is still unclear why cells discard important metabolizable energy sources in the form of acetylated compounds. <b><i>Methods:</i></b> We describe the purification and characterization of an approximately 28-kDa intracellular carboxylesterase (YjfP) and the analysis of gene and protein expression by qRT-PCR and Western blot. <b><i>Results:</i></b> qRT-PCR and Western blot, respectively, showed that y<i>jfP</i> is upregulated during the diauxic lag in cells growing with a mixture of glucose and lactose. The β-galactosidase activity in the &#x0394;<i>yjfP</i> strain was both delayed and half the magnitude of that of the wild-type strain. YjfP-hyperproducing strains displayed a long lag phase when cultured with glucose and then challenged to grow with lactose or galactose as the sole carbon source. <b><i>Conclusion:</i></b> Our results suggest that YjfP controls the intracellular concentration of acetyl sugars by redirecting them to the main metabolic circuits. Instead of detoxification, we propose that sugar acetylation is utilized by the cell for protection and to prevent the metabolism of a necessary minimal intracellular sugar pool. Those sugars can eventually be exported as a side effect of these mechanisms.

Natural Variation in Preparation for Nutrient Depletion Reveals a Cost-Benefit Tradeoff

Maximizing growth and survival in the face of a complex, time-varying environment is a common problem for single-celled organisms in the wild. When offered two different sugars as carbon sources, microorganisms first consume the preferred sugar, then undergo a transient growth delay, the “diauxic lag”, while inducing genes to metabolize the less preferred sugar. This delay is commonly assumed to be an inevitable consequence of selection to maximize use of the preferred sugar. Contrary to this view, we found that many natural isolates of Saccharomyces cerevisiae display short or non-existent diauxic lags when grown in mixtures of glucose (preferred) and galactose. These strains induce galactose-utilization (GAL) genes hours before glucose exhaustion, thereby “preparing” for the transition from glucose to galactose metabolism. The extent of preparation varies across strains, and seems to be determined by the steady-state response of GAL genes to mixtures of glucose and galactose rather than by induction kinetics. Although early GAL induction gives strains a competitive advantage once glucose runs out, it comes at a cost while glucose is still present. Costs and benefits correlate with the degree of preparation: strains with higher expression of GAL genes prior to glucose exhaustion experience a larger upfront growth cost but also a shorter diauxic lag. Our results show that classical diauxic growth is only one extreme on a continuum of growth strategies constrained by a cost-benefit tradeoff. This type of continuum is likely to be common in nature, as similar tradeoffs can arise whenever cells evolve to use mixtures of nutrients.

Development of a semi-defined medium for high cell density cultivation of Escherichia coli in shake flask culture system: Part 2

The characteristics of the culture vessel determine, to a large extent, the type of growth medium suitable for use. For example, most growth media for high cell density cultivation are designed for expensive bioreactors operating either in continuous or fed-batch mode, where provision of additional nutrients and/or removal of metabolic waste products from the basal medium – comprising salts, buffer components and small amount of carbon and nitrogen sources - help increase biomass yield by maintaining culture conditions within the range conducive for growth. The inexpensive and ubiquitous shake flask, in contrast, is usually operated in batch mode and contains, at the outset, a comprehensive medium with all necessary nutrients for conversion into biomass and, also serves as a repository for secreted metabolites - some of which are detrimental for cell growth. Thus, designing medium for high cell density cultivation in shake-flask is an optimization process aiming to increase biomass formation while reducing toxic metabolite secretion. This preprint reports improvements to a previously reported semi-defined medium (Wenfa Ng, 2013, https://peerj.com/preprints/115v1) for high cell density aerobic cultivation of Escherichia coli DH5α (ATCC 53868) in shake flask. Specifically, by reducing the concentrations of glucose (from 6.0 to 4.0 g/L) and ammonium chloride (from 1.5 to 1.0 g/L), the following improvements were obtained: a shorter diauxic lag phase (3 versus 5 hours); a higher maximal optical density (12.0 versus 11.0) in a shorter total culture period (27 versus 48 hours), and smaller pH variation during cultivation (6.0 to 7.6 versus 5.5 to 7.8). Similar to the earlier study, glucose and yeast extract served as principal carbon sources in separate growth phases for E. coli in the improved formulated medium (FMimproved). Specifically, an OD600nm of 6.6 was attained after 9 hours of growth on glucose at 37 oC. Following a lag phase of 3 hours, growth resumed on yeast extract and the OD600nm reached 12.0 after 27 hours. The broth’s pH decreased from 7.1 to 6.0 during the first growth phase, whereupon it gradually rose to 7.6 at the end of culture. A smaller pH decrease along with higher biomass yield in the first growth phase suggested that the lower glucose concentration in FMimproved might have prevented overflow metabolism - and associated negative effects on growth - thus, resulting in a shorter diauxic lag phase and total culture period. Collectively, increase in cell yield, as well as decrease in total culture time and a shorter diauxic lag phase arise from a small reduction in glucose concentration - which suggested that an optimum exist, beyond which, occurrence of overflow metabolism would reduce cell yield and biomass formation.