The yield of experimental yeast populations declines during selection

The trade-off between growth rate and yield can limit population productivity. Here we tested for this life-history trade-off in replicate haploid and diploid populations of Saccharomyces cerevisiae propagated in glucose-limited medium in batch cultures for 5000 generations. The yield of single clones isolated from the haploid lineages, measured as both optical and population density at the end of a growth cycle, declined during selection and was negatively correlated with growth rate. Initially, diploid populations did not pay this cost of adaptation but haploidized after about 1000–3000 generations of selection, and this ploidy transition was associated with a decline in yield caused by reduced cell size. These results demonstrate the experimental evolution of a trade-off between growth rate and yield, caused by antagonistic pleiotropy, during adaptation in haploids and after an adaptive transition from diploidy to haploidy.     

Evolution of Escherichia coli during Growth in a Constant Environment

Populations of Escherichia coli, initiated with a single clone and maintained for long periods in glucose-limited continuous culture, developed extensive polymorphisms. In one population, examined after 765 generations, two majority and two minority types were identified. Stable mixed populations were reestablished from the isolated strains. Factors involved in the development of this polymorphism included differences in the maximum specific growth rate and in the transport of glucose, and excretion of metabolites by some clones which were utilized by minority clones.     

The effect of the dilution rate on CHO cell physiology and recombinant interferon-gamma production in glucose-limited chemostat culture

The physiology of a recombinant Chinese hamster ovary cell line in glucose-limited chemostat culture was studied over a range of dilution rates (D = 0.008 to 0.20 h(-1)). The specific growth rate (mu) deviated from D at low dilution rates due to an increased specific death rate. Extrapolation of these data suggested a minimum specific growth rate of 0.011 h(-1) (mu(max) = 0.025 h(-1)) The metabolism at each steady state was characterized by determining the metabolic quotients for glucose, lactate, ammonia, amino acids, and interferon-gamma (IFN-gamma). The specific rate of glucose uptake increased linearly with mu, and the saturation constant for glucose (K(s)) was calculated to be 59.6 muM. There was a linear increase in the rate of lactate production with a higher yield of lactate from glucose at high growth rates. The decline in the rate of production of lactate, alanine, and serine at low growth rate was consistent with the limitation of the glycolytic pathway by glucose. The specific rate of IFN-gamma production increased with mu in a manner indicative of a growth-related product. Despite changes in the IFN-gamma production rate and cell physiology, the pattern of IFN-gamma glycosylation was similar at all except the lowest growth rates where there was increased production of nonglycosylated IFN-gamma. (c) 1993 John Wiley & Sons, Inc.     

Microbial Evolution in a Simple Unstructured Environment: Genetic Differentiation in Escherichia Coli

Populations of Escherichia coli initiated with a single clone and maintained for long periods in glucose-limited continuous culture, become polymorphic. In one population, three clones were isolated and by means of reconstruction experiments were shown to be maintained in stable polymorphism, although they exhibited substantial differences in maximum specific growth rates and in glucose uptake kinetics. Analysis of these three clones revealed that their stable coexistence could be explained by differential patterns of the secretion and uptake of two alternative metabolites acetate and glycerol. Regulatory (constitutive and null) mutations in acetyl-coenzyme A synthetase accounted for different patterns of acetate secretion and uptake seen. Altered patterns in glycerol uptake are most likely explained by mutations which result in quantitative differences in the induction of the glycerol regulon and/or structural changes in glycerol kinase that reduce allosteric inhibition by effector molecules associated with glycolysis. The evolution of resource partitioning, and consequent polymorphisms which arise may illustrate incipient processes of speciation in asexual organisms.     

Hexose uptake and control of fibroblast proliferation

The role of glucose uptake in control of cell growth was studied by experimentally varying the rate of glucose uptake and examining the subsequent effect on initiation and cessation of cell proliferation. The rate of glucose uptake was varied by adjusting the concentration of glucose in the culture medium. This permitted analysis of two changes in rate of glucose uptake which are closely related to the regulation of cell growth: (1) the rapid increase in glucose uptake that can be detected within several minutes after mitogenic stimulation of quiescent fibroblasts and (2) the decrease in glucose uptake which accompanies growth to a quiescent state. Quiescent cultures of mouse 3T3, human diploid foreskin and secondary chick embryo cells were switched to fresh serum-containing medium with either the normal amount of glucose or a reduced level that lowered the rate of glucose uptake below the rate characteristic of quiescent control cells. The subsequent increases in cell number were equal in both media, demonstrating that the increase in glucose uptake, commonly observed after mitogenic stimulation, was not necessary for initiation of cell division. Measurements of intracellular D-glucose pools after serum stimulation of quiescent cells revealed that the increase in glucose uptake was not accompanied by a detectable change in the intracellular concentration of glucose. Nonconfluent growing cultures of mouse 3T3, human diploid foreskin and secondary chick embryo cells were switched to low glucose media, lowering the rate of glucose uptake below levels observed for quiescent cells. This did not affect rates of DNA synthesis or cell division over a several-day period. Thus, the decrease in glucose uptake, which usually occurs at about the same time as the decrease in DNA synthesis as cells grow to quiescence, does not cause the decline in cell proliferation. Experiments indicated that there was no set temporal relationship between the decline in glucose uptake and DNA synthesis as cells grew to quiescence. The sequence was variable and probably depended on the cell type as well as culture conditions. Measurements of intracellular D-glucose pools in secondary chick embryo cells demonstrated that the internal concentration of glucose in these cells did not significantly vary during growth to quiescence. Taken together, our results show that these fluctuations in the rate of glucose uptake do not lead to detectable changes in the intracellular concentration of glucose and that they do not control cell proliferation rates under usual culture conditions.     

Identification of adaptive changes in an evolving population of Escherichia coli: the role of changes with regulatory and highly pleiotropic effects

A population of Escherichia coli initiated with a single clone developed extensive morphological and physiological polymorphism after being maintained for 773 generations in glucose-limited continuous culture. To understand the mechanisms of adaptation to this environment, total protein patterns of four adaptive clones and of the parent strains were examined by two-dimensional gel electrophoresis. Approximately 20% of the proteins (approximately 160 in absolute numbers) showed significantly different levels of expression in pairwise comparisons of parent and adapted clones. The extent of these changes points to the importance of mutations with regulatory and/or highly pleiotropic effects in the adaptive process. The four evolved clones all expressed fewer proteins than did the parent strain, supporting the hypothesis of energy conservation during evolutionary change. Forty-two proteins that could be assigned to known cellular functions were identified. The changes in some of them indicated that the evolved clones developed different adaptive mechanisms to glucose-limited environment. Changes were observed in the expression levels of proteins associated with translation, membrane composition, shock response, and active transport. A fraction of the changes could not be either explained or predicted from a consideration of the nature of the environment in which the clones evolved.     

The generation of multiple co-existing mal-regulatory mutations through polygenic evolution in glucose-limited populations of Escherichia coli

The multicomponent glucose transport system of Escherichia coli was used to study the polygenic basis of increased fitness in prolonged nutrient-limited, continuous cultures. After 280 generations of glucose-limited growth, nearly all bacteria in four independent chemostat populations exhibited increased glucose transport and contained multiple, stable mutations. Fitter bacteria increased outer membrane permeability for glucose through overexpression of the LamB glycoporin. Three classes of mutation influenced LamB levels as well as regulation of other mal genes. Low-level mal/lamB constitutivity resulted from mlc mutations acquired in all populations as well as changes at another uncharacterized locus. Larger increases in transporter content resulted from widespread acquisition of a regulatory malT–con mutation in fit isolates. The malT mutations sequenced from 67 adapted isolates were all single base substitutions resulting in amino acid replacements in the N-terminal third of the MalT activator protein. Analysis of malT–con sequences revealed a mutational spectrum distinct from that found in plate-selected malT mutants, suggesting that mutational pathways were affected by environmental factors. A second major finding was the remarkable allele diversity in malT within a population derived from a single clone, with at least 11 different alleles co-existing in a population. The multiplicity of alleles (as well as those found in adaptive mgl changes in the accompanying study) suggest that the periodic selection events observed previously in such populations are not a major factor in reducing genetic diversity. A simple model is presented for the generation of genetic heterogeneity in bacterial populations undergoing polygenic selection.     

Transient Responses of Glucose-Limited Cultures of Cytophaga johnsonae to Nutrient Excess and Starvation

Cells from glucose-limited chemostat cultures of Cytophaga johnsonae were subjected to a sudden relaxation of substrate limitation by injecting the cells into fresh batch cultures. Starvation experiments were carried out by injecting glucose-limited cells into batch cultures lacking glucose. Transient responses of biomass, glucose uptake and mineralization, ATP content, and viability on different agar media were monitored during these nutrient-shift experiments. Cells reacted differently depending on growth rate and time spent in the chemostat. Fast-growing cells showed an immediate adaptation to the new growth conditions, despite some initial overshoot reactions in ATP and uptake potential. In contrast, slowly growing cells and long-term-adapted cells showed extensive transient growth responses. Glucose uptake and mineralization potentials changed considerably during the transient growth phase before reaching new levels. During the starvation experiments, all cell types displayed a fast decrease in ATP, but the responses of the substrate uptake and mineralization potentials were strongly dependent upon the previous growth rate. Both potentials decreased rapidly in cells with high growth rates. On the other hand, cells with low growth rates maintained 80% of their uptake and mineralization potentials after 8 h of starvation. Thus, slowly growing cells are much better adapted for starvation than are fast-growing cells.     

Adaptive mgl-regulatory mutations and genetic diversity evolving in glucose-limited Escherichia coli populations

The mutational adaptation of E. coli to low glucose concentrations was studied in chemostats over 280 generations of growth. All members of six independent populations acquired increased fitness through the acquisition of mutations at the mgl locus, increasing the binding protein-dependent transport of glucose. These mutations provided a strong fitness advantage (up to 10-fold increase in glucose affinity) and were present in most isolates after 140 generations. mgl constitutivity in some isolates was caused by base substitution, short duplication, small deletion and IS1 insertion in the 1041 bp gene encoding the repressor of the mgl system, mglD (galS). But an unexpectedly large proportion of mutations were located in the short mgl operator sequence (mglO), and the majority of mutations were in mglO after 280 generations of selection. The adaptive mglO substitutions in several independent populations were at exactly the positions conserved in the two 8 bp half-sites of the mgl operator, with the nature of the base changes also completely symmetrical. Either mutations were directed to the operator or the particular operator mutations had a selective advantage under glucose limitation. Indeed, isolates carrying mglO mutations showed greater rates of transport for glucose and galactose at low concentrations than those carrying mglD null mutations. mglO mutations avoid cross-talk by members of the GalR-Lacl repressor family, reducing transporter expression and providing a competitive advantage in a glucose-limited environment. Another interesting aspect of these results was that each adapted population acquired multiple mgl alleles, with several populations containing at least six different mgl-regulatory mutations co-existing after 200 generations. The diversity of mutations in the mglO/mglD region, generally in combination with mutations at other loci regulating glucose uptake (malT, mlc, ptsG), provided evidence for multiple clones in each population. Increased fitness was accompanied by the generation of genetic diversity and not the evolution of a single winner clone, as predicted by the periodic selection model of bacterial populations.     

Xylose and Glucose Utilization by Bacteroides xylanolyticus X5-1 Cells Grown in Batch and Continuous Culture

During cultivation on a mixture of xylose and glucose, Bacteroides xylanolyticus X5-1 showed neither diauxic growth nor a substrate preference. Xylose-limited continuous-culture cells were able to consume xylose and glucose both as single substrates and as mixed substrates without any lag phase. When glucose was the growth-limiting substrate, the microorganism was unable to consume xylose. However, in the presence of a small amount of glucose or pyruvate, xylose was utilized after a short lag phase. In glucose-limited cells, xylose isomerase was present at low activity but xylulose kinase activity could not be detected. On addition of a mixture of xylose and glucose, xylose isomerase was induced immediately and xylulose kinase was induced after about 30 min. The induction of the two enzymes was sensitive to chloramphenicol, showing de novo synthesis. Xylose uptake in glucose-grown cells was very low, but the uptake rate could be increased when incubated with a xylose-glucose mixture. The increase in the uptake rate was not affected by chloramphenicol, indicating that a constitutive uptake system had to be activated. The inability of B. xylanolyticus X5-1 cells undergoing glucose-limited continuous culture to induce the xylose catabolic pathway after the addition of only xylose probably was caused by energy limitation.     

Defined media optimization for growth of recombinant Escherichia coli X90

An optimized, defined minimal medium was developed to support balanced growth of Escherichia coli X90 harboring a recombinant plasmid. Foreign protein expression was repressed in these studies. A pulse injection technique was used to identify the growth responses to nutrients in a chemostat. Once the nutrients essential for growth had been identified, the yield coefficients for individual medium components. These yield coefficients were used to develop an optimized, glucose-limited defined minimal medium that supports balanced cell growth in chemostat culture. The biomass and substrate concentrations follow the Monod chemostat model. The maximum specific growth rate determined in a washout experiment is 0.87 h(-1) for this strain in the optimized medium. the glucose yield factor is 0.42 g DCW/g glucose and the maintenance coefficient is zero in the glucose-limited chemostat culture. (c) 1993 John Wiley & Sons, Inc.     

Kinetics of substrate utilization in adenine-limited chemostat cultures of Bacillus subtilis KYA741.

The specific rates of limiting substrate utilization were investigated in adenine- or glucose-limited chemostat cultures of Bacillus subtilis KYA741, an adenine-requiring strain, at 37 degrees C. With the glucose-limited cultures, the specific rate of glucose consumption versus dilution rate gave a linear relationship from which the true growth yield and maintenance coefficient were determined to be 0.09 mg of bacteria per mg of glucose and 0.2 mg of glucose per mg of bacteria per h, respectively. With the adenine-limited cultures, adenine as the limiting substrate was not completely consumed at lower dilution rates (e.g., D less than 0.1), unlike in the glucose-limited cultures. When a linear relationship of specific rate of adenine consumption versus dilution rate was extrapolated to zero dilution rate, a negative value for the specific rate of adenine consumption, -0.01 mg of adenine per mg of bacteria per h, was obtained, giving a true growth yield for adenine of 5.2 mg of bacteria per mg of adenine. On the other hand, the maintenance coefficient of oxygen uptake gave a positive value of 8.1 x 10(-3) mmol/mg of bacteria per h. Based on previous results showing that adenine is resupplied by lysing cells, we developed kinetic models of adenine utilization and cell growth that gave a good estimation of the peculiar behavior of cell growth and adenine utilization in adenine-limited chemostat cultures.     

Multiple duplications of yeast hexose transport genes in response to selection in a glucose-limited environment

When microbes evolve in a continuous, nutrient-limited environment, natural selection can be predicted to favor genetic changes that give cells greater access to limiting substrate. We analyzed a population of baker's yeast that underwent 450 generations of glucose-limited growth. Relative to the strain used as the inoculum, the predominant cell type at the end of this experiment sustains growth at significantly lower steady-state glucose concentrations and demonstrates markedly enhanced cell yield per mole glucose, significantly enhanced high-affinity glucose transport, and greater relative fitness in pairwise competition. These changes are correlated with increased levels of mRNA hybridizing to probe generated from the hexose transport locus HXT6. Further analysis of the evolved strain reveals the existence of multiple tandem duplications involving two highly similar, high- affinity hexose transport loci, HXT6 and HXT7. Selection appears to have favored changes that result in the formation of more than three chimeric genes derived from the upstream promoter of the HXT7 gene and the coding sequence of HXT6. We propose a genetic mechanism to account for these changes and speculate as to their adaptive significance in the context of gene duplication as a common response of microorganisms to nutrient limitation.      

Biochemical Properties of Haploid and Diploid Strains of Penicillium chrysogenum

An intensive parasexual genetics program in which industrial strains of Penicillium chrysogenum were used culminated in the isolation of a number of heterozygous diploid strains. The diploid clones were selected from heterokaryons formed from matings between mutant strains having complementary biochemical and conidial color markers. Several diploid cultures were compared with their haploid wild-type parents and other distantly related production strains on the basis of a variety of cultural and physiological criteria. The diploid strains characteristically produced conidia of larger volume and higher deoxyribonucleic acid content. Some were vigorous with respect to growth rate and onset and degree of conidiation. One diploid strain (WC-9) had a 46% greater oxygen uptake rate and oxidized glucose at a 57% greater rate than its haploid parent (M-2). It also produced 33% higher concentrations of β-galactosidase, 66% more alkaline protease, and 53% more glucose oxidase than the M-2 haploid parent. The selection of rare stable diploid mold cultures through the use of parasexual genetics offers a unique approach to the direct selection of mutants with potential for increased enzyme formation.     

Escherichia coli responds with a rapid and large change in growth rate upon a shift from glucose-limited to glucose-excess conditions

Glucose pulse experiments at seconds time scale resolution were performed in aerobic glucose-limited Escherichia coli chemostat cultures. The dynamic responses of oxygen-uptake and growth rate at seconds time scale were determined using a new method based on the dynamic liquid-phase mass balance for oxygen and the pseudo-steady-state ATP balance. Significant fold changes in metabolites (10-1/10) and fluxes (4-1/4) were observed during the short (200 s) period of glucose excess. During glucose excess there was no secretion of by-products and the increased glucose uptake rate led within 40s to a 3.7 fold increase in growth rate. Also within 40-60s a new pseudo-steady-state was reached for both metabolite levels and fluxes. Flux changes of reactions were strongly correlated to the concentrations of involved compounds. Surprisingly the 3.7 fold increase in growth rate and hence protein synthesis rate was not matched by a significant increase in amino acid concentrations. This poses interesting questions for the kinetic factors, which drive protein synthesis by ribosomes.     

Kinetic study of hybridoma cell growth in continuous culture. I. A model for non-producing cells

The kinetic behavior of a nonproducing hybridoma clone AFP-27-NP was investigated in continuous culture under glucose-limited conditions. A total of more than 21, 000 h of cultures were operated at dilution rates ranging from 0.01 to 0.06 h(-1). The viable cell concentrations, dead cell concentrations, and cell volumes all varied with the dilution rate. A steady-state model was developed based on the biomass concentration and the glucose concentration. The specific growth rate as a function of glucose concentration is described by a model similar to the Monod model with a threshold glucose concentration and a minimum specific growth rate incorporated; the model is meaningful only at glucose concentrations and specific growth rates above these levels. A death rate is included in the model which is described by an inverted Monod-type function of glucose concentration. The yield coefficient based on glucose is constant in the lower range of specific growth rates and changes to a new constant value in the upper region of specific growth rates. No maintenance term for glucose consumption was needed; in the plot of specific glucose consumption rate vs. specific growth rate, the line intercepted the specific growth rate axis at a value close to the minimum growth rate. The values for the model parameters were determined from regression analysis of the steady-state data. The model predictions and experimental results fit very well.     

Modelling of the protonophoric uncoupling by phenoxyacetic acid of the plasma membrane potential of Penicillium chrysogenum

Physiological effects of phenoxyacetic acid, the penicillin V side-chain precursor, on steady-state continuous cultures of Penicillium chrysogenum have been studied both theoretically and experimentally. Theoretical calculations show that at an extracellular pH of 6.50, phenoxyacetic acid has negligible influence on the growth energetics due to protonophoric uncoupling of membrane potentials by passive diffusive uptake. On the other hand, when the extracellular pH is lowered to 5.00, a severe maintenance-related uncoupling effect of phenoxyacetic acid is calculated. These findings were confirmed experimentally by steady-state continuous cultivations with a high-yielding penicillin strain of P. chrysogenum performed on a chemically defined and glucose-limited medium at pH 6.50 and pH 5.00, both with and without phenoxyacetic acid present. The yield and maintenance coefficients were determined from steady-state measurements of the specific uptake rates of glucose and oxygen and the specific production rate of carbon dioxide as functions of the specific growth rate. Combining these data with a simple stoichiometric model for the primary metabolism of P. chrysogenum allows quantitative information to be extracted on the growth energetics in terms of ATP spent in maintenance- and growth-related processes, i.e. mATP and YxATP. The increased maintenance-related ATP consumption when adding phenoxyacetic acid at pH 5.00 agrees with the theoretical calculations on the uncoupling effect of phenoxyacetic acid. When YxATP is compared with earlier reported values for the theoretical ATP requirement for biosynthesis of P. chrysogenum, i.e. YxATP, growth, it is found that YxATP,growth is only 40-50% of YxATP, which stresses that a large amount of ATP is wasted in turnover of macromolecules, leaks, and futile cycles.     

The role of energy-spilling reactions in the growth of Klebsiella aerogenes NCTC 418 in aerobic chemostat culture.

When cell-saturating amounts of glucose and phosphate were added to steady state cultures of Klebsiella aerogenes that were, respectively, glucose- and phosphate-limited, the organisms responded immediately with an increased oxygen consumption rate. This suggested that in neither case was glucose transport the rate-limiting process, and also that organisms must possess effective mechanisms for spilling the excess energy initially generated when a growth-limitation is temporarily relieved. Steady state cultures of mannitol- or glucose-limited organisms also seemingly generated energy at a greater rate than was required for cell synthesis since gluconate-limited cultures consumed oxygen at a lower rate, at each corresponding growth rate, than did mannitol- or glucose-limited cultures, and therefore expressed a higher YO value. Thus, mannitol- and glucose-limitations must be essentially carbon (and not energy) limitations. The excess energy generated by glucose metabolism is one component of "maintenance" and could be used at lower growth rates to maintain an increased solute gradient across the cell membrane, imposed by the addition of 2%, w/v, NaCl to the growth environment. The maintenance rates of oxygen consumption of K. aerogenes also could be caused to increase by adding glucose discontinuously (drop-wise) to a glucose-limited chemostat culture, or by exchanging nitrate for ammonia as the sole utilizable nitrogen source. The significance of these findings to an assessment of the physiological factors circumscribing energy-spilling reactions in aerobic cultures of K. aerogenes is discussed.     

Kinetic study of hybridoma cell growth in continuous culture: II. Behavior of producers and comparison to nonproducers

A hybridoma cell line, AFP-27-P, was cultivated in continuous culture under glucose-limited conditions. The viable cell concentration, dead-cell concentration, and cell volume all varied with the dilution rate. A model previously developed for a nonproducing clone of the same cell line, AFP-27-NP, was extended to describe the behavior of the cells. The relationship between the specific growth rate and glucose concentration is described by a function similar to the Monod model. A threshold glucose concentration and a minimum specific growth rate are incorporated; the model is meaningful only at glucose concentration and a minimum specific growth rate are incorporated; the model is meaningful only at glucose concentrations and specific growth rates above these levels. The relationship between the death rate and the glucose concentration is described by an inverted Monod-type function. Furthermore, the yield coefficient based on glucose is constant in the lower range of specific growth rates and changes to a new constant value in the upper range of specific growth rates. No maintenance term for glucose consumption is used; in the plot of specific glucose consumption rate vs. specific growth rate, the line intercepts the specific growth rate at a value close to the minimum growth rate. The productivity of antibody as a function of the specific growth rate is described by a mixed type model with a noon-growth-associated term and a negative-growth-associated term. The values for the model parameters were determined from regression analysis of the steady state data.     

Adaptation of Salmonella typhimurium mutants containing uncoupled enzyme IIGlc to glucose-limited conditions.

Uncoupled enzyme IIGlc of the phosphoenolpyruvate (PEP):glucose phosphotransferase system (PTS) in Salmonella typhimurium is able to catalyze glucose transport in the absence of PEP-dependent phosphorylation. As a result of the ptsG mutation, the apparent Km of the system for glucose transport is increased about 1,000-fold (approximately 18 mM) compared with wild-type PTS-mediated glucose transport. An S. typhimurium mutant containing uncoupled enzyme IIGlc as the sole system for glucose uptake was grown in glucose-limited chemostat cultures. Selective pressure during growth in the chemostat resulted in adaptation to the glucose-limiting conditions in two different ways. At first, mutations appeared that led to a decrease in Km value of uncoupled enzyme IIGlc. These results suggested that uncoupled enzyme IIGlc had significant control on the growth rate under glucose-limiting conditions. More efficient glucose uptake enabled a mutant to outgrow its parent and caused a decrease in the steady-state glucose concentration in the chemostat. At very low glucose concentrations (10 microM), mutants arose that contained a constitutively synthesized methyl-beta-galactoside permease. Apparently, further changes in the uncoupled enzyme IIGlc did not lead to a substantial increase in growth rate at very low glucose concentrations.