The amino acid sensitive TOR pathway from yeast to mammals

The target of rapamycin (TOR) is an ancient effector of cell growth that integrates signals from growth factors and nutrients. Two downstream effectors of mammalian TOR, the translational components S6K1 and 4EBP1, are commonly used as reporters of mTOR activity. The conical signaling cascade initiated by growth factors is mediated by PI3K, PKB, TSC1/2 and Rheb. However, the process through which nutrients, i.e., amino acids, activate mTOR remains largely unknown. Evidence exists for both an intracellular and/or a membrane bound sensor for amino acid mediated mTOR activation. Research in eukaryotic models, has implicated amino acid transporters as nutrient sensors. This review describes recent advances in nutrient signaling that impinge on mTOR and its targets including hVps34, class III PI3K, a transducer of nutrient availability to mTOR.     

General approach to bacterial nutrition: growth factor requirements of Moraxella nonliquefaciens.

A general procedure was devised for the determination of growth factor requirements of heterotrophic bacteria based upon identification of individual nutrients as they are successively depleted from a limited quantity of complex medium. By using this approach, it was possible to develop a defined medium for growth of Moraxella nonliquefaciens that contained nine amino acids and three vitamins. Three of the amino acids, proline, serine, and cysteine, were required in unusually high concentrations to obtain optimal growth. Methionine had a sparing action on the requirements for serine and cysteine. Glycine could substitute for serine. Although a required nutrient, cysteine was inhibitory for growth, but this inhibitory action was antagonized by valine or leucine. The requirement for cysteine was satisfied by cystine, glutathione, or sodium sulfide. M. nonliquefaciens could not use ammonia as a nitrogen source but could use glutamate or aspartate for this purpose. With the exception of 1 auxotrophic strain, the growth factor requirements of 23 independently isolated strains of M. nonliquefaciens were essentially the same.     

Adequacy of soil testing for predicting fertilizer requirements

Summary Routine soil-testing methods are excellent tools for monitoring soil fertility. However, if used for predicting optimal fertilizer requirements they will frequently fail. This applies especially to soils having high fertility levels needed for commercial farming with many other factors affecting plant growth. Explanatory examples are given for crop-specific nutrient requirements, for nutrient transport within the rhizosphere, for nutrient supply from subsoil and for interactions of nutrients. Soil-testing methods must become more comprehensive with regard to the number of nutrients analysed and to the rates of nutrient supply and the magnitudes of nutrient reserves.     

Improving amino acid nutrition to prevent intrauterine growth restriction in mammals

Intrauterine growth restriction (IUGR) is one of the most common concerns in human obstetrics and domestic animal production. It is usually caused by placental insufficiency, which decreases fetal uptake of nutrients (especially amino acids) from the placenta. Amino acids are not only building blocks for protein but also key regulators of metabolic pathways in fetoplacental development. The enhanced demands of amino acids by the developing conceptus must be met via active transport systems across the placenta as normal pregnancy advances. Growing evidence indicates that IUGR is associated with a reduction in placental amino acid transport capacity and metabolic pathways within the embryonic/fetal development. The positive relationships between amino acid concentrations in circulating maternal blood and placental amino acid transport into fetus encourage designing new therapies to prevent or treat IUGR by enhancing amino acid availability in maternal diets or maternal circulation. Despite the positive effects of available dietary interventions, nutritional therapy for IUGR is still in its infancy. Based on understanding of the underlying mechanisms whereby amino acids promote fetal growth and of their dietary requirements by IUGR, supplementation with functional amino acids (e.g., arginine and glutamine) hold great promise for preventing fetal growth restriction and improving health and growth of IUGR offspring.     

A MAP4 kinase related to Ste20 is a nutrient-sensitive regulator of mTOR signalling

The mTOR (mammalian target of rapamycin) signalling pathway is a key regulator of cell growth and is controlled by growth factors and nutrients such as amino acids. Although signalling pathways from growth factor receptors to mTOR have been elucidated, the pathways mediating signalling by nutrients are poorly characterized. Through a screen for protein kinases active in the mTOR signalling pathway in Drosophila we have identified a Ste20 family member (MAP4K3) that is required for maximal S6K (S6 kinase)/4E-BP1 [eIF4E (eukaryotic initiation factor 4E)-binding protein 1] phosphorylation and regulates cell growth. Importantly, MAP4K3 activity is regulated by amino acids, but not the growth factor insulin and is not regulated by the mTORC1 inhibitor rapamycin. Our results therefore suggest a model whereby nutrients signal to mTORC1 via activation of MAP4K3.     

New insights in insect prey choice by chimpanzees and gorillas in southeast Cameroon: the role of nutritional value

The insect diet of chimpanzees and gorillas living at the northern periphery of the Dja Biosphere Reserve in southeast Cameroon and its nutritional contribution is described. We analyzed fecal samples and recorded additional evidence of insectivory. A detailed prey species list is presented for both apes. We carried out nutritional analyses (macronutrients, macro- and micro-minerals) on 11 important and eight nonimportant, but accessible, ant and termite prey species, and estimated the average nutrient intake/day through insects. Although gorillas ate insects more frequently, the average prey biomass intake/day by chimpanzees was twice that by gorillas. The lack of tool-use by gorillas cannot be the main reason for the small overlap of important prey species. Both apes did not seem to consume ant prey for one or more specific nutrients. Also other factors, such as medicinal use, should be considered. Termites, on the other hand, seemed to be selected for particular nutrients. Gorilla intake of the important termite prey, Cubitermes and Thoracotermes, met with estimated iron requirements. Their potential role as antidiarrheal treatment is as yet unclear. Chimpanzee intake of the important termite prey, Macrotermes spp., met with estimated manganese requirements and the protein intake/day (mean: 2 g/d) reached significant values (>20 g/d). To fully understand the importance of nutritional contributions of insects to ape diets in Cameroon, the chemical composition and nutrient intake of fruit and foliage in their diets should be investigated.     

Multiple-carbon-source-limited growth kinetics of a marine coryneform bacterium.

The steady-state growth rate of a marine isolate was related to the concentrations of several carbon and energy source substrates when these substrates limited growth simultaneously in continuous culture. Glucose limitation was characterized by a threshold of 0.21 mg/liter for growth, a half-maximal growth rate at 0.48 mg/liter, U-shaped curves in extractable pool concentration-versus-growth velocity plots, and slow maximal growth rates. Arginine addition reduced the glucose threshold to 0.008 mg/liter, more than doubled the maximal growth rate, and stabilized pool concentrations at low growth rates. Addition of a third substrate, glutamate, caused further reduction of the glucose concentration a steady state. Maximal reduction of the glucose concentration was effected by adding a mixture of 20 amino acids. Steady-state limiting nutrient concentration was dependent on the specific identity of the auxiliary nutrients and on the concentration ratio at which they were supplied. When glucose was supplemented with an equal quantity of an amino acid mixture, the external steady-state glucose remained below 10 mug/liter. When 1 mug of glucose was added to a 2.5-mg/liter amino acid mixture, at least 70% of it was consumed at steady state in spite of the threshold observed. Lack of crossover between metabolic pathways, suggested by the absence of glucose carbon in pool glutamate of arginine-glucose-grown cells, may have been partly responsible for the mixed carbon source stimulation of nutrient accumulation observed. The affinity observed is sufficient to account for normal growth at a total organic substrate concentration of only 0.11 mg/liter when supplied from a suitable mixture.     

Modeling the bacterial contribution to planktonic community respiration in the regulation of solar energy and nutrient availability

In planktonic ecosystems, algae and bacteria exhibit complex interrelationships, as algae provide an important organic matter source for microbial growth while microbial metabolism recycles limiting nutrients for algae in a loose commensalism. However, algae and bacteria can also compete for available nutrients if supplies of organic matter are sufficient to satisfy bacterial demand. We developed a stoichiometrically explicit model of bacteria–algae interactions that incorporated realistic assumptions about algal light and nutrient utilization, algal exudation of organic matter, and bacterial processing of organic matter and nutrients. The model makes specific predictions about how the relative balance of algae and bacteria should change in response to varied nutrient and light availability seen in lakes and oceans. The model successfully reproduces published empirical data and indicates that, under moderate nutrient supply, the bacterial percentage of total respiration should be maximal at intermediate light intensity.     

Prediction of effects of amino acid supplementation on growth of E. coli B/r

A mathematical model for the growth of a single cell of E. coli on medium containing amino acid is presented. A mixture of purified amino acids (glutamate, aspartate, serine, tyrosine, and leucine) combined in the ratios found in a natural digest (casein) were employed as the nitrogen source. Each of these amino acids is the representative of a different family of amino acids. The transport mechanisms and assimilation routes for each amino acid were inserted into the prototype model. The enzyme activities and saturation constants used in the model were based on literature data. The maximum velocities for uptake systems were calculated from experimental data. The formation and homeostasis of amino acid pools were regulated through cross-control of the activities of biosynthetic enzymes and of membrane transport of exogenous nutrients. The size of each amino acid pool was determined with mass balance equations that included terms for a transport system, a biosynthesis system, a transaminase enzyme system for interchange between the amino acid families, and a consumption system. The predictions of the extended model with regard to nutrient concentrations and growth rates compared well with the experimental data.     

Nutrient regulation in the pea aphid Acyrthosiphon pisum: application of a novel geometric framework to sugar and amino acid consumption

Abstract. A recently developed framework was applied to investigate the responses of newborn pea aphids, Acyrthosiphon pisum (Harris) (Homoptera: Aphididae), to simultaneous variations in dietary suerose and amino acid levels. The location of functional 'targets' for intake and growth were determined experimentally using performance criteria. Behavioural rules employed by insects to approach these targets were investigated by reference to the geometry of arrays of intake and growth across a range of diets. First-instar aphids were fed one of thirty-one chemically-defined diets ranging in sucrose concentration from 200 to 1000 mM and amino acid concentration from 25 to 250 mM. Insect survivorship, dry weight, protein- and carbohydrate-derived growth were high for all diets except those with the lowest nutrient levels. Peaks in final dry weight and protein content identified the intake target as the point reached by larvae fed the 600 mM sucrose, 75 mM amino acid diet. Aphids regulated sucrose primarily by consumption (i.e. behaviourally), whereas amino acids were regulated post-ingestively (i.e. by physiological mechanisms).     

Control of the Balb/c-3T3 cell cycle by nutrients and serum factors: analysis using platelet-derived growth factor and platelet-poor plasma.

Much controversy regarding the relationship between nutrients and serum in regulation of cell growth can be reconciled by recognizing that serum contains multiple factors which regulate different events in the cell cycle. Serum was fractionated into a platelet-derived growth factor (PDGF), which induces cells to become competent to synthesize DNA, and plasma which allows competent cells to traverse G0/G1 and enter the S phase. Nutrients are not required for the cellular response to PDGF; however amino acids are required for plasma to promote the entry of PDGF-treated, competent cells into S phase. The nutrient independent, PDGF-modulated, growth regulatory event (competence) is located 12 hours prior to the G1/S phase boundary in quiescent, density-arrested Balb/c-3T3 cells. The nutrient dependent, plasma-modulated event is located six hours prior to the G1/S phase boundary and corresponds in concentration of amino acids required for DNA synthesis. Infection of density-arrested Balb/c3T3 cells with SV40 overrides both the nutrient independent and the nutrient dependent growth regulatory events.     

Additive and interactive effects of nutrient classes on longevity, reproduction, and diet consumption in the Queensland fruit fly (Bactrocera tryoni)

Insect lifespan is often closely linked to diet, and diet manipulations have been central to studies of ageing. Recent research has found that lifespan for some flies is maximised on a very low yeast diet, but once all yeast is removed, lifespan drops precipitously. Although effects of yeast availability on lifespan are commonly interpreted in terms of protein, yeast is a complex mix of nutrients and provides a rich source of vitamins, minerals and sterols. Elucidating which components of yeast are involved in this lifespan drop provides insights into more specific nutritional requirements and also provides a test for the commonplace interpretation of yeast in terms of protein. To this end, we fed Queensland fruit flies (Bactrocera tryoni) one of eight experimental diets that differed in the nutrient group(s) found in yeast that were added to sucrose: none, vitamins, minerals, amino acids, cholesterol, vitamin+mineral+cholesterol (VMC), vitamin+mineral+cholesterol+amino acids (VMCA), and yeast. We measured survival rates and egg production in single sex and mixed sex cages, as well as nutrient intake of individual flies. We found that the addition of minerals increased lifespan of both male and female flies housed in single sex cages by decreasing baseline mortality. The addition of just amino acids decreased lifespan in female flies; however, when combined with other nutrient groups found in yeast, amino acids increased lifespan by decreasing both baseline mortality and age-specific mortality. Flies on the yeast and VMCA diets were the only ones to show significant egg production. We conclude that the drop in lifespan observed when all yeast is removed is explained by missing micronutrients (vitamins, minerals and cholesterol) as well as the absence of protein in females, whereas minerals alone can explain the pattern for males. These results indicate a need for caution when interpreting effects of dietary yeast as effects of protein.     

Regulation of development in Dictyostelium discoideum: I. Initiation of the growth to development transition by amino acid starvation

Evidence is presented which indicates that amino acid starvation is the specific stimulus initiating the developmental phase of the life cycle of Dictyostelium discoideum: (i) Amoebae were washed free of complex growth medium and placed in buffer supplemented with specific nutrients; amino acids were the only nutrients that specifically inhibited the initiation of development. (ii) A partially defined growth medium allowing selective starvation for amino acids or glucose during growth is described. Amoebae initiated development only when starved for amino acids. Any effect of glucose on the primary control of the initiation of development is an indirect result of its utilization as a source of precursors for endogenously synthesized amino acids.     

Impact of plant nutrients on the relationship between a herbivorous insect and its symbiotic bacteria

The interactions between herbivorous insects and their symbiotic micro-organisms can be influenced by the plant species on which the insects are reared, but the underlying mechanisms are not understood. Here, we identify plant nutrients, specifically amino acids, as a candidate factor affecting the impact of symbiotic bacteria on the performance of the phloem-feeding aphid Aphis fabae. Aphis fabae grew more slowly on the labiate plant Lamium purpureum than on an alternative host plant Vicia faba, and the negative effect of L. purpureum on aphid growth was consistently exacerbated by the bacterial secondary symbionts Regiella insecticola and Hamiltonella defensa, which attained high densities in L. purpureum-reared aphids. The amino acid content of the phloem sap of L. purpureum was very low; and A. fabae on chemically defined diets of low amino acid content also grew slowly and had elevated secondary symbiont densities. It is suggested that the phloem nutrient profile of L. purpureum promotes deleterious traits in the secondary symbionts and disturbs insect controls over bacterial abundance.     

Predicting the profile of nutrients available for absorption: from nutrient requirement to animal response and environmental impact

Current feed evaluation systems for dairy cattle aim to match nutrient requirements with nutrient intake at pre-defined production levels. These systems were not developed to address, and are not suitable to predict, the responses to dietary changes in terms of production level and product composition, excretion of nutrients to the environment, and nutrition related disorders. The change from a requirement to a response system to meet the needs of various stakeholders requires prediction of the profile of absorbed nutrients and its subsequent utilisation for various purposes. This contribution examines the challenges to predicting the profile of nutrients available for absorption in dairy cattle and provides guidelines for further improved prediction with regard to animal production responses and environmental pollution.The profile of nutrients available for absorption comprises volatile fatty acids, long-chain fatty acids, amino acids and glucose. Thus the importance of processes in the reticulo-rumen is obvious. Much research into rumen fermentation is aimed at determination of substrate degradation rates. Quantitative knowledge on rates of passage of nutrients out of the rumen is rather limited compared with that on degradation rates, and thus should be an important theme in future research. Current systems largely ignore microbial metabolic variation, and extant mechanistic models of rumen fermentation give only limited attention to explicit representation of microbial metabolic activity. Recent molecular techniques indicate that knowledge on the presence and activity of various microbial species is far from complete. Such techniques may give a wealth of information, but to include such findings in systems predicting the nutrient profile requires close collaboration between molecular scientists and mathematical modellers on interpreting and evaluating quantitative data. Protozoal metabolism is of particular interest here given the paucity of quantitative data.Empirical models lack the biological basis necessary to evaluate mitigation strategies to reduce excretion of waste, including nitrogen, phosphorus and methane. Such models may have little predictive value when comparing various feeding strategies. Examples include the Intergovernmental Panel on Climate Change (IPCC) Tier II models to quantify methane emissions and current protein evaluation systems to evaluate low protein diets to reduce nitrogen losses to the environment. Nutrient based mechanistic models can address such issues. Since environmental issues generally attract more funding from governmental offices, further development of nutrient based models may well take place within an environmental framework.     

Review: Nutrient requirements of the modern high-producing lactating sow,with an emphasis on amino acid requirements

Sow productivity improvements continue to increase metabolic demands during lactation. During the peripartum period, energy requirements increase by 60%, and amino acid needs increase by 150%. As litter size has increased, research on peripartum sows has focused on increasing birth weight, shortening farrowing duration to reduce stillbirths and improving colostrum composition and yield. Dietary fibre can provide short-chain fatty acids to serve as an energy source for the uterus prior to farrowing; however, fat and glucose appear to be the main energy sources used by the uterus during farrowing. Colostrum immunoglobulin G concentration can be improved by increasing energy and amino acid availability prior to farrowing; however, the influence of nutrient intake on colostrum yield is unequivocal. As sows transition to the lactation period, nutrient requirements increase with milk production demands to support large, fast-growing litters. The adoption of automated feed delivery systems has increased feed supply and intake of lactating sows; however, sows still cannot consume enough feed to meet energy and amino acid requirements during lactation. Thus, sows typically catabolise body fat and protein to meet the needs for milk production. The addition of energy sources to lactation diets increases energy intake and energy output in milk, leading to a reduction in BW loss and an improvement in litter growth rate. The supply of dietary amino acids and CP close to the requirements improves milk protein output and reduces muscle protein mobilisation. The amino acid requirements of lactating sows are variable as a consequence of the dynamic body tissue mobilisation during lactation; however, lysine (Lys) is consistently the first-limiting amino acid. A regression equation using published data on Lys requirement of lactating sows predicted a requirement of 27 g/day of digestible Lys intake for each 1 kg of litter growth, and 13 g/day of Lys mobilisation from body protein reserves. Increases in dietary amino acids reduce protein catabolism, which historically leads to improvements in subsequent reproductive performance. Although the connection between lactation catabolism and subsequent reproduction remains a dogma, recent literature with high-producing sows is not as clear on this response. Many practical aspects of meeting the nutrient requirements of lactating sows have not changed. Sows with large litters should approach farrowing without excess fat reserves (e.g. <18 mm backfat thickness), be fed ad libitum from farrowing to weaning, be housed in a thermoneutral environment and have their skin wetted to remove excess heat when exposed to high temperatures.     

Development of improved defined media for Clostridium botulinum serotypes A, B, and E

The minimal nutritional growth requirements were determined for strains Okra B and Iwanai E, which are representatives of groups I and II, respectively, of Clostridium botulinum. These type B and E strains differed considerably in their nutrient requirements. The organic growth factors required in high concentrations by the Okra B strain (group I) were arginine and phenylalanine. Low concentrations (less than or equal to 0.1 g/liter) of eight amino acids (methionine, leucine, valine, isoleucine, glycine, histidine, tryptophan, and tyrosine) and of five vitamins (pyridoxamine, p-aminobenzoic acid, biotin, nicotinic acid, and thiamine) were also essential for biosynthesis. The 10 required amino acids could be replaced by intact protein of known composition by virtue of the bacterium's ability to synthesize proteases. Glucose or other carbohydrates were not essential for Okra B, although they did stimulate growth. Quantitatively, the most essential nutrients for Okra B were arginine and phenylalanine. In contrast, the nonproteolytic strain, Iwanai E (group II), did not require either arginine or phenylalanine. It required glucose or another carbohydrate energy source for growth and did not utilize arginine or intact protein as a substitute source of energy. Iwanai E utilized ammonia as a nitrogen source, although growth was stimulated significantly by organic nitrogenous nutrients, especially glutamate and asparagine. Iwanai E also required biosynthesis levels of seven amino acids (histidine, isoleucine, leucine, tryptophan, tyrosine, valine, and serine), adenine, and six vitamins (biotin, thiamine, pyridoxamine, folic acid, choline, and nicotinamide). Calcium pantothenate also stimulated growth. On the basis of the nutritional requirements, chemically defined minimal media have been constructed for C. botulinum serotypes A, B, E, and F (proteolytic).(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular regulation of fibroblast multiplication: A direct kinetic approach to analysis of role of low molecular weight nutrients and serum growth factors

The principles of enzyme kinetic analysis were applied to quantitate the relationships among serum-derived growth factors, nutrients, and the rate of survival and multiplication of human fibroblasts in culture. The survival or multiplication rate of a population of cells plotted against an increasing concentration of a growth factor or nutrient in the medium exhibited a hyperbolic pattern that is characteristic of a dissociable, saturable interaction between cells and the ligands. Parameters equivalent to the K_m and V_max of enzyme kinetics were assigned to nutrients and growth factors. When all nutrient concentrations were optimized and in steady state, serum factors accelerated the rate of multiplication of a normal cell population. The same set of nutrients that supported a maximal rate of multiplication in the presence of serum factors supported the maintenance of non-proliferating cells in the absence of serum factors. Therefore, under this condition, serum factors are required for cell division and play a purely regulatory iole in multiplication of the cell population. The quantitative requirement for 18 nutrients of 29 that were examined was significantly higher (P < 0.001) for cell multiplication in the presence of serum factors than for cell maintenance in the absence of serum factors. This indicated specific nutrients that may be quantitatively important in cell division processes as well as in cell maintenance. The quantitative requirement for Ca²⁺, Mg²⁺, K⁺, Pi, and 2-oxocarboxylic acid for cell multiplication was modified by serum factors and other purified growth factors. The requirement for over 30 other nutrients could not clearly be related to the level of serum factors in the medium. Serum factors also determined the Ca²⁺, K⁺, and 2-oxocarboxylic acid requirement for maintenance of non-proliferating cells. Therefore, when either Ca²⁺, K⁺, or 2-oxocarboxylic acid concentration was limiting, factors in serum played a role as cell “survival or maintenance” factors in addition to their role in cell division as “growth regulatory” factors. However, with equivalent levels of serum factors in the medium, the requirement for Ca²⁺, K⁺, and 2-oxocarboxylic acids was still much higher for multiplication than for maintenance. Kinetic analysis revealed that the concentrations of individual nutrients modify the quantitative requirement for others for cell multiplication in a specific pattern. Thus, specific quantitative relationships among different nutrients in the medium are important in the control of the multiplication rate of the cell population. When all nutrient concentrations were optimal for multiplication of normal cells, the multiplication response of SV40-virus-transformed cells to serum factors was similar to that of normal cells. When serum factors were held constant, transformed cells required significantly less (P < 0.001) of 12 of the 26 nutrients examined. Therefore, the transformed cells only have a growth advantage when the external concentration of specific nutrients limits the multiplication rate of normal cells. Taken together, the results suggest that the control of cell multiplication is intimately related to external concentrations of nutrients. Specific growth regulatory factors may stimulate cell proliferation by modification of the response of normal cells to nutrients. Transforming agents may confer a selective growth advantage on cells by a constitutive alteration of their response to extracellular nutrients.     

Development of improved defined media for Clostridium botulinum serotypes A, B, and E.

The minimal nutritional growth requirements were determined for strains Okra B and Iwanai E, which are representatives of groups I and II, respectively, of Clostridium botulinum. These type B and E strains differed considerably in their nutrient requirements. The organic growth factors required in high concentrations by the Okra B strain (group I) were arginine and phenylalanine. Low concentrations (less than or equal to 0.1 g/liter) of eight amino acids (methionine, leucine, valine, isoleucine, glycine, histidine, tryptophan, and tyrosine) and of five vitamins (pyridoxamine, p-aminobenzoic acid, biotin, nicotinic acid, and thiamine) were also essential for biosynthesis. The 10 required amino acids could be replaced by intact protein of known composition by virtue of the bacterium's ability to synthesize proteases. Glucose or other carbohydrates were not essential for Okra B, although they did stimulate growth. Quantitatively, the most essential nutrients for Okra B were arginine and phenylalanine. In contrast, the nonproteolytic strain, Iwanai E (group II), did not require either arginine or phenylalanine. It required glucose or another carbohydrate energy source for growth and did not utilize arginine or intact protein as a substitute source of energy. Iwanai E utilized ammonia as a nitrogen source, although growth was stimulated significantly by organic nitrogenous nutrients, especially glutamate and asparagine. Iwanai E also required biosynthesis levels of seven amino acids (histidine, isoleucine, leucine, tryptophan, tyrosine, valine, and serine), adenine, and six vitamins (biotin, thiamine, pyridoxamine, folic acid, choline, and nicotinamide). Calcium pantothenate also stimulated growth. On the basis of the nutritional requirements, chemically defined minimal media have been constructed for C. botulinum serotypes A, B, E, and F (proteolytic).(ABSTRACT TRUNCATED AT 250 WORDS)