An alternate method of calculating the heat of growth of Escherichia coli K-12 on succinic acid

The DeltaH(f) (0) unit weight of a complex substance such as a biological macromolecule is almost always obtained by means of combustion analysis. In theory, this can also be done by summing the DeltaH(f) (0) values for the monomers comprising the macromolecule plus the enthalpic energies involved in their polymerization. The enthalpy of formation of one unit-carbon formula weight of dried Escherichia coli K-12 cells was determined by summing the values of the enthalpies of formation of the quantities of monomers in the major classes of macromolecules substances comprising the cellular biomass and the enthalpic energies involved in their polymerizations. To this value was added the enthalpy of formation of the cellular ions in their aqueous standard states, per unit-carbon formula weight of cellular substance and the enthalpy change with respect to the ionization of the protein amino acid side chains. If it is assumed that the cellular fabric is insoluble and that the ions are soluble, the sum of the enthalpies of formation of all the cellular components should closely approximate the enthalpy of formation of one unit-carbon formula weight equivalent of living cells. Using this value, a calculation of the enthalpy change accompanying anabolism shows this latter to be effectively zero, indicating that the heat of growth (anabolism plus catabolism) is equal to that calculated for catabolism alone. This conclusion is in accord with those of several investigators who have used manometry or direct calorimetry.     

On the enthalpy of formation of Escherichia coli K-12 cells

An examination is made of five methods for obtaining values of the enthalpy of formation of a unit mass of living Escherichia coli K-12 cells. The values obtained by these methods ranged from -88.95 kJ to -99.55 kJ, the gross average being 96.01 kJ, per unit carbon formula weight equivalent of living, hydrated cells. Although theoretically the growth of this organism in a microcalorimeter should provide the best value, the value obtained by this method (-88.95 kJ per UCFW equivalent) is not in close agreement with those of the other four methods, the values from which form a cluster averaging -97.8 +/- 1.0 kJ (-23.4 +/- 0.2 kcal)/UCFW equivalent. Calculations using this value indicate that the enthalpy change accompanying anabolism (as this is represented) is zero, or very nearly so, and that the heat of growth is that from catabolism alone.     

Calculation of thermodynamic properties of protein in Escherichia coli K-12 grown on succinic acid, energy changes accompanying protein anabolism, and energetic role of ATP in protein synthesis

Using an average of the results from three methods of calculation, estimations are made of the thermodynamic properties of a unit carbon formula weight (UCFW) of Escherichia coli K-12 protein. These resulted in values fro DeltaG(f) of -38.09 kJ (-9.10 kcal)/ UCFW, for DeltaH(f) of -68.18 kJ (-16.29 kcal)/UCFW, and for DeltaS(f) of -94.2 J (-22.5 cal)/UCFW deg. The absolute entropy of one UCFW of E. coli K-12 protein is calculated to be 73.8 J/UCFW deg. Using these values, the corresponding changes in thermodynamic properties accompanying the anabolism of protein by this microorganism to from one UCFW of protein by this microorganism to from one UCFW of protein are calculated to be 1.97 kJ (0.47 kcal)/UCFW for DeltaG, 0.75 kJ (0.18 kcal)/UCFW for DeltaH, and -4.09 J (-0.98 cal)/UCFW deg for DeltaS. All these values are sufficiently close to zero that they may be considered to be so. The question is raised as to the quantity of ATP energy conserved within the substance of the protein as it is synthesized from succinic acid. It is calculated that only 3.8% of the total free energy available from ATP that is required during protein anabolism can have been conserved within the substance of the protein, there being a net conversion of the remaninder into heat and entropy.     

Microcalorimetric study of Escherichia coli aerobic growth: theoretical aspects of growth on succinic acid.

Two methods of investigation were used to evaluate the heat quantity associated with anabolic processes (qan) during the aerobic growth of Escherichia coli in a minimal medium containing succinic acid as the sole energy and carbon source. The study of the contribution of biosynthetic reactions from succinic acid and ammonia were investigated by both methods. The two qan values obtained were in excellent agreement and were found to be significant. Thus it was demonstrated that the contribution of anabolism strongly influenced the quantity of heat associated with microbial aerobic growth. The qan calculated as above explained the experimental enthalpy change which was recently reported.     

Calculation of entropy change accompanying growth of Escherichia coli K-12 on succinic acid

The ΔS of one unit carbon formula weight of Escherichia coli K-12 cells, when grown on succinic acid, was calculated to be ?80.13 J/deg. This value could then be used to calculate the entropy change accompanying the anabolism and metabolism of succinic acid to be 30.82 J/deg and 32.40 J/mol deg, respectively. The entropy of one unit carbon formula weight of dried E. Coli K-12 cells is calculated to be 94.40 J/deg, which when divided by the mass of these cells becomes 3.90 J/g deg. The corresponding entropy of succinic acid is 2.77 J/g deg, making it apparent that the entropy per unit mass of the cells is greater than that of the substrate. It might be thought that because the cells appear to be so much more complex than the substrate, the cells should have a lesser entropy per unit mass than the substrate. That this does not appear to be true leads to the conclusion that the macromolecular organization (informational content?) of the cells contributes only in a very minor way to the total physical entropy of cells. © 1993 John Wiley & Sons, Inc.     

Structure of Escherichia coli ribose-5-phosphate isomerase: a ubiquitous enzyme of the pentose phosphate pathway and the Calvin cycle

Ribose-5-phosphate isomerase A (RpiA; EC 5.3.1.6) interconverts ribose-5-phosphate and ribulose-5-phosphate. This enzyme plays essential roles in carbohydrate anabolism and catabolism; it is ubiquitous and highly conserved. The structure of RpiA from Escherichia coli was solved by multiwavelength anomalous diffraction (MAD) phasing, and refined to 1.5 A resolution (R factor 22.4%, R(free) 23.7%). RpiA exhibits an alpha/beta/(alpha/beta)/beta/alpha fold, some portions of which are similar to proteins of the alcohol dehydrogenase family. The two subunits of the dimer in the asymmetric unit have different conformations, representing the opening/closing of a cleft. Active site residues were identified in the cleft using sequence conservation, as well as the structure of a complex with the inhibitor arabinose-5-phosphate at 1.25 A resolution. A mechanism for acid-base catalysis is proposed.     

Alternate method of calculating the free-energy change accompanying the growth of saccharomyces cerevisiae (Hansen) on three substrates.

A method is described for determining the free energy of formation of the cells of Saccharomyces cerevisiae (Hansen) that are formed as the result of anaerobic growth on glucose, and aerobic growth on glucose and ethanol. The method is based on the direct relationship that exists between the enthalpy changes and the free-energy changes that accompany the oxidation of 1 g cellular material formed during these growth reactions and the degree of reduction of the same material. When the results of these calculations are used together with the free energies of formation of the reactants and of other products of a given growth reaction, it becomes possible to calculate the free-energy change accompanying this reaction. These free-energy changes are in excellent agreement with those calculated by another method based on the hypothesis that the free-energy change accompanying the conversion of the substrate plus other reactants into cellular material plus other products is equal to zero.     

The calorimetric-respirometric ratio is an on-line marker of enthalpy efficiency of yeast cells growing on a non-fermentable carbon source

Although on-line calorimetry has been widely used to detect transitions in global metabolic activity during the growth of microorganisms, the relationships between oxygen consumption flux and heat production are poorly documented. In this work, we developed a respirometric and calorimetric approach to determine the enthalpy efficiency of respiration-linked energy transformation of isolated yeast mitochondria and yeast cells under growing and resting conditions. On isolated mitochondria, the analysis of different phosphorylating and non-phosphorylating steady states clearly showed that the simultaneous measurements of heat production and oxygen consumption rates can lead to the determination of both the enthalpy efficiency and the ATP/O yield of oxidative phosphorylation. However, these determinations were made possible only when the net enthalpy change associated with the phosphorylating system was different from zero. On whole yeast cells, it is shown that the simultaneous steady state measurements of the heat production and oxygen consumption rates allow the enthalpy growth efficiency (i.e. the amount of energy conserved as biomass compared to the energy utilised for complete catabolism plus anabolism) to be assessed. This method is based on the comparison between the calorimetric-respirometric ratio (CR ratio) determined under growth versus resting conditions during a purely aerobic metabolism. Therefore, in contrast to the enthalpy balance approach, this method does not rely on the exhaustive and tedious determinations of the metabolites and elemental composition of biomass. Thus, experiments can be performed in the presence of non-limiting amounts of carbon substrate, an approach which has been successfully applied to slow growing cells such as yeast cells expressing wild-type or a mutant rat uncoupling protein-1.     

Model of energy uncoupling for substrate-sufficient culture

The growth yields (Y(obs)) are greater under substrate-limited conditions than those under substrate-sufficient conditions in continuous cultures. This indicates that the excess substrate should cause uncoupling between anabolism and catabolism, which leads to energy spilling. Although the uncoupling between anabolism and catabolism has already been recognized in the microbiology literature, how to quantitatively describe such uncoupling remains unclear. Based on a balance on substrate reaction, a growth yield model was developed in relation to residual substrate concentration for substrate-sufficient continuous cultures. On the basis of that yield model, the concept of an uncoupling coefficient between anabolism and catabolism is defined in this work. A model describing the effect of the residual substrate concentration on the uncoupling coefficient of anabolism to catabolism is proposed. This model agrees very well with literature data. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 571-576, 1997.     

Photothermal studies of CO photodissociation from mixed valence Escherichia coli cytochrome bo3

Here, we report the volume and enthalpy changes accompanying CO photodissociation from the mixed valence form of cytochrome bo3 oxidase from Escherichia coli. The results of photoacoustic calorimetry indicate two kinetic phases with distinct volume and enthalpy changes accompanying CO photodissociation from heme o3 and its transfer to CuB. The first phase occurring on a timescale of <50 ns is characterized by a volume decrease of -1.3+/-0.3 mL mol-1 and enthalpy change of 32+/-1.6 kcal mol-1. Subsequently, a volume increase of 2.9 mL mol-1 with an enthalpy change of -5.3+/-2.5 kcal mol-1 is observed with the lifetime of approximately 250 ns (this phase has not been detected in previous optical studies). These volume and enthalpy changes differ from the volume and enthalpy changes observed for CO dissociation from fully reduced cytochrome bo3 oxidase indicating that the heme o3/CuB active site dynamics are affected by the redox state of heme b.     

Charges of nicotinamide adenine nucleotides and adenylate energy charge as regulatory parameters of the metabolism in Escherichia coli.

Methods for measurements of catabolic reduction charge (defined as NADH/(NADH+NAD+)) and anabolic reduction charge (defined as NADPH/(NADPH + NADP+)) are described using [14C]nicotinamide labeling of Escherichia coli cultures. Together with these parameters the adenylate energy charge (ATP + 1/2ADP)/(ATP + ADP + AMP) was measured using labeling with [2-3H]adenine. These three charges were found under different exponential growth conditions to have values independent of the growth conditions: catabolic reduction charge, 0.05; anabolic reduction charge, 0.45; and adenylate energy charge, 0.9. The charges were examined during interruption of growth primarily affecting catabolism, respiration, or anabolism, leading to changes of the charges. The changes of charges are evaluated as a possible regulation of the metabolic rates utilizing or producing the nucleotides by their respective charges.     

Genes for ribitol and D-arabitol catabolism in Escherichia coli: their loci in C strains and absence in K-12 and B strains.

Escherichia coli C strains can grow at the expense of the two natural pentitols ribitol and D-arabitol, sugar alcohols previously thought not to be utilized by E. coli. E. coli strains K-12 and B cannot utilize either compound. The genetic loci responsible for pentitol catabolism in E. coli C, designated rtl and atl, are separate and closely linked. Each lies between metG and his and is highly co-transducible with metG and with a P2 prophage attachment site. rtl and atl readily can be transduced into E. coli K-12 or B strains, in which they integrate at, or very near, their E. coli C location. Transduction also can be used to insert rtl and atl into certain E. coli K-12 F' plasmids. No recombination between E. coli C strains and either K-12 or B strains occurs within the rtl-atl genetic region after interstrain conjugations or transductions. No cryptic rtl or atl genes in K-12 or B strains can be detected by complementation, recombination, or mutagenesis. These results are consistent with the view that the rtl-atl portion of the E. coli C chromosome has no counterpart in E. coli K-12 or B and may have been obtained from an extrageneric source. Detailed biochemical and genetic comparisons of penitol utilization in E. coli and Klebsiella aerogenes are in progress. The ability to catabolize xylitol is conferred upon E. coli C strains by a mutation at or adjacent to the rtl locus, whereas in E. coli K-12 or B strains harboring rtl an additional mutation at a separate locus is required for xylitol utilization.     

Accumulation of 3-(N-morpholino)propanesulfonate by osmotically stressed Escherichia coli K-12.

We found that exogenous morpholinopropanesulfonate (MOPS) is concentrated approximately fivefold in the free volume of the cytoplasm of Escherichia coli K-12 (strain MG1665) when grown at high osmolarity (1.1 OsM) in two different media containing 40 mM MOPS. MOPS was not accumulated by E. coli grown in low-osmolarity MOPS-buffered medium or in 1.1 OsM MOPS-buffered medium containing the osmoprotectant glycine betaine. Salmonella typhimurium LT2 did not accumulate MOPS under any condition examined. We infer that accumulation of MOPS by E. coli K-12 is not due to passive equilibration but rather to transport, possibly involving an as yet uncharacterized porter not present in S. typhimurium. Glutamate and MOPS were the only anionic osmolytes we observed by 13C nuclear magnetic resonance in E. coli K-12 grown in MOPS-buffered medium. The increase in positive charge accompanying the increase in the steady-state amount of K+ in cells shifted from low to high external osmolarity appeared to be compensated for by changes in the amounts of putrescine, glutamate, and MOPS. MOPS is not an osmoprotectant, because its accumulation did not increase cell growth rate.     

Metabolism of D-arabinose: a new pathway in Escherichia coli

Several growth characteristics of Escherichia coli K-12 suggest that growth on l-fucose results in the synthesis of all the enzymes necessary for growth on d-arabinose. Conversely, when a mutant of E. coli is grown on d-arabinose, all of the enzymes necessary for immediate growth on l-fucose are present. Three enzymes of the l-fucose pathway in E. coli, l-fucose isomerase, l-fuculokinase, and l-fuculose-l-phospháte aldolase possess activity on d-arabinose, d-ribulose, and d-ribulose-l-phosphate, respectively. The products of the aldolase, with d-ribulose-l-phosphate as substrate, are dihydroxyacetone phosphate and glycolaldehyde. l-Fucose, but not d-arabinose, is capable of inducing these activities in wild-type E. coli. In mutants capable of utilizing d-arabinose as sole source of carbon and energy, these activities are induced in the presence of d-arabinose and in the presence of l-fucose. Mutants unable to utilize l-fucose, selected from strains capable of growth on d-arabinose, are found to have lost the ability to grow on d-arabinose. Enzymatic analysis of cell-free extracts, prepared from cultures of these mutants, reveals that a deficiency in any of the l-fucose pathway enzymes results in the loss of ability to utilize d-arabinose. Thus, the pathway of d-arabinose catabolism in E. coli K-12 is believed to be: d-arabinose right harpoon over left harpoon d-ribulose --> d-ribulose-l-phosphate right harpoon over left harpoon dihydroxyacetone phosphate plus glycolaldehyde. Evidence is presented which suggests that the glycolaldehyde is further oxidized to glycolate.     

Energetics of heparin binding to human acidic fibroblast growth factor

The binding of low-molecular-weight heparin to an amino-terminal-truncated, 132-amino-acid, human acidic fibroblast growth factor form has been studied by isothermal titration calorimetry. This technique yields values for the enthalpy change and equilibrium constant, from which the Gibbs energy and entropy change are also calculated. Experiments in different buffers and pH values show that the protonic balance during the reaction is negligible. Experiments made at pH 7.0 with NaCl concentrations ranging from 0.20 to 0.60 M revealed changes in enthalpy and Gibbs energy in the range of -30- -17 and -27- -24 kJ x mol(-1), respectively. Isothermal titration calorimetry was also performed at different temperatures to obtain a value for the heat-capacity change at pH 7.0 and 0.4 M NaCl concentration of -96 J K- x mol(-1). A change in the length of heparin brought about no change in the thermodynamic parameters at 25 degrees C under the same experimental conditions. Changes upon ligand binding in the range of -50- -200 A2 in both polar and non-polar solvent-accessible surface areas were calculated from thermodynamic data by using different parametric equations taken from the literature. These values suggest a negligible overall conformational change in the protein when it binds to heparin and no formation of any protein-protein interface.     

Characterization of unexpected growth of Escherichia coli O157:H7 by modeling

Modeling of batch kinetics in minimal synthetic medium was used to characterize Escherichia coli O157:H7 growth, which appeared to be different from the exponential growth expected in minimal synthetic medium and observed for E. coli K-12. The turbidimetric kinetics of 14 of the 15 O157:H7 strains tested (93%) were nonexponential, whereas 25 of the 36 other E. coli strains tested (70%) exhibited exponential kinetics. Moreover, the anomaly was almost corrected when the minimal medium was supplemented with methionine. These observations were confirmed with two reference strains by using plate count monitoring. In mixed cultures, E. coli K-12 had a positive effect on E. coli O157:H7 and corrected its growth anomaly. This demonstrated that commensalism occurred, as the growth curve for E. coli K-12 was not affected. The interaction could be explained by an exchange of methionine, as the effect of E. coli K-12 on E. coli O157:H7 appeared to be similar to the effect of methionine.     

Structural energetics of barstar studied by differential scanning microcalorimetry.

The energetics of barstar denaturation have been studied by CD and scanning microcalorimetry in an extended range of pH and salt concentration. It was shown that, upon increasing temperature, barstar undergoes a transition to the denatured state that is well approximated by a two-state transition in solutions of high ionic strength. This transition is accompanied by significant heat absorption and an increase in heat capacity. The denaturational heat capacity increment at approximately 75 degrees C was found to be 5.6 +/- 0.3 kJ K-1 mol-1. In all cases, the value of the measured enthalpy of denaturation was notably lower than those observed for other small globular proteins. In order to explain this observation, the relative contributions of hydration and the disruption of internal interactions to the total enthalpy and entropy of unfolding were calculated. The enthalpy and entropy of hydration were found to be in good agreement with those calculated for other proteins, but the enthalpy and entropy of breaking internal interactions were found to be among the lowest for all globular proteins that have been studied. Additionally, the partial specific heat capacity of barstar in the native state was found to be 0.37 +/- 0.03 cal K-1 g-1, which is higher than what is observed for most globular proteins and suggests significant flexibility in the native state. It is known from structural data that barstar undergoes a conformational change upon binding to its natural substrate barnase.(ABSTRACT TRUNCATED AT 250 WORDS)