BioNumberOfTheMonth

=Bionumber of the Month - December 2008= compiled and written by Phillip Mongiovi and Ron Milo

ATP requirement for the creation of an E. coli cell
(with glucose as carbon and energy source, cell of 0.28 pg dry mass, ~40 minutes division time. Theoretical value does not include any maintenance costs, experimental cost supposed to deduct maintenance costs that are constant per unit time)

Experimental: 	12 billion-20 billion 

Theoretical: 		6 billion-11 billion 

The main energy currency of the cell is ATP. One can ask how many ATPs are required to make a cell ? Here we discuss the measurements and analysis performed on this question for a growing e.coli cell. Cells require energy to grow and divide as well as for many maintenance processes. Before a cell divides it essentially must duplicate each of its components so that the two daughter cells will have the same constituents as the mother cell. Most chemical reactions and pathways that allow the cell to biosynthesize these new cellular materials have associated energetic costs that have been determined. Adenosine triphosphate (ATP) is the primary provider of energy for these chemical reactions. It is hydrolyzed to form adenosine diphosphate (ADP) and inorganic phosphate, which results in the release of approximately 50 kJ/mol of energy under physiological conditions (BNID: 101964).

Empirical versus Theoretical estimates of the ATP cost per cell

There are two complementary methods for answering the question – one experimental, the other theoretical. The amount of ATP used by a population of cells as they grow can be experimentally determined based on the rate of substrate consumption. The average amount of ATP required per cell can then be determined. In the theoretical approach, the amount of ATP necessary for creating all the macromolecules can be calculated from all the known biochemical pathways within a cell. This calculation is based on knowledge of the composition of an E. coli cell, i.e. how much of each amino acid, nucleotide, lipid, etc. exists in a cell or gram of cells. This theoretical computation tests our understanding of the energy-consuming processes of a cell.

To compare experimental and theoretical calculations, the value of YATPmax is commonly used. It is derived from YATP, which is defined as the number of grams of cells (dry weight) that are produced by 1 mole of ATP (Bauchop and Elsden 1960). (see description below on how it is derived). For example, at the slow growth rate of 0.087/hr, and under anaerobic conditions on glucose, an E. coli population produces ~3 g cells/mole of ATP (Hempfling and Mainzer 1975). All numbers presented refer to the gram(s) of dry cell weight. 96.1% of the cell’s dry weight is made of macromolecules (Neidhardt et al. 1990)(BNID 101436).

To arrive at the number of ATP molecules per cell, one needs to know the dry weight of an average E. coli, which is taken to be 0.28 pg (Neidhardt et al. 1990)(BNID 100008) or 3.6 trillion cells/g. Cell size is dependent on growth rate and the value of 0.28 pg is for a growth rate of 0.66, (doubling time 40 minutes).

Experimental determination of the ATP cost under anaerobic conditions

For experimental calculations one needs to know how to convert from the amount of carbon source consumed to the amount of ATP produced. It is easiest to calculate YATP under anaerobic conditions because ATP is only generated outside the mitochondria (substrate phosphorylation). The amount of ATP produced per substrate molecule is well established (Stouthamer 1979). For example, the anaerobic breakdown of glucose into pyruvate (glycolysis) yields 2 ATP. E. coli can produce another ATP by further conversion of the carbon source into acetate, for a total of 3 ATP/glucose. However, it is important to remember that not all glucose is used to create ATP; glucose is both the energy and the carbon source in the experimental data presented below. By knowing the carbon content of the cell biomass, one can infer the net amount of carbons that become part of the cell’s dry mass, and therefore the remaining part is the amount of glucose which goes into the creation of ATP for energy. The basic input into these calculations is the amount of dry cell weight produced per mole of substrate consumed, also known as Yglu for a glucose substrate. The amount of substrate consumed can be measured experimentally, which enables the transformation of Yglu to YATP (Stouthamer and Bettenhausen 1973).

YATP values were found to be dependent on the growth rate. At higher growth rates, less ATP was needed to form cells. With growth rates ranging from 0.570 to 0.087 per hour, the YATP values ranged from 7.4 to 3.0 grams of cells per mole of ATP, respectively, on glucose and minimal media in continuous culture (Hempfling and Mainzer 1975). The slower the growth rate, the more ATP it takes to make one gram of cells. Unless the composition of cells is changing at different growth rates, this increased ATP requirement/cell most likely reflects the longer duplication time, which requires much higher maintenance costs per cell duplication. These numbers need to be corrected for the extra maintenance costs implicit in longer doubling times. It was recognized that the slope of a plot of 1/YATP vs. 1/μ, where μ is the growth rate, is equivalent to a value of the growth-rate-independent energy requirement, or maintenance energy (Pirt 1965). This is used to calculate a value of YATPmax, the maximum yield of grams of cells per mol of ATP which aims not to include the maintenance energy costs.

When YATP is corrected for maintenance costs, the value for duplicating a cell’s contents (now referred to as YATPmax after the correction) is 10.3 grams of cells per mole of ATP (Hempfling and Mainzer 1975). This is equivalent to 16.4 billion ATP/cell (BNID 101983). Under similar conditions (glucose-limited anaerobic growth in a chemostat), a different set of authors determined a YATPmax value of 8.5 (19.8 billion ATP/cell) (Stouthamer and Bettenhausen 1977). Therefore, under glucose-limited anaerobic growth, it requires ~16-20 billion ATP molecules to generate a new E. coli.

Experimental determination of the ATP cost under aerobic conditions

Some small adjustments need to be made to the above-mentioned description in order to calculate the YATP in aerobic conditions. The idea remains the same but now the amount of ATP produced by oxidative phosphorylation must be included. This requires a measurement of the oxygen consumption and the number of ATP produced per oxygen (Stouthamer 1973).

Under aerobic, carbon-source-limited conditions, YATPmax was calculated to be 13.9 g cells/mol ATP on glucose and minimal media, and down to 7.1 g cells/mol ATP on acetate (Farmer and Jones 1976). This corresponds to 12.1 billion ATP/cell for glucose and 24.8 billion ATP/cell for acetate (BNID 101981 and 101982). It is not clear what are the reasons for the difference between aerobic and anaerobic energetic costs.

Theoretical ATP requirement per cell under aerobic and anaerobic conditions

When performing theoretical calculations, it is possible to break down the costs into the major factors, for example that assist in biosynthetic reactions by providing the necessary oxidizing or reducing power. These key factors include the adenine dinucleotides NAD(H), NADP(H), and FAD(H2). In some of the theoretical calculations, the metabolic cost of these was included (e.g. Niedhardt et al. 1990; Feist et al. 2007), but not in all cases (e.g. Stouthamer 1973, 1977). Also, other nucleotide triphosphates such as guanosine triphosphate (GTP) are used in some reactions, such as protein synthesis. One GTP was considered to be equivalent to one ATP in all calculations. It is more difficult to convert the adenine dinucleotide factors into ATP equivalents; different research groups have used different numbers in calculating these values. In general, the equivalent of one adenine dinucleotide will be on the order of two, three, or four ATP (Atkinson 1977; Akashi and Gojobori 2002). Consequently, the number of ATP used to make an E. coli cell would be stated more accurately as the number of ATP equivalents used to make an E. coli cell, since not all of the biosynthetic reactions use ATP as the sole energy source. For simplicity, ATP is listed as the unit of measure in all cases presented here.

The issue of whether the E. coli is growing with oxygen (aerobic) or without oxygen (anaerobic, fermentation) is not a very important factor in the theoretical calculation of YATPmax. Aerobic conditions allow E. coli to perform oxidative phosphorylation, which results in a much greater production of ATP per carbon substrate than fermentation. However, that fact would not affect the amount of biomass produced per mole of ATP.

In contrast, the energy requirement is heavily dependent on other growth conditions of the bacteria, particularly on the carbon/energy source. One important aspect of the cost of the carbon source is transport. It is believed that energy is not needed for glucose transport into the cell apart from its subsequent phosphorylation; it enters via passive transport. Other carbon sources typically require the energy of one ATP per molecule of carbon substrate for transport into the cell. This alone makes a great difference in the total amount of ATP required for the biosynthesis of one cell. There is also typically a greater energy input for the conversion of other carbon substrates into cell materials because other carbon sources are contain less energy.

The additional biological components of the growth media are an important consideration in the energetic cost determination. For example, it requires much less energy to transport cellular building blocks such as nucleic acid bases inside the bacteria from the media than to build them from precursor metabolites that are derived from the breakdown of the carbon source. On average, the biosynthesis of amino acids is not as costly, energetically speaking, as the polymerization of amino acids into protein. In contrast, the majority of the energy required to make DNA and RNA molecules in the cell is due to the synthesis of the individual nucleotides, not polymerization. The synthesis of nucleoside monophosphates of DNA requires 0.86 mmol ATP per gram of cells, while polymerization only requires 0.19 mmol ATP per gram of cells when grown on glucose (Stouthamer 1973). The difference in energetic cost of the two is greater for other carbon sources. Therefore, the addition of nucleic acid monomers to the growth media will reduce the overall energy cost much more than adding amino acids. In fact, the cost of transporting amino acids into the cells was calculated to be greater than the cost of their biosynthesis (Stouthamer 1973). Other sources have estimated greater costs for the biosynthesis of amino acids, so it is not clear whether the addition of amino acids to the media would reduce the overall energetic cost (Akashi and Gojobori 2002; Neidhardt et al. 1990; Atkinson 1977).

Adding up all the individual energetic costs of formation and polymerization of the macromolecules of the cell, and including mRNA turnover and some ion transport costs, gives a total of ~35 mmol of ATP per gram of cells formed on glucose and inorganic salts (Stouthamer 1973). These conditions are comparable to the experimental conditions of Farmer and Jones. This is equivalent to 5.9 billion ATP/cell. (For a table of ATP yields, see BNID 101637). These conditions(but not the value…) are comparable to the experimental conditions of Farmer and Jones, who measured an experimental YATPmax of 12.1 billion ATP/cell (Farmer and Jones 1976).

If the glucose is supplemented with nucleic acid bases or amino acids and nucleic acid bases, then the total ATP requirement is reduced by approximately 10% to 5.3 billion ATP/cell. At the other end of the spectrum is growth on the carbon source acetate. The formation of a gram of biomass on acetate and inorganic salts requires 99.5 mmol of ATP per gram of cells or 16.8 billion ATP/cell. (Stouthamer 1973) 42 mmol of ATP/g cells are required according to Neidhardt’s calculation for the average E. coli cell growing on glucose and minimal media (Neidhardt et al. 1990). This is equivalent to 7.2 billion ATP/cell. Akashi and Gojobori (2002) state that 20 billion to 60 billion ATP/cell are required for the biosynthesis of one E. coli cell. They state that they based their calculations on Stouthamer’s data from 1973, although it is unclear how the calculation was performed.

The most recent theoretical calculations performed by the Palsson group in 2007, state that the cost of growth, excluding core metabolic activity, is ~60 mmol ATP/g cells under conditions of aerobic growth on glucose. They also determined non-growth maintenance costs under these conditions to be 8.4 mmol ATP/g cells. Therefore, the energy cost without core metabolic energy costs is 68 mmol ATP/g cell, or 11.5 billion ATP/cell (Feist et al. 2007).

Decomposing experimental costs into biosynthetic and maintenance costs 

There exists a large difference between theoretical YATPmax and experimental YATPmax (Farmer and Jones 1976). Cells seem unable to reach the theoretical maximum yield. The maintenance energy cost component accounts only for some energy costs that are constant per unit time but not other maintenance costs. Some ATP consumption is not being accounted for by theoretical analysis. Ideally, maintenance energy would include all the energy costs of transport and turnover of cell materials. These are variable with the growth rate. The transport of ions to keep the membrane in an energized state is believed to be a very substantial energetic cost that is not taken into account in theoretical measurements. Stouthamer and Bettenhausen found in their 1977 study based on experimental work with mutants that under anaerobic conditions, the cost of maintaining electrochemical gradients across the membrane was estimated to be about 51% of the total energy cost. They compared the amount of ATP necessary for growth under anaerobic conditions for wild-type E. coli to the amount of ATP required for growth of ATPase-negative E. coli under aerobic conditions. The ATPase-negative mutant cannot use ATP to energize the membrane because it lacks a functional ATPase, so the process of using ATP is separated from the process of energizing the membrane. The anaerobically growing E. coli must spend their ATP to energize the membrane as well as to grow, so they require more ATP than the ATPase-negative mutant. With the assumption that the efficiency of biomass formation is the same under both conditions, the difference between the two values is the cost of membrane energization, which was equal to 61 mmol ATP, or 51% of the total energy requirement under anaerobic conditions.

This summary is partial and limited, comments and additions will be appreciated. Please send to ron.milo@weizmann.ac.il

References

Akashi, H. and Gojobori, T. Metabolic efficiency and amino acid composition in the proteomes of Escherichia coli and Bacillus subtilis. (2002) PNAS. 99(6): 3695-3700.

Atkinson, D.E. Cellular Energy Metabolism and Its Regulation. New York: Academic Press, 1977. p. 45.

Bauchop, T. and Elsden, S.R. The growth of microorganisms in relation to their energy supply. (1960) J. Gen. Microbiol. 23: 457-469. Farmer, I.S. and Jones, C.W. The energetics of Escherichia coli during aerobic growth in continuous culture. (1976) Eur. J. Biochem. 67(1):115-22. Feist, A.M., Henry, C.S., Reed, J.L., Krummenacker, M., Joyce, A.R., Karp, P.D., Broadbelt, L.J., Hatzimanikatis, V., Palsson, B.Ø. A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information. (2007) Molecular Systems Biology. 3:121. Hempfling, W. and Mainzer, S. Effects of Varying the Carbon Source Limiting Growth on Yield and Maintenance Characteristics of Escherichia coli in Continuous Culture. (1975) 	J. Bacteriol. 123(3): 1076-1087.

Neidhardt, F.C., Ingraham, J.L., and Schaechter, M. Physiology of the Bacterial Cell: A Molecular Approach. Sunderland, MA: Sinauer Associates, Inc., 1990.

Pirt, S.J. The Maintenance Energy of Bacteria in Growing Cultures. (1965) Proc R Soc Lond B Biol Sci. 163(991):224-31.

Stouthamer, A.H. A theoretical study on the amount of ATP required for synthesis of microbial cell material. (1973) Antonie van Leeuwenhoek. 39: 545-565.

Stouthamer, A.H. The search for correlation between theoretical and experimental growth yields. (1979) Int. Rev. Biochem. Microb. Biochem. 21:1-47 ed. J.R. Quayle. University Park Press.

Stouthamer, A.H. and Bettenhausen, C.W. Utilization of Energy for Growth and Maintenance in Continuous and Batch Cultures of Microorganisms. (1973) Biochim. Biophys. Acta. 301: 53-70.

Stouthamer, A.H. and Bettenhausen, C.W. A Continuous Culture Study of an ATPase-Negative Mutant of Escherichia coli. (1977) Arch. Microbiol. 113: 185-189.

Tran, Q.H. and Unden, G. Changes in the proton potential and the cellular energetics of Escherichia coli during growth by aerobic and anaerobic respiration or by fermentation. (1998) Eur. J. Biochem. 251, 538-543.