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Chapter 107

Growth Yield and Energy Distribution

OENSE M. NEIJSSEL, M. JOOST TEIXEIRA de MATTOS, and DAVID W. TEMPEST

 

INTRODUCTION

Although quantitative studies of microbial growth and of growth energetics were in progress some 60 years ago or more (52, 59), it is widely acknowledged that present-day theories regarding the relationships between substrate concentration, growth rate, and yield value stem from the classical studies of Monod (33). In these studies, quantitative measurements of the growth of Escherichia coli, Salmonella typhimurium, and Bacillus subtilis in batch cultures revealed that the equivalent dry weight of organisms formed per gram of carbon substrate metabolized (the yield value) was remarkably constant. Thus, when growing in a simple salts medium on a variety of related carbon substrates (hexoses, pentoses, polyalcohols, and disaccharides), E. coli expressed yield values that ranged between 0.21 and 0.28 g (equivalent dry weight) of cells formed per g of substrate consumed. Corresponding cultures of S. typhimurium expressed slightly (though consistently) lower values. The constancy of these data indicated the presence of mechanisms that precisely partition the flow of carbon substrate between catabolic (energy-generating) and anabolic (energy-consuming) processes such as to allow growth to proceed with a fixed overall efficiency. In this connection, however, Monod (33) realized that not all of the energy generated by catabolism necessarily would be consumed by anabolic processes and postulated that a small amount might be needed for cell maintenance ("ration d’entretien"). But, from his observations, he was forced to conclude that, with actively growing cultures, the latter requirement was too small to be detected by the methods then available.

The relationship between substrate concentration, growth rate, and respiration rate was subsequently studied by Schulze and Lipe (45), who used continuous-culture techniques that allowed growth rate to be varied over a wide range. From these studies on an unnamed strain of E. coli, they concluded that the maintenance rate of glucose consumption was indeed small but sufficient to affect markedly the yield value of a glucose-limited chemostat culture at low dilution rates. When corrected for maintenance, the (maximum) growth yield was found to be 0.53 g of cells per g of glucose. This value was more than double that reported by Monod (33), but is in accord with many subsequent measurements made with different strains of E. coli growing in glucose-limited chemostat culture (Table 1).

The results obtained by Schulze and Lipe (45) confirmed and extended those of Herbert (18) and Marr et al. (30), who had shown that the yield value with respect to the carbon substrate consumed was not a constant but varied with the growth rate (Table 1). A similar variation of oxygen yield was found, not surprisingly, and was also ascribed to a requirement of metabolic energy for cell maintenance (41). Thus, it became clear that meaningful comparisons of yield values could only be made by taking maintenance into account. Unfortunately, the precise nature of these maintenance processes and their minimum energetic requirements were then (as now) largely unresolved. Hence, it is still not clear whether maintenance energy requirements are quantitatively independent of growth rate, as originally assumed by Pirt (41) and others (17, 18, 30), or whether they vary with growth rate, as is now thought probable (38, 42, 59, 62). Thus it is not easy to assess the significance of small differences in the yield value expressed by related organisms growing under comparable conditions, but large consistent differences ought to be interpretable in physiological terms.

It is the purpose of this chapter to concentrate on those conditions that markedly affect the growth yield with respect to carbon substrate and oxygen consumption (Y _sub and , respectively) or ATP generation (Y _ATP) and to assess their physiological implications. It is useful to consider first the overall relationship between catabolism and anabolism which the yield value embodies. The uptake of a carbon substrate and its subsequent metabolism generate intermediary metabolites and reductant. Aerobically, part of the reductant so formed is oxidized by the respiratory chain to generate ATP; this ATP, along with the remaining reductant, is used to convert intermediary metabolites into monomers and polymers. A shortfall of reductant or ATP can be avoided by oxidation of some of the intermediary metabolites to CO₂. However, because the concentrations of the main carriers of reductant (the pyridine nucleotides) and of energy (the adenine nucleotides) are relatively low (15), surplus reductant or energy must be rapidly disposed of. Thus, with organisms growing aerobically a potential redox imbalance can be circumvented by a higher respiration rate, but if respiration is coupled to ATP generation, surplus energy is generated that cannot be stored as adenine nucleotides. This is not a hypothetical problem, for there are at least two circumstances in which catabolism is extensively dissociated from anabolism: first, washed cell suspensions often oxidize carbon substrate at a high rate without growing; second, carbon substrate-sufficient cultures frequently catabolize substrate far more rapidly than corresponding carbon-limited cultures (4, 34, 37, 38, 53, 54). In both of these cases, excess energy is generated that must be dissipated at a high rate (presumably as heat) by growth-unassociated processes. Therefore, microbial cells must have a capacity either to uncouple respiration from ADP phosphorylation or to turn over the ATP pool at a high rate in the absence of biosynthesis. This assumption raises questions regarding the regulation of energy fluxes in microorganisms and the nature of energy coupling between catabolic and anabolic processes in actively growing cells. To answer these questions, we consider the following: (i) the mechanisms of energy generation and how they might be caused to vary, (ii) the multifarious nature of energy-consuming processes, and (iii) the possible nature of energy-spilling reactions.

 

ENERGY-GENERATING SYSTEMS

 

Oxidative Phosphorylation

The efficiency of respiratory energy conservation (moles of ATP equivalents generated per 0.5 mol of oxygen reduced) depends upon the composition of the respiratory chain and the number of proton-translocating segments. Extensive studies of the aerobic respiratory chain components of several E. coli strains (24, 26) suggest that they lack a c-type cytochrome and hence contain only two proton-translocating segments. A schematic representation of the aerobic respiratory chain of E. coli is shown in Fig. 1. (For more detailed information, see chapter 17). Briefly, NADH can be oxidized via either of two dehydrogenases, NADH dehydrogenase 1 (NDH-1) and NADH dehydrogenase 2 (NDH-2). NDH-1 catalyzes the generation of a proton motive force during NADH oxidation, whereas NDH-2 does not. From the quinone pool there are two branches that transport electrons to oxygen. Under fully aerobic conditions a bo-type oxidase is operational, and this enzyme has an H⁺/e ratio of 2. At low oxygen tensions, the bd-type oxidase, which has an H⁺/e ratio of 1, operates.

Apart from the NADH-linked dehydrogenases and those linked to flavins, E. coli and S. typhimurium have been found to synthesize a glucose dehydrogenase apo-enzyme that is active in the presence of 2,7,9-tricarboxy-1H-pyrrolo-(2,3f)-quinoline quinone (PQQ) (3, 20, 21). The active center of this enzyme faces the periplasmic space, and the activity of glucose dehydrogenase of E. coli or S. typhimurium can be reconstituted when PQQ is added to the medium (3, 20, 21). Thus, in the presence of PQQ, and not in its absence, whole cells or cell homogenates of these organisms oxidize glucose directly to gluconate. Strains of Klebsiella pneumoniae, in contrast, are able to synthesize PQQ and have a functionally competent glucose dehydrogenase (3, 39). It has now been established unequivocally that PQQH₂ reacts with the quinone pool, because a cyo as well as a cyd mutant of E. coli oxidize glucose to gluconate in the presence of PQQ (R. de Jonge, M. J. Teixeira de Mattos, and O. M. Neijssel, unpublished data).

It is interesting to know how the electron transport fluxes are partitioned over these different branches in the growing wild-type organism. This problem was investigated by Calhoun et al. (5), who determined the specific rates of oxygen consumption of wild-type and mutant strains of E. coli in which one or two genes encoding components of the respiratory chain (ndh, cyo, or cyd) were deleted. The organisms were grown in a fully aerobic, glucose-limited chemostat culture at different dilution rates. According to the classical theory, the growth rate (μ), specific rate of oxygen consumption (q _O2), and growth yield on oxygen (Y _O2) are interrelated according to: μ = Y _O2 · q _O2, and when maintenance requirement is taken into account, μ =

· (q _O2 – q_m), where q_m is the specific rate of oxygen consumption at zero growth rate.

The results obtained by Calhoun et al. (5) (Fig. 2; Table 2) lead to interesting conclusions. First, the specific rates of oxygen consumption by the ndh cyo strain and the ndh strain differ significantly. Assuming that the synthesis of cytochrome bd by the ndh mutant, like that of the wild type, is repressed under fully aerobic conditions, the results show that the cytochrome bo branch conserves more energy than the cytochrome bd branch. But the bd branch must contribute to energy conservation, because the growth yield on oxygen of the ndh cyo strain is higher than would be expected if no energy were conserved. In this latter case, the specific rates of oxygen consumption of the ndh cyo strain should have been twofold higher than those of the ndh strain.

Another surprising result was the observation that NDH-2 catalyzes part of the electron transport in the wild-type organism. This could be deduced from the fact that an ndh mutant had lower oxygen uptake rates (= higher growth yields) than the wild type when both organisms were grown at similar growth rates. The possible physiological significance of this result will be discussed later, but this result strongly suggests that maximization of growth yield has not been a selective factor during the evolution of E. coli.

The effect of the presence of PQQ in the medium on growth yields of E. coli has been studied by Hommes et al. (22). When E. coli was grown in a glucose-limited chemostat culture, no effect of PQQ could be observed. This is not surprising because the PQQ-linked glucose dehydrogenase has a rather low affinity for glucose (K_m = 0.9 to 5 mM [1, 21]), and the glucose concentration in glucose-limited cultures is in the micromolar region (45). When the organism was grown under carbon-excess conditions (glucose concentration, >5 mM), addition of PQQ to the growth medium invariably caused the production of gluconate, with a concomitant stoichiometric increase in the specific rates of oxygen and glucose consumption (thus, lower growth yields on oxygen and glucose, respectively), but no further effects could be noted. This result shows again that when heterotrophic organisms are grown under carbon-excess conditions their rate of consumption of the carbon and energy source is not strictly proportional to growth rate.

 

Substrate-Level Phosphorylation

When growing anaerobically on glucose, in the absence of an added electron acceptor, E. coli effects a mixed-acid fermentation in which the principal products are lactate, ethanol, acetate, and formate (or H₂ plus CO₂ at acid pH values). Thus, energy is conserved (as ATP) principally at the levels of 3-phosphoglycerate kinase, pyruvate kinase, and acetate kinase (Fig. 3). Significant amounts of succinate derived from endogenously generated fumarate also are often found (Table 3), and, because fumarate reduction is coupled to respiratory chain-linked oxidation of NADH or formate, the formation of succinate is accompanied by the generation of a proton motive force. It follows, therefore, that whereas the formation of acetate is accompanied by a net production of 2 mol of ATP per mol, the formation of lactate, ethanol, and succinate is accompanied by just 1 mol of ATP per mol.

It would be advantageous (in terms of net ATP gain) to ferment glucose solely to acetate and formate. Redox considerations, however, require a concomitant formation of a product more reduced than lactate because acetate production is accompanied by a net formation of NADH. Production of ethanol fulfills this requirement in E. coli and K. pneumoniae. Hence, in fermenting glucose, a maximal ATP gain would be achieved in producing equal amounts of ethanol and acetate and would amount to 3 mol of ATP per mol of glucose fermented. It follows, therefore, that because anaerobic cultures generally are considered to be limited in their growth by the availability of energy (ATP), one would expect glucose to be fermented entirely to ethanol and acetate (plus formate, of course). Surprisingly, this was not found with batch cultures of E. coli (Table 3) or Aerobacter cloacae (19) or with glucose-sufficient chemostat cultures of K. pneumoniae (54). This condition was apparent only with glucose-limited chemostat cultures of E. coli, though small amounts of succinate and lactate were still formed (47). Taken at face value, these observations suggest that organisms growing on glucose anaerobically in batch culture (as in glucose-sufficient chemostat culture) are not energy limited. On the other hand, a high rate of cell synthesis, such as is manifest in batch cultures, demands a high rate of ATP generation, and the presence of branched fermentation capacity in an organism may actually serve to facilitate this high rate of ATP generation, albeit with a concomitant decrease in the efficiency of catabolic energy conservation (in terms of net moles of ATP formed per mole of glucose fermented).

 

ENERGY-CONSUMING REACTIONS

We now review the major energy-consuming processes that lead directly to the synthesis of biomass or extracellular products.

 

Cell Synthesis

The principal known energy-consuming reactions occurring in the growing microbial cell are those associated with solute transport, monomer formation, and most particularly, polymer synthesis, which all lead to a net increase in biomass. A detailed analysis of the theoretical ATP requirements for these processes, for organisms growing in a simple salts medium on a variety of carbon substrates, was provided by Stouthamer (48, 49) (Table 4).

On the basis of a rather atypical macromolecular composition of E. coli (i.e., a high polysaccharide content and a relatively low protein content) it was concluded that ATP needed for cell synthesis varied from 34.8 mmol/g (dry weight) of cells for growth on glucose to 99.5 mmol/g (dry weight) of cells for growth on acetate. Translated into yield coefficients, these values gave theoretical

values ranging from 28.8 to 10 g (dry weight) of cells per mol of ATP. These values are, respectively, 207% and 43% higher than the experimentally derived values for for E. coli growing aerobically on glucose and acetate, assuming two sites of respiratory chain energy conservation (5, 11). Hence either the energy requirements for transport and for monomer and polymer synthesis had been grossly underestimated or, as seems more probable, a substantial amount of energy is needed for growth-associated processes other than those specified above.

It is now known that certain biosynthetic processes are energetically more expensive than thought previously. For instance, the ATP consumption by molecular chaperones during the folding of proteins had not been taken into account (16). Martin et al. (31) determined the ATP requirement of GroEL during the folding of denatured bovine liver rhodanese in vitro. They arrived at the figure of 130 ± 20 molecules of ATP hydrolyzed per molecule of rhodanese folded at GroEL in the presence of GroES. This would account for 5 to 10% of the total ATP requirement for rhodanese synthesis. If this result were the same for all proteins synthesized in the E. coli cell, and this is highly unlikely, the total costs of protein synthesis as calculated by Stouthamer have to be increased by only 5 to 10%.

A similar discrepancy between theoretical and probable

values previously had been noted by Gunsalus and Shuster (13). They postulated dissipation of energy by ATPase mechanisms. Other authors (14, 64) also addressed this question and postulated that a major part of this discrepancy is explained by thermodynamic effects (see below).

We are aware of no definitive experiments that identify unequivocally how this apparent excess energy is consumed. There are, however, other processes that consume energy and lead to an apparent lowering of the yield values.

 

Extracellular Polymer Formation

Whereas most of the polymers synthesized by microbial cells are contained within the plasma membrane and envelope structure, some may be secreted into the medium. This is particularly the case with polysaccharides, which under some conditions and with particular species may account for a substantial part of the carbon substrate consumed. For example, ammonia-limited chemostat cultures of K. pneumoniae growing on glucose at a rate of 0.17 h^–1 excreted more than one-third of the substrate consumed into the medium as polysaccharide (37). With similarly limited cultures of E. coli (PC-1000), also growing at a low rate (0.18 h^–1), less extracellular polysaccharide was excreted, but polysaccharide still accounted for 14% of the total glucose consumed (Table 5). Though polyglucose is energetically less expensive to synthesize than protein, RNA, and DNA (i.e., 12.4 mmol of ATP per g as compared with, respectively, 39.1, 37.3, and 33.0 mmol of ATP per g [49]), substantial production of this compound would consume a significant portion of the ATP generated by catabolism.

Proteins are not commonly secreted by E. coli and K. pneumoniae. Nevertheless, it is conceivable that enzymes and binding proteins normally located in the periplasmic space may, under some conditions, diffuse into the extracellular fluids. Thus, protein was found to accumulate in the extracellular fluids of chemostat cultures of K. pneumoniae that were sulfate, magnesium, or potassium limited when growing on glucose at a low rate (57). The extracellular protein from the sulfate-limited culture was shown not to be a product of lysis because it was composed of an excess of acidic amino acids over basic amino acids and contained virtually no methionine or cysteine.

 

Excretion of Low-Molecular-Weight Metabolites

Batch cultures of E. coli, or carbon substrate-limited chemostat cultures growing aerobically in a defined simple salts medium, generally metabolize substrate solely to cells and CO₂. However, carbon substrate-sufficient chemostat cultures often metabolize substrate inefficiently, accumulating substantial amounts of products in the medium (Table 5). Acetate almost invariably is found (37), along with various amounts of pyruvate, 2-oxoglutarate, and d-lactate. Hence, the yield value with respect to the carbon substrate is markedly lowered. In general, formation of these so-called overflow metabolites does not consume energy. On the contrary, these metabolites often (perhaps invariably) are associated with an increased energy flux, as manifested by an increased rate of respiration.

When cells are growing anaerobically by fermentation, accumulation of metabolites in the extracellular fluids is a sine qua non. However, when growing at a fixed rate, glucose-sufficient chemostat cultures of E. coli and K. pneumoniae consumed glucose at enhanced rates as compared to glucose-limited cultures (54; J. L. Snoep, Ph.D. thesis, University of Amsterdam, Amsterdam, The Netherlands, 1992). Under conditions of glucose excess, a branched fermentation pattern was manifest, with a concomitant lowering of the overall efficiency of ATP generated (i.e., net moles of ATP formed per mole of glucose fermented). Nevertheless, this decrease in the efficiency factor was smaller than the increase in glucose consumption rate, and again, just as with aerobic cultures, the apparent Y _ATP values were low.

It remains a matter for speculation whether, in these anaerobic cultures, the formation of some products is energetically favorable, neutral, or unfavorable. For example, the formation of d-lactate might be accompanied by a net ATP gain (as would be the case if it arose from the reduction of pyruvate or if lactate excretion generated a proton motive force [32]), or it might cause a severe energy drain if it arose via dihydroxyacetone phosphate and methylglyoxal (8, 9).

 

ENERGY-DISSIPATING PROCESSES

In view of the above considerations, there must be endergonic processes that do not lead to a net increase of biomass or the excretion of a metabolic product into the culture fluid. We call these energy-dissipating processes.

 

Maintenance Functions

As mentioned, Monod (33) conceived that a small amount of energy must be dissipated in the maintenance of cell integrity and viability. But although this concept now has been repeatedly validated experimentally, it still is not clear what precise physiological functions constitute maintenance. This issue is clouded and confused by the widely disparate maintenance values cited in the literature for different strains of the same species growing in putatively identical media (Table 1), as well as for a single strain growing on a range of different carbon substrates (17). In this latter study, exceptionally high values of the apparent oxygen consumption rate for maintenance (m_O) were reported for the growth of E. coli B on glycerol, succinate, glutamate, and acetate (respectively, 10.2, 13.1, 17.1, and 25.9 mmol of O per h per g [dry weight] of cells) as compared with values of 0.9 and 1.8 mmol of O per h per g (dry weight) of cells for growth on glucose and galactose, respectively. Moreover, at the other extreme, microcalorimetric studies of glucose-limited chemostat cultures of E. coli K-12 (25) indicated a maintenance rate of heat evolution of only 0.02 kcal/h per g (dry weight) of cells (1 cal = 4.184 J), which would equate with an oxygen consumption rate of no more than 0.36 mmol of O per h per g (dry weight) of cells. As yet, there is no rational explanation for these widely differing maintenance rates. If the quantitatively major components of maintenance were turnover of macromolecules, maintenance of ion gradients, and motility, there is no obvious reason why the maintenance requirement of an acetate-limited culture should be almost 30-fold higher than that of a glucose-limited one.

High apparent maintenance rates of oxygen or carbon substrate consumption or both are commonplace with chemostat cultures in which growth is limited by the availability of an anabolic substrate (e.g., source of nitrogen, sulfur, or phosphorus) or an essential cation (K⁺ or Mg²⁺). Here, the question becomes how the uptake of carbon substrate can be modulated to meet the low biosynthetic and bioenergetic demands of cell synthesis. Because washed suspensions of bacteria, which clearly cannot grow, often oxidize substrates such as glucose at a high rate, one may conclude that such regulation, if present, is by no means stringent. It seems reasonable that the high rates of oxygen and/or carbon substrate consumption by slowly growing carbon substrate-sufficient chemostat cultures do not necessarily result from some enhanced specific maintenance energy requirement, but more likely from partial uncoupling of catabolism from anabolism.

To analyze yield values in physiological terms one needs to know more about mechanisms that might dissociate catabolism from anabolism and how they might be modulated. We address these questions below.

 

Regulatory Processes

Regulatory processes are an important class of energy-consuming reactions that do not contribute directly to net biomass synthesis. One could consider energy consumption by these reactions as part of the maintenance energy requirement, but in view of their special nature we will deal with these regulatory processes separately.

Energy Flux Regulation.

Several researchers have proposed that biological energy converters have evolved toward maximal energy output (biomass formation) and consequently function at reduced efficiency (14, 28, 50). This hypothesis is based on the application of nonequilibrium thermodynamics to energy conversion in biological systems. Westerhoff and van Dam (63) have analyzed this problem in detail, and they, like their predecessors, concluded that maximal efficiency of energy generation is possible only when the rate of energy generation is zero. Maximal output power is attained at an efficiency of free energy conservation of 50%. This could explain the large difference between calculated and experimentally derived growth yields (see above). The interesting question therefore arises of whether and how the efficiency of energy conversion is regulated.

There are two mechanisms by which a microbe could decrease its efficiency of energy conservation: first, ATPase reactions, and second, mechanisms that lower the efficiency of energy generation by the respiratory chain or substrate-level phosphorylation. Several lines of evidence indicate that at high proton motive force values, energy dissipation occurs by leakage of protons through the membrane into the cytoplasm or by a slip in the proton pumps (for review, see reference 64). The uncoupled NADH dehydrogenase (NDH-2) could also play a role: electron transport catalyzed by this enzyme would lead to a 50% lower efficiency of energy generation when cytochrome bo is the terminal oxidase and 2/3 lower when cytochrome bd is the terminal oxidase. Experimental evidence for this physiological role of NDH-2 is not available. The ndh strain of E. coli used by Calhoun et al. (5) grew in a glucose-limited chemostat culture without apparent difficulty, but its response to sudden changes in the concentration of the growth-limiting nutrient and its behavior under energy-excess conditions were not tested.

Under fermentative conditions, NADH oxidation and ADP phosphorylation can be uncoupled by the reactions of the methylglyoxal bypass (Fig. 3) (53). Methylglyoxal synthase, the first enzyme of the pathway, is strongly inhibited by moderate concentrations of phosphate (<1 mM); it has been suggested that this enzyme recycles phosphate when the intracellular phosphate concentration is low (9). In slowly growing glucose-limited anaerobic cultures of K. pneumoniae, the products of glucose fermentation were acetate and ethanol (plus formate, CO₂, and H₂). But when suddenly exposed to a cell-saturating concentration of glucose, 50% of the extra glucose was converted to d-lactate. Because in the short term (20 min) there was a large increase in the glucose consumption rate but no increase in the growth rate, and because the key enzymes of the methylglyoxal bypass were present in substantial amounts, it was concluded that the formation of d-lactate occurred exclusively via methylglyoxal. The other products formed, after the glucose pulse, were succinate and 2,3-butanediol, and by drawing out the fermentation scheme (Fig. 4) it became clear that this extra glucose was indeed being fermented with no concomitant net formation of ATP. In other words, fermenting this extra glucose through the methylglyoxal bypass generates no ATP. Because the enzymes that effect a conversion of dihydroxyacetone phosphate to d-lactate, via methylglyoxal, are formed constitutively in E. coli (8, 9), there is little doubt that this organism also can totally uncouple glycolysis from ADP phosphorylation. The fact that E. coli as well as K. pneumoniae can synthesize d-lactate from either pyruvate (via a nicotinamide adenine dinucleotide [NAD]-linked lactate dehydrogenase [51]) or dihydroxyacetone phosphate (via methylglyoxal synthase and glyoxylase) renders difficult an accurate assessment of the energy balance in those anaerobic cultures in which this metabolite is a major product. For example, a homolactic fermentation of glucose (to d-lactate) could yield, maximally, 2 mol of ATP per mol of glucose (if only the NAD-linked lactate dehydrogenase was active) or, minimally, zero ATP if 50% of the lactate arose via methylglyoxal. Anaerobically, no more than 50% of the glucose carbon can be converted to d-lactate via the methylglyoxal bypass, since the conversion of glucose to the two triose phosphates consumes 2 mol of ATP and this must be recovered by glycolysis. Aerobically, however, all the glucose carbon could be converted to d-lactate via methylglyoxal, and the d-lactate could then be oxidized to pyruvate to generate ATP for phosphorylating glucose and fructose 6-phosphate. Indeed, since the oxidation of d-lactate to pyruvate by the respiratory chain-linked dehydrogenase invokes just one site of energy conservation, glucose could be aerobically catabolized solely to pyruvate without any net gain of biologically usable energy.

Relevant in the context mentioned above is the observation that glucose-limited aerobic chemostat cultures of K. pneumoniae and E. coli can immediately accelerate the rate of glucose consumption when suddenly relieved of the growth limitation (36). There was a concomitant marked increase in the respiration rate but no corresponding increase in the growth rate, indicating that excess energy was being dissipated. Significantly, the extra glucose consumed was not oxidized completely; substantial amounts of pyruvate and acetate were excreted into the medium (M. P. M. Leegwater, Ph.D. thesis, University of Amsterdam, Amsterdam, The Netherlands, 1983). Clearly, if the pyruvate arose from d-lactate one could partially account for the energetic uncoupling; however, the formation of acetate from pyruvate would yield NADH, and therefore a question arises as to whether this could be oxidized without generating a proton motive force. We know of no such mechanism or postulated mechanism, but both K. pneumoniae and E. coli have constitutively two enzymes (an NAD-linked lactate dehydrogenase that generates d-lactate and a d-lactate dehydrogenase which is flavoprotein) that together can oxidize NADH without invoking site 1 of the respiratory chain. The NAD-linked lactate dehydrogenase is homotropic with respect to pyruvate; hence, an accumulation of pyruvate within the cell would promote this bypass reaction.

Metabolic Flux Regulation.

Several biochemical pathways serve more than one function. For example, the respiratory chain generates energy and reoxidizes cofactors. Similarly, glycolysis provides reducing equivalents, energy via substrate level phosphorylation, carbon skeletons for anabolism, and pyruvate as fuel for the tricarboxylic acid cycle. One can imagine growth conditions in which these different functions are not optimally compatible. The coupling of these functions could be loosened if there were a mechanism to regulate the flux through glycolysis such that one process (say, anabolism) is provided optimally with the essential ingredients while another (say, energy generation) is diminished. Futile cycles might optimize the regulation of metabolic fluxes (27, 40). When two compounds are interconverted by two reactions in which one consumes more energy than the other generates, there is futile cycling which dissipates energy without changing metabolite concentrations.

One possible futile cycle in E. coli is the interconversion of fructose 6-phosphate and fructose 1,6-bisphosphate, effected by phosphofructokinase and fructose-1,6-bisphosphatase, respectively (6, 10). However, little if any futile cycling could be observed in batch cultures growing on either glucose or gluconeogenic substrates. But the growth conditions used were not those which one would expect to favor extensive futile cycling. Scrutton and Utter (46) found that phosphofructokinase is inhibited by ATP and activated by AMP, whereas fructose-1,6-bisphosphatase is inhibited by AMP and activated by ATP. Hence, rapid ATP generation by respiration when ATP utilization by biosynthetic reactions is constrained might well activate this futile cycle.

E. coli synthesizes both glutamine synthetase and glutaminase, which also could cause futile cycling (43). Because glutamine synthetase is highly active in ammonia-limited cells (29) and because significant amounts of glutaminase also are present (56), futile cycling must occur unless the activity of glutaminase is constrained in some way. Activities of both enzymes are markedly influenced by the energy charge (43); however, the patterns of regulation suggest that when the energy charge is high the glutaminase activity would be low (i.e., about 20% of the maximal activity), though significant. A possible explanation for the presence of glutaminase in ammonia-limited cells is that, by releasing ammonia, it allows the key enzyme of ammonia assimilation (glutamine synthetase) to continue working at a significant rate in the virtual absence of exogenous ammonia. This, one might argue, permits the uptake system to remain highly active during transient changes in the supply of growth-limiting nutrient. This hypothesis could be tested by determining whether the intracellular pool level of ammonia is maintained at a significant value after an interruption in the supply of nutrients to an ammonia-limited culture. To the best of our knowledge, this experiment has not been performed.

If some futile cycles are involved in regulating the rate of uptake of growth-limiting nutrients, one might expect similar reactions to be associated with common nutrients such as sulfate, phosphate, or potassium. Indeed, it has been shown that E. coli has two enzymes [adenosine-5'-phosphosulfate kinase and 3'(2'),5'-diphosphonucleoside 3'-phosphohydrolase] that are potentially capable of acting as a futile cycle (61). Similarly, phosphatases are synthesized to high levels by phosphate-limited cells (60).

The specific rates of oxygen consumption of potassium-limited cultures of K. pneumoniae or E. coli are extremely high (4, 58). The same effect can be observed in glucose-limited cultures with a low [K⁺] input, and the respiration rate varies and appears to correlate closely with the transmembrane K⁺ gradient (23). It was proposed that much respiratory energy was dissipated as a consequence of an induced K⁺ leakage current (58). Subsequently, Mulder et al. (34) postulated that futile cycling of K⁺ ions in K-limited E. coli cells was caused by the uptake of this ion via the Kdp system (the high-affinity K⁺ uptake system, which is derepressed under these growth conditions) and leakage via the low-affinity Trk system.

Buurman et al. (4) investigated this cycle further and found that the very high rates of oxygen consumption (17.1 mmol of O₂ per g [dry weight] of cells per h, at D = 0.3 h^–1) observed in potassium-limited chemostat cultures of E. coli (with ammonium chloride as the nitrogen source) are caused by the Kdp system, which also pumps NH₄ ⁺ ions into the cell. This can be explained by the similar size and charge of the K⁺ and NH₄ ⁺ ions (ionic radii: 1.33 Å [0.133 nm] and 1.43 Å, respectively). Intracellularly, NH₄ ⁺ dissociates into NH₃ and a proton. Since the cell membrane of E. coli is permeable to NH₃, this molecule will diffuse out if the extracellular [NH₃] is lower. This is the case when the culture pH value is lower than the cytoplasmic pH, or when the Kdp activity is high. In addition, the maintenance of cytoplasmic pH homeostasis requires extrusion of the proton formed by the dissociation of NH₄ ⁺, an energy-consuming process. When alanine was used as the nitrogen source (at concentrations that did not cause the production of ammonia due to deamination), the specific rate of oxygen consumption of a potassium-limited E. coli culture was similar to those of phosphate or sulfate-limited cultures (10 to 12 mmol of O₂ per g [dry weight] of cells per h at D = 0.3 h–1). Similarly, a mutant strain, in which the Kdp was not active, showed the same lower oxygen uptake rates, indicating that futile cycling of NH4⁺ ions no longer occurred.

Signal Transduction.

Changes in the growth environment can be detected by two-component regulatory systems (see chapter 80). These systems consist of a sensor protein, embedded in the membrane, which can phosphorylate an activator protein that subsequently binds at the appropriate place in the genome. Thus detecting a signal from the growth environment costs energy, although no data on the amount of energy consumed by these systems are known. Possibly, processes such as DNA editing, protein folding, and signal transduction are very expensive terms energetically and are responsible for a large part of the difference between the energetic costs of polymer synthesis and the synthesis of a living cell.

 

GENERAL CONCLUSIONS

Microbial growth is the product of a large number of interconnected enzyme-catalyzed reactions; the fact that, in any particular environment, cell synthesis proceeds with a more or less constant efficiency indicates that a substantial measure of control must be exercised over the fluxes of intermediary metabolites and precursor substances involved in polymer synthesis. Moreover, in chemoheterotrophic organisms such as E. coli and S. typhimurium, processes of regulation are further complicated by the fact that the energy needed for biosynthesis necessarily must be derived from the breakdown of carbon substrate that simultaneously is being assimilated into cell substance. Hence, one might expect mechanisms operating in these cells to precisely apportion the flow of intermediary metabolites between catabolic and anabolic reactions. Thus, control systems might act at specific branch points between catabolic and anabolic pathways which would be "tuned" to the overall energy status of the cell. That some such controls indeed are present within the microbial cell is abundantly obvious from the widely reported involvement of adenine nucleotides as control elements in intermediary metabolism (7). Indeed, the mode of action of these regulatory processes (e.g., allosteric effectors) suggests a stringent coupling between ATP synthesis and growth which manifests itself in a precise yield value. However, such a concept is untenable because of observations extending back over many years (13, 44, 55); energy (ATP) generation can occur at a high rate when cell synthesis is severely constrained. Clearly, there is no obligatory coupling between catabolism and anabolism, and herein lies the source of much difficulty in attempting to interpret yield data in energetic terms. It is obvious, therefore, that further progress in evaluating the energetics of microbial growth (particularly aerobic growth) hinges critically on the acquisition of a better understanding both of those energy-spilling processes extant within the cell and of their associated regulatory mechanisms. In this chapter we have attempted to make a start along these lines.