The authors propose that prokaryotic metabolism is fundamentally constrained by the cytoplasmic membrane surface area available for protein expression, and show that this constraint can explain previously puzzling physiological phenomena, including respiro-fermentation.
We propose that prokaryotic cellular metabolism is fundamentally constrained by the finite cytoplasmic membrane surface area available for protein expression.A metabolic model of Escherichia coli updated to include a cytoplasmic membrane constraint is capable of predicting a variety of puzzling phenomena in this organism, including the respiro-fermentation phenomenon.Because the surface area to volume ratio is directly related to the morphology of the cell, this constraint provides a direct link between prokaryotic morphology and physiology.The potential relevance of this constraint to eukaryotes is discussed.
Many heterotrophs can produce ATP through both respiratory and fermentative pathways, allowing them to survive with or without oxygen. Since the molar ATP yield (molar ATP yield: mole of ATP produced/mole of substrate consumed) from respiration is about 15-fold higher than that from fermentation, ATP production via respiration is more efficient. Surprisingly, at high catabolic rate, many facultative aerobic organisms employ fermentative pathways simultaneously with respiration, even in the presence of abundant oxygen to produce ATP (Pfeiffer et al, 2001; Vemuri et al, 2006; Molenaar et al, 2009). This leads to an observable tradeoff between the ATP yield and the catabolic rate (Pfeiffer et al, 2001; Vemuri et al, 2006). This respiro-fermentation physiology is commonly observed in microorganisms, including Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae (Molenaar et al, 2009), as well as cancer cells (Vander Heiden et al, 2009). Despite extensive research, existing theories (Majewski and Domach, 1990; Varma and Palsson, 1994; Pfeiffer et al, 2001; Vazquez et al, 2008; Molenaar et al, 2009) cannot fully explain the respiro-fermentation phenomenon.
The membrane economics theory
We propose the hypothesis that the prokaryotic metabolism is fundamentally constrained by the finite cytoplasmic surface area available for protein expression—in order to maximize fitness, prokaryotic organisms such as E. coli must economically manage the expression of membrane proteins based on the membrane cost and the fitness benefit of the proteins. This hypothesis is proposed based on theoretical considerations (in this work), numerical analysis (Phillips and Milo, 2009), and experimental observation that the overexpression of non-respiratory membrane protein significantly reduces the oxygen consumption rate and induces aerobic fermentation (Wagner et al, 2007). Such a constraint on transmembrane protein expression may have significant physiological consequences in prokaryotes, such as E. coli, at higher catabolic rates. First, since both substrate transporters and respiratory enzymes are localized on the cytoplasmic membrane in prokaryotes, increased substrate uptake rates necessitates a decrease in the respiratory rate. This decrease in the respiratory rate, forces prokaryotes to process the additional substrate through the fermentative pathways, which are not catalyzed by transmembrane proteins, for continued ATP production. Furthermore, since the membrane requirement of an enzyme is inversely related to its turnover rate (see Materials and methods section in the manuscript), the faster and inefficient respiratory enzymes (such as Cyd-I and Cyd-II in E. coli) might be preferred over the slower and efficient enzymes (such as Cyo in E. coli), leading to an altered respiratory stoichiometry at higher catabolic rates. Finally, the absence of the respiratory enzymes under anaerobic conditions explains why the maximum glucose uptake rate (GUR) of E. coli is much higher.
Applying membrane economics theory to E. coli
To illustrate that the ‘membrane economics' theory could satisfactorily explain the physiological changes associated with the respiro-fermentation phenomenon in E. coli, we modified the genome-scale metabolic model of E. coli (Feist et al, 2007) to include a cytoplasmic membrane occupancy constraint. Using ‘relative membrane costs' calculated from experimental data, the new modeling framework—FBA with membrane economics (FBAME)—predicted that wild-type E. coli has a GUR of 10.7 mmol/gdw/h, an oxygen uptake rate (OUR) of 15.8 mmol/gdw/h, and a specific growth rate of 0.69 per hour during aerobic growth with excess glucose. FBAME also predicted that under the same growth condition, an E. coli knockout strain with no cytochromes has a GUR of 18 mmol/gdw/h and growth rate of 0.42. These values agree very well with the reported experimental values for E. coli grown in batch cultures (Vemuri et al, 2006; Portnoy et al, 2008), which supports our hypothesis that the higher GUR of E. coli during glucose-excess anaerobiosis than under aerobic conditions is due to the absence of the respiratory enzymes. We also simulated the aerobic growth of E. coli in glucose-limited chemostat using both conventional FBA and FBAME. FBAME successfully predicted the growth rate and yield changes with respect to increasing GUR (Figure 2A and B), as well as the aerobic production of acetate (Figure 2C) and concomitant repression of oxygen uptake (Figure 2D). On the other hand, traditional FBA significantly overestimated the growth rate and yield at higher GURs (this overestimation cannot be explained by varying the growth-associated maintenance (GAM) energy parameter; Figure 2A), and failed to predict the decrease in yield independent of acetate overflow and reduction in oxygen uptake at higher GURs (Figure 2). In addition, FBAME was able to predict the reduction of the TCA cycle activities at higher uptake rates (Figure 3C and D) as well as the selective expression of Cyo and Cyd-II at lower uptake rates (Figure 3A and B), whereas conventional FBA cannot predict the expression of inefficient Cyd-II. These predictions agree with the gene expression data from glucose-limited chemostat (Figure 3). Given the simplicity of the constraint we imposed, our model predictions agree surprisingly well with experimental observations, lending strong credibility to the membrane economics hypothesis.
Although it has been long suggested that cellular evolution are governed by non-adjustable mechanistic constraints (Palsson, 2000; Papin et al, 2005; Novak et al, 2006), to date, most metabolic models rely on empirically derived parameters such as glucose and OUR. In this article, we showed that complex phenomena, such as the respiro-fermentation in E. coli, could be satisfactorily explained and accurately predicted by using constraint-based optimization by introducing a simple mechanistic constraint on membrane enzyme occupancy. Given that the cytoplasmic membrane occupancy constraint is directly related to the surface area to volume (S/V) ratio of the cell, it is possible that this constraint resulted in the evolution of mitochondria in eukaryotes as mitochondria allows for a significantly increased S/V ratio. Further efforts to elucidate such fundamental cellular constraints as well as the underlying design principles could significantly improve our understanding of the regulation and evolution of metabolism.
The simultaneous utilization of efficient respiration and inefficient fermentation even in the presence of abundant oxygen is a puzzling phenomenon commonly observed in bacteria, yeasts, and cancer cells. Despite extensive research, the biochemical basis for this phenomenon remains obscure. We hypothesize that the outcome of a competition for membrane space between glucose transporters and respiratory chain (which we refer to as economics of membrane occupancy) proteins influences respiration and fermentation. By incorporating a sole constraint based on this concept in the genome-scale metabolic model of Escherichia coli, we were able to simulate respiro-fermentation. Further analysis of the impact of this constraint revealed differential utilization of the cytochromes and faster glucose uptake under anaerobic conditions than under aerobic conditions. Based on these simulations, we propose that bacterial cells manage the composition of their cytoplasmic membrane to maintain optimal ATP production by switching between oxidative and substrate-level phosphorylation. These results suggest that the membrane occupancy constraint may be a fundamental governing constraint of cellular metabolism and physiology, and establishes a direct link between cell morphology and physiology.