Bacillus subtilis Metabolism and Energetics in Carbon-Limited and Excess-Carbon Chemostat Culture

Abstract

The energetic efficiency of microbial growth is significantly reduced in cultures growing under glucose excess compared to cultures growing under glucose limitation, but the magnitude to which different energy-dissipating processes contribute to the reduced efficiency is currently not well understood. We introduce here a new concept for balancing the total cellular energy flux that is based on the conversion of energy and carbon fluxes into energy equivalents, and we apply this concept to glucose-, ammonia-, and phosphate-limited chemostat cultures of riboflavin-producing Bacillus subtilis. Based on [U-13 C 6 ]glucose-labeling experiments and metabolic flux analysis, the total energy flux in slow-growing, glucose-limited B. subtilis is almost exclusively partitioned in maintenance metabolism and biomass formation. In excess-glucose cultures, in contrast, uncoupling of anabolism and catabolism is primarily achieved by overflow metabolism, while two quantified futile enzyme cycles and metabolic shifts to energetically less efficient pathways are negligible. In most cultures, about 20% of the total energy flux could not be assigned to a particular energy-consuming process and thus are probably dissipated by processes such as ion leakage that are not being considered at present. In contrast to glucoseor ammonia-limited cultures, metabolic flux analysis revealed low tricarboxylic acid (TCA) cycle fluxes in phosphate-limited B. subtilis, which is consistent with CcpA-dependent catabolite repression of the cycle and/or transcriptional activation of genes involved in overflow metabolism in the presence of excess glucose. ATPdependent control of in vivo enzyme activity appears to be irrelevant for the observed differences in TCA cycle fluxes.The very basis of microbial growth resides in balanced fluxes through anabolic and catabolic reactions. These metabolic fluxes are highly variable and change with the environmental conditions and the rate of growth, since faster-growing cells demand a higher rate of metabolism. To delineate these influences, metabolic flux responses are typically studied in chemostat cultures that are maintained under different nutrient limitations. When microorganisms are limited for their energy source (usually the carbon source), catabolism is tightly coupled to anabolism and high biomass yields on the carbon source are achieved (40). Compared to those with carbon (C) limitation, excess-C cultures exhibit generally high rates of carbon consumption and low yields of biomass and thus have a low energetic growth efficiency (11,31,32,57). Most frequently excess-C cultures in chemostats are limited by other cellular macroelements such as nitrogen (N) and phosphorus (P) but also potassium, the predominant intracellular cation (11). Potassium or P limitation invokes usually a strong uncoupling of catabolic and anabolic processes that lead consequently to low biomass yields on the energy source, while N limitation has more moderate effects on cellular physiology (31, 68). Although mostly studied in the gr...

Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC‐MS

“…4), and overflow metabolism, but to some extent also by the genetic background of the wild-type strains (Fig. 5), as was noted previously for different organisms [7,16,26,35,36]. Generally, anaplerosis is high under conditions that invoke overflow metabolism, as acetate formation reduces the fraction of intact two carbon units entering the TCA cycle.…”

Analysis of Growth of Lactobacillus plantarum WCFS1 on a Complex Medium Using a Genome-scale Metabolic Model

“…The identified flexibility in the metabolic network of L. plantarum for ATP production and consumption may help in explaining a largely unresolved question in the case of metabolic uncoupling: where does all the ATP go (18,25)? Isotopic labeling studies have failed to identify a clear primary futile cycle that can consume all the excess ATP generated by catabolism under glucose excess (34), although high fluxes through certain futile cycles have been observed under specific (often, paradoxically, glucose-limited) conditions, in particular involving pyruvate metabolism (35)(36)(37), and proton (38) or potassium leakage currents over the cell membrane (39). Our analyses identified many more futile cycles that could potentially contribute to ATP dissipation.…”

A Novel Metabolic Cycle Catalyzes Glucose Oxidation and Anaplerosis in Hungry Escherichia coli

“…Considering that catabolism of one PEP molecule generates a total of 7-11 ATP equivalents in the tricarboxylic acid cycle and oxidative phosphorylation (27), the loss of one ATP equivalent does not seem to be a major energetic consequence. This is particularly true because aerobic microbial metabolism does not optimize the yield of ATP per se (30 -32), not even under conditions of hunger (33).…”