Bacillus subtilis metabolism and energetics in carbon-limited and excess-carbon chemostat culture

The energetic efficiency of microbial growth is significantly reduced in cu ltures growing under glucose excess compared to cultures growing under gluc ose limitation, but the magnitude to which different energy-dissipating pro cesses contribute to the reduced efficiency is currently not well understoo d. 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 energ y equivalents, and we apply this concept to glucose-, ammonia-, and phospha te-limited chemostat cultures of riboflavin-producing Bacillus subtilis. Ba sed on [U-C-13(6)] glucose-labeling experiments and metabolic flux analysis , the total energy flux in slow-growing, glucose-limited B. subtilis is alm ost exclusively partitioned in maintenance metabolism and biomass formation . In excess-glucose cultures, in contrast, uncoupling of anabolism and cata bolism is primarily achieved by overflow metabolism, while two quantified f utile enzyme cycles and metabolic shifts to energetically less efficient pa thways are negligible. In most cultures, about 20% of the total energy flux could not be assigned to a particular energy-consuming process and thus ar e probably dissipated by processes such as ion leakage that are not being c onsidered at present. In contrast to glucose- or ammonia-limited cultures, metabolic flux analysis revealed low tricarboxylic acid (TCA) cycle fluxes in phosphate-limited B. subtilis, which is consistent with CcpA-dependent c atabolite repression of the cycle and/or transcriptional activation of gene s involved in overflow metabolism in the presence of excess glucose. ATP-de pendent control of in vivo enzyme activity appears to be irrelevant for the observed differences in TCA cycle fluxes.