Metabolic Fluxes during Strong Carbon Catabolite Repression by Malate in Bacillus subtilis

Abstract

Commonly glucose is considered to be the only preferred substrate in Bacillus subtilis whose presence represses utilization of other alternative substrates. Because recent data indicate that malate might be an exception, we quantify here the carbon source utilization hierarchy. Based on physiology and transcriptional data during co-utilization experiments with eight carbon substrates, we demonstrate that malate is a second preferred carbon source for B. subtilis, which is rapidly co-utilized with glucose and strongly represses the uptake of alternative substrates. From the different hierarchy and degree of catabolite repression exerted by glucose and malate, we conclude that both substrates might act through different molecular mechanisms. To obtain a quantitative and functional network view of how malate is (co)metabolized, we developed a novel approach to metabolic flux analysis that avoids error-prone, intuitive, and ad hoc decisions on 13 C rearrangements. In particular, we developed a rigorous approach for deriving reaction reversibilities by combining in vivo intracellular metabolite concentrations with a thermodynamic feasibility analysis. The thus-obtained analytical model of metabolism was then used for network-wide isotopologue balancing to estimate the intracellular fluxes. These 13 C-flux data revealed an extraordinarily high malate influx that is primarily catabolized via the gluconeogenic reactions and toward overflow metabolism. Furthermore, a considerable NADPH-producing malic enzyme flux is required to supply the biosynthetically required NADPH in the presence of malate. Co-utilization of glucose and malate resulted in a synergistic decrease of the respiratory tricarboxylic acid cycle flux.Typically, microbes prefer a single carbon source whose presence represses utilization of alternative substrates. The classical example is the diauxic shift of Escherichia coli with subsequent growth on first glucose and then lactose (1). This widespread phenomenon, referred to as carbon catabolite repression, has been intensively studied in many chemoorganotrophic microbes, and in the vast majority of documented cases, the preferred carbon source is glucose (2, 3). Apart from repressing the co-utilization of alternative substrates, preferred carbon sources are normally associated with a high specific growth rate and substantial secretion of overflow metabolites such as ethanol, lactate, or acetate. For the Grampositive model bacterium Bacillus subtilis, glucose has long been recognized to be preferred, but recent data indicated that glucose might not be the only preferred carbon source (4, 5).According to the current model of the global carbon catabolite repression mechanism in B. subtilis, glucose-induced catabolite repression is mediated by the HPr kinase-catalyzed phosphorylation of HPr at its Ser-46 residue (2). This effect is propagated throughout the cell by the pleiotropic transcription factor CcpA (6), whose binding to the target sites for catabolite repression is controlled by the presence of HPr(Ser...

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