Genome-scale metabolic models represent the entirety of metabolic reactions of an organism based on the annotation of the respective genome. These models commonly allow all reactions to proceed concurrently, disregarding the fact that at no point all proteins will be present in a cell. The metabolic reaction space can be constrained to a more physiological state using experimentally obtained information on enzyme abundances. However, high-quality, genome-wide protein measurements have been challenging and typically transcript abundances have been used as a surrogate for protein measurements. With recent developments in mass spectrometry-based proteomics, exemplified by SWATH-MS, the acquisition of highly quantitative proteome-wide data at reasonable throughput has come within reach. Here we present methodology to integrate such proteome-wide data into genome-scale models. We applied this methodology to study cellular changes in Enterococcus faecalis during adaptation to low pH. Our results indicate reduced proton production in the central metabolism and decreased membrane permeability for protons due to different membrane composition. We conclude that proteomic data constrain genome-scale models to a physiological state and, in return, genome-scale models are useful tools to contextualize proteomic data.

dIncreasing antibiotic resistance in pathogenic bacteria necessitates the development of new medication strategies. Interfering with the metabolic network of the pathogen can provide novel drug targets but simultaneously requires a deeper and more detailed organism-specific understanding of the metabolism, which is often surprisingly sparse. In light of this, we reconstructed a genome-scale metabolic model of the pathogen Enterococcus faecalis V583. The manually curated metabolic network comprises 642 metabolites and 706 reactions. We experimentally determined metabolic profiles of E. faecalis grown in chemically defined medium in an anaerobic chemostat setup at different dilution rates and calculated the net uptake and product fluxes to constrain the model. We computed growth-associated energy and maintenance parameters and studied flux distributions through the metabolic network. Amino acid auxotrophies were identified experimentally for model validation and revealed seven essential amino acids. In addition, the important metabolic hub of glutamine/glutamate was altered by constructing a glutamine synthetase knockout mutant. The metabolic profile showed a slight shift in the fermentation pattern toward ethanol production and increased uptake rates of multiple amino acids, especially L-glutamine and L-glutamate. The model was used to understand the altered flux distributions in the mutant and provided an explanation for the experimentally observed redirection of the metabolic flux. We further highlighted the importance of gene-regulatory effects on the redirection of the metabolic fluxes upon perturbation. The genome-scale metabolic model presented here includes gene-protein-reaction associations, allowing a further use for biotechnological applications, for studying essential genes, proteins, or reactions, and the search for novel drug targets. Enterococcus faecalis plays an important role in both biotechnology and medicine (1, 2). Some strains are used in the dairy industry for food fermentation and flavor production. At the same time, other strains play an increasingly important role as pathogens, especially in hospital-acquired infections (1). E. faecalis strains show multiple antibiotic resistances and are therefore the cause of serious complications. Alternative strategies for combating multiple resistant bacteria such as E. faecalis are urgently needed. Targeting vulnerable points in the bacterial metabolism could offer such an alternative route and has been discussed as a promising strategy (3, 4). However, surprisingly little is known about the detailed metabolism of E. faecalis, and it is therefore mandatory to explore this in a more elaborate fashion than previously reported.E. faecalis shows a high stress tolerance and is adapted to a variety of different native environments ranging from soil up to human or animal digestive tracts (1, 2). This environmental variability requires a highly flexible metabolic system to quickly adapt to diverse and changing environmental conditions. Therefore, strategies...

Brassinosteroids (BR) are involved in the control of several developmental processes ranging from root elongation to senescence and adaptation to environmental cues. Thus, BR perception and signaling have to be precisely regulated. One regulator is BRI1‐associated kinase 1 (BAK1)‐interacting receptor‐like kinase 3 (BIR3). In the absence of BR, BIR3 forms complexes with BR insensitive 1 (BRI1) and BAK1. However, the biophysical and energetic requirements for complex formation in the absence of the ligand have yet to be determined. Using computational modeling, we simulated the potential complexes between the cytoplasmic domains of BAK1, BRI1 and BIR3. Our calculations and experimental data confirm the interaction of BIR3 with BAK1 and BRI1, with the BAK1 BIR3 interaction clearly favored. Furthermore, we demonstrate that BIR3 and BRI1 share the same interaction site with BAK1. This suggests a competition between BIR3 and BRI1 for binding to BAK1, which results in preferential binding of BIR3 to BAK1 in the absence of the ligand thereby preventing the active participation of BAK1 in BR signaling. Our model also suggests that BAK1 and BRI1 can interact even while BAK1 is in complex with BIR3 at an additional binding site of BAK1 that does not allow active BR signaling.

Brassinosteroids (BR) are key hormonal regulators of plant development. However, whereas the individual components of BR perception and signaling are well characterized experimentally, the question of how they can act and whether they are sufficient to carry out the critical function of cellular elongation remains open. Here, we combined computational modeling with quantitative cell physiology to understand the dynamics of the plasma membrane (PM)-localized BR response pathway during the initiation of cellular responses in the epidermis of the Arabidopsis root tip that are be linked to cell elongation. The model, consisting of ordinary differential equations, comprises the BR induced hyperpolarization of the PM, the acidification of the apoplast and subsequent cell wall swelling. We demonstrate that the competence of the root epidermal cells for the BR response predominantly depends on the amount and activity of H+-ATPases in the PM. The model further predicts that an influx of cations is required to compensate for the shift of positive charges caused by the apoplastic acidification. A potassium channel was subsequently identified and experimentally characterized, fulfilling this function. Thus, we established the landscape of components and parameters for physiological processes potentially linked to cell elongation, a central process in plant development.