Essentiality of c-di-AMP in Bacillus subtilis: Bypassing mutations converge in potassium and glutamate homeostasis.

In order to adjust to changing environmental conditions, bacteria use nucleotide second messengers to transduce external signals and translate them into a specific cellular response. Cyclic di-adenosine monophosphate (c-di-AMP) is the only known essential nucleotide second messenger. In addition to the well-established role of this second messenger in the control of potassium homeostasis, we observed that glutamate is as toxic as potassium for a c-di-AMP-free strain of the Gram-positive model bacterium Bacillus subtilis. In this work, we isolated suppressor mutants that allow growth of a c-di-AMP-free strain under these toxic conditions. Characterization of glutamate resistant suppressors revealed that they contain pairs of mutations, in most cases affecting glutamate and potassium homeostasis. Among these mutations, several independent mutations affected a novel glutamate transporter, AimA (Amino acid importer A, formerly YbeC). This protein is the major transporter for glutamate and serine in B. subtilis. Unexpectedly, some of the isolated suppressor mutants could suppress glutamate toxicity by a combination of mutations that affect phospholipid biosynthesis and a specific gain-of-function mutation of a mechanosensitive channel of small conductance (YfkC) resulting in the acquisition of a device for glutamate export. Cultivation of the c-di-AMP-free strain on complex medium was an even greater challenge because the amounts of potassium, glutamate, and other osmolytes are substantially higher than in minimal medium. Suppressor mutants viable on complex medium could only be isolated under anaerobic conditions if one of the two c-di-AMP receptor proteins, DarA or DarB, was absent. Also on complex medium, potassium and osmolyte toxicity are the major bottlenecks for the growth of B. subtilis in the absence of c-di-AMP. Our results indicate that the essentiality of c-di-AMP in B. subtilis is caused by the global impact of the second messenger nucleotide on different aspects of cellular physiology.

Inducible expression of (pp)pGpp synthetases in Staphylococcus aureus is associated with activation of stress response genes.

The stringent response is characterized by the synthesis of the messenger molecules pppGpp, ppGpp or pGpp (here collectively designated (pp)pGpp). The phenotypic consequences resulting from (pp)pGpp accumulation vary among species and can be mediated by different underlying mechanisms. Most genome-wide analyses have been performed under stress conditions, which often mask the immediate effects of (pp)pGpp-mediated regulatory circuits. In Staphylococcus aureus, (pp)pGpp can be synthesized via the RelA-SpoT-homolog, RelSau upon amino acid limitation or via one of the two small (pp)pGpp synthetases RelP or RelQ upon cell wall stress. We used RNA-Seq to compare the global effects in response to induction of the synthetase of rel-Syn (coding for the enzymatic region of RelSau) or relQ without the need to apply additional stress conditions. Induction of rel-Syn resulted in changes in the nucleotide pool similar to induction of the stringent response via the tRNA synthetase inhibitor mupirocin: a reduction in the GTP pool, an increase in the ATP pool and synthesis of pppGpp, ppGpp and pGpp. Induction of all three enzymes resulted in similar changes in the transcriptome. However, RelQ was less active than Rel-Syn and RelP, indicating strong restriction of its (pp)pGpp-synthesis activity in vivo. (pp)pGpp induction resulted in the downregulation of many genes involved in protein and RNA/DNA metabolism. Many of the (pp)pGpp upregulated genes are part of the GTP sensitive CodY regulon and thus likely regulated through lowering of the GTP pool. New CodY independent transcriptional changes were detected including genes involved in the SOS response, iron storage (e.g. ftnA, dps), oxidative stress response (e.g., perR, katA, sodA) and the psmα1-4 and psmß1-2 operons coding for cytotoxic, phenol soluble modulins (PSMs). Analyses of the ftnA, dps and psm genes in different regulatory mutants revealed that their (pp)pGpp-dependent regulation can occur independent of the regulators PerR, Fur, SarA or CodY. Moreover, psm expression is uncoupled from expression of the quorum sensing system Agr, the main known psm activator. The expression of central genes of the oxidative stress response protects the bacteria from anticipated ROS stress derived from PSMs or exogenous sources. Thus, we identified a new link between the stringent response and oxidative stress in S. aureus that is likely crucial for survival upon phagocytosis.

Genoscapist: online exploration of quantitative profiles along genomes via interactively customized graphical representations.

SUMMARY: Genoscapist is a tool to design web interfaces generating high-quality images for interactive visualization of hundreds of quantitative profiles along a reference genome together with various annotations. Relevance is demonstrated by deployment of two websites dedicated to large condition-dependent transcriptome datasets available for Bacillus subtilis and Staphylococcus aureus. AVAILABILITY AND IMPLEMENTATION: Websites and source code freely accessible at https://genoscapist.migale.inrae.fr.

Impact of high salinity and the compatible solute glycine betaine on gene expression of Bacillus subtilis.

The Gram-positive bacterium Bacillus subtilis is frequently exposed to hyperosmotic conditions. In addition to the induction of genes involved in the accumulation of compatible solutes, high salinity exerts widespread effects on B. subtilis physiology, including changes in cell wall metabolism, induction of an iron limitation response, reduced motility and suppression of sporulation. We performed a combined whole-transcriptome and proteome analysis of B. subtilis 168 cells continuously cultivated at low or high (1.2 M NaCl) salinity. Our study revealed significant changes in the expression of more than one-fourth of the protein-coding genes and of numerous non-coding RNAs. New aspects in understanding the impact of high salinity on B. subtilis include a sustained low-level induction of the SigB-dependent general stress response and strong repression of biofilm formation under high-salinity conditions. The accumulation of compatible solutes such as glycine betaine aids the cells to cope with water stress by maintaining physiologically adequate levels of turgor and also affects multiple cellular processes through interactions with cellular components. Therefore, we additionally analysed the global effects of glycine betaine on the transcriptome and proteome of B. subtilis and revealed that it influences gene expression not only under high-salinity, but also under standard growth conditions.

Analyses of competent and non-competent subpopulations of Bacillus subtilis reveal yhfW, yhxC and ncRNAs as novel players in competence.

Upon competence-inducing nutrient-limited conditions, only part of the Bacillus subtilis population becomes competent. Here, we separated the two subpopulations by fluorescence-assisted cell sorting (FACS). Using RNA-seq, we confirmed the previously described ComK regulon. We also found for the first time significantly downregulated genes in the competent subpopulation. The downregulated genes are not under direct control by ComK but have higher levels of corresponding antisense RNAs in the competent subpopulation. During competence, cell division and replication are halted. By investigating the proteome during competence, we found higher levels of the regulators of cell division, MinD and Noc. The exonucleases SbcC and SbcD were also primarily regulated at the post-transcriptional level. In the competent subpopulation, yhfW was newly identified as being highly upregulated. Its absence reduces the expression of comG, and has a modest, but statistically significant effect on the expression of comK. Although expression of yhfW is higher in the competent subpopulation, no ComK-binding site is present in its promoter region. Mutants of yhfW have a small but significant defect in transformation. Metabolomic analyses revealed significant reductions in tricarboxylic acid (TCA) cycle metabolites and several amino acids in a ΔyhfW mutant. RNA-seq analysis of ΔyhfW revealed higher expression of the NAD synthesis genes nadA, nadB and nadC.

Large-scale reduction of the Bacillus subtilis genome: consequences for the transcriptional network, resource allocation, and metabolism.

Understanding cellular life requires a comprehensive knowledge of the essential cellular functions, the components involved, and their interactions. Minimized genomes are an important tool to gain this knowledge. We have constructed strains of the model bacterium, Bacillus subtilis, whose genomes have been reduced by ∼36%. These strains are fully viable, and their growth rates in complex medium are comparable to those of wild type strains. An in-depth multi-omics analysis of the genome reduced strains revealed how the deletions affect the transcription regulatory network of the cell, translation resource allocation, and metabolism. A comparison of gene counts and resource allocation demonstrates drastic differences in the two parameters, with 50% of the genes using as little as 10% of translation capacity, whereas the 6% essential genes require 57% of the translation resources. Taken together, the results are a valuable resource on gene dispensability in B. subtilis, and they suggest the roads to further genome reduction to approach the final aim of a minimal cell in which all functions are understood.

Staphylococcus aureus Transcriptome Architecture: From Laboratory to Infection-Mimicking Conditions.

Staphylococcus aureus is a major pathogen that colonizes about 20% of the human population. Intriguingly, this Gram-positive bacterium can survive and thrive under a wide range of different conditions, both inside and outside the human body. Here, we investigated the transcriptional adaptation of S. aureus HG001, a derivative of strain NCTC 8325, across experimental conditions ranging from optimal growth in vitro to intracellular growth in host cells. These data establish an extensive repertoire of transcription units and non-coding RNAs, a classification of 1412 promoters according to their dependence on the RNA polymerase sigma factors SigA or SigB, and allow identification of new potential targets for several known transcription factors. In particular, this study revealed a relatively low abundance of antisense RNAs in S. aureus, where they overlap only 6% of the coding genes, and only 19 antisense RNAs not co-transcribed with other genes were found. Promoter analysis and comparison with Bacillus subtilis links the small number of antisense RNAs to a less profound impact of alternative sigma factors in S. aureus. Furthermore, we revealed that Rho-dependent transcription termination suppresses pervasive antisense transcription, presumably originating from abundant spurious transcription initiation in this A+T-rich genome, which would otherwise affect expression of the overlapped genes. In summary, our study provides genome-wide information on transcriptional regulation and non-coding RNAs in S. aureus as well as new insights into the biological function of Rho and the implications of spurious transcription in bacteria.

Changes of DNA topology affect the global transcription landscape and allow rapid growth of a Bacillus subtilis mutant lacking carbon catabolite repression.

Bacteria are able to prioritize preferred carbon sources from complex mixtures. This is achieved by the regulatory phenomenon of carbon catabolite repression. To allow the simultaneous utilization of multiple carbon sources and to prevent the time-consuming adaptation to each individual nutrient in biotechnological applications, mutants lacking carbon catabolite repression can be used. However, such mutants often exhibit pleiotropic growth defects. We have isolated and characterized mutations that overcome the growth defect of Bacillus subtilis ccpA mutants lacking the major regulator of catabolite repression, in particular their glutamate auxotrophy. Here we show, that distinct mutations affecting the essential DNA topoisomerase I (TopA) cause glutamate prototrophy of the ccpA mutant. These suppressing variants of the TopA enzyme exhibit increased activity resulting in enhanced relaxation of the DNA. Reduced DNA supercoiling results in enhanced expression of the gltAB operon encoding the biosynthetic glutamate synthase. This is achieved by a significant re-organization of the global transcription network accompanied by re-routing of metabolism, which results in inactivation of the glutamate dehydrogenase. Our results provide a link between DNA topology, the global transcriptional network, and glutamate metabolism and suggest that specific topA mutants may be well suited for biotechnological purposes.

From the genome sequence via the proteome to cell physiology - Pathoproteomics and pathophysiology of Staphylococcus aureus.

While the genome sequence is the blueprint of life, functional genomics is required to transfer the genome sequence to cell physiology. Among the Omics technologies, proteomics holds a privileged position because it deals with the main players of life, the proteins. For the model organism Staphylococcus aureus comprehensive coverage of the proteome was accomplished and used to address physiological and pathophysiological questions. This review article demonstrates that the proteomic view of physiology and pathophysiology of S. aureus unveils cellular processes in an unprecedented manner. These new insights into bacterial adaptation are starting points for detailed follow-up studies aiming at a deep and comprehensive understanding of metabolism, stress responses and virulence of this dangerous pathogen. In vivo proteomics uncovered the life style of S. aureus under infection related conditions, namely after internalization by eukaryotic cells, and in infection settings. However, further analytical advances will improve capabilities for in vivo studies, particularly in murine and human tissue specimen and in this way support the identification of new targets for therapeutic interventions. Furthermore, a comprehensive set of cell surface-associated proteins required for biofilm formation and host cell invasion as well as secreted proteins, among them many proteins of still unknown function, was described. The identification of the functions of these proteins will help to better understand the molecular mechanisms of the different diseases caused by S. aureus, thus leading to a more complete understanding of its pathogenicity. Finally, immunoproteomics can visualize the perception of the pathogen by the immune system and host defense mechanisms and may pave the way to the development of new vaccination approaches, which are urgently required.

AureoWiki ̵ The repository of the Staphylococcus aureus research and annotation community.

In light of continuously accumulating data and knowledge on major human pathogens, comprehensive and up-to-date sources of easily accessible information are urgently required. The AureoWiki database (http://aureowiki.med.uni-greifswald.de) provides detailed information on the genes and proteins of clinically and experimentally relevant S. aureus strains, currently covering NCTC 8325, COL, Newman, USA300_FPR3757, and N315. By implementing a pan-genome approach, AureoWiki facilitates the transfer of knowledge gained in studies with different S. aureus strains, thus supporting functional annotation and better understanding of this organism. All data related to a given gene or gene product is compiled on a strain-specific gene page. The gene pages contain sequence-based information complemented by data on, for example, protein function and localization, transcriptional regulation, and gene expression. The information provided is connected via links to other databases and published literature. Importantly, orthologous genes of the individual strains, which are linked by a pan-genome gene identifier and a unified gene name, are presented side by side using strain-specific tabs. The respective pan-genome gene page contains an orthologue table for 32 S. aureus strains, a multiple-strain genome viewer, a protein sequence alignment as well as other comparative information. The data collected in AureoWiki is also accessible through various download options in order to support bioinformatics applications. In addition, based on two large-scale gene expression data sets, AureoWiki provides graphical representations of condition-dependent mRNA levels and protein profiles under various laboratory and infection-related conditions.

SubtiWiki 2.0--an integrated database for the model organism Bacillus subtilis.

To understand living cells, we need knowledge of each of their parts as well as about the interactions of these parts. To gain rapid and comprehensive access to this information, annotation databases are required. Here, we present SubtiWiki 2.0, the integrated database for the model bacterium Bacillus subtilis (http://subtiwiki.uni-goettingen.de/). SubtiWiki provides text-based access to published information about the genes and proteins of B. subtilis as well as presentations of metabolic and regulatory pathways. Moreover, manually curated protein-protein interactions diagrams are linked to the protein pages. Finally, expression data are shown with respect to gene expression under 104 different conditions as well as absolute protein quantification for cytoplasmic proteins. To facilitate the mobile use of SubtiWiki, we have now expanded it by Apps that are available for iOS and Android devices. Importantly, the App allows to link private notes and pictures to the gene/protein pages. Today, SubtiWiki has become one of the most complete collections of knowledge on a living organism in one single resource.

Small regulatory RNA-induced growth rate heterogeneity of Bacillus subtilis.

Isogenic bacterial populations can consist of cells displaying heterogeneous physiological traits. Small regulatory RNAs (sRNAs) could affect this heterogeneity since they act by fine-tuning mRNA or protein levels to coordinate the appropriate cellular behavior. Here we show that the sRNA RnaC/S1022 from the Gram-positive bacterium Bacillus subtilis can suppress exponential growth by modulation of the transcriptional regulator AbrB. Specifically, the post-transcriptional abrB-RnaC/S1022 interaction allows B. subtilis to increase the cell-to-cell variation in AbrB protein levels, despite strong negative autoregulation of the abrB promoter. This behavior is consistent with existing mathematical models of sRNA action, thus suggesting that induction of protein expression noise could be a new general aspect of sRNA regulation. Importantly, we show that the sRNA-induced diversity in AbrB levels generates heterogeneity in growth rates during the exponential growth phase. Based on these findings, we hypothesize that the resulting subpopulations of fast- and slow-growing B. subtilis cells reflect a bet-hedging strategy for enhanced survival of unfavorable conditions.

Synthesis of the compatible solute proline by Bacillus subtilis: point mutations rendering the osmotically controlled proHJ promoter hyperactive.

The ProJ and ProH enzymes of Bacillus subtilis catalyse together with ProA (ProJ-ProA-ProH), osmostress-adaptive synthesis of the compatible solute proline. The proA-encoded gamma-glutamyl phosphate reductase is also used for anabolic proline synthesis (ProB-ProA-ProI). Transcription of the proHJ operon is osmotically inducible whereas that of the proBA operon is not. Targeted and quantitative proteome analysis revealed that the amount of ProA is not limiting for the interconnected anabolic and osmostress-responsive proline production routes. A key player for enhanced osmostress-adaptive proline production is the osmotically regulated proHJ promoter. We used site-directed mutagenesis to study the salient features of this stress-responsive promoter. Two important features were identified: (i) deviations of the proHJ promoter from the consensus sequence of SigA-type promoters serve to keep transcription low under non-inducing growth conditions, while still allowing a finely tuned induction of transcriptional activity when the external osmolarity is increased and (ii) a suboptimal spacer length for SigA-type promoters of either 16-bp (the natural proHJ promoter), or 18-bp (a synthetic promoter variant) is strictly required to allow regulation of promoter activity in proportion to the external salinity. Collectively, our data suggest that changes in the local DNA structure at the proHJ promoter are important determinants for osmostress-inducibility of transcription.

Translation elicits a growth rate-dependent, genome-wide, differential protein production in Bacillus subtilis.

Complex regulatory programs control cell adaptation to environmental changes by setting condition-specific proteomes. In balanced growth, bacterial protein abundances depend on the dilution rate, transcript abundances and transcript-specific translation efficiencies. We revisited the current theory claiming the invariance of bacterial translation efficiency. By integrating genome-wide transcriptome datasets and datasets from a library of synthetic gfp-reporter fusions, we demonstrated that translation efficiencies in Bacillus subtilis decreased up to fourfold from slow to fast growth. The translation initiation regions elicited a growth rate-dependent, differential production of proteins without regulators, hence revealing a unique, hard-coded, growth rate-dependent mode of regulation. We combined model-based data analyses of transcript and protein abundances genome-wide and revealed that this global regulation is extensively used in B. subtilis We eventually developed a knowledge-based, three-step translation initiation model, experimentally challenged the model predictions and proposed that a growth rate-dependent drop in free ribosome abundance accounted for the differential protein production.

Adaptive immune response to lipoproteins of Staphylococcus aureus in healthy subjects.

Staphylococcus aureus is a frequent commensal but also a dangerous pathogen, causing many forms of infection ranging from mild to life-threatening conditions. Among its virulence factors are lipoproteins, which are anchored in the bacterial cell membrane. Lipoproteins perform various functions in colonization, immune evasion, and immunomodulation. These proteins are potent activators of innate immune receptors termed Toll-like receptors 2 and 6. This study addressed the specific B-cell and T-cell responses directed to lipoproteins in human S. aureus carriers and non-carriers. 2D immune proteomics and ELISA approaches revealed that titers of antibodies (IgG) binding to S. aureus lipoproteins were very low. Proliferation assays and cytokine profiling data showed only subtle responses of T cells; some lipoproteins did not elicit proliferation. Hence, the robust activation of the innate immune system by S. aureus lipoproteins does not translate into a strong adaptive immune response. Reasons for this may include inaccessibility of lipoproteins for B cells as well as ineffective processing and presentation of the antigens to T cells.

Global analysis of the impact of linezolid onto virulence factor production in S. aureus USA300.

The translation inhibitor linezolid is an antibiotic of last resort against Gram-positive pathogens including methicillin resistant strains of the nosocomial pathogen Staphylococcus aureus. Linezolid is reported to inhibit production of extracellular virulence factors, but the molecular cause is unknown. To elucidate the physiological response of S. aureus to linezolid in general and the inhibition of virulence factor synthesis in particular a holistic study was performed. Linezolid was added to exponentially growing S. aureus cells and the linezolid stress response was analyzed with transcriptomics and quantitative proteomics methods. In addition, scanning and transmission electron microscopy experiments as well as fluorescence microscopy analyses of the cellular DNA and membrane were performed. As previously observed in studies on other translation inhibitors, S. aureus adapts its protein biosynthesis machinery to the reduced translation efficiency. For example the synthesis of ribosomal proteins was induced. Also unexpected results like a decline in the amount of extracellular and membrane proteins were obtained. In addition, cell shape and size changed after linezolid stress and cell division was diminished. Finally, the chromosome was condensed after linezolid stress and lost contact to the membrane. These morphological changes cannot be explained by established theories. A new hypothesis is discussed, which suggests that the reduced amount of membrane and extracellular proteins and observed defects in cell division are due to the disintegration of transertion complexes by linezolid.

Comprehensive absolute quantification of the cytosolic proteome of Bacillus subtilis by data independent, parallel fragmentation in liquid chromatography/mass spectrometry (LC/MS(E)).

In the growing field of systems biology, the knowledge of protein concentrations is highly required to truly understand metabolic and adaptational networks within the cells. Therefore we established a workflow relying on long chromatographic separation and mass spectrometric analysis by data independent, parallel fragmentation of all precursor ions at the same time (LC/MS(E)). By prevention of discrimination of co-eluting low and high abundant peptides a high average sequence coverage of 40% could be achieved, resulting in identification of almost half of the predicted cytosolic proteome of the Gram-positive model organism Bacillus subtilis (>1,050 proteins). Absolute quantification was achieved by correlation of average MS signal intensities of the three most intense peptides of a protein to the signal intensity of a spiked standard protein digest. Comparative analysis with heavily labeled peptides (AQUA approach) showed the use of only one standard digest is sufficient for global quantification. The quantification results covered almost four orders of magnitude, ranging roughly from 10 to 150,000 copies per cell. To prove this method for its biological relevance selected physiological aspects of B. subtilis cells grown under conditions requiring either amino acid synthesis or alternatively amino acid degradation were analyzed. This allowed both in particular the validation of the adjustment of protein levels by known regulatory events and in general a perspective of new insights into bacterial physiology. Within new findings the analysis of "protein costs" of cellular processes is extremely important. Such a comprehensive and detailed characterization of cellular protein concentrations based on data independent, parallel fragmentation in liquid chromatography/mass spectrometry (LC/MS(E)) data has been performed for the first time and should pave the way for future comprehensive quantitative characterization of microorganisms as physiological entities.

Deletion of membrane-associated Asp23 leads to upregulation of cell wall stress genes in Staphylococcus aureus.

With about 25 000 molecules per cell, Asp23 is one of the most abundant proteins in Staphylococcus aureus. Asp23 has been characterized as a protein that, following an alkaline shock, accumulates in the soluble protein fraction. Transcription of the asp23 gene is exclusively regulated by the alternative sigma factor σ(B) , which controls the response of the bacterium to environmental stress. Sequence analysis identified Asp23 as a member of the widely distributed Pfam DUF322 family, precluding functional predictions based on its sequence. Using fluorescence microscopy we found that Asp23 colocalized with the cell membrane of Staphylococcus aureus. Since Asp23 has no recognizable transmembrane spanning domains, we initiated a search for proteins that link Asp23 to the cell membrane. We identified SAOUHSC_02443 as the Asp23 membrane anchor and have renamed it AmaP (Asp23 membrane anchoring protein). Deletion of the asp23 gene led to an upregulation of the cell wall stress response. In summary, we have identified Asp23 as a membrane-associated protein and we suggest a function for Asp23 in cell envelope homoeostasis.

Global impact of protein arginine phosphorylation on the physiology of Bacillus subtilis.

Reversible protein phosphorylation is an important and ubiquitous protein modification in all living cells. Here we report that protein phosphorylation on arginine residues plays a physiologically significant role. We detected 121 arginine phosphorylation sites in 87 proteins in the gram-positive model organism Bacillus subtilis in vivo. Moreover, we provide evidence that protein arginine phosphorylation has a functional role and is involved in the regulation of many critical cellular processes, such as protein degradation, motility, competence, and stringent and stress responses. Our results suggest that in B. subtilis the combined activity of a protein arginine kinase and phosphatase allows a rapid and reversible regulation of protein activity and that protein arginine phosphorylation can play a physiologically important and regulatory role in bacteria.

Adaptation of Bacillus subtilis carbon core metabolism to simultaneous nutrient limitation and osmotic challenge: a multi-omics perspective.

The Gram-positive bacterium Bacillus subtilis encounters nutrient limitations and osmotic stress in its natural soil ecosystem. To ensure survival and sustain growth, highly integrated adaptive responses are required. Here, we investigated the system-wide response of B. subtilis to different, simultaneously imposed stresses. To address the anticipated complexity of the cellular response networks, we combined chemostat experiments under conditions of carbon limitation, salt stress and osmoprotection with multi-omics analyses of the transcriptome, proteome, metabolome and fluxome. Surprisingly, the flux through central carbon and energy metabolism is very robust under all conditions studied. The key to achieve this robustness is the adjustment of the biocatalytic machinery to compensate for solvent-induced impairment of enzymatic activities during osmotic stress. Specifically, increased production of several enzymes of central carbon metabolism compensates for their reduced activity in the presence of high salt. A major response of the cell during osmotic stress is the production of the compatible solute proline. This is achieved through the concerted adjustment of multiple reactions around the 2-oxoglutarate node, which drives metabolism towards the proline precursor glutamate. The fine-tuning of the transcriptional and metabolic networks involves functional modules that overarch the individual pathways.