Vincent Fromion

Condition-Dependent Transcriptome Reveals High-Level Regulatory Architecture in Bacillus subtilis

Outside In Acquisition and analysis of large data sets promises to move us toward a greater understanding of the mechanisms by which biological systems are dynamically regulated to respond to external cues. Now, two papers explore the responses of a bacterium to changing nutritional conditions (see the Perspective by Chalancon et al.). Nicolas et al. (p. 1103) measured transcriptional regulation for more than 100 different conditions. Greater amounts of antisense RNA were generated than expected and appeared to be produced by alternative RNA polymerase targeting subunits called sigma factors. One transition, from malate to glucose as the primary nutrient, was studied in more detail by Buescher et al. (p. 1099) who monitored RNA abundance, promoter activity in live cells, protein abundance, and absolute concentrations of intracellular and extracellular metabolites. In this case, the bacteria responded rapidly and largely without transcriptional changes to life on malate, but only slowly adapted to use glucose, a shift that required changes in nearly half the transcription network. These data offer an initial understanding of why certain regulatory strategies may be favored during evolution of dynamic control systems. A horizontal analysis reveals the breadth of genes turned on and off as nutrients change. Bacteria adapt to environmental stimuli by adjusting their transcriptomes in a complex manner, the full potential of which has yet to be established for any individual bacterial species. Here, we report the transcriptomes of Bacillus subtilis exposed to a wide range of environmental and nutritional conditions that the organism might encounter in nature. We comprehensively mapped transcription units (TUs) and grouped 2935 promoters into regulons controlled by various RNA polymerase sigma factors, accounting for ~66% of the observed variance in transcriptional activity. This global classification of promoters and detailed description of TUs revealed that a large proportion of the detected antisense RNAs arose from potentially spurious transcription initiation by alternative sigma factors and from imperfect control of transcription termination.

Processive Movement of MreB-Associated Cell Wall Biosynthetic Complexes in Bacteria

Bacteria elongation involves moving synthetic complexes around the cell wall. The peptidoglycan cell wall and the actin-like MreB cytoskeleton are major determinants of cell shape in rod-shaped bacteria. The prevailing model postulates that helical, membrane-associated MreB filaments organize elongation-specific peptidoglycan-synthesizing complexes along sidewalls. We used total internal reflection fluorescence microscopy to visualize the dynamic relation between MreB isoforms and cell wall synthesis in live Bacillus subtilis cells. During exponential growth, MreB proteins did not form helical structures. Instead, together with other morphogenetic factors, they assembled into discrete patches that moved processively along peripheral tracks perpendicular to the cell axis. Patch motility was largely powered by cell wall synthesis, and MreB polymers restricted diffusion of patch components in the membrane and oriented patch motion.

Reconstruction and analysis of the genetic and metabolic regulatory networks of the central metabolism of Bacillus subtilis

BackgroundFew genome-scale models of organisms focus on the regulatory networks and none of them integrates all known levels of regulation. In particular, the regulations involving metabolite pools are often neglected. However, metabolite pools link the metabolic to the genetic network through genetic regulations, including those involving effectors of transcription factors or riboswitches. Consequently, they play pivotal roles in the global organization of the genetic and metabolic regulatory networks.ResultsWe report the manually curated reconstruction of the genetic and metabolic regulatory networks of the central metabolism of Bacillus subtilis (transcriptional, translational and post-translational regulations and modulation of enzymatic activities). We provide a systematic graphic representation of regulations of each metabolic pathway based on the central role of metabolites in regulation. We show that the complex regulatory network of B. subtilis can be decomposed as sets of locally regulated modules, which are coordinated by global regulators.ConclusionThis work reveals the strong involvement of metabolite pools in the general regulation of the metabolic network. Breaking the metabolic network down into modules based on the control of metabolite pools reveals the functional organization of the genetic and metabolic regulatory networks of B. subtilis.

Nonlinear performance of a PI controlled missile: an explanation

The primary aim of this paper is to investigate the practical interest of the incremental norm approach for analysing (realistic) nonlinear dynamical systems. In this framework indeed, incremental stability, a stronger notion than ℒ2-gain stability, ensures suitable qualitative and quantitative properties. On the one hand, the qualitative properties essentially correspond to (steady-state) input/output properties, which are not necessarily obtained when ensuring only ℒ2-gain stability. On the other hand, it is possible to analyse quantitative robustness performance properties using the notion of (nonlinear) incremental performance, the latter being defined in the continuity of the (linear) H∞ performance (i.e. through the use of a weighting function). As testing incremental properties is a difficult problem, stronger, but computationally more attractive, notions are introduced, namely quadratic incremental stability and performance. Testing these properties reduces indeed to solving convex optimization problems over Linear Matrix Inequalities (LMIs). As an illustration, we consider a classical missile problem, which was already treated using several (linear and nonlinear) approaches. We focus here on the analysis of the nonlinear behavior of this PI controlled missile: using the notions of quadratic incremental stability and performance, the closed loop nonlinear missile is proved to meet desirable control specifications. Copyright

H/sub /spl infin// control of an irrigation canal pool with a mixed control politics

This paper presents a method to design efficient automatic controllers for an irrigation canal pool, that realize a compromise between the water resource management and the performance in terms of rejecting unmeasured perturbations. This mixed controller design is casted into the H/sub /spl infin// optimization framework, and experimentally tested on a real canal located in Portugal. The experimental results show the effectiveness of the method. We also interpret classical control politics for an irrigation canal (local upstream and distant downstream control) using automatic control tools, and show that our method enables to combine both classical politics, keeping the distant downstream control water management while recovering the local upstream control real-time performance with respect to the user.

Comprehensive Absolute Quantification of the Cytosolic Proteome of Bacillus subtilis by Data Independent, Parallel Fragmentation in Liquid Chromatography/Mass Spectrometry (LC/MSE)

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/MSE). 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/MSE) data has been performed for the first time and should pave the way for future comprehensive quantitative characterization of microorganisms as physiological entities.

A constraint-based model analysis of the metabolic consequences of increased NADPH oxidation in Saccharomyces cerevisiae.

Controlling the amounts of redox cofactors to manipulate metabolic fluxes is emerging as a useful approach to optimizing byproduct yields in yeast biotechnological processes. Redox cofactors are extensively interconnected metabolites, so predicting metabolite patterns is challenging and requires in-depth knowledge of how the metabolic network responds to a redox perturbation. Our aim was to analyze comprehensively the metabolic consequences of increased cytosolic NADPH oxidation during yeast fermentation. Using a genetic device based on the overexpression of a modified 2,3-butanediol dehydrogenase catalyzing the NADPH-dependent reduction of acetoin into 2,3-butanediol, we increased the NADPH demand to between 8 and 40-fold the anabolic demand. We developed (i) a dedicated constraint-based model of yeast fermentation and (ii) a constraint-based modeling method based on the dynamical analysis of mass distribution to quantify the in vivo contribution of pathways producing NADPH to the maintenance of redox homeostasis. We report that yeast responds to NADPH oxidation through a gradual increase in the flux through the PP and acetate pathways, providing 80% and 20% of the NADPH demand, respectively. However, for the highest NADPH demand, the model reveals a saturation of the PP pathway and predicts an exchange between NADH and NADPH in the cytosol that may be mediated by the glycerol-DHA futile cycle. We also reveal the contribution of mitochondrial shuttles, resulting in a net production of NADH in the cytosol, to fine-tune the NADH/NAD(+) balance. This systems level study helps elucidate the physiological adaptation of yeast to NADPH perturbation. Our findings emphasize the robustness of yeast to alterations in NADPH metabolism and highlight the role of the glycerol-DHA cycle as a redox valve, providing additional NADPH from NADH under conditions of very high demand.

A comparative transcriptomic, fluxomic and metabolomic analysis of the response of Saccharomyces cerevisiae to increases in NADPH oxidation

BackgroundRedox homeostasis is essential to sustain metabolism and growth. We recently reported that yeast cells meet a gradual increase in imposed NADPH demand by progressively increasing flux through the pentose phosphate (PP) and acetate pathways and by exchanging NADH for NADPH in the cytosol, via a transhydrogenase-like cycle. Here, we studied the mechanisms underlying this metabolic response, through a combination of gene expression profiling and analyses of extracellular and intracellular metabolites and 13 C-flux analysis.ResultsNADPH oxidation was increased by reducing acetoin to 2,3-butanediol in a strain overexpressing an engineered NADPH-dependent butanediol dehydrogenase cultured in the presence of acetoin. An increase in NADPH demand to 22 times the anabolic requirement for NADPH was accompanied by the intracellular accumulation of PP pathway metabolites consistent with an increase in flux through this pathway. Increases in NADPH demand were accompanied by the successive induction of several genes of the PP pathway. NADPH-consuming pathways, such as amino-acid biosynthesis, were upregulated as an indirect effect of the decrease in NADPH availability. Metabolomic analysis showed that the most extreme modification of NADPH demand resulted in an energetic problem. Our results also highlight the influence of redox status on aroma production.ConclusionsCombined 13 C-flux, intracellular metabolite levels and microarrays analyses revealed that NADPH homeostasis, in response to a progressive increase in NADPH demand, was achieved by the regulation, at several levels, of the PP pathway. This pathway is principally under metabolic control, but regulation of the transcription of PP pathway genes can exert a stronger effect, by redirecting larger amounts of carbon to this pathway to satisfy the demand for NADPH. No coordinated response of genes involved in NADPH metabolism was observed, suggesting that yeast has no system for sensing NADPH/NADP+ ratio. Instead, the induction of NADPH-consuming amino-acid pathways in conditions of NADPH limitation may indirectly trigger the transcription of a set of PP pathway genes.

Boundary control of hyperbolic conservation laws using a frequency domain approach

The paper uses a frequency domain method for boundary control of hyperbolic conservation laws. We show that the transfer function of the hyperbolic system belongs to the Callier-Desoer algebra, for which the Nyquist theorem provides necessary and sufficient conditions for input-output closed-loop stability. We examine the link between input-output stability and exponential stability of the state. Specific results are then derived for the case of proportional boundary controllers. The results are illustrated in the case of boundary control of open-channel flow

An expanded protein–protein interaction network in Bacillus subtilis reveals a group of hubs: Exploration by an integrative approach

We have generated a protein–protein interaction network in Bacillus subtilis focused on several essential cellular processes such as cell division, cell responses to various stresses, the bacterial cytoskeleton, DNA replication and chromosome maintenance by careful application of the yeast two‐hybrid approach. This network, composed of 793 interactions linking 287 proteins with an average connectivity of five interactions per protein, represents a valuable resource for future functional analyses. A striking feature of the network is a group of highly connected hubs (GoH) linking many different cellular processes. Most of the proteins of the GoH have unknown functions and are associated to the membrane. By the integration of available knowledge, in particular of transcriptome data sets, the GoH was decomposed into subgroups of party hubs corresponding to protein complexes or regulatory pathways expressed under different conditions. At a global level, the GoH might function as a very robust group of date hubs having partially redundant functions to integrate information from the different cellular pathways. Our analyses also provide a rational way to study the highly redundant functions of the GoH by a genetic approach.