Johannes Geiselmann

Parallel Changes in Global Protein Profiles During Long-Term Experimental Evolution in Escherichia coli

Twelve populations of Escherichia coli evolved in and adapted to a glucose-limited environment from a common ancestor. We used two-dimensional protein electrophoresis to compare two evolved clones, isolated from independently derived populations after 20,000 generations. Exceptional parallelism was detected. We compared the observed changes in protein expression profiles with previously characterized global transcription profiles of the same clones; this is the first time such a comparison has been made in an evolutionary context where these changes are often quite subtle. The two methodologies exhibited some remarkable similarities that highlighted two different levels of parallel regulatory changes that were beneficial during the evolution experiment. First, at the higher level, both methods revealed extensive parallel changes in the same global regulatory network, reflecting the involvement of beneficial mutations in genes that control the ppGpp regulon. Second, both methods detected expression changes of identical gene sets that reflected parallel changes at a lower level of gene regulation. The protein profiles led to the discovery of beneficial mutations affecting the malT gene, with strong genetic parallelism across independently evolved populations. Functional and evolutionary analyses of these mutations revealed parallel phenotypic decreases in the maltose regulon expression and a high level of polymorphism at this locus in the evolved populations.

Shared control of gene expression in bacteria by transcription factors and global physiology of the cell

Gene expression is controlled by the joint effect of (i) the global physiological state of the cell, in particular the activity of the gene expression machinery, and (ii) DNA‐binding transcription factors and other specific regulators. We present a model‐based approach to distinguish between these two effects using time‐resolved measurements of promoter activities. We demonstrate the strength of the approach by analyzing a circuit involved in the regulation of carbon metabolism in E. coli. Our results show that the transcriptional response of the network is controlled by the physiological state of the cell and the signaling metabolite cyclic AMP (cAMP). The absence of a strong regulatory effect of transcription factors suggests that they are not the main coordinators of gene expression changes during growth transitions, but rather that they complement the effect of global physiological control mechanisms. This change of perspective has important consequences for the interpretation of transcriptome data and the design of biological networks in biotechnology and synthetic biology.

The carbon assimilation network in Escherichia coli is densely connected and largely sign-determined by directions of metabolic fluxes.

Gene regulatory networks consist of direct interactions but also include indirect interactions mediated by metabolites and signaling molecules. We describe how these indirect interactions can be derived from a model of the underlying biochemical reaction network, using weak time-scale assumptions in combination with sensitivity criteria from metabolic control analysis. We apply this approach to a model of the carbon assimilation network in Escherichia coli. Our results show that the derived gene regulatory network is densely connected, contrary to what is usually assumed. Moreover, the network is largely sign-determined, meaning that the signs of the indirect interactions are fixed by the flux directions of biochemical reactions, independently of specific parameter values and rate laws. An inversion of the fluxes following a change in growth conditions may affect the signs of the indirect interactions though. This leads to a feedback structure that is at the same time robust to changes in the kinetic properties of enzymes and that has the flexibility to accommodate radical changes in the environment.

Hybrid Modeling and Simulation of Genetic Regulatory Networks: A Qualitative Approach

The study of genetic regulatory networks has received a major impetus from the recent development of experimental techniques allowing the measurement of patterns of gene expression in a massively parallel way. This experimental progress calls for the development of appropriate computer tools for the modeling and simulation of gene regulation processes. We present a method for the hybrid modeling and simulation of genetic regulatory networks, based on a class of piecewiselinear (PL) differential equations that has been well-studied in mathematical biology. Distinguishing characteristics of the method are that it makes qualitative predictions of the behavior of regulatory systems and that it deals with discontinuities in the right-hand side of the differential equations. The simulation method has been implemented in Java in the computer tool Genetic Network Analyzer (GNA). The method and the tool have been used to analyze several networks of biological interest, including the network underlying the initiation of sporulation in Bacillus subtilis.

Experimental and computational validation of models of fluorescent and luminescent reporter genes in bacteria.

BackgroundFluorescent and luminescent reporter genes have become popular tools for the real-time monitoring of gene expression in living cells. However, mathematical models are necessary for extracting biologically meaningful quantities from the primary data.ResultsWe present a rigorous method for deriving relative protein synthesis rates (mRNA concentrations) and protein concentrations by means of kinetic models of gene expression. We experimentally and computationally validate this approach in the case of the protein Fis, a global regulator of transcription in Escherichia coli. We show that the mRNA and protein concentration profiles predicted from the models agree quite well with direct measurements obtained by Northern and Western blots, respectively. Moreover, we present computational procedures for taking into account systematic biases like the folding time of the fluorescent reporter protein and differences in the half-lives of reporter and host gene products. The results show that large differences in protein half-lives, more than mRNA half-lives, may be critical for the interpretation of reporter gene data in the analysis of the dynamics of regulatory systems.ConclusionsThe paper contributes to the development of sound methods for the interpretation of reporter gene data, notably in the context of the reconstruction and validation of models of regulatory networks. The results have wide applicability for the analysis of gene expression in bacteria and may be extended to higher organisms.

Symbolic reachability analysis of genetic regulatory networks using discrete abstractions

We use hybrid-systems techniques for the analysis of reachability properties of a class of piecewise-affine (PA) differential equations that are particularly suitable for the modeling of genetic regulatory networks. More specifically, we introduce a hyperrectangular partition of the state space that forms the basis for a discrete abstraction preserving the sign of the derivatives of the state variables. The resulting discrete transition system provides a qualitative description of the network dynamics that is well-adapted to available experimental data and that can be efficiently computed in a symbolic manner from inequality constraints on the parameters.

Qualitative analysis and verification of hybrid models of genetic regulatory networks: nutritional stress response in Escherichia coli

The switch-like character of the dynamics of genetic regulatory networks has attracted much attention from mathematical biologists and researchers on hybrid systems alike. We extend our previous work on a method for the qualitative analysis of hybrid models of genetic regulatory networks, based on a class of piecewise-affine differential equation (PADE) models, in two directions. First, we present a refinement of the method using a discrete or qualitative abstraction that preserves stronger properties of the dynamics of the PA systems, in particular the sign patterns of the derivatives of the concentration variables. The discrete transition system resulting from the abstraction is a conservative approximation of the dynamics of the PA system and can be computed symbolically. Second, we apply the refined method to a regulatory system whose functioning is not yet well-understood by biologists, the nutritional stress response in the bacterium Escherichia coli.

The Crl-RpoS Regulon of Escherichia coli

The RpoS subunit of RNA polymerase controls the expression of numerous genes involved in stationary phase and in response to different stress conditions. The regulatory protein Crl increases the activity of RpoS by direct interaction with the RpoS holoenzyme. To define the extent of the Crl regulon, we used two-dimensional SDS-PAGE to measure the role of Crl in regulating the expression of the Escherichia coliproteome in stationary phase at 30 °C. By comparing the proteome of four strains (wild type, crl−, rpoS−, and crl−rpoS−), we observed that the intensity of 74 spots was modified in at least one mutant context. 62 spots were identified by mass spectrometry and correspond to 40 distinct proteins. They were classified in four main categories: DNA metabolism, central metabolism, response to environmental modifications, and miscellaneous. Three proteins were specifically involved in quorum sensing: TnaA (the tryptophanase that converts tryptophan to indole), WrbA (Trp repressor-binding protein), and YgaG (homologous to LuxS, autoinducer-2 synthase). Because little is known about the regulation of Crl expression, we investigated the influence of diffusible molecules on the expression of Crl. Using Western blotting experiments, we showed that, at 30 °C, a diffusible molecule(s) produced during the transition phase between the exponential and stationary phases induces a premature expression of Crl. Indole was tested as one of the potential candidates: at 37 °C, it is present in the extracellular medium at a constant concentration, but at 30 °C, its concentration peaks during the transition phase. When indole was added to the culture medium, it also induced prematurely the expression of Crl at both the transcriptional and translational levels in a Crl-dependent manner. Crl may thus be considered a new environmental sensor via the indole concentration.

Participation of IHF and a distant UP element in the stimulation of the phage lambda PL promoter.

We have previously identified a UP element in the phage λ PL promoter, centred at position −90 from the transcription start site. Integration host factor (IHF), a heterodimeric DNA‐binding and ‐bending protein, binds upstream of the λ PL promoter in a region overlapping the UP element. Stimulation of transcription by IHF requires an intact αCTD and affects the initial binding of RNA polymerase to the promoter. We propose a model for the stimulation of PL by IHF in which IHF bends the DNA to bring the distal UP sequence in closer proximity to the promoter core sequences to allow the docking of the αCTD of RNA polymerase. Furthermore, IHF may also participate in protein–protein interactions with the αCTD. In support of this model, we found that alanine substitutions in αCTD at positions 265, 268, 270 and 275 reduced PL promoter activity. Mutations in the IHF DNA binding site, as well as IHF mutant proteins exhibiting a decreased ability to bend the DNA, were both defective in stimulating the PL promoter. In addition, some of the mutated IHF residues are clustered at a protein surface that interacts with the UP DNA sequence. These residues may also participate in protein–protein interactions with the αCTD.

The tRNA function of SsrA contributes to controlling repression of bacteriophage Mu prophage

The small regulatory RNA SsrA has both tRNA and mRNA activities. It charges alanine and interacts with stalled ribosomes, allowing for translation to resume on the SsrA mRNA moiety. Hence, unfinished peptides carry a short amino acid tag, which serves as a signal for degradation by energy-dependent proteases. In SsrA-defective Escherichia coli strains, thermoinducible mutants of the transposable bacteriophage Mu (Mucts) are no longer induced at high temperature. Here we show that truncated forms of the key regulator of Mu lysogeny, the repressor Repc, accumulate in the absence of SsrA. These forms resemble C-terminally truncated dominant Mu repressor mutants previously isolated from Mucts, which are no longer thermoinducible and bind operator DNA with a high affinity even at high temperature. Using various ssrA alleles, we demonstrate the importance of SsrA charging on the ribosome for controlling Mu prophage repression. Our results thus substantiate the previous observation that trans-translation is not the only function of the SsrA. The alternative function of SsrA appears to influence the stability of Mu lysogens by controlling the translation of the C-terminal domain of the repressor protein, which modulates the affinity of the protein for DNA and its susceptibility to proteolytic degradation.