Chemotaxis receptors and associated signalling proteins in Escherichia coli form clusters that consist of thousands of molecules and are the largest native protein complexes described to date in bacteria. Clusters are located at the cell poles and laterally along the cell body, and play an important role in signal transduction. Much work has been done to study the structure and function of receptor clusters, but the significance of their positioning and the underlying mechanisms are not understood. Here, we used fluorescence imaging to study cluster distribution and follow cluster dynamics during cell growth. Our data show that lateral clusters localise to specific periodic positions along the cell body, which mark future division sites and are involved in the localisation of the replication machinery. The chemoreceptor cluster positioning is thus intricately related to the overall structure and division of an E. coli cell.

SummaryChemotactic stimuli in bacteria are sensed by large sensory complexes, or receptor clusters, that consist of tens of thousands of proteins. Receptor clusters appear to play a key role in signal processing, but their structure remains poorly understood. Here we used fluorescent protein fusions to study in vivo formation of the cluster core, which consists of receptors, a kinase CheA and an assisting protein CheW. We show that receptors aggregate through their cytoplasmic domains even in the absence of other chemotaxis proteins. Clustering is further enhanced by the binding of CheW. Surprisingly, we observed that some fragments of CheA bind receptor clusters well in the absence of CheW, although the latter does assist the binding of full-length CheA. The resulting mode of receptor cluster formation is consistent with an experimentally observed flexible stoichiometry of chemosensory complexes and with assumptions of recently proposed computer models of signal processing in chemotaxis.

Protein-protein interactions play key roles in virtually all cellular processes, often forming complex regulatory networks. A powerful tool to study interactions in vivo is fluorescence resonance energy transfer (FRET), which is based on the distance-dependent energy transfer from an excited donor to an acceptor fluorophore. Here, we used FRET to systematically map all protein interactions in the chemotaxis signaling pathway in Escherichia coli, one of the most studied models of signal transduction, and to determine stimulation-induced changes in the pathway. Our FRET analysis identified 19 positive FRET pairs out of the 28 possible protein combinations, with 9 pairs being responsive to chemotactic stimulation. Six stimulation-dependent and five stimulation-independent interactions were direct, whereas other interactions were apparently mediated by scaffolding proteins. Characterization of stimulation-induced responses revealed an additional regulation through activity dependence of interactions involving the adaptation enzyme CheB, and showed complex rearrangement of chemosensory receptors. Our study illustrates how FRET can be efficiently employed to study dynamic protein networks in vivo.

Signal processing in bacterial chemotaxis relies on large sensory complexes consisting of thousands of protein molecules. These clusters create a scaffold that increases the efficiency of pathway reactions and amplifies and integrates chemotactic signals. The cluster core in Escherichia coli comprises a ternary complex composed of receptors, kinase CheA, and adaptor protein CheW. All other chemotaxis proteins localize to clusters by binding either directly to receptors or to CheA. Here, we used fluorescence recovery after photobleaching (FRAP) to investigate the turnover of chemotaxis proteins at the cluster and their mobility in the cytoplasm. We found that cluster exchange kinetics were proteinspecific and took place on several characteristic time scales that correspond to excitation, adaptation, and cell division, respectively. We further applied analytical and numerical data fitting to analyze intracellular protein diffusion and to estimate the rate constants of cluster equilibration in vivo. Our results indicate that the rates of protein turnover at the cluster have evolved to ensure optimal performance of the chemotaxis pathway.T he relatively simple chemotaxis signaling pathway in Escherichia coli, with analogues of its components-receptors, kinase, phosphatase, and adaptation system-common to many other networks, is an ideal model system for studying general principles of signal transduction (1-3). In E. coli, allosteric interactions among receptors in chemosensory arrays or clusters (Fig. 1), where receptors of different ligand specificities are intermixed (4, 5), integrate and amplify chemotactic stimuli. The networked receptors regulate the autophosphorylation activity of an associated kinase, CheA, which in turn controls the phosphorylation state of a small response regulator protein, CheY, to modulate the cell's flagellar motors. The signaling pathway also includes CheZ, a phosphatase of CheY-P. Excitatory signaling is rapid: changes in CheY phosphorylation level upon repellent or attractant stimulation take place in several hundreds of milliseconds (6-9).In addition, the pathway includes an adaptation system, comprising methyltransferase CheR and methylesterase CheB, that adjusts the activity and sensitivity of the sensory complex by methylating and demethylating receptors. The adaptation system uses feedback from receptor and kinase activity to return CheY phosphorylation to a preset level even in the presence of high levels of chemoeffectors. The time course of the adaptation process depends on stimulus strength (10, 11), varying from several seconds for weak stimuli to several minutes for strong stimuli.Most of the reaction rates and binding constants for chemotaxis proteins have been measured in vitro, and the average intracellular protein concentrations under standard growth conditions were determined (12,13). This abundance of biochemical data has inspired multiple attempts at detailed kinetic analysis of the chemotaxis pathway (9, 13-17), making it the most thoroughly modeled signaling pathw...

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Shigella flexneri proliferate in infected human epithelial cells at exceptionally high rates. This vigorous growth has important consequences for rapid progression to life-threatening bloody diarrhea, but the underlying metabolic mechanisms remain poorly understood. Here, we used metabolomics, proteomics, and genetic experiments to determine host and Shigella metabolism during infection in a cell culture model. The data suggest that infected host cells maintain largely normal fluxes through glycolytic pathways, but the entire output of these pathways is captured by Shigella, most likely in the form of pyruvate. This striking strategy provides Shigella with an abundant favorable energy source, while preserving host cell ATP generation, energy charge maintenance, and survival, despite ongoing vigorous exploitation. Shigella uses a simple three-step pathway to metabolize pyruvate at high rates with acetate as an excreted waste product. The crucial role of this pathway for Shigella intracellular growth suggests targets for antimicrobial chemotherapy of this devastating disease.infectious diseases | host-pathogen interactions

Conditional gene expression systems have developed into essential tools for the study of gene functions. However, their utility is often limited by the difficulty of identifying clonal cell lines, in which transgene control can be realized to its full potential. Here, we describe HeLa cell lines, in which we have identified—by functional analysis—genomic loci, from which the expression of transgenes can be tightly controlled via tetracycline-regulated expression. These loci can be re-targeted by recombinase-mediated cassette exchange. Upon exchange of the gene of interest, the resulting cell line exhibits the qualitative and quantitative properties of controlled transgene expression characteristic for the parent cell line. Moreover, by using an appropriate promoter, these cell lines express the tetracycline controlled transcription activator rtTA2-M2 uniformly throughout the entire cell population. The potential of this approach for functional genomics is highlighted by utilizing one of our master cell lines for the efficient microRNA-mediated knockdown of the endogenous human lamin A/C gene.

Metabolomics has emerged as a powerful tool for addressing biological questions. Liquid chromatography coupled with mass spectrometry (LC-MS) is widely used for metabolic characterization, including targeted and untargeted approaches. Despite recent innovations, a crucial aspect of this technique is the sample preparation for accurate data analyses. In this protocol, we present a robust and adaptable workflow for metabolic analyses of mammalian cells from adherent cell cultures, which is particularly suited for qualitative and quantitative central metabolite characterization by LC-MS. Each sample consists of 600,000 mammalian cells grown on cover glasses, allowing for fast and complete transfer of the cells for metabolite extraction or medium exchange, e.g., for labeling experiments. The sampling procedure includes a fast and efficient washing step in liquid flow in water, which reduces cross-contamination and matrix effects while minimizing perturbation of the metabolic steady state of the cells; it is followed by quenching cell metabolism. The latter is achieved by using a -20 °C cold methanol acetonitrile mixture acidified with formic acid, followed by freeze drying, metabolite extraction and LC-MS. The protocol requires 2 s for cell sampling until quenching, and the entire protocol takes a total of 1.5 h per sample when the provided nanoscale LC-MS method is applied.