21 research outputs found

    Predicting Inter-Species Cross-Talk in Two-Component Signalling Systems

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    Phosphosignalling pathways are an attractive option for the synthetic biologist looking for a wide repertoire of modular components from which to build. We demonstrate that two-component systems can be used in synthetic biology. However, their potential is limited by the fact that host cells contain many of their own phosphosignalling pathways and these may interact with, and cross-talk to, the introduced synthetic components. In this paper we also demonstrate a simple bioinformatic tool that can help predict whether interspecies cross-talk between introduced and native two-component signalling pathways will occur and show both in vitro and in vivo that the predicted interactions do take place. The ability to predict potential cross-talk prior to designing and constructing novel pathways or choosing a host organism is essential for the promise that phosphosignalling components hold for synthetic biology to be realised

    Localization of MreB in Rhodobacter sphaeroides under Conditions Causing Changes in Cell Shape and Membrane Structure

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    MreB is thought to be a bacterial actin homolog that defines the morphology of rod-shaped bacteria. Rhodobacter sphaeroides changes shape, from a rod to coccobacillus, and undergoes extensive cytoplasmic membrane invagination when it switches from aerobic to photoheterotrophic growth. The role of MreB in defining R. sphaeroides shape was therefore investigated. Attempts at deleting or insertionally inactivating mreB were unsuccessful under all growth conditions. Immunofluorescence microscopy showed MreB localized to mid-cell in elongating cells under both aerobic and photoheterotrophic conditions. Three-dimensional reconstruction showed that MreB formed a ring at mid-cell. MreB remained at mid-cell as septation began but localized to new sites in the daughter cells before the completion of septation. MreB localized to putative septation sites in cephalexin-treated filamentous cells. Genomic single-copy mreB was replaced with gfp-mreB, and green fluorescent protein (GFP)-MreB localized in the same pattern, as seen with immunofluorescence microscopy. Some of the cells expressing GFP-MreB were abnormal, principally displaying an increase in cell width, suggesting that the fusion was not fully functional in all cells. GFP-MreB localized to swellings at mid-cell in cells treated with the penicillin-binding protein 2 inhibitor amdinocillin. These data suggest that MreB is essential in R. sphaeroides, performing a role at mid-cell in elongating cells, and in early septation, putatively in the cytoplasmic control of the peptidoglycan synthetic complexes

    Phosphorylation assays of EnvZ/OmpR and RSP0203/RSP1138.

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    <p>Both histidine kinases were autophophorylated and tested for phosphotransfer to the response regulators after different time intervals. <b>A</b> Phosphotransfer from EnvZ to OmpR. <b>B</b> Phosphotransfer from EnvZ to RSP1138. <b>C</b> Phosphotransfer from RSP0203 to OmpR. <b>D</b> Phosphotransfer from RSP0203 to RSP1138.</p

    Fluorescence output data.

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    <p>Fluorescence output data of wild type <i>R. sphaeroides</i> (WS8N) and the reporter strain <i>cheA1::PompC-yfp</i> (JPA1800) expressing OmpR or the non-phosphorylatable OmpRD55A Cells were grown under photosynthetic conditions for 72 h and analysed in a plate reader (Tecan, Austria). Transcription from the plasmids pIND4, pINDOmpR and pINDD55A was induced with 10 µM IPTG.</p
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