Combining two optimized and affordable methods to assign chemoreceptors to a specific signal

International audienceChemotaxis allows bacteria to detect specific compounds and move accordingly. This pathway involves signal detection by chemoreceptors (MCPs). Attributing a chemoreceptor to a ligand is difficult because there is a lot of redundancy in the MCPs that recognize a single ligand. We propose a methodology to define which chemoreceptors bind a given ligand. First, an MCP is overproduced to increase sensitivity to the ligand(s) it recognizes, thus promoting accumulation of cells around an agarose plug containing a low attractant concentration. Second, the ligand-binding domain (LBD) of the chemoreceptor is fused to maltose-binding protein (MBP), which facilitates purification and provides a control for a thermal shift assay (TSA). An increase in the melting temperature of the LBD in the presence of the ligand indicates that the chemoreceptor directly binds it. We showed that overexpression of two Shewanella oneidensis chemoreceptors (SO_0987 and SO_1056) promoted swimming toward an agarose plug containing a low concentration of chromate. The LBD of each of the two chemoreceptors was fused to MBP. A TSA revealed that only the LBD from SO_1056 had its melting temperature increased by chromate. In conclusion, we describe an efficient approach to define chemoreceptor-ligand pairs before undertaking more-sophisticated biochemical and structural studies

The phosphorylated regulator of chemotaxis is crucial throughout biofilm biogenesis in Shewanella oneidensis

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The Tol-Pal system of Escherichia coli plays an unexpected role in the import of the oxyanions chromate and phosphate

Chromate is a toxic metal that enters bacteria by using oxyanion importers. Here, we show that each mutant of the Tol-Pal system of Escherichia coli exhibited increased chromate resistance. This system, which spans the cell envelope, plays a major role in envelope integrity and septation. The ΔtolQR mutant accumulated three-fold less chromate than the wild-type. Addition of phosphate but not sulfate to rich medium drastically reduced chromate toxicity and import in the wild-type strain. Furthermore, the intracellular concentration of free inorganic phosphate was significantly reduced for the ΔtolR mutant in comparison to the wild-type strain. Moreover, extracellular labelled phosphate was significantly less incorporated into the ΔtolR mutant. Finally, two distinct TolQR mutant complexes, specifically affected in Tol-Pal energization without affecting the TolQRA complex structure, did not complement the ΔtolQR mutant for inorganic phosphate accumulation. We thus propose that, while the Pst system is well known to import inorganic phosphate, the Tol-Pal system participates to phosphate uptake in particular at medium to high extracellular phosphate concentrations. Since mutations disabling the Tol-Pal system lead to pleiotropic effects, chromate resistance and reduced inorganic phosphate import could occur from an indirect effect of mutations in components of the Tol-Pal system

ChrASO, the chromate efflux pump of Shewanella oneidensis, improves chromate survival and reduction

International audienceThe chromate efflux pump encoding gene chrA SO was identified on the chromosome of She-wanella oneidensis MR1. Although chrA SO is expressed without chromate, its expression level increases when Cr(VI) is added. When deleted, the resulting mutant ΔchrA SO exhibits a chromate sensitive phenotype compared to that of the wild-type strain. Interestingly, heter-ologous expression of chrA SO in E. coli confers resistance to high chromate concentration. Moreover, expression of chrA SO in S. oneidensis and E. coli significantly improves Cr(VI) reduction. This effect could result either from extracytoplasmic chromate reduction or from a better cell survival leading to enhanced Cr(VI) reduction

Defining Two Chemosensory Arrays in Shewanella oneidensis

International audienceShewanella oneidensis has 2 functional chemosensory systems named Che1 and Che3, and 27 chemoreceptors. Che3 is dedicated to chemotaxis while Che1 could be involved in RpoS posttranslational regulation. In this study, we have shown that two chemoreceptors Aer2so and McpAso, genetically related to the Che1 system, form distinct core-signaling units and signal to Che1 and Che3, respectively. Moreover, we observed that Aer2so is a cytoplasmic dynamic chemoreceptor that, when in complex with CheA1 and CheW1, localizes at the two poles and the centre of the cells. Altogether, the results obtained indicate that Che1 and Che3 systems are interconnected by these two chemoreceptors allowing a global response for bacterial survival

Multiple detection of both attractants and repellents by the dCache ‐chemoreceptor SO_1056 of Shewanella oneidensis

International audienceChemoreceptors are usually transmembrane proteins dedicated to the detection of compound gradients or signals in the surroundings of a bacterium. After detection, they modulate the activation of CheA-CheY, the core of the chemotactic pathway, to allow cells to move upwards or downwards depending on whether the signal is an attractant or a repellent, respectively. Environmental bacteria such as Shewanella oneidensis harbour dozens of chemoreceptors or MCPs (methyl-accepting chemotaxis proteins). A recent study revealed that MCP SO_1056 of S. oneidensis binds chromate. Here, we show that this MCP also detects an additional attractant (L-malate) and two repellents (nickel and cobalt). The experiments were performed in vivo by the agarose-in-plug technique after overproducing MCP SO_1056 and in vitro, when possible, by submitting the purified ligand-binding domain (LBD) of SO_1056 to a thermal shift assay (TSA) coupled to isothermal titration calorimetry (ITC). ITC assays revealed a K D of 3.4 lM for L-malate and of 47.7 lM for nickel. We conclude that MCP SO_1056 binds attractants and repellents of unrelated composition. The LBD of SO_1056 belongs to the double Cache_1 family and is highly homologous to PctA, a chemoreceptor from Pseudomonas aeruginosa that detects several amino acids. Therefore, LBDs of the same family can bind diverse compounds, confirming that experimental approaches are required to define accurate LBD-binding molecules or signals

Small membranous proteins of the TorE/NapE family, crutches for cognate respiratory systems in Proteobacteria

Abstract In this report, we investigate small proteins involved in bacterial alternative respiratory systems that improve the enzymatic efficiency through better anchorage and multimerization of membrane components. Using the small protein TorE of the respiratory TMAO reductase system as a model, we discovered that TorE is part of a subfamily of small proteins that are present in proteobacteria in which they play a similar role for bacterial respiratory systems. We reveal by microscopy that, in Shewanella oneidensis MR1, alternative respiratory systems are evenly distributed in the membrane contrary to what has been described for Escherichia coli. Thus, the better efficiency of the respiratory systems observed in the presence of the small proteins is not due to a specific localization in the membrane, but rather to the formation of membranous complexes formed by TorE homologs with their c-type cytochrome partner protein. By an in vivo approach combining Clear Native electrophoresis and fluorescent translational fusions, we determined the 4:4 stoichiometry of the complexes. In addition, mild solubilization of the cytochrome indicates that the presence of the small protein reinforces its anchoring to the membrane. Therefore, assembly of the complex induced by this small protein improves the efficiency of the respiratory system

A Network of Paralogous Stress Response Transcription Factors in the Human Pathogen Candida glabrata

International audienceThe yeast Candida glabrata has become the second cause of systemic candidemia in humans. However, relatively few genome-wide studies have been conducted in this organism and our knowledge of its transcriptional regulatory network is quite limited. In the present work, we combined genome-wide chromatin immunoprecipitation (ChIP-seq), transcriptome analyses, and DNA binding motif predictions to describe the regulatory interactions of the seven Yap (Yeast AP1) transcription factors of C. glabrata. We described a transcriptional network containing 255 regulatory interactions and 309 potential target genes. We predicted with high confidence the preferred DNA binding sites for 5 of the 7 CgYaps and showed a strong conservation of the Yap DNA binding properties between S. cerevisiae and C. glabrata. We provided reliable functional annotation for 3 of the 7 Yaps and identified for Yap1 and Yap5 a core regulon which is conserved in S. cerevisiae, C. glabrata, and C. albicans. We uncovered new roles for CgYap7 in the regulation of iron-sulfur cluster biogenesis, for CgYap1 in the regulation of heme biosynthesis and for CgYap5 in the repression of GRX4 in response to iron starvation. These transcription factors define an interconnected transcriptional network at the crossroads between redox homeostasis, oxygen consumption, and iron metabolism

Expression of <i>lacZ</i> fusions in the presence of chromate.

<p>The wild-type <i>S</i>. <i>oneidensis</i> strain containing either the plasmid <i>pchrA</i>_<i>SO</i>::<i>lacZ</i> (transcriptional fusion between the promoter of <i>chrA</i>_<i>SO</i> and the <i>lacZ</i> reporter gene; light grey bars) or the plasmid p<i>mxd</i>_<i>450</i>::<i>lacZ</i> (transcriptional fusion between the promoter of <i>mxdA</i> and the <i>lacZ</i> reporter gene; dark grey bars), used as a control, was grown during 16 hours in the presence of increasing concentrations of chromate (0, 0.05, 0.1 and 0.2 mM) before β-galactosidase activity was determined. The MC1061 <i>E</i>. <i>coli</i> strain containing the plasmid <i>pchrA</i>_<i>SO</i>::<i>lacZ</i> was grown in similar conditions and β-galactosidase activity was also determined (black bars in the insert). [Cr(VI)] indicates the concentration of chromate during growth. β-galactosidase activity is expressed as Miller arbitrary units (AU). Values are means ± standard deviations (error bars) from at least three experiments.</p