14 research outputs found

    Isotopic composition of E. coli cells measured with EA-IRMS.

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    <p>Isotopic composition of untreated, fixed, and fixed/hybridized <i>E. coli</i> cells measured with EA-IRMS on dried cells pellets. Standard deviation is given in parentheses.</p

    Mean isotopic composition of individual cells measured with nanoSIMS.

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    <p>Mean isotopic composition of untreated and fixed/hybridized <i>E. coli</i> cells measured with nanoSIMS on individual cells. Standard deviation is given in parentheses.</p

    NanoSIMS images obtained from a double-labelled complex sample (<sup>13</sup>C-labeled methanol degrading anaerobic digester).

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    <p>NanoSIMS images obtained from acomplex sample(<sup>13</sup>C-labeled methanol degrading anaerobic digester) labelled with a generalist bacterial iodinated probe (EUBI) and a Methanosarcina genera specific brominated probe (MS1414). Panel (a) shows the secondary ion of <sup>32</sup>S<sup>−</sup> image as an image of total biomass. Panel (b) shows the secondary ion of <sup>81</sup>Br<sup>−</sup> image as an indication of archaeal cell identity. Panel (c) shows the secondary ion of <sup>127</sup>I<sup>−</sup> image as an indication of total bacteria. Panels (d) shows the <sup>13</sup>C Isotopic abundance map.</p

    NanoSIMS images obtained for mixed untreated and fixed/hybridized cells grown in enriched culture media.

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    <p>NanoSIMS images obtained for mixed untreated and fixed/hybridized cells grown in enriched culture media with nominal <sup>13</sup>C abundance of 10.9, 20.7, 40.3 and 79.4%. Panel (a) shows the secondary ion of <sup>32</sup>S<sup>−</sup> image as an image of total biomass (scale bar : 5 µm). Panel (b) shows the secondary ion of <sup>127</sup>I<sup>−</sup> image as an indication of hybridized cells. (c) <sup>13</sup>C Isotopic abundance map of corresponding area.</p

    Carbon accumulation and storage within cyanophycin over time.

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    <p>(<b>A</b>) Isotopic enrichment in cyanophycin granules and cell cytosol after 60 and 120 mins of incubation (see images “<b>60 mins</b>” and “<b>120 mins</b>”). (<b>B</b>) Labeled carbon in cyanophycin granules expressed as the percentage of total labeled carbon in samples shown in (<b>60 mins, 120 mins</b>). (<b>C</b>) Percentage of cellular area that is occupied by cyanophycin granules in (<b>60 mins, 120 mins</b>). (<b>60 mins</b>) Nano-scale SIMS image of large cyanophycin granules in filamentous cyanobacteria ∼120 µm below the surface of the cone after 60 min of incubation in the labeling medium. (<b>120 mins</b>) Nano-scale SIMS image of cyanophycin granules in filamentous cyanobacteria ∼120 µm below the surface of the cone after 120 mins of incubation in the labeling medium. Cyanophycin granules in (II) contain more labeled carbon and are larger. Scale bar is 5 µm for (<b>D</b>, <b>E</b>).</p

    Cyanophycin Mediates the Accumulation and Storage of Fixed Carbon in Non-Heterocystous Filamentous Cyanobacteria from Coniform Mats

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    <div><p>Thin, filamentous, non-heterocystous, benthic cyanobacteria (Subsection III) from some marine, lacustrine and thermal environments aggregate into macroscopic cones and conical stromatolites. We investigate the uptake and storage of inorganic carbon by cone-forming cyanobacteria from Yellowstone National Park using high-resolution stable isotope mapping of labeled carbon (H<sup>13</sup>CO<sub>3</sub><sup>−</sup>) and immunoassays. Observations and incubation experiments in actively photosynthesizing enrichment cultures and field samples reveal the presence of abundant cyanophycin granules in the active growth layer of cones. These ultrastructurally heterogeneous granules rapidly accumulate newly fixed carbon and store 18% of the total particulate labeled carbon after 120 mins of incubation. The intracellular distribution of labeled carbon during the incubation experiment demonstrates an unexpectedly large contribution of PEP carboxylase to carbon fixation, and a large flow of carbon and nitrogen toward cyanophycin in thin filamentous, non-heterocystous cyanobacteria. This pattern does not occur in obvious response to a changing N or C status. Instead, it may suggest an unusual interplay between the regulation of carbon concentration mechanisms and accumulation of photorespiratory products that facilitates uptake of inorganic C and reduces photorespiration in the dense, surface-attached communities of cyanobacteria from Subsection III.</p></div

    Temporal dynamics of <sup>13</sup>CN<sup>−</sup> in cyanophycin.

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    <p>(<b>A</b>) Average accumulation of labeled carbon within the cells (N>10) at different time points. (<b>B</b>) Accumulation of labeled carbon within cyanophycin after 30 min of incubation. The <sup>13</sup>CN<sup>−/12</sup>CN<sup>−</sup> ratio was 2.5% (i.e., twice above the 1.25% background). Cyanophycin is marked by a red arrow. Labeled carbon is also present in or close to cell envelopes (white arrow). Scale bar is 3 µm.</p

    Requirement of AnsP2 and AnsA for <i>M. tuberculosis</i> resistance to acid in host macrophages.

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    <p>(A,B) Exogenous asparagine accumulates in the mycobacterial phagosome. (A) Images from a representative infected cell showing the locations of <i>M. tuberculosis</i> (<sup>13</sup>C% map, middle) and <sup>15</sup>N-asparagine uptake (<sup>15</sup>N/<sup>14</sup>N ratio map, right), as derived from secondary ion mass spectrometry (SIMS) analysis of <i>M. tuberculosis</i> H37Rv-infected mouse bone marrow-derived macrophages (BMMs). <sup>13</sup>C-labeled bacteria were used to infect BMMs at a multiplicity of infection of 10 bacteria per cell. At 20 h post-infection, infected cells were pulsed for 4 h with 5 mM <sup>15</sup>N<sub>1 (amine)</sub>-asparagine, and <sup>13</sup>C and <sup>15</sup>N isotope proportions were analyzed. (B) Quantification of <sup>15</sup>N isotope enrichment in surface areas chosen in the intracellular <sup>13</sup>C-labeled bacteria; “background” indicates the level of enrichment measured in the host cell cytoplasm. For more details about the technique, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003928#ppat.1003928-Gouzy1" target="_blank">[30]</a>. (C) IFNγ- and LPS-activated BMMs were infected with <i>M. tuberculosis</i> wild type (H37Rv), the <i>ansA</i>-KO mutant or its complemented strain (Compl.) at a multiplicity of infection (MOI) of 0.1 bacterium/cell for 4 h at 37°C. Cells were washed and further incubated with fresh medium for 0, 2 or 5 days. At the indicated time-points, cells lysates were plated for CFU scoring. (D) Confocal microscopy analysis of activated BMMs infected for 1 h with Alexa Fluor 488-labeled <i>M. tuberculosis</i> wild type (H37Rv), the <i>ansA</i>-KO mutant or its complemented strain (Compl.) (green), and stained with LysoTracker Red DND-99 (red) and DAPI (blue) to visualize nuclei. Bar represents 10 µm. Arrowheads point to example phagosomes considered positive for LysoTracker staining. (E) Quantification of LysoTracker-positive phagosomes in samples prepared as in (h) 2 or 4 h after infection. Colocalisation events were recorded in ≈300 phagosomes observed in ≈10 different fields. (F) Phagosomal pH measured by flow cytometry in activated BMMs infected with <i>M. tuberculosis</i> wild type (H37Rv), the <i>ansA</i>-KO mutant or its complemented counterpart (Compl.). (G) Cells were pre-incubated with 100 nM bafilomycin A1 for 1 h, infected as in (C) and bafilomycin A1 was removed after 24 h. All data are representative of at least three independent experiments. In (C), (E) and (G), data represent mean±s.d. of triplicate samples, and were analyzed using the Student's <i>t</i> test. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.</p

    The function and <i>in vivo</i> relevance of AnsP2 and AnsA in asparagine utilization in <i>M. tuberculosis</i>.

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    <p>(A) U-<sup>14</sup>C-Asn uptake assay with <i>M. tuberculosis</i> H37Rv, the <i>ansP2</i>-KO mutant and its complemented strains (Compl.). Bacteria previously grown in 7H9 with 5 mM Asn, were harvested and resuspended in an uptake buffer containing a mix of <sup>14</sup>C-labeled and non-labeled asparagine to obtain a final concentration of 20 µM asparagine. Bacteria were incubated at 37°C and samples were removed and bacteria-associated <sup>14</sup>C radioactivity was quantified at the indicated time points. Data are expressed as the percentage of the number of disintegrations per minute (DPM) per total protein concentration (<sup>14</sup>C-Asn (DPM). µg protein<sup>−1</sup>), as compared to the values obtained at t<sub>0</sub>. (B) Growth of <i>M. tuberculosis</i> H37Rv and the <i>ansP2</i>-KO mutant strain in the presence of asparagine as sole nitrogen source. (C) C57BL/6 mice were infected intranasally with 1,000 CFUs <i>M. tuberculosis</i> wild type (H37Rv) or the <i>ansP2</i>-KO mutant. Three weeks later, lungs and spleen were recovered, homogenized and plated onto agar for CFU scoring. (D) Western blotting analysis of total protein extracts (Tot) or a Ni-NTA purified fraction (Pur) from <i>M. smegmatis</i> containing a pVV16 control plasmid (pVV16) or an <i>ansA-his<sub>6</sub></i> cassette cloned into pVV16 (pVV16 <i>ansA-his<sub>6</sub></i>), using an anti-HIS<sub>6</sub> monoclonal antibody. 1 µg of proteins were loaded in the “Tot” lanes, 0.5 µg of proteins were loaded in the “Pur” lanes. The expected molecular weight of recombinant AnsA-HIS<sub>6</sub> fusion protein is of 34 kDa. (E) Asparaginase activity, as monitored by NADPH disappearance at OD<sub>340</sub> (see Materials & Methods), of recombinant AnsA in the presence of asparagine (Asn) or glutamine (Gln). Control reactions lack (w/o) substrate or enzyme. (F) Growth of <i>M. tuberculosis</i> H37Rv, the <i>ansA</i>-KO mutant strain, and the <i>ansA</i>-KO complemented strain (Compl.) in minimal medium containing 5 mM asparagine as sole nitrogen source. (G) C57BL/6 mice were infected intranasally with 1,000 CFUs <i>M. tuberculosis</i> wild type (H37Rv), the <i>ansA</i>-KO mutant or its complemented strain (Compl.). Three weeks later, lungs and spleen were recovered, homogenized and plated onto agar for CFU scoring. All data are representative of at least two independent experiments. In (A), (C), (F) and (G), data represent mean±s.d. of triplicate samples and were analyzed using the Student's <i>t</i> test; *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.</p
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