7 research outputs found

    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

    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