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Vital dye reaction and granule localization in periplasm of Escherichia coli.

Ping L, Mavridou DA, Emberly E, Westermann M, Ferguson SJ - PLoS ONE (2012)

Bottom Line: However, pervasive reduction did not result in a random distribution of formazan aggregates.We observed that formazan granules formed in the periplasm after reduction of tetrazolium, which calls for re-evaluation of previous studies using cell-free systems that liberate inaccessible intracellular reductant and potentially generate artifacts.In living bacteria, the seeds formed at or migrated to the new pole would become visible only when that new pole already became an old pole, because of the relatively slow growth rate of granules relative to cell division.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, Jena, Germany. lping@ice.mpg.de

ABSTRACT

Background: Tetrazolium salts are widely used in biology as indicators of metabolic activity - hence termed vital dyes - but their reduction site is still debated despite decades of intensive research. The prototype, 2,3,5- triphenyl tetrazolium chloride, which was first synthesized a century ago, often generates a single formazan granule at the old pole of Escherichia coli cells after reduction. So far, no explanation for their pole localization has been proposed.

Method/principal findings: Here we provide evidence that the granules form in the periplasm of bacterial cells. A source of reducing power is deduced to be thiol groups destined to become disulfides, since deletion of dsbA, coding for thiol-oxidase, enhances the formation of reduced formazan. However, pervasive reduction did not result in a random distribution of formazan aggregates. In filamentous cells, large granules appear at regular intervals of about four normal cell-lengths, consistent with a diffusion-to-capture model. Computer simulations of a minimal biophysical model showed that the pole localization of granules is a spontaneous process, i.e. small granules in a normal size bacterium have lower energy at the poles. This biased their diffusion to the poles. They kept growing there and eventually became fixed.

Conclusions: We observed that formazan granules formed in the periplasm after reduction of tetrazolium, which calls for re-evaluation of previous studies using cell-free systems that liberate inaccessible intracellular reductant and potentially generate artifacts. The localization of formazan granules in E. coli cells can now be understood. In living bacteria, the seeds formed at or migrated to the new pole would become visible only when that new pole already became an old pole, because of the relatively slow growth rate of granules relative to cell division.

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Transmission electron microscopy images of E. coli cells containing formazan granules.Scale bars equal 400 nm. A, Longitudinal section of a control cell growing in medium without tetrazolium. B, Longitudinal section of a cell growing in the presence of TTC. The formazan granule is located at the right pole of the cell. C, Expanded view of the boxed area in panel. The cytoplasmic membrane is indicated by a closed arrow. B. The small lateral granule is indicated by an open arrow. D, Expanded view of the pole area of the control cell. E, Expanded view of the pole of a cell growing in TTC. The concentric contour lines on the cutting surface of the granule are indicated by open arrowheads. F, A further expanded picture of a cell pole, showing the concentric contour lines (open arrowhead) and the cytoplasmic membrane (closed arrow). G, An ultrathin section in which three control cells were cut at different positions, the bottom one being at the tip of the pole. H, Ultrathin sections of poles of cells growing in a medium containing tetrazolium for 12 h. (upper panels: longitudinal sections; lower panels: transverse sections). I, Sections of the poles of cells growing in the presence of tetrazolium for 18 h (upper panels: longitudinal sections; lower panels: transverse sections).
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pone-0038427-g001: Transmission electron microscopy images of E. coli cells containing formazan granules.Scale bars equal 400 nm. A, Longitudinal section of a control cell growing in medium without tetrazolium. B, Longitudinal section of a cell growing in the presence of TTC. The formazan granule is located at the right pole of the cell. C, Expanded view of the boxed area in panel. The cytoplasmic membrane is indicated by a closed arrow. B. The small lateral granule is indicated by an open arrow. D, Expanded view of the pole area of the control cell. E, Expanded view of the pole of a cell growing in TTC. The concentric contour lines on the cutting surface of the granule are indicated by open arrowheads. F, A further expanded picture of a cell pole, showing the concentric contour lines (open arrowhead) and the cytoplasmic membrane (closed arrow). G, An ultrathin section in which three control cells were cut at different positions, the bottom one being at the tip of the pole. H, Ultrathin sections of poles of cells growing in a medium containing tetrazolium for 12 h. (upper panels: longitudinal sections; lower panels: transverse sections). I, Sections of the poles of cells growing in the presence of tetrazolium for 18 h (upper panels: longitudinal sections; lower panels: transverse sections).

Mentions: Formazan granules are often found at the old pole of E. coli cells. We have already shown that the granules refracted fluorescence from GFP fused to the membrane serine receptor Tsr, making the pole look dimmer than non-treated cells [11], suggesting an out-of-membrane localization. When E. coli cells were fractionated as described by Ausubel et al.[22] with the EDTA (ethylenediaminetetraacetate) treatment step omitted, after digestion of the outer cell membrane and release of the periplasm; the insoluble granules were obviously floating in the periplasmic fraction (Data not shown). Transmission electron microscopy (TEM) performed on strain LMG194 that was employed for studying granules localization before [11] confirmed that large formazan granules were mainly formed in the periplasm and localized at the poles of the cell (Fig. 1). Using TEM we were also able to detect randomly distributed tiny lateral granules (Fig. 1C), which would not be observed by light microscopy. It is worth noting that the formazan granules are electron lucent due to their chemical composition, and even slightly lighter than the areas of the periplasm devoid of granules on TEM images. We only consider that a tiny granule is present when we observe a particle with low uniform electron density that is at least twice as thick as the width of the periplasm and is clearly bound by the electron dense cytoplasmic membrane and cell wall. In some cases, concentric contour lines on the cutting surface of the granule were also visible (Fig. 1E), suggesting that formazan had precipitated layer by layer. At many locations on the TEM images, the triple-layered cytoplasmic membrane was clearly visible (Fig. 1F), demonstrating that neither the granule formation nor the sample preparation caused any mechanical damage or dissolution of the cytoplasmic membrane [23]. Granules that were not large enough to be visualized under the light microscope were cap-shaped (Fig. 1H). The centers of these granules were often located on one side of the pole cap. The large granules visible under the light microscope were round on the tip side and exhibited random extrusions on the side facing the cytoplasmic membrane (Fig. 1I).


Vital dye reaction and granule localization in periplasm of Escherichia coli.

Ping L, Mavridou DA, Emberly E, Westermann M, Ferguson SJ - PLoS ONE (2012)

Transmission electron microscopy images of E. coli cells containing formazan granules.Scale bars equal 400 nm. A, Longitudinal section of a control cell growing in medium without tetrazolium. B, Longitudinal section of a cell growing in the presence of TTC. The formazan granule is located at the right pole of the cell. C, Expanded view of the boxed area in panel. The cytoplasmic membrane is indicated by a closed arrow. B. The small lateral granule is indicated by an open arrow. D, Expanded view of the pole area of the control cell. E, Expanded view of the pole of a cell growing in TTC. The concentric contour lines on the cutting surface of the granule are indicated by open arrowheads. F, A further expanded picture of a cell pole, showing the concentric contour lines (open arrowhead) and the cytoplasmic membrane (closed arrow). G, An ultrathin section in which three control cells were cut at different positions, the bottom one being at the tip of the pole. H, Ultrathin sections of poles of cells growing in a medium containing tetrazolium for 12 h. (upper panels: longitudinal sections; lower panels: transverse sections). I, Sections of the poles of cells growing in the presence of tetrazolium for 18 h (upper panels: longitudinal sections; lower panels: transverse sections).
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getmorefigures.php?uid=PMC3366950&req=5

pone-0038427-g001: Transmission electron microscopy images of E. coli cells containing formazan granules.Scale bars equal 400 nm. A, Longitudinal section of a control cell growing in medium without tetrazolium. B, Longitudinal section of a cell growing in the presence of TTC. The formazan granule is located at the right pole of the cell. C, Expanded view of the boxed area in panel. The cytoplasmic membrane is indicated by a closed arrow. B. The small lateral granule is indicated by an open arrow. D, Expanded view of the pole area of the control cell. E, Expanded view of the pole of a cell growing in TTC. The concentric contour lines on the cutting surface of the granule are indicated by open arrowheads. F, A further expanded picture of a cell pole, showing the concentric contour lines (open arrowhead) and the cytoplasmic membrane (closed arrow). G, An ultrathin section in which three control cells were cut at different positions, the bottom one being at the tip of the pole. H, Ultrathin sections of poles of cells growing in a medium containing tetrazolium for 12 h. (upper panels: longitudinal sections; lower panels: transverse sections). I, Sections of the poles of cells growing in the presence of tetrazolium for 18 h (upper panels: longitudinal sections; lower panels: transverse sections).
Mentions: Formazan granules are often found at the old pole of E. coli cells. We have already shown that the granules refracted fluorescence from GFP fused to the membrane serine receptor Tsr, making the pole look dimmer than non-treated cells [11], suggesting an out-of-membrane localization. When E. coli cells were fractionated as described by Ausubel et al.[22] with the EDTA (ethylenediaminetetraacetate) treatment step omitted, after digestion of the outer cell membrane and release of the periplasm; the insoluble granules were obviously floating in the periplasmic fraction (Data not shown). Transmission electron microscopy (TEM) performed on strain LMG194 that was employed for studying granules localization before [11] confirmed that large formazan granules were mainly formed in the periplasm and localized at the poles of the cell (Fig. 1). Using TEM we were also able to detect randomly distributed tiny lateral granules (Fig. 1C), which would not be observed by light microscopy. It is worth noting that the formazan granules are electron lucent due to their chemical composition, and even slightly lighter than the areas of the periplasm devoid of granules on TEM images. We only consider that a tiny granule is present when we observe a particle with low uniform electron density that is at least twice as thick as the width of the periplasm and is clearly bound by the electron dense cytoplasmic membrane and cell wall. In some cases, concentric contour lines on the cutting surface of the granule were also visible (Fig. 1E), suggesting that formazan had precipitated layer by layer. At many locations on the TEM images, the triple-layered cytoplasmic membrane was clearly visible (Fig. 1F), demonstrating that neither the granule formation nor the sample preparation caused any mechanical damage or dissolution of the cytoplasmic membrane [23]. Granules that were not large enough to be visualized under the light microscope were cap-shaped (Fig. 1H). The centers of these granules were often located on one side of the pole cap. The large granules visible under the light microscope were round on the tip side and exhibited random extrusions on the side facing the cytoplasmic membrane (Fig. 1I).

Bottom Line: However, pervasive reduction did not result in a random distribution of formazan aggregates.We observed that formazan granules formed in the periplasm after reduction of tetrazolium, which calls for re-evaluation of previous studies using cell-free systems that liberate inaccessible intracellular reductant and potentially generate artifacts.In living bacteria, the seeds formed at or migrated to the new pole would become visible only when that new pole already became an old pole, because of the relatively slow growth rate of granules relative to cell division.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, Jena, Germany. lping@ice.mpg.de

ABSTRACT

Background: Tetrazolium salts are widely used in biology as indicators of metabolic activity - hence termed vital dyes - but their reduction site is still debated despite decades of intensive research. The prototype, 2,3,5- triphenyl tetrazolium chloride, which was first synthesized a century ago, often generates a single formazan granule at the old pole of Escherichia coli cells after reduction. So far, no explanation for their pole localization has been proposed.

Method/principal findings: Here we provide evidence that the granules form in the periplasm of bacterial cells. A source of reducing power is deduced to be thiol groups destined to become disulfides, since deletion of dsbA, coding for thiol-oxidase, enhances the formation of reduced formazan. However, pervasive reduction did not result in a random distribution of formazan aggregates. In filamentous cells, large granules appear at regular intervals of about four normal cell-lengths, consistent with a diffusion-to-capture model. Computer simulations of a minimal biophysical model showed that the pole localization of granules is a spontaneous process, i.e. small granules in a normal size bacterium have lower energy at the poles. This biased their diffusion to the poles. They kept growing there and eventually became fixed.

Conclusions: We observed that formazan granules formed in the periplasm after reduction of tetrazolium, which calls for re-evaluation of previous studies using cell-free systems that liberate inaccessible intracellular reductant and potentially generate artifacts. The localization of formazan granules in E. coli cells can now be understood. In living bacteria, the seeds formed at or migrated to the new pole would become visible only when that new pole already became an old pole, because of the relatively slow growth rate of granules relative to cell division.

Show MeSH
Related in: MedlinePlus