Limits...
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.

Show MeSH

Related in: MedlinePlus

Formazan granule formation in different E. coli mutants.Scale bars equal 20 µm. Granules were false-colored in dark magenta. The top two lines show the parental strain (MC1000) and its derivatives: the dsbD deletion, the dsbA deletion, and the dsbA/dsbD double mutant strains. The third line shows the parental strain (MC1061) and the Ccm  mutant derived from it. Photos of the corresponding cell cultures after growth in the presence of TTC are shown at the top-right corner of each DIC image. The percentage of cells containing different numbers of granules for each strain is compared in the histogram at the bottom of the figure.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3366950&req=5

pone-0038427-g002: Formazan granule formation in different E. coli mutants.Scale bars equal 20 µm. Granules were false-colored in dark magenta. The top two lines show the parental strain (MC1000) and its derivatives: the dsbD deletion, the dsbA deletion, and the dsbA/dsbD double mutant strains. The third line shows the parental strain (MC1061) and the Ccm mutant derived from it. Photos of the corresponding cell cultures after growth in the presence of TTC are shown at the top-right corner of each DIC image. The percentage of cells containing different numbers of granules for each strain is compared in the histogram at the bottom of the figure.

Mentions: We tested mutants in the Dsb pathway that blocked either the reducing power inlet (ΔdsbD) or the oxidizing power outlet (ΔdsbA). The accumulation of formazan granules in ΔdsbD strain showed no difference from the parental strain (Fig. 2). However, the reduction of TTC was significantly enhanced in the ΔdsbA strain under identical growth conditions. The extensive accumulation of granules in the dsbA deletion strain was obvious even by simply examining the intense red color of the cell culture (inset). In the dsbA and dsbD double deletion strain, the formation of granules was at the same levels as in the parental strain. When the whole ccm operon was deleted, no change in granule formation was observed (Fig. 2).


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

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

Formazan granule formation in different E. coli mutants.Scale bars equal 20 µm. Granules were false-colored in dark magenta. The top two lines show the parental strain (MC1000) and its derivatives: the dsbD deletion, the dsbA deletion, and the dsbA/dsbD double mutant strains. The third line shows the parental strain (MC1061) and the Ccm  mutant derived from it. Photos of the corresponding cell cultures after growth in the presence of TTC are shown at the top-right corner of each DIC image. The percentage of cells containing different numbers of granules for each strain is compared in the histogram at the bottom of the figure.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366950&req=5

pone-0038427-g002: Formazan granule formation in different E. coli mutants.Scale bars equal 20 µm. Granules were false-colored in dark magenta. The top two lines show the parental strain (MC1000) and its derivatives: the dsbD deletion, the dsbA deletion, and the dsbA/dsbD double mutant strains. The third line shows the parental strain (MC1061) and the Ccm mutant derived from it. Photos of the corresponding cell cultures after growth in the presence of TTC are shown at the top-right corner of each DIC image. The percentage of cells containing different numbers of granules for each strain is compared in the histogram at the bottom of the figure.
Mentions: We tested mutants in the Dsb pathway that blocked either the reducing power inlet (ΔdsbD) or the oxidizing power outlet (ΔdsbA). The accumulation of formazan granules in ΔdsbD strain showed no difference from the parental strain (Fig. 2). However, the reduction of TTC was significantly enhanced in the ΔdsbA strain under identical growth conditions. The extensive accumulation of granules in the dsbA deletion strain was obvious even by simply examining the intense red color of the cell culture (inset). In the dsbA and dsbD double deletion strain, the formation of granules was at the same levels as in the parental strain. When the whole ccm operon was deleted, no change in granule formation was observed (Fig. 2).

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