Limits...
Subcellular Min oscillations as a single-cell reporter of the action of polycations, protamine, and gentamicin on Escherichia coli.

Downing BP, Rutenberg AD, Touhami A, Jericho M - PLoS ONE (2009)

Bottom Line: We found rapid and substantial increases in the average MinD oscillation periods in the presence of any of these polyvalent cations.We also found striking interdependence in the action of the small cations with protamine or gentamicin, distorted oscillations under the action of intermediate levels of gentamicin and Ca(++), and reversible freezing of the Min oscillation at high cationic concentrations.Intracellular Min oscillations provide a fast single-cell reporter of bacterial response to extracellular polycations, which can be explained by the penetration of polycations into cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and Atmospheric Science, Dalhousie University, Halifax, Nova Scotia, Canada.

ABSTRACT

Background: In Escherichia coli, MinD-GFP fusion proteins show rapid pole to pole oscillations. The objective was to investigate the effects of extracellular cations on the subcellular oscillation of cytoplasmic MinD within Escherichia coli.

Methodology/principal findings: We exposed bacteria to the extracellular cations Ca(++), Mg(++), the cationic antimicrobial peptide (CAP) protamine, and the cationic aminoglycoside gentamicin. We found rapid and substantial increases in the average MinD oscillation periods in the presence of any of these polyvalent cations. For Ca(++) and Mg(++) the increases in period were transient, even with a constant extracellular concentration, while increases in period for protamine or gentamicin were apparently irreversible. We also found striking interdependence in the action of the small cations with protamine or gentamicin, distorted oscillations under the action of intermediate levels of gentamicin and Ca(++), and reversible freezing of the Min oscillation at high cationic concentrations.

Conclusions/significance: Intracellular Min oscillations provide a fast single-cell reporter of bacterial response to extracellular polycations, which can be explained by the penetration of polycations into cells.

Show MeSH

Related in: MedlinePlus

Description of the flow cell.Experimental chamber used for the observation of MinD oscillations in the presence of cations and antimicrobial peptides, as described in the text. Bacteria were observed over a 18 mm×13 mm area on the chamber bottom. The field of illumination and view per image was 0.25 mm2, so that a large number of non-overlapping images could be taken.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2749335&req=5

pone-0007285-g001: Description of the flow cell.Experimental chamber used for the observation of MinD oscillations in the presence of cations and antimicrobial peptides, as described in the text. Bacteria were observed over a 18 mm×13 mm area on the chamber bottom. The field of illumination and view per image was 0.25 mm2, so that a large number of non-overlapping images could be taken.

Mentions: Experiments were carried out in flow cells with dimensions of 18×13×0.8 mm, as illustrated in Fig. 1. The bottoms of the flow cells consisted of microscope cover slips that were supported by thin metal plates with openings for viewing and imaging of the bacteria. The flow cells were inoculated with bacteria through a small rubber plug. Prior to inoculation the cells were filled with control solution. This was either un-buffered 5 mM NaCl solution or 10 mM HEPES buffer. After inoculation, flow cells were flushed with buffer and remaining bacteria were allowed to settle in the flow cell for at least an hour in order to enhance the number of bacteria attached to the cell bottom. Following bacterial attachment, cationic solution was drawn through the cell with a syringe. Preliminary tests with dyed water showed that all visual traces of the dye disappeared after pulling 20 ml of fluid through the flow cell. All experiments were therefore carried out with that quantity of ionic solution. Fluid exchange flexed the thin bottoms of the chambers, temporarily moving attached bacteria out of focus. Depending on the size of the opening in the cover slip support plate, and hence the degree of cover slip flexing, imaging was delayed for 2–10 minutes after fluid exchange to allow the chamber bottom to flatten. This delay also ensured that ion diffusion was given more than sufficient time to homogenize the extracellular environment in the boundary layers (of thickness of a few microns) at the flow cell walls. A small thermocouple near the flow cell monitored the ambient temperature, between 24 and 26°C, during experiments.


Subcellular Min oscillations as a single-cell reporter of the action of polycations, protamine, and gentamicin on Escherichia coli.

Downing BP, Rutenberg AD, Touhami A, Jericho M - PLoS ONE (2009)

Description of the flow cell.Experimental chamber used for the observation of MinD oscillations in the presence of cations and antimicrobial peptides, as described in the text. Bacteria were observed over a 18 mm×13 mm area on the chamber bottom. The field of illumination and view per image was 0.25 mm2, so that a large number of non-overlapping images could be taken.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2749335&req=5

pone-0007285-g001: Description of the flow cell.Experimental chamber used for the observation of MinD oscillations in the presence of cations and antimicrobial peptides, as described in the text. Bacteria were observed over a 18 mm×13 mm area on the chamber bottom. The field of illumination and view per image was 0.25 mm2, so that a large number of non-overlapping images could be taken.
Mentions: Experiments were carried out in flow cells with dimensions of 18×13×0.8 mm, as illustrated in Fig. 1. The bottoms of the flow cells consisted of microscope cover slips that were supported by thin metal plates with openings for viewing and imaging of the bacteria. The flow cells were inoculated with bacteria through a small rubber plug. Prior to inoculation the cells were filled with control solution. This was either un-buffered 5 mM NaCl solution or 10 mM HEPES buffer. After inoculation, flow cells were flushed with buffer and remaining bacteria were allowed to settle in the flow cell for at least an hour in order to enhance the number of bacteria attached to the cell bottom. Following bacterial attachment, cationic solution was drawn through the cell with a syringe. Preliminary tests with dyed water showed that all visual traces of the dye disappeared after pulling 20 ml of fluid through the flow cell. All experiments were therefore carried out with that quantity of ionic solution. Fluid exchange flexed the thin bottoms of the chambers, temporarily moving attached bacteria out of focus. Depending on the size of the opening in the cover slip support plate, and hence the degree of cover slip flexing, imaging was delayed for 2–10 minutes after fluid exchange to allow the chamber bottom to flatten. This delay also ensured that ion diffusion was given more than sufficient time to homogenize the extracellular environment in the boundary layers (of thickness of a few microns) at the flow cell walls. A small thermocouple near the flow cell monitored the ambient temperature, between 24 and 26°C, during experiments.

Bottom Line: We found rapid and substantial increases in the average MinD oscillation periods in the presence of any of these polyvalent cations.We also found striking interdependence in the action of the small cations with protamine or gentamicin, distorted oscillations under the action of intermediate levels of gentamicin and Ca(++), and reversible freezing of the Min oscillation at high cationic concentrations.Intracellular Min oscillations provide a fast single-cell reporter of bacterial response to extracellular polycations, which can be explained by the penetration of polycations into cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and Atmospheric Science, Dalhousie University, Halifax, Nova Scotia, Canada.

ABSTRACT

Background: In Escherichia coli, MinD-GFP fusion proteins show rapid pole to pole oscillations. The objective was to investigate the effects of extracellular cations on the subcellular oscillation of cytoplasmic MinD within Escherichia coli.

Methodology/principal findings: We exposed bacteria to the extracellular cations Ca(++), Mg(++), the cationic antimicrobial peptide (CAP) protamine, and the cationic aminoglycoside gentamicin. We found rapid and substantial increases in the average MinD oscillation periods in the presence of any of these polyvalent cations. For Ca(++) and Mg(++) the increases in period were transient, even with a constant extracellular concentration, while increases in period for protamine or gentamicin were apparently irreversible. We also found striking interdependence in the action of the small cations with protamine or gentamicin, distorted oscillations under the action of intermediate levels of gentamicin and Ca(++), and reversible freezing of the Min oscillation at high cationic concentrations.

Conclusions/significance: Intracellular Min oscillations provide a fast single-cell reporter of bacterial response to extracellular polycations, which can be explained by the penetration of polycations into cells.

Show MeSH
Related in: MedlinePlus