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Multilayered distribution of peptidoglycan in the periplasmic space of Escherichia coli
1Unité Associée du CNRS, Biochimie Moléculaire et Cellulaire, Université Paris-Sud, Orsay, France.
Abstract:
When a staining technique using phosphotungstic acid (PTA) in 10% (w/v) chromic acid was applied to cells of Escherichia coli, the periplasmic space was seen as a dark 15-nm-thick layer of uniform appearance and constant width. Our observations are consistent with peptidoglycan being the main material stained. Isolated sacculi as well as purified peptidoglycan (protein free) were also stained by the same procedure, the thickness of the peptidoglycan being 8.8 +/- 1.8 and 6.6 +/- 1.5 nm, respectively. The increased thickness of the PTA-stained layer in stationary phase cells correlated well with the increased thickness of isolated sacculi or purified peptidoglycan and with the increased amount of peptidoglycan in such cells. Thickness measurements on isolated peptidoglycan were compatible with a two to three layer structure for material from exponential phase cells and with a four to five layer structure for that from stationary phase cells. Furthermore, the results indicated an uneven distribution of peptidoglycan material in the periplasmic space, the peptidoglycan spanning the space from the inner to the outer membrane.
Insights
Phosphotungstic acid staining reveals Escherichia coli
Area of Science:
- Microbiology
- Cell Biology
- Biochemistry
Background:
- The bacterial cell envelope is crucial for structural integrity and survival.
- Peptidoglycan forms a vital layer within the periplasmic space of Gram-negative bacteria.
- Understanding peptidoglycan structure and distribution is key to bacterial physiology.
Purpose of the Study:
- To visualize and quantify the peptidoglycan layer in Escherichia coli using phosphotungstic acid staining.
- To investigate the structural organization and thickness of peptidoglycan in different growth phases.
- To determine the distribution of peptidoglycan within the periplasmic space.
Main Methods:
- Application of phosphotungstic acid (PTA) staining in 10% chromic acid to Escherichia coli cells.
- Microscopic analysis of stained cells to visualize the periplasmic space and peptidoglycan layer.
- Isolation and purification of bacterial sacculi and peptidoglycan for thickness measurements.
Main Results:
- PTA staining clearly delineated a 15-nm-thick layer in the periplasmic space, consistent with peptidoglycan.
- Thickness of isolated peptidoglycan varied, with stationary phase cells showing thicker layers (8.8 +/- 1.8 nm) than exponential phase cells (6.6 +/- 1.5 nm).
- Peptidoglycan distribution was uneven, spanning the periplasmic space from the inner to the outer membrane.
Conclusions:
- Phosphotungstic acid is effective for visualizing the peptidoglycan layer in Escherichia coli.
- Peptidoglycan thickness increases in stationary phase, correlating with increased cellular peptidoglycan content.
- The study provides insights into the structural organization and layered nature of bacterial peptidoglycan.