Spheroplasts are microbial cells where the rigid outer cell wall is partially or largely removed. They are derived from bacteria or yeast, and retain their inner cell membrane. This modification transforms the cell into a spherical shape, making it a valuable tool for various scientific investigations.
Understanding Spheroplasts
Spheroplasts are characterized by the absence of a fully intact cell wall, which normally provides structural support and protection to the cell. Despite this, their cell membrane remains whole and functional. The term “spheroplast” originates from the spherical form these cells adopt once their cell wall is compromised, a shape that helps them cope with osmotic pressures.
They are commonly generated from Gram-negative bacteria, such as Escherichia coli, and from yeast. Gram-negative bacteria have an outer membrane in addition to their peptidoglycan cell wall. In spheroplasts, the peptidoglycan layer is largely removed, but the outer membrane may remain. This structural change makes spheroplasts highly susceptible to osmotic and mechanical stress, requiring them to be maintained in isotonic solutions to prevent bursting or shriveling.
How Spheroplasts Are Made
The formation of spheroplasts involves enzymatic digestion of the cell wall. For bacteria, the enzyme lysozyme is used, as it breaks down the peptidoglycan layer. For Gram-negative bacteria, a membrane permeabilizer like ethylenediaminetetraacetate (EDTA) might be used alongside lysozyme to help the enzyme reach the peptidoglycan layer by easing its passage through the outer membrane.
For yeast cells, enzymes such as chitinase, lyticase, or beta-glucuronidase degrade their cell wall components. Another method, particularly for Gram-negative bacteria, involves treatment with antibiotics that inhibit cell wall synthesis, such as penicillin or other beta-lactam antibiotics. Maintaining an isotonic solution is essential during spheroplast preparation to prevent lysis from osmotic shock.
Applications of Spheroplasts
Spheroplasts are used in scientific research due to their compromised cell wall and accessible cell membrane. A primary application is in gene transfer, also known as transformation or transfection, where foreign DNA can be introduced into cells with greater ease than with intact cells. Their ability to fuse with other cells, facilitated by agents like polyethylene glycol (PEG), allows for the transfer of genetic material into animal cells with nearly 100% efficiency.
These modified cells are also useful for studying cell membrane function and protein transport, as the absence of the rigid cell wall provides a clearer view of membrane processes. Researchers use spheroplasts in patch-clamp analysis to investigate bacterial ion channels. This method allows for the direct measurement of current through individual ion channels.
Spheroplasts aid in antibiotic discovery and characterization, particularly for identifying antibiotics that target cell wall synthesis. If a bacterium forms a spheroplast after drug treatment, it indicates that the antibiotic interferes with cell wall biosynthesis. This approach has led to the discovery of antibiotics like fosfomycin and carbapenems. Spheroplasts can also facilitate cell lysis, making it easier to extract cellular proteins from organisms like yeast that are normally protected by a thick cell wall.
Spheroplasts Versus Protoplasts
While both spheroplasts and protoplasts are microbial cells with altered cell walls, a distinction lies in the extent of cell wall removal and their cellular origin. Spheroplasts originate from Gram-negative bacteria and may retain some residual cell wall material, specifically the outer membrane layer. This means spheroplasts from Gram-negative bacteria possess two membranes: the inner cytoplasmic membrane and an outer membrane.
In contrast, protoplasts are derived from plant cells, fungi, or Gram-positive bacteria, where the cell wall is completely removed. Gram-positive bacteria possess only a single cytoplasmic membrane. Protoplasts, regardless of their origin, are bounded by a single membrane. Both cell types adopt a spherical shape and are sensitive to osmotic changes, requiring isotonic solutions.