A spheroplast (or sphaeroplast in British usage) is a microbial cell from which the cell wall has been almost completely removed, as by the action of penicillin or lysozyme. According to some definitions, the term is used to describe Gram-negative bacteria.
When used to describe Gram-negative bacteria, the term spheroplast refers to cells from which the peptidoglycan component but not the outer membrane component of the cell wall has been removed.
Spheroplast formation
Antibiotic-induced spheroplasts
Various
antibiotics convert Gram-negative bacteria into spheroplasts. These include
peptidoglycan synthesis inhibitors such as
fosfomycin,
vancomycin, moenomycin,
lactivicin and the β-lactam antibiotics.
Antibiotics that inhibit biochemical pathways directly upstream of peptidoglycan synthesis induce spheroplasts too (e.g.
fosmidomycin, phosphoenolpyruvate).
In addition to the above antibiotics, inhibitors of protein synthesis (e.g. chloramphenicol, oxytetracycline, several ) and inhibitors of folic acid synthesis (e.g. trimethoprim, sulfamethoxazole) also cause Gram-negative bacteria to form spheroplasts.
Enzyme-induced spheroplasts
The enzyme
lysozyme causes Gram-negative bacteria to form spheroplasts, but only if a membrane permeabilizer such as
lactoferrin or ethylenediaminetetraacetate (EDTA) is used to ease the enzyme's passage through the outer membrane.
EDTA acts as a permeabilizer by binding to divalent ions such as Ca
2+ and removing them from the outer membrane.
The yeast Candida albicans can be converted to spheroplasts using the enzymes zymolyase, chitinase and β-glucuronidase.
Uses and applications
Antibiotic discovery
From the 1960s into the 1990s, Merck and Co. used a spheroplast screen as a primary method for
Drug discovery of antibiotics that inhibit cell wall biosynthesis. In this screen devised by Eugene Dulaney, growing bacteria were exposed to test substances under hypertonic conditions. Inhibitors of cell wall synthesis caused growing bacteria to form spheroplasts. This screen enabled the discovery of fosfomycin,
Cephamycin,
thienamycin and several
carbapenems.
Patch clamping
Specially prepared giant spheroplasts of
Gram-negative bacteria can be used to study the function of bacterial
ion channels through a technique called
patch clamp, which was originally designed for characterizing the behavior of
neurons and other excitable cells. To prepare giant spheroplasts, bacteria are treated with a septation inhibitor (e.g.
cephalexin). This causes the bacteria to form
Filamentation, elongated cells that lack internal cross-walls.
After a period of time, the cell walls of the filaments are digested, and the bacteria collapse into very large spheres surrounded by just their
Cell membrane and outer membranes. The membranes can then be analyzed on a
patch clamp apparatus to determine the
phenotype of the ion channels embedded in it. It is also common to
overexpress a particular channel to amplify its effect and make it easier to characterize.
The technique of patch clamping giant E. coli spheroplasts has been used to study the native mechanosensitive channels (MscL, MscS, and MscM) of E. coli.
Cell lysis
Yeast cells are normally protected by a thick
cell wall which makes extraction of cellular proteins difficult. Enzymatic digestion of the cell wall with zymolyase, creating spheroplasts, renders the cells vulnerable to easy
lysis with detergents or rapid osmolar pressure changes.
Transfection
Bacterial spheroplasts, with suitable
recombinant DNA inserted into them, can be used to
transfection animal cells. Spheroplasts with recombinant DNA are introduced into the media containing animal cells and are fused by polyethylene glycol (PEG). With this method, nearly 100% of the animal cells may take up the foreign DNA.
Upon conducting experiments following a modified
Douglas Hanahan protocol using calcium chloride in
E. coli, it was determined that spheroplasts may be able to transform at 4.9x10
−4.
See also
External links
