A spheroplast is a cell from which the cell wall has been almost completely removed, as by the action of penicillin. The name stems from the fact that after a microbe's cell wall is digested, membrane tension causes the cell to acquire a characteristic spherical shape. Spheroplasts are osmotically fragile, and will lyse if transferred to a hypotonic solution.
Uses and applications
From the 1960s into the 1990s Merck and Co. used a spheroplast screen as a primary method for 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 C, thienamycin and several carbapenems.
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 grown in a medium containing chemicals that prevent the cells from dividing completely. This causes bacteria to form long "snakes" that share a single membrane and cytoplasm. After a period of time, the cell walls of the "snakes" are digested, and the bacteria collapse into very large spheres surrounded by a single lipid bilayer. The membrane 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 extensively for studying the native mechanosensitive channels (MscL, MscS, and MscM) of E. coli since 1987. Recently, it has been extended to study other heterologously expressed ion channels and it has been shown that the giant E. coli spheroplast can be used as an ion-channel expression system comparable to the Xenopus oocyte.
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.
Gram-negative bacteria, such as E.coli are a subject to outer cell lysis by osmotic shock and the action of enzyme lysozyme and ethylenediaminetetraacetate (EDTA), the chelating agent, with subsequent release of periplasmic gap content and spheroplasts containing inner cell content into the sucrose medium. Lysozyme catalyzes the hydrolysis of the peptidoglycan layer, while EDTA destroys the outer membrane facilitating enzyme's access to the inner layers of the cell wall. The periplasmic enzymes released during the rupture of E.coli outer cell wall include: an alkaline phosphatase, a cyclic phosphodiesterase, an acid phosphatase, and a 5’-nucleotidase. Sucrose-tris(hydroxymethyl)aminomethane-HCl medium is essential to the preservation of the integrity of the enzymes upon lysis. EDTA aids cell hydrolysis by binding to divalent ions, such as Ca2+, and removing them from the wall thus softening the wall for further lysozyme action.
Bacterial spheroplasts, with suitable recombinant DNA inserted into it, can be used to transfect animal cells. Spheroplasts with recombinant DNA are introduced into the media containing animal cells and are fused by polyethylene glycol (PEG). With this methodology, nearly 100% of the animal cells may take up the foreign DNA. Upon conducting experiments following a modified Hanahan protocol using calcium chloride in E.Coli, it was determined that spheroplasts may be able to transform at 4.9x10−4.
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