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Spheroplast

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A spheroplast (or sphaeroplast in British usage) 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.

Spheroplast formation

Gram-negative bacteria attempting to grow and divide in the presence of peptidoglycan synthesis-inhibiting antibiotics fail to do so, and instead end up forming spheroplasts.[1][2]

Various chemical and biological agents can convert bacteria into spheroplasts. These include peptidoglycan synthesis-inhibiting antibiotics such as fosfomycin, vancomycin, moenomycin, lactivicin and the β-lactam antibiotics.[1][2] Antibacterial compounds that inhibit biochemical pathways directly upstream of peptidoglycan synthesis sometimes induce spheroplasts too (eg. fosmidomycin, phosphoenolpyruvate),[1] as do some protein synthesis inhibitors (eg. chloramphenicol, oxytetracycline, several aminoglycosides).[2] The enzyme lysozyme also induces spheroplast formation, but only if a membrane permeabilizer such as lactoferrin or EDTA is used to ease the enzyme’s passage through the outer membrane.[2]

Uses and applications

Antibiotic discovery

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.[1]

Patch clamping

An E.coli spheroplast patched with a glass pipette.

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 (eg. cephalexin) that disrupts cell division. This causes the bacteria to form filaments, elongated cells that lack internal cross-walls.[3] After a period of time, the cell walls of the filaments 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.[4][5] 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.[6][7][8][9]

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.

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.[10] 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.[11] 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.[12]

Transfection

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.[13] 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.[14]

See also

References

  1. ^ a b c d Silver, L.L (2011). "Chapter 2, Rational approaches to antibiotic discovery: pre-genomic directed and phenotypic screening". In Dougherty, T.; Pucci, M.J. (eds.). Antibiotic Discovery and Development. Springer. pp. 33–75. ISBN 978-1-4614-1400-1. {{cite book}}: Cite has empty unknown parameter: |lastauthoramp= (help); External link in |chapterurl= (help); Unknown parameter |chapterurl= ignored (|chapter-url= suggested) (help)
  2. ^ a b c d Cushnie, T.P.; O’Driscoll, N.H.; Lamb, A.J. (2016). "Morphological and ultrastructural changes in bacterial cells as an indicator of antibacterial mechanism of action". Cellular and Molecular Life Sciences. 73 (23): 4471–4492. doi:10.1007/s00018-016-2302-2. PMID 27392605.
  3. ^ Kikuchi, K.; Sugiura, M.; Nishizawa-Harada, C.; Kimura, T. (2015). "The application of the Escherichia coli giant spheroplast for drug screening with automated planar patch clamp system" (PDF). Biotechnology Reports. 7: 17–23. doi:10.1016/j.btre.2015.04.007. PMID 28626710.
  4. ^ Martinac, B., Buechner, M., Delcour, A. H., Adler, J., and Kung, C. (1987) Pressure-sensitive ion channel in Escherichia coli. Proc. Natl. Acad. Sci. USA 84, 2297-2301.
  5. ^ Blount, P., Sukharev, S. I., Moe, P. C., Martinac, B., and Kung, C. (1999) Mechanosensitive channels of bacteria. Methods in Enzymology 294, 458-482.
  6. ^ Santos, J. S., Lundby, A., Zazueta, C., and Montal, M. (2006) Molecular template for a voltage sensor in a novel K+ channel. I. Identification and functional characterization of KvLm, a voltage-gated K+ channel from Listeria monocytogenes. Journal of General Physiology 128(3), 283-292.
  7. ^ Nakayama, Y., Fujiu, K., Sokabe, M., and Yoshimura, K. (2007) Molecular and electrophysiological characterization of a mechanosensitive channel expressed in the chloroplasts of Chlamydomonas. Proc. Natl. Acad. Sci. USA 104, 5883-5888.
  8. ^ Kuo, M. M.-C., Baker, K. A., Wong, L., and Choe, S. (2007) Dynamic oligomeric conversions of the cytoplasmic RCK domains mediate MthK potassium channel activity. Proc. Natl. Acad. Sci. USA 104, 2151-2156.
  9. ^ Kuo, M. M.-C., Saimi, Y., Kung, C., and Choe, S. (2007). Patch-clamp and phenotypic analyses of a prokaryotic cyclic nucleotide-gated K+ channel using Escherichia Coli as a host. J. Biol. Chem. 282, 24294-24301.
  10. ^ Tortora, Gerard; Funke, Berdell; Case, Christine (2016). Microbiology: An Introduction. United States: Pearson. p. 84. ISBN 0-321-92915-2.
  11. ^ Neu, Harold C.; Heppel, Leon A. (September 1, 1965). "The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts". The Journal of Biological Chemistry. 240 (9): 3685–3692. PMID 4284300.
  12. ^ Ninfa, Alexander J.; Ballou, David P.; Benore, Marilee (2010). Fundamental Laboratory Approaches for biochemistry and Biotechnology (2nd ed.). United States of America: John Wiley & Sons, Inc. p. 234. ISBN 978-0-470-08766-4.
  13. ^ Gietz, R. D.; Woods, R. A. (2001-04-01). "Genetic transformation of yeast". BioTechniques. 30 (4): 816–820, 822–826, 828 passim. ISSN 0736-6205. PMID 11314265.
  14. ^ "The Effect of Spheroplast Formation on the Transformation Efficiency in Escherichia coli DH5α". ResearchGate. Retrieved 2017-05-07.
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