Spheroplast

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Gram-negative bacteria attempting to grow and divide in the presence of peptidoglycan synthesis-inhibiting antibiotics (e.g. penicillin) fail to do so, and instead end up forming spheroplasts. Penicillin spheroplast generation.svg
Gram-negative bacteria attempting to grow and divide in the presence of peptidoglycan synthesis-inhibiting antibiotics (e.g. penicillin) fail to do so, and instead end up forming spheroplasts.

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. [3] [4] According to other definitions, the term also encompasses yeasts. [5] [6] The name spheroplast stems from the fact that after the microbe's cell wall is digested, membrane tension causes the cell to acquire a characteristic spherical shape. [4] Spheroplasts are osmotically fragile, and will lyse if transferred to a hypotonic solution. [5]

Contents

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. [2] [5]

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. [1] [2] Antibiotics that inhibit biochemical pathways directly upstream of peptidoglycan synthesis induce spheroplasts too (e.g. fosmidomycin, phosphoenolpyruvate). [1] [2]

In addition to the above antibiotics, inhibitors of protein synthesis (e.g. chloramphenicol, oxytetracycline, several aminoglycosides) and inhibitors of folic acid synthesis (e.g. trimethoprim, sulfamethoxazole) also cause Gram-negative bacteria to form spheroplasts. [2]

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. [2] [7] EDTA acts as a permeabilizer by binding to divalent ions such as Ca2+ and removing them from the outer membrane. [8]

The yeast Candida albicans can be converted to spheroplasts using the enzymes lyticase, chitinase and β-glucuronidase. [9]

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. Patchclamp Spheroplast1.jpg
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 (e.g. cephalexin). This causes the bacteria to form filaments, elongated cells that lack internal cross-walls. [10] 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 cytoplasmic 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. [11] [12] 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. [13] [14] [15] [16]

Cell lysis

Yeast cells are normally protected by a thick cell wall which makes extraction of cellular proteins difficult.[ citation needed ] Enzymatic digestion of the cell wall with zymolyase, creating spheroplasts, renders the cells vulnerable to easy lysis with detergents or rapid osmolar pressure changes. [9]

Transfection

Bacterial spheroplasts, with suitable recombinant DNA inserted into them, 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 method, nearly 100% of the animal cells may take up the foreign DNA. [17] 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. [18]

See also

Related Research Articles

<span class="mw-page-title-main">Cell wall</span> Outermost layer of some cells

A cell wall is a structural layer that surrounds some cell types, found immediately outside the cell membrane. It can be tough, flexible, and sometimes rigid. Primarily, it provides the cell with structural support, shape, protection, and functions as a selective barrier. Another vital role of the cell wall is to help the cell withstand osmotic pressure and mechanical stress. While absent in many eukaryotes, including animals, cell walls are prevalent in other organisms such as fungi, algae and plants, and are commonly found in most prokaryotes, with the exception of mollicute bacteria.

<span class="mw-page-title-main">Gram stain</span> Investigative procedure in microbiology

Gram stain, is a method of staining used to classify bacterial species into two large groups: gram-positive bacteria and gram-negative bacteria. It may also be used to diagnose a fungal infection. The name comes from the Danish bacteriologist Hans Christian Gram, who developed the technique in 1884.

<span class="mw-page-title-main">Gram-negative bacteria</span> Group of bacteria that do not retain the Gram stain used in bacterial differentiation

Gram-negative bacteria are bacteria that unlike gram-positive bacteria do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation. Their defining characteristic is their cell envelope, which consists of a thin peptidoglycan cell wall sandwiched between an inner (cytoplasmic) membrane and an outer membrane. These bacteria are found in all environments that support life on Earth.

Peptidoglycan or murein is a unique large macromolecule, a polysaccharide, consisting of sugars and amino acids that forms a mesh-like peptidoglycan layer (sacculus) that surrounds the bacterial cytoplasmic membrane. The sugar component consists of alternating residues of β-(1,4) linked N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). Attached to the N-acetylmuramic acid is an oligopeptide chain made of three to five amino acids. The peptide chain can be cross-linked to the peptide chain of another strand forming the 3D mesh-like layer. Peptidoglycan serves a structural role in the bacterial cell wall, giving structural strength, as well as counteracting the osmotic pressure of the cytoplasm. This repetitive linking results in a dense peptidoglycan layer which is critical for maintaining cell form and withstanding high osmotic pressures, and it is regularly replaced by peptidoglycan production. Peptidoglycan hydrolysis and synthesis are two processes that must occur in order for cells to grow and multiply, a technique carried out in three stages: clipping of current material, insertion of new material, and re-crosslinking of existing material to new material.

<span class="mw-page-title-main">FtsZ</span> Protein encoded by the ftsZ gene

FtsZ is a protein encoded by the ftsZ gene that assembles into a ring at the future site of bacterial cell division. FtsZ is a prokaryotic homologue of the eukaryotic protein tubulin. The initials FtsZ mean "Filamenting temperature-sensitive mutant Z." The hypothesis was that cell division mutants of E. coli would grow as filaments due to the inability of the daughter cells to separate from one another. FtsZ is found in almost all bacteria, many archaea, all chloroplasts and some mitochondria, where it is essential for cell division. FtsZ assembles the cytoskeletal scaffold of the Z ring that, along with additional proteins, constricts to divide the cell in two.

The periplasm is a concentrated gel-like matrix in the space between the inner cytoplasmic membrane and the bacterial outer membrane called the periplasmic space in gram-negative bacteria. Using cryo-electron microscopy it has been found that a much smaller periplasmic space is also present in gram-positive bacteria, between cell wall and the plasma membrane. The periplasm may constitute up to 40% of the total cell volume of gram-negative bacteria, but is a much smaller percentage in gram-positive bacteria.

<span class="mw-page-title-main">Bacterial outer membrane</span> Plasma membrane found in gram-negative bacteria

The bacterial outer membrane is found in gram-negative bacteria. Gram-negative bacteria form two lipid bilayers in their cell envelopes - an inner membrane (IM) that encapsulates the cytoplasm, and an outer membrane (OM) that encapsulates the periplasm.

<span class="mw-page-title-main">Filamentation</span> Type of bacteria growth

Filamentation is the anomalous growth of certain bacteria, such as Escherichia coli, in which cells continue to elongate but do not divide. The cells that result from elongation without division have multiple chromosomal copies.

<span class="mw-page-title-main">Penicillin-binding proteins</span> Class of proteins

Penicillin-binding proteins (PBPs) are a group of proteins that are characterized by their affinity for and binding of penicillin. They are a normal constituent of many bacteria; the name just reflects the way by which the protein was discovered. All β-lactam antibiotics bind to PBPs, which are essential for bacterial cell wall synthesis. PBPs are members of a subgroup of enzymes called transpeptidases. Specifically, PBPs are DD-transpeptidases.

A bacterium, despite its simplicity, contains a well-developed cell structure which is responsible for some of its unique biological structures and pathogenicity. Many structural features are unique to bacteria and are not found among archaea or eukaryotes. Because of the simplicity of bacteria relative to larger organisms and the ease with which they can be manipulated experimentally, the cell structure of bacteria has been well studied, revealing many biochemical principles that have been subsequently applied to other organisms.

<span class="mw-page-title-main">Large-conductance mechanosensitive channel</span> Group of transport proteins

Large conductance mechanosensitive ion channels (MscLs) (TC# 1.A.22) are a family of pore-forming membrane proteins that are responsible for translating stresses at the cell membrane into an electrophysiological response. MscL has a relatively large conductance, 3 nS, making it permeable to ions, water, and small proteins when opened. MscL acts as stretch-activated osmotic release valve in response to osmotic shock.

<span class="mw-page-title-main">Bactoprenol</span> Chemical compound

Bactoprenol also known as dolichol-11 and C55-isoprenyl alcohol (C55-OH) is a lipid first identified in certain species of lactobacilli. It is a hydrophobic alcohol that plays a key role in the growth of cell walls (peptidoglycan) in Gram-positive bacteria.

<span class="mw-page-title-main">L-form bacteria</span> Bacterial growth form that lack cell walls, derived from different bacteria

L-form bacteria, also known as L-phase bacteria, L-phase variants or cell wall-deficient bacteria (CWDB), are growth forms derived from different bacteria. They lack cell walls. Two types of L-forms are distinguished: unstable L-forms, spheroplasts that are capable of dividing, but can revert to the original morphology, and stable L-forms, L-forms that are unable to revert to the original bacteria.

Mechanosensitive channels (MSCs), mechanosensitive ion channels or stretch-gated ion channels are membrane proteins capable of responding to mechanical stress over a wide dynamic range of external mechanical stimuli. They are present in the membranes of organisms from the three domains of life: bacteria, archaea, and eukarya. They are the sensors for a number of systems including the senses of touch, hearing and balance, as well as participating in cardiovascular regulation and osmotic homeostasis (e.g. thirst). The channels vary in selectivity for the permeating ions from nonselective between anions and cations in bacteria, to cation selective allowing passage Ca2+, K+ and Na+ in eukaryotes, and highly selective K+ channels in bacteria and eukaryotes.

Small conductance mechanosensitive ion channels (MscS) provide protection against hypo-osmotic shock in bacteria, responding both to stretching of the cell membrane and to membrane depolarization. In eukaryotes, they fulfill a multitude of important functions in addition to osmoregulation. They are present in the membranes of organisms from the three domains of life: bacteria, archaea, fungi and plants.

Bacterial morphological plasticity refers to changes in the shape and size that bacterial cells undergo when they encounter stressful environments. Although bacteria have evolved complex molecular strategies to maintain their shape, many are able to alter their shape as a survival strategy in response to protist predators, antibiotics, the immune response, and other threats.

<span class="mw-page-title-main">Lipid II</span> Chemical compound

Lipid II is a precursor molecule in the synthesis of the cell wall of bacteria. It is a peptidoglycan, which is amphipathic and named for its bactoprenol hydrocarbon chain, which acts as a lipid anchor, embedding itself in the bacterial cell membrane. Lipid II must translocate across the cell membrane to deliver and incorporate its disaccharide-pentapeptide "building block" into the peptidoglycan mesh. Lipid II is the target of several antibiotics.

The bacterial murein precursor exporter (MPE) family is a member of the cation diffusion facilitator (CDF) superfamily of membrane transporters. Members of the MPE family are found in a large variety of Gram-negative and Gram-positive bacteria and facilitate the translocation of lipid-linked murein precursors. A representative list of proteins belonging to the MPE family can be found in the Transporter Classification Database.

The multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) flippase superfamily is a group of integral membrane protein families. The MOP flippase superfamily includes twelve distantly related families, six for which functional data are available:

  1. One ubiquitous family (MATE) specific for drugs - (TC# 2.A.66.1) The Multi Antimicrobial Extrusion (MATE) Family
  2. One (PST) specific for polysaccharides and/or their lipid-linked precursors in prokaryotes - (TC# 2.A.66.2) The Polysaccharide Transport (PST) Family
  3. One (OLF) specific for lipid-linked oligosaccharide precursors of glycoproteins in eukaryotes - (TC# 2.A.66.3) The Oligosaccharidyl-lipid Flippase (OLF) Family
  4. One (MVF) lipid-peptidoglycan precursor flippase involved in cell wall biosynthesis - (TC# 2.A.66.4) The Mouse Virulence Factor (MVF) Family
  5. One (AgnG) which includes a single functionally characterized member that extrudes the antibiotic, Agrocin 84 - (TC# 2.A.66.5) The Agrocin 84 Antibiotic Exporter (AgnG) Family
  6. And finally, one (Ank) that shuttles inorganic pyrophosphate (PPi) - (TC# 2.A.66.9) The Progressive Ankylosis (Ank) Family
<span class="mw-page-title-main">Divisome</span> A protein complex in bacteria responsible for cell division

The divisome is a protein complex in bacteria that is responsible for cell division, constriction of inner and outer membranes during division, and peptidoglycan (PG) synthesis at the division site. The divisome is a membrane protein complex with proteins on both sides of the cytoplasmic membrane. In gram-negative cells it is located in the inner membrane. The divisome is nearly ubiquitous in bacteria although its composition may vary between species.

References

  1. 1 2 3 4 Silver, L.L (2011). "Rational Approaches to Antibacterial Discovery: Pre-Genomic Directed and Phenotypic Screening". In Dougherty, T.; Pucci, M.J. (eds.). Antibiotic Discovery and Development. United States of America: Springer. pp. 33–75. doi:10.1007/978-1-4614-1400-1_2. ISBN   978-1-4614-1400-1.
  2. 1 2 3 4 5 6 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. hdl: 10059/2129 . PMID   27392605. S2CID   2065821.
  3. ↑ "Spheroplast". www.dictionary.com. Dictionary.com. 2019. Retrieved July 21, 2019.
  4. 1 2 "Spheroplast". ahdictionary.com. The American Heritage Dictionary of the English Language. 2019. Retrieved July 21, 2019.
  5. 1 2 3 "Protoplasts and spheroplasts". www.encyclopedia.com. Encyclopedia.com. 2016. Retrieved July 21, 2019.
  6. ↑ "Definition of spheroplast". www.merriam-webster.com. Merriam-Webster. 2019. Retrieved July 21, 2019.
  7. ↑ Tortora, G.; Funke, B.; Case, C. (2016). "Chapter 4, Functional anatomy of prokaryotic and eukaryotic cells". Microbiology: An Introduction (12th ed.). United States of America: Pearson. p. 84. ISBN   978-0-321-92915-0.
  8. ↑ Ninfa, A.J.; Ballou, D.P.; Benore, M. (2009). 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.
  9. 1 2 Calvert, C.M.; Sanders, D. (1995). "Inositol trisphosphate-dependent and -independent Ca2+ mobilization pathways at the vacuolar membrane of Candida albicans". The Journal of Biological Chemistry. 270 (13): 7272–80. doi: 10.1074/jbc.270.13.7272 . PMID   7706267.
  10. ↑ 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". Biotechnology Reports. 7: 17–23. doi:10.1016/j.btre.2015.04.007. PMC   5466043 . PMID   28626710.
  11. ↑ Martinac, B.; Buechner, M.; Delcour, A.H.; Adler, J.; Kung, C. (1987). "Pressure-sensitive ion channel in Escherichia coli". Proceedings of the National Academy of Sciences of the United States of America. 84 (8): 2297–2301. Bibcode:1987PNAS...84.2297M. doi: 10.1073/pnas.84.8.2297 . PMC   304637 . PMID   2436228.
  12. ↑ Blount, P.; Sukharev, S.I.; Moe, P.C.; Kung, C. (1999). Mechanosensitive channels of bacteria. Methods in Enzymology. Vol. 294. pp. 458–482. doi:10.1016/s0076-6879(99)94027-2. PMID   9916243.
  13. ↑ Santos, J.S.; Lundby, A.; Zazueta, C.; 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. doi:10.1085/jgp.200609572. PMC   2151562 . PMID   16908725.
  14. ↑ Nakayama, Y.; Fujiu, K.; Sokabe, M.; Yoshimura, K. (2007). "Molecular and electrophysiological characterization of a mechanosensitive channel expressed in the chloroplasts of Chlamydomonas". Proceedings of the National Academy of Sciences of the United States of America. 104 (14): 5883–5888. Bibcode:2007PNAS..104.5883N. doi: 10.1073/pnas.0609996104 . PMC   1851586 . PMID   17389370.
  15. ↑ Kuo, M. M.-C.; Baker, K. A.; Wong, L.; Choe, S. (2007). "Dynamic oligomeric conversions of the cytoplasmic RCK domains mediate MthK potassium channel activity". Proceedings of the National Academy of Sciences of the United States of America. 104 (7): 2151–2156. Bibcode:2007PNAS..104.2151K. doi: 10.1073/pnas.0609085104 . PMC   1892972 . PMID   17287352.
  16. ↑ Kuo, M. M.-C.; Saimi, Y.; Kung, C.; Choe, S. (2007). "Patch-clamp and phenotypic analyses of a prokaryotic cyclic nucleotide-gated K+ channel using Escherichia coli as a host". Journal of Biological Chemistry. 282 (33): 24294–24301. doi: 10.1074/jbc.M703618200 . PMC   3521034 . PMID   17588940.
  17. ↑ Gietz, R.D.; Woods, R.A. (2001). "Genetic transformation of yeast". BioTechniques. 30 (4): 816–820, 822–826, 828. doi: 10.2144/01304rv02 . PMID   11314265.
  18. ↑ Liu, I.; Liu, M.; Shergill, K. (2006). "The effect of spheroplast formation on the transformation efficiency in Escherichia coli DH5α" (PDF). Journal of Experimental Microbiology and Immunology. 9: 81–85.
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