A spheroplast (or sphaeroplast in British usage) is a microbial cell from which the

cell wall A cell wall is a structural layer surrounding some types of cells, just outside the cell membrane. It can be tough, flexible, and sometimes rigid. It provides the cell with both structural support and protection, and also acts as a filtering mec ...

has been almost completely removed, as by the action of

penicillin Penicillins (P, PCN or PEN) are a group of β-lactam antibiotics originally obtained from ''Penicillium'' moulds, principally '' P. chrysogenum'' and '' P. rubens''. Most penicillins in clinical use are synthesised by P. chrysogenum using ...

or lysozyme. According to some definitions, the term is used to describe

Gram-negative bacteria Gram-negative bacteria are bacteria that do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation. They are characterized by their cell envelopes, which are composed of a thin peptidoglycan cell wa ...

. According to other definitions, the term also encompasses

yeast Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are estimated to consti ...

s. 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. Spheroplasts are osmotically fragile, and will lyse if transferred to a

hypotonic In chemical biology, tonicity is a measure of the effective osmotic pressure gradient; the water potential of two solutions separated by a partially-permeable cell membrane. Tonicity depends on the relative concentration of selective membrane-i ...

solution. When used to describe Gram-negative bacteria, the term spheroplast refers to cells from which the

peptidoglycan Peptidoglycan or murein is a unique large macromolecule, a polysaccharide, consisting of sugars and amino acids that forms a mesh-like peptidoglycan layer outside the plasma membrane, the rigid Cell wall#Bacterial_cell_walls, cell wall (murein sac ...

component but not the outer membrane component of the cell wall has been removed.

Spheroplast formation

Antibiotic-induced spheroplasts

Various

antibiotics An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important type of antibacterial agent for fighting bacterial infections, and antibiotic medications are widely used in the treatment and prevention ...

convert Gram-negative bacteria into spheroplasts. These include

synthesis inhibitors such as

fosfomycin Fosfomycin, sold under the brand name Monurol among others, is an antibiotic primarily used to treat lower UTI. It is not indicated for kidney infections. Occasionally it is used for prostate infections. It is generally taken by mouth. Common ...

vancomycin Vancomycin is a glycopeptide antibiotic medication used to treat a number of bacterial infections. It is recommended intravenously as a treatment for complicated skin infections, bloodstream infections, endocarditis, bone and joint infection ...

, moenomycin, lactivicin and the β-lactam antibiotics. Antibiotics that inhibit biochemical pathways directly upstream of peptidoglycan synthesis induce spheroplasts too (e.g.

fosmidomycin Fosmidomycin is an antibiotic that was originally isolated from culture broths of bacteria of the genus ''Streptomyces''. It specifically inhibits DXP reductoisomerase, a key enzyme in the non-mevalonate pathway of isoprenoid biosynthesis. It is ...

phosphoenolpyruvate Phosphoenolpyruvate (2-phosphoenolpyruvate, PEP) is the ester derived from the enol of pyruvate and phosphate. It exists as an anion. PEP is an important intermediate in biochemistry. It has the highest-energy phosphate bond found (−61.9 kJ/ ...

). In addition to the above antibiotics, inhibitors of protein synthesis (e.g.

chloramphenicol Chloramphenicol is an antibiotic An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important type of antibacterial agent for fighting pathogenic bacteria, bacterial infections, and antibiotic medica ...

oxytetracycline Oxytetracycline is a broad-spectrum tetracycline antibiotic, the second of the group to be discovered. Oxytetracycline works by interfering with the ability of bacteria to produce essential proteins. Without these proteins, the bacteria cannot g ...

, several

aminoglycoside Aminoglycoside is a medicinal and bacteriologic category of traditional Gram-negative antibacterial medications that inhibit protein synthesis and contain as a portion of the molecule an amino-modified glycoside ( sugar). The term can also refer ...

s) and inhibitors of folic acid synthesis (e.g.

trimethoprim Trimethoprim (TMP) is an antibiotic used mainly in the treatment of bladder infections. Other uses include for middle ear infections and travelers' diarrhea. With sulfamethoxazole or dapsone it may be used for ''Pneumocystis'' pneumonia in pe ...

sulfamethoxazole Sulfamethoxazole (SMZ or SMX) is an antibiotic. It is used for bacterial infections such as urinary tract infections, bronchitis, and prostatitis and is effective against both gram negative and positive bacteria such as ''Listeria monocytogenes' ...

) also cause Gram-negative bacteria to form spheroplasts.

Enzyme-induced spheroplasts

The enzyme

lysozyme Lysozyme (EC 3.2.1.17, muramidase, ''N''-acetylmuramide glycanhydrolase; systematic name peptidoglycan ''N''-acetylmuramoylhydrolase) is an antimicrobial enzyme produced by animals that forms part of the innate immune system. It is a glycosid ...

causes Gram-negative bacteria to form spheroplasts, but only if a membrane permeabilizer such as

lactoferrin Lactoferrin (LF), also known as lactotransferrin (LTF), is a multifunctional protein of the transferrin family. Lactoferrin is a globular glycoprotein with a molecular mass of about 80 kDa that is widely represented in various secretory fluids, ...

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 Ca²⁺ and removing them from the outer membrane. The yeast ''

Candida albicans ''Candida albicans'' is an opportunistic pathogenic yeast that is a common member of the human gut flora. It can also survive outside the human body. It is detected in the gastrointestinal tract and mouth in 40–60% of healthy adults. It is usua ...

'' can be converted to spheroplasts using the enzymes lyticase,

chitinase Chitinases (EC 3.2.1.14, chitodextrinase, 1,4-β-poly-N-acetylglucosaminidase, poly-β-glucosaminidase, β-1,4-poly-N-acetyl glucosamidinase, poly ,4-(N-acetyl-β-D-glucosaminide)glycanohydrolase, (1→4)-2-acetamido-2-deoxy-β-D-glucan glycano ...

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

discovery Discovery may refer to: * Discovery (observation), observing or finding something unknown * Discovery (fiction), a character's learning something unknown * Discovery (law), a process in courts of law relating to evidence Discovery, The Discover ...

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.

Patch clamping

Specially prepared giant spheroplasts of

Gram-negative Gram-negative bacteria are bacteria that do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation. They are characterized by their cell envelopes, which are composed of a thin peptidoglycan cell wa ...

bacteria Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were am ...

can be used to study the function of bacterial

ion channels Ion channels are pore-forming membrane proteins that allow ions to pass through the channel pore. Their functions include establishing a resting membrane potential, shaping action potentials and other electrical signals by gating the flow of i ...

through a technique called

patch clamp The patch clamp technique is a laboratory technique in electrophysiology used to study ionic currents in individual isolated living cells, tissue sections, or patches of cell membrane. The technique is especially useful in the study of excitabl ...

, which was originally designed for characterizing the behavior of

neurons A neuron, neurone, or nerve cell is an electrically excitable cell that communicates with other cells via specialized connections called synapses. The neuron is the main component of nervous tissue in all animals except sponges and placozoa. ...

and other excitable cells. To prepare giant spheroplasts, bacteria are treated with a

septation In biology, a septum (Latin for ''something that encloses''; plural septa) is a wall, dividing a cavity or structure into smaller ones. A cavity or structure divided in this way may be referred to as septate. Examples Human anatomy * Interatri ...

inhibitor (e.g. cephalexin). This causes the bacteria to form filaments, 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

cytoplasmic In cell biology, the cytoplasm is all of the material within a eukaryotic cell, enclosed by the cell membrane, except for the cell nucleus. The material inside the nucleus and contained within the nuclear membrane is termed the nucleoplasm. T ...

and outer membranes. The membranes can then be analyzed on a

apparatus to determine the

phenotype In genetics, the phenotype () is the set of observable characteristics or traits of an organism. The term covers the organism's morphology or physical form and structure, its developmental processes, its biochemical and physiological prop ...

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 Mechanosensitive channels, mechanosensitive ion channels or stretch-gated ion channels (not to be confused with mechanoreceptors). They are present in the membranes of organisms from the three domains of life: bacteria, archaea, and eukarya. They ...

(MscL, MscS, and MscM) of ''E. coli''. 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 ''Xenopus'' () (Gk., ξενος, ''xenos''=strange, πους, ''pous''=foot, commonly known as the clawed frog) is a genus of highly aquatic frogs native to sub-Saharan Africa. Twenty species are currently described within it. The two best-kno ...

oocyte An oocyte (, ), oöcyte, or ovocyte is a female gametocyte or germ cell involved in reproduction. In other words, it is an immature ovum, or egg cell. An oocyte is produced in a female fetus in the ovary during female gametogenesis. The femal ...

Cell lysis

Yeast Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are estimated to consti ...

cells are normally protected by a thick

which makes extraction of cellular proteins difficult. Enzymatic digestion of the cell wall with zymolyase, creating spheroplasts, renders the cells vulnerable to easy

lysis Lysis ( ) is the breaking down of the membrane of a cell, often by viral, enzymic, or osmotic (that is, "lytic" ) mechanisms that compromise its integrity. A fluid containing the contents of lysed cells is called a ''lysate''. In molecular bio ...

with detergents or rapid osmolar pressure changes.

Transfection

Bacterial spheroplasts, with suitable

recombinant DNA Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating sequences that would not otherwise be fou ...

inserted into them, can be used to

transfect Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. It may also refer to other methods and cell types, although other terms are often preferred: "transformation" is typically used to desc ...

animal cells. Spheroplasts with recombinant DNA are introduced into the media containing animal cells and are fused by

polyethylene glycol Polyethylene glycol (PEG; ) is a polyether compound derived from petroleum with many applications, from industrial manufacturing to medicine. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular w ...

(PEG). With this method, 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.

References

External links

* {{MeshName, Spheroplasts Bacteria