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

cell wall A cell wall is a structural layer that surrounds some Cell type, 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, ...

has been almost completely removed, as by the action of

penicillin Penicillins (P, PCN or PEN) are a group of beta-lactam antibiotic, β-lactam antibiotics originally obtained from ''Penicillium'' Mold (fungus), moulds, principally ''Penicillium chrysogenum, P. chrysogenum'' and ''Penicillium rubens, P. ru ...

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

Gram-negative bacteria Gram-negative bacteria are bacteria that, unlike gram-positive bacteria, do not retain the Crystal violet, crystal violet stain used in the Gram staining method of bacterial differentiation. Their defining characteristic is that their cell envelo ...

. According to other definitions, the term also encompasses

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

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 memb ...

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 layer (sacculus) that surrounds the bacterial cytoplasmic membrane. The sugar component consists of alternating ...

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 pathogenic bacteria, bacterial infections, and antibiotic medications are widely used in the therapy ...

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 urinary tract infections. It is not indicated for kidney infections. Occasionally it is used for prostate infections. It is generally ...

vancomycin Vancomycin is a glycopeptide antibiotic medication used to treat certain bacterial infections. It is administered intravenously ( injection into a vein) to treat complicated skin infections, bloodstream infections, endocarditis, bone an ...

, moenomycin,

lactivicin Lactivicin is a non-beta-lactam antibiotic that is active against a range of Gram-positive and Gram-negative bacteria. Lactivicin demonstrates a similar affinity for penicillin-binding proteins to beta-lactam antibiotics and is also susceptible t ...

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

phosphoenolpyruvate Phosphoenolpyruvate (2-phosphoenolpyruvate, PEP) is the carboxylic acid derived from the enol of pyruvate and a phosphate anion. It exists as an anion. PEP is an important intermediate in biochemistry. It has the high-energy phosphate, highest-e ...

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

chloramphenicol Chloramphenicol is an antibiotic useful for the treatment of a number of bacterial infections. This includes use as an eye ointment to treat conjunctivitis. By mouth or by intravenous, injection into a vein, it is used to treat meningitis, pl ...

, oxytetracycline, 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 ...

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 ''Escherichia coli' ...

) also cause Gram-negative bacteria to form spheroplasts.

Enzyme-induced spheroplasts

The enzyme

lysozyme Lysozyme (, 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 glycoside hydrolase ...

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 proteins, globular glycoprotein with a molecular mass of about 80 Atomic mass unit, kDa that is widely repre ...

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 usu ...

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

chitinase Chitinases (, 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 glycanohydrola ...

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 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.

Patch clamping

Specially prepared giant spheroplasts of

Gram-negative 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 that their cell envelope consists ...

bacteria Bacteria (; : bacterium) are ubiquitous, mostly free-living organisms often consisting of one Cell (biology), biological cell. They constitute a large domain (biology), domain of Prokaryote, prokaryotic microorganisms. Typically a few micr ...

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 ...

through a technique called

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

, which was originally designed for characterizing the behavior of

neurons A neuron (American English), neurone (British English), or nerve cell, is an membrane potential#Cell excitability, excitable cell (biology), cell that fires electric signals called action potentials across a neural network (biology), neural net ...

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. 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 The cytoplasm describes all the material within a eukaryotic or prokaryotic cell, enclosed by the cell membrane, including the organelles and excluding the nucleus in eukaryotic cells. The material inside the nucleus of a eukaryotic cell and ...

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 (physical form and structure), its developmental processes, its biochemical and physiological propert ...

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 (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 mem ...

(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 with ...

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 female ger ...

Cell lysis

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

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 ( ; from Greek 'loosening') 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 ...

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 fo ...

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 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 wei ...

(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