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 mech ...

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 wall ...

. 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 constitut ...

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 Lyse may refer to: * Lyse Abbey, a former Cistercian abbey in Norway * Lyse, an alternative name of Lysebotn, Norway * Lyse Energi, a Norwegian power company * Łyse, Masovian Voivodeship, a village in east-central Poland * Łyse, Podlaskie Voivode ...

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-imp ...

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 (murein sacculus) characteristic of most ...

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 o ...

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. Comm ...

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 infections, ...

, 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 antibiotic β-lactam antibiotics (beta-lactam antibiotics) are antibiotics that contain a beta-lactam ring in their chemical structure. This includes penicillin derivatives (penams), cephalosporins and cephamycins (cephems), monobactams, carbapenems and c ...

s. Antibiotics that inhibit

biochemical pathways In biochemistry, a metabolic pathway is a linked series of chemical reactions occurring within a cell. The reactants, products, and intermediates of an enzymatic reaction are known as metabolites, which are modified by a sequence of chemical reac ...

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 ...

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

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 peop ...

, sulfamethoxazole) 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 glycoside ...

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, s ...

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 us ...

'' 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 Beta-glucuronidases are members of the glycosidase family of enzymes that catalyze breakdown of complex carbohydrates. Human β-glucuronidase is a type of glucuronidase (a member of glycosidase Family 2) that catalyzes hydrolysis of β-D-glucur ...

Uses and applications

Antibiotic discovery

From the 1960s into the 1990s,

Merck and Co. Merck & Co., Inc. is an American Multinational corporation, multinational pharmaceutical company headquartered in Rahway, New Jersey, and is named for Merck Group, founded in Germany in 1668, of whom it was once the American arm. The company doe ...

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 Cephamycins are a group of β-lactam antibiotics. They are very similar to cephalosporins, and the cephamycins are sometimes classified as cephalosporins. Like cephalosporins, cephamycins are based upon the cephem nucleus. Unlike most cephalospor ...

thienamycin Thienamycin (also known as thienpenem) is one of the most potent naturally produced antibiotics known thus far, discovered in '' Streptomyces cattleya'' in 1976. Thienamycin has excellent activity against both Gram-positive and Gram-negative bact ...

and several

carbapenems Carbapenems are a class of very effective antibiotic agents most commonly used for the treatment of severe bacterial infections. This class of antibiotics is usually reserved for known or suspected multidrug-resistant (MDR) bacterial infections. ...

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 wall ...

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 among ...

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 io ...

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. N ...

and other excitable cells. To prepare giant spheroplasts, bacteria are treated with a septation inhibitor (e.g.

cephalexin Cefalexin, also spelled cephalexin, is an antibiotic that can treat a number of bacterial infections. It kills gram-positive and some gram-negative bacteria by disrupting the growth of the bacterial cell wall. Cefalexin is a beta-lactam antibi ...

). 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. Th ...

and

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