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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.[1][2]

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]

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

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Antibiotic-induced spheroplasts

[edit]

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

[edit]

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

[edit]

Antibiotic discovery

[edit]

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

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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

[edit]

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

[edit]

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

[edit]

References

[edit]
[edit]
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Spheroplast

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A spheroplast is an enlarged, spherical form of a bacterial cell, primarily derived from Gram-negative species such as Escherichia coli, in which the peptidoglycan layer of the cell wall has been partially or completely removed, leaving the outer membrane intact but rendering the cell osmotically fragile.[1] Unlike protoplasts, which are produced from Gram-positive bacteria and lack any outer membrane after complete cell wall digestion, spheroplasts retain this additional membrane layer, distinguishing their structure and stability in hypotonic environments.[2] These structures typically measure 2–5 µm in diameter, significantly larger than unaltered bacteria (around 1 µm), due to the absence of the rigid cell wall that normally constrains shape and size.[1] Spheroplasts are generated through enzymatic or chemical treatments that target the cell wall while maintaining osmotic support to prevent lysis; for instance, lysozyme digests the peptidoglycan, often in combination with EDTA to permeabilize the outer membrane, and stabilizers like sucrose provide the necessary hypertonic conditions.[3] Factors influencing successful formation and enlargement include precise osmotic pressure (to avoid rupture) and the presence of divalent cations such as calcium or magnesium ions, which stabilize the membrane.[2] Although spheroplasts cannot divide, they can grow in size under controlled conditions, offering a model for studying bacterial physiology without the confounding influence of the cell wall.[2] In microbiology and biotechnology, spheroplasts serve critical roles in research, including the visualization of antimicrobial peptide localization to membranes, protein extraction via osmotic shock, and investigations into mechanosensitive channels or envelope biogenesis.[1] They also facilitate gene transfer techniques and the production of cell wall-deficient variants, such as L-forms, which mimic certain pathogenic states or enable enhanced catalytic activities in engineered systems.[3]

Definition and Distinctions

Core Definition

A spheroplast is a microbial cell, primarily bacterial but also fungal or plant in origin, in which the cell wall has been partially removed, leading to a characteristic spherical morphology bounded by the cytoplasmic membrane and remnants of the cell wall.[4] In bacteria, this partial degradation disrupts the rigid peptidoglycan structure that maintains the cell's typical rod or coccoid shape, allowing osmotic pressure to force the membrane into a rounded form.[5] In non-bacterial microbes, analogous wall components (such as chitin and glucans in fungi, or cellulose in plants) are partially digested to achieve similar spherical forms.[4] In Gram-negative bacteria, such as Escherichia coli, spheroplasts form through the specific loss of the peptidoglycan layer while retaining portions of the outer membrane, providing some structural support compared to fully wall-less forms.[5] In Gram-positive bacteria, spheroplast formation involves greater removal of the thicker peptidoglycan wall, often resulting in structures with minimal residual wall material.[3] The term "spheroplast" emerged in the 1950s amid research on cell wall synthesis inhibitors, notably penicillin, which induces these forms by blocking peptidoglycan cross-linking during bacterial growth.[4] Seminal work by Joshua Lederberg demonstrated penicillin's role in generating protoplast-like spherical cells from E. coli, highlighting their osmotic fragility and potential for reversion to normal morphology upon removal of the inhibitor.[6] Spheroplasts can arise naturally in bacterial populations under stress conditions such as antibiotic exposure, serving as a survival mechanism by temporarily shedding wall components to cope with environmental pressures.[7] This phenomenon has been observed in diverse Gram-negative species, such as Pseudomonas aeruginosa, where cell wall-deficient forms enhance persistence in challenging habitats like soil or host tissues.[7]

Comparison to Protoplasts

Protoplasts are microbial cells, typically derived from gram-positive bacteria or plant cells, in which the cell wall is completely removed through enzymatic digestion, such as with lysozyme for bacteria or cellulases for plants, resulting in structures bounded solely by the plasma membrane and lacking any rigid components.[5] In contrast, spheroplasts are primarily formed from gram-negative bacteria where the peptidoglycan layer is extensively but not fully degraded, retaining a small amount of residual peptidoglycan along with the outer membrane, which provides partial structural support under isotonic conditions.[3][8] A representative example illustrates this distinction: spheroplasts can be generated from Escherichia coli (gram-negative) via lysozyme treatment in the presence of EDTA to permeabilize the outer membrane, leaving vestigial peptidoglycan fragments, whereas protoplasts from Bacillus subtilis (gram-positive) require lysozyme digestion supplemented with chelators like EDTA to achieve complete wall removal, as there is no outer membrane.[9] This partial retention in spheroplasts allows for somewhat greater mechanical integrity compared to protoplasts, which are highly prone to lysis without support.[8] Regarding stability, spheroplasts exhibit enhanced robustness over protoplasts due to their residual wall elements, yet both remain osmotically fragile and necessitate hypertonic media to prevent bursting, as explored further in sections on osmotic properties.[3][5]

Properties and Behavior

Morphological and Structural Features

Spheroplasts undergo a pronounced morphological transition from the rod-like shape typical of bacilli or the coccal form of certain bacteria to a spherical configuration upon removal of the peptidoglycan layer, which provides structural rigidity and determines the native cell geometry.[5] This change occurs because the loss of peptidoglycan exposes the underlying cytoplasmic membrane to turgor pressure, forcing the cell to adopt a spherical shape that balances internal osmotic forces and minimizes surface tension.[10] In Gram-negative bacteria, such as Escherichia coli, this results in smooth, refractile spherical bodies observable under light microscopy, with the outer membrane remaining intact to enclose the modified periplasmic space.[5] Ultrastructural alterations in spheroplasts, revealed through electron microscopy, include cytoplasmic condensation appearing as phase-dense regions and membrane irregularities such as blebbing, which arise from the sudden relaxation of peptidoglycan constraints and subsequent membrane stress.[11] In Gram-negative spheroplasts, the periplasmic space is retained due to the preservation of the outer membrane, though it expands without the intervening peptidoglycan scaffold, leading to a more voluminous compartment between the inner and outer membranes.[5] Transmission electron microscopy of E. coli spheroplasts often shows these features as spherical profiles with occasional invaginations in the inner membrane and a lack of the dense peptidoglycan layer, highlighting the shift to a more fluid, tension-dependent architecture.[11] The formation of spheroplasts is accompanied by a significant increase in cell size, with diameters expanding up to 2-3 times the original dimensions in certain mutants or under controlled osmotic conditions, accompanied by surface area expansion that induces membrane tension.[11] For instance, in certain mutants of E. coli spheroplasts generated via lysozyme treatment, phase-contrast microscopy reveals ghost-like appearances with central phase-dense cores and peripheral invaginations, reflecting the internal reorganization and osmotic adjustments following wall removal.[11] These morphological traits underscore the spheroplast's reliance on membrane integrity for maintaining shape under hypotonic stress, though they contribute to heightened osmotic fragility.[10]

Osmotic Fragility and Viability

Spheroplasts display pronounced osmotic fragility owing to the diminished cell wall, rendering them prone to lysis upon exposure to hypotonic conditions where water influx exceeds membrane tolerance.[5] To mitigate this, spheroplasts necessitate maintenance in hypertonic media, typically containing 0.5-1 M sucrose or sorbitol, which counteracts osmotic pressure and preserves structural integrity.[12][13] Without such stabilization, rapid lysis occurs, as evidenced by significant turbidity reduction and DNA release within minutes in dilute buffers.[12] Viability of spheroplasts remains high in the short term, allowing metabolic activity to persist in supportive environments.[14] Long-term survival depends on regeneration, wherein spheroplasts regrow their cell walls in permissive hypertonic media, restoring functionality and enabling colony formation.[15] Key factors influencing viability include optimal temperatures of 25-30°C, neutral pH around 7.0-7.4, and the inclusion of divalent cations such as Mg²⁺ at concentrations of 0.001-0.01 M, which enhance membrane stability and prevent premature lysis.[15][16] The regeneration process involves de novo cell wall synthesis, typically commencing within 1.5-2 hours and completing structural reformation over 5-8 hours, thereby reverting spherical forms to the original rod shape in bacteria such as E. coli.[15] This regrowth is facilitated by resuming peptidoglycan assembly under isotonic conditions post-inhibitor removal, with viable spheroplasts dividing and elongating to yield normal progeny.[11]

Formation Mechanisms

Antibiotic-Induced Formation

Antibiotic-induced formation of spheroplasts primarily occurs through the action of β-lactam antibiotics, such as penicillins (e.g., ampicillin) and cephalosporins (e.g., cephaloridine, cefsulodin), which target penicillin-binding proteins (PBPs) essential for peptidoglycan synthesis in the bacterial cell wall.[5] These antibiotics covalently bind to the transpeptidase active sites of PBPs, preventing the cross-linking of peptidoglycan strands that provide structural rigidity to the cell wall.[17] In Gram-negative bacteria like Escherichia coli, PBPs 1a and 1b are particularly critical targets, as their inhibition disrupts the elongase complex responsible for lateral wall expansion, leading to progressive weakening of the peptidoglycan layer without immediate lysis under appropriate conditions.[5] The underlying mechanism involves the accumulation of uncross-linked peptidoglycan precursors, such as UDP-MurNAc-pentapeptide, due to blocked transpeptidation. This buildup triggers the activation of endogenous autolysins—autolytic enzymes that normally remodel the cell wall during growth but become dysregulated here, resulting in hydrolysis of existing peptidoglycan and further wall degradation.[18][19] The imbalance between new synthesis and autolysin-mediated breakdown creates a futile cycle, converting rod-shaped cells into spherical forms as internal turgor pressure reshapes the osmotically stabilized protoplasm.[18] This process is distinct from full lysis, as spheroplasts remain viable if maintained in hypertonic media, though they exhibit heightened osmotic fragility.[5] Optimal conditions for spheroplast formation emphasize actively growing cells, with log-phase bacteria being most susceptible due to high rates of cell wall synthesis.[20] Sub-lethal doses, typically 1× to 10× the minimum inhibitory concentration (MIC), are used to inhibit synthesis without overwhelming autolysis, often in osmotically stabilized media like sucrose-supplemented broth to prevent bursting.[5] Incubation durations of 1–2 hours at 37°C suffice for significant conversion, as longer exposure risks complete lysis. For instance, in E. coli, treatment with ampicillin at 1× MIC (approximately 2–4 μg/ml) during log phase yields 20–70% spheroplast conversion after 60 minutes, depending on the strain and precise conditions.[5][20]

Enzyme-Induced Formation

Enzyme-induced formation of spheroplasts involves the controlled enzymatic degradation of the cell wall, primarily using hydrolytic enzymes to remove peptidoglycan or analogous structures while preserving the plasma membrane and osmotic integrity.[21] Lysozyme, derived from sources such as hen egg white, is the primary enzyme for bacterial spheroplasts, as it specifically hydrolyzes the β-1,4 glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine residues in peptidoglycan, the key structural component of bacterial cell walls.[21] This action weakens the cell wall, leading to the transition from rod-shaped bacilli to spherical forms when conducted in an isotonic environment.[1] The standard protocol for bacterial spheroplast preparation entails suspending cells in an isotonic buffer, such as 0.5 M sucrose in Tris-HCl (pH 7.8–8.0), supplemented with magnesium chloride for stabilization, followed by the addition of lysozyme at concentrations of 20–50 µg/mL.[9] Incubation typically occurs at 37°C for 30–60 minutes with gentle shaking, though shorter times (5–15 minutes at room temperature) suffice for some strains, with progress monitored by phase-contrast microscopy to observe morphological rounding and osmotic fragility.[1] For gram-negative bacteria like Escherichia coli, lysozyme is combined with EDTA (0.125–1 mM) to chelate divalent cations and permeabilize the outer membrane, enabling enzyme access to the peptidoglycan layer; this pretreatment often includes a brief heat shock or calcium ion exposure to enhance efficiency, achieving over 98% conversion to viable spheroplasts with minimal enzyme (approximately 1,000 molecules per cell).[22] These methods ensure high-fidelity spheroplast generation for downstream applications, with morphological outcomes including uniform spherical cells 2–5 µm in diameter.[1]

Research and Biotechnological Applications

Antibiotic Testing and Discovery

Spheroplasts play a crucial role in the discovery of antibiotics that target bacterial cell wall synthesis, particularly β-lactams and other inhibitors of peptidoglycan formation. In screening assays, bacteria are grown in hypertonic media and exposed to potential drug candidates; compounds inhibiting cell wall biosynthesis induce spheroplast formation, rendering the cells osmotically fragile. These spheroplasts subsequently lyse upon transfer to hypotonic conditions, providing a clear phenotypic readout for identifying active inhibitors.[23] This approach, pioneered by Eugene Dulaney at Merck & Co. in the 1960s, facilitated the detection of compounds like fosfomycin (a MurA inhibitor) and cycloserine (an alanine racemase/D-alanine:D-alanine ligase inhibitor).[23] Testing methods for antibiotic efficacy often involve spheroplast-based assays that measure the minimum concentration required to induce lysis or inhibit spheroplast stability, which correlates closely with the minimum inhibitory concentration (MIC) observed in intact bacterial cells. These assays offer advantages over standard MIC tests, including faster visualization of effects through osmotic lysis and the ability to bypass outer membrane efflux pumps in Gram-negative bacteria, allowing direct evaluation of drug potency against cell wall targets without permeability barriers.[23] Historically, spheroplasts from Staphylococcus aureus were instrumental in elucidating penicillin's mode of action during the 1960s. Studies demonstrated that penicillin exposure under osmotic stabilization led to incomplete cell walls in growing staphylococci, forming spheroplasts that revealed disruptions in peptidoglycan cross-linking and turnover, confirming the drug's inhibition of transpeptidase activity.[24] This work, building on earlier observations of wall-deficient forms, established spheroplast models as key tools for mechanistic studies of β-lactam antibiotics.[25]

Electrophysiological Analysis

Spheroplasts, particularly giant forms derived from Escherichia coli overexpressing ion channels, have become a valuable model for patch-clamp electrophysiology to investigate bacterial membrane proteins at the single-channel level.[26] The removal of the cell wall facilitates the formation of high-resistance giga-ohm seals (typically 1–5 GΩ) between the recording pipette and the membrane, enabling precise measurements of ionic currents without the mechanical interference posed by intact cell walls.[27] This approach is especially useful for studying inner membrane proteins, such as porins and transporters, which are otherwise challenging to access in native bacteria due to their small size (0.8–2 μm) and rigid envelope.[26] A key advantage of spheroplasts over intact cells lies in their enlarged dimensions and enhanced membrane accessibility, which reduce turgor pressure artifacts and allow for cleaner isolation of channel activities.[27] Unlike whole bacteria, where the cell wall can distort pipette seals and obscure inner membrane events, spheroplasts provide a more direct interface for electrophysiological probing, promoting reliable single-channel recordings with minimal background noise.[26] Protocols for patch-clamp studies typically begin with the generation of giant spheroplasts through cephalexin-induced filamentation of E. coli followed by enzymatic digestion using lysozyme, EDTA, and DNase in hypertonic media (e.g., 0.8–1 M sucrose or glucose) to maintain osmotic stability.[26] Smaller spheroplasts (initially ~1–2 μm) are then fused via dielectrophoresis under an alternating current field combined with direct current pulses, yielding giant structures 10–50 μm in diameter suitable for patching.[27] These are perfused in artificial solutions, such as 200–250 mM KCl with 40–90 mM MgCl₂ and 5–15 mM HEPES (pH 7.2), adjusted to ~800 mOsm with sucrose to prevent lysis, allowing inside-out or whole-cell configurations for current recordings under voltage steps or pressure pulses.[28][26] Seminal studies using this system, dating from the late 1980s onward, have elucidated the function of voltage-gated channels and transporters in bacteria. For instance, early patch-clamp recordings on E. coli spheroplasts identified pressure-sensitive ion channels, such as the mechanosensitive channel of large conductance (MscL), with activation thresholds around -195 mmHg. Subsequent work revealed voltage-dependent K⁺ channels in Listeria monocytogenes spheroplasts, exhibiting outward rectification and single-channel conductances of ~10 pS.[27] Similarly, investigations into K⁺ uptake systems in methanogenic bacteria like Methanobacterium thermoautotrophicum demonstrated cyclic nucleotide-gated K⁺ channels with conductances up to 100 pS, highlighting their role in osmotic regulation and pH homeostasis.[27] These findings underscore spheroplasts' utility in dissecting bacterial electrophysiology, with applications extending to heterologous expression of eukaryotic channels in bacterial hosts.[29]

Membrane Isolation and Genetic Transformation

Spheroplasts provide a valuable model for isolating bacterial plasma membranes due to their lack of cell wall, allowing gentle hypotonic lysis to generate sealed vesicles enriched in inner membrane components for subsequent lipid and protein analyses.[30] This approach involves suspending spheroplasts in a low-osmolarity buffer, such as dilute phosphate or Tris-EDTA, to induce osmotic bursting while preserving membrane integrity, followed by differential centrifugation to separate vesicles from soluble cytoplasmic contents.[31] The resulting vesicles maintain functional transport proteins and lipid bilayers, enabling biochemical studies of membrane composition without contamination from cell wall peptidoglycan.[30] In genetic transformation, spheroplasts exhibit enhanced DNA uptake compared to intact bacterial cells, primarily because the removed cell wall reduces physical barriers to nucleic acid entry.[32] Methods such as polyethylene glycol (PEG) treatment or electroporation exploit this fragility; for instance, PEG-mediated protocols involve mixing spheroplasts with plasmid DNA in an osmotic stabilizer like 0.5-1 M sorbitol, followed by incubation to promote fusion and internalization.[33] Electroporation applies short electrical pulses to further permeabilize the membrane, achieving transformation frequencies of 10^3 to 10^5 transformants per microgram of DNA in species like Escherichia coli and Lactobacillus.[32] Post-transformation, spheroplasts are typically plated on selective media containing osmotic support (e.g., sorbitol or sucrose) to allow regeneration of cell walls and expression of introduced genes, with viability rates often exceeding 10-20% under optimized conditions.[33] These techniques have been instrumental in gene cloning and studies of wall-deficient mutants since the 1970s, particularly in bacteria such as Salmonella typhimurium, where spheroplasts enable the introduction of plasmids to investigate peptidoglycan-independent growth and genetic complementation.[34] Early applications included transforming spheroplasts with recombinant DNA to restore functions in cell wall mutants, facilitating the mapping of genes involved in envelope biogenesis and osmotic regulation.[35] This approach has supported the creation of stable wall-deficient strains for long-term genetic analysis, bypassing limitations of standard electroporation in walled cells.[7]

Emerging Biocatalytic Uses

Spheroplasts derived from Escherichia coli have emerged as versatile "cell factories" in biocatalysis, particularly for terpenoid production, due to their semi-permeable nature that retains essential cofactors within the cell while facilitating substrate diffusion through the compromised outer membrane.[36] This hybrid system leverages the protective intracellular environment of whole cells alongside the accessibility of cell-free extracts, enabling efficient enzymatic conversions without the need for additional permeabilization agents.[36] A key advancement came in 2022 with studies demonstrating the use of E. coli spheroplasts expressing squalene-hopene cyclase (SHC) enzymes, which achieved up to 99% conversion of substrates like squalene to hopene, with turnover frequencies (TOFs) improved by 5- to 98-fold compared to cell lysates or whole cells.[36] For instance, in the cyclization of E-geranyl acetone to its bicyclic product, spheroplasts exhibited a 5-fold increase in productivity (0.19 g/L) over lysates, highlighting their superior catalytic efficiency.[36] These spheroplasts are typically prepared via enzymatic or antibiotic-induced removal of the outer membrane, as detailed in formation mechanisms.[36] The advantages of this approach include minimized protease degradation of the enzyme, as the inner membrane and cytoplasm provide a stable milieu, combined with enhanced substrate access that boosts overall yields in terpenoid syntheses.[36] Notable examples encompass the production of hopanoids, such as hopene from squalene, where spheroplasts enabled near-complete conversions unattainable in intact cells.[36] This methodology shows promise for scalable industrial biocatalysis, offering a cost-effective alternative for high-value terpenoid compounds used in pharmaceuticals and materials.[36]

References

  1. Production and Visualization of Bacterial Spheroplasts and ... - NIH
  2. Factors That Affect the Enlargement of Bacterial Protoplasts and ...
  3. Spheroplast - an overview | ScienceDirect Topics
  4. What are Spheroplasts? - News-Medical.Net
  5. Morphological and ultrastructural changes in bacterial cells as an ...
  6. https://doi.org/10.1073/pnas.42.9.574
  7. Cell Wall Deficiency as a Coping Strategy for Stress - ScienceDirect
  8. Escherichia coli spheroplasts in a Croatian patient misclassified by ...
  9. Production and Ultrastructure of Lysozyme and - ASM Journals
  10. Article Physical Properties of Escherichia coli Spheroplast Membranes
  11. The Rcs Stress Response and Accessory Envelope Proteins Are ...
  12. induced spheroplasts of escherichia coli - ASM Journals
  13. Rapid Formation of Protoplasts of Streptomyces griseoflavus and ...
  14. Analyses of the Effect of Peptidoglycan on Photocatalytic ... - MDPI
  15. Regeneration of Escherichia coli Giant Protoplasts to Their Original ...
  16. None
  17. Beta-Lactam Antibiotics - StatPearls - NCBI Bookshelf
  18. Article Beta-Lactam Antibiotics Induce a Lethal Malfunctioning of the ...
  19. β-Lactam antibiotic targets and resistance mechanisms
  20. Antibacterial Compounds of Canadian Honeys Target Bacterial Cell ...
  21. Lysozyme - an overview | ScienceDirect Topics
  22. A highly efficient procedure for the quantitative formation of intact ...
  23. [PDF] Factors Affecting Spheroplast Formation of
  24. Production of an Antifungal Chitinase from Enterobacter sp. NRG4 ...
  25. Improvements in Protoplast Isolation Protocol and Regeneration of ...
  26. An Efficient Procedure for Protoplast Isolation from Mesophyll Cells ...
  27. Does the cell wall of bacteria remain a viable source of targets for ...
  28. https://doi.org/10.1016/0002-9343(65
  29. How penicillin kills bacteria: progress and problems - Journals
  30. Patch Clamp Electrophysiology for the Study of Bacterial Ion ...
  31. (PDF) Patch clamp recording from giant bacterial spheroplasts
  32. [PDF] Application Note Preparation and recordings on Nanion´s Port-a ...
  33. Analyzing plant mechanosensitive ion channels expressed in giant ...
  34. Improved membrane isolation in the purification of beta 2 ... - PubMed
  35. Improved Membrane Isolation in the Purification of β2 ...
  36. [22] Bacterial transformation using temperature-sensitive mutants ...
  37. Development of an efficient spheroplast transformation procedure ...
  38. Growth and transformation of phage T4 in Escherichia coli B4 ...
  39. Transfection of Escherichia coli and Salmonella typhimurium ...
  40. Spheroplasts preparation boosts the catalytic potential of a squalene ...
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