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The twin arginine translocation (Tat) pathway transports folded protein across the cytoplasmic membrane in bacteria, archaea, and across the thylakoid membrane in plants as well as the inner membrane in some mitochondria. In plant chloroplasts, the Tat pathway utilizes the protonmotive force (PMF) to drive protein translocation. However, in bacteria, it has been shown that Tat transport depends only on the transmembrane electrical potential (Δψ) component of PMF in vitro. To investigate the comprehensive PMF requirement in Escherichia coli, we have developed the first real-time assay to monitor Tat transport utilizing the NanoLuc Binary Technology in E. coli spheroplasts. This luminescence assay allows for continuous monitoring of Tat transport with high-resolution, making it possible to observe subtle changes in transport in response to different treatments. By applying the NanoLuc assay, we report that, under acidic conditions (pH = 6.3), ΔpH, in addition to Δψ, contributes energetically to Tat transport in vivo in E. coli spheroplasts. These results provide novel insight into the mechanism of energy utilization by the Tat pathway.

Saccharomyces cerevisiae is a genetically tractable, affordable, and extensively documented eukaryotic single-cell model organism. This budding yeast is amenable for the development of genetic and biochemical experiments and is frequently used to investigate the function, activity, and mechanism of mammalian proteins. However, yeast contains a cell wall that hinders select assays including organelle isolation. Lytic enzymes, with Zymolyase as the most effective and frequently used tool, are utilized to weaken the yeast cell wall resulting in yeast spheroplasts. Spheroplasts are easily lysed by, for example, osmotic-shock conditions to isolate yeast nuclei or mitochondria. However, during our studies of the DNA repair enzyme tyrosyl-DNA phosphodiesterase I (Tdp1), we encountered a negative effect of Zymolyase. We observed that Zymolyase treatment affected the steady-state protein levels of Tdp1. This was revealed by inconsistencies in technical and biological replicate lysates of plasmid-born galactose-induced expression of Tdp1. This off-target effect of Zymolyase is rarely discussed in articles and affects a select number of intracellular proteins, including transcription factors and assays such as chromatin immunoprecipitations. Following extensive troubleshooting, we concluded that the culprit is the Ser-protease, Zymolyase B, component of the Zymolyase enzyme mixture that causes the degradation of Tdp1. In this study, we report the protocols we have used, and our final protocol with an easy, affordable adaptation to any assay/protocol involving Zymolyase. [ABSTRACT FROM AUTHOR]

Phospholipid translocation occurs ubiquitously in biological membranes and primarily is protein catalyzed. Lipid flippases mediate the net translocation of specific phospholipids from one leaflet of a membrane to the other. In the inner (cytoplasmic) membrane (IM) of Gram-negative bacteria, lysophospholipid translocase (LplT) and cytosolic bifunctional acyl-acyl carrier protein (ACP) synthetase/2-acylglycerolphosphoethanolamine acyltransferase (Aas) form a glycerophospholipid regeneration system, which is capable of facilitating rapid retrograde translocation of lyso forms of phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL) but not exogenous (host-derived) phosphatidylcholine (PC) across the IM of Gram-negative diderm (two-membraned) bacteria in consequential order lyso-PE = lyso-PG > > lysophosphatidic acid (lyso-PA) >> lyso-PC. Although several flippases that bind and move non-glycerophosphatidyl lipids across the IM are characterized in Gram-negative bacteria, LplT appears to be the first example of a bacterial protein capable of facilitating the rapid translocation of monoacylated glycerophospholipids. On the cytoplasmic surface, Aas restores the lysophospholipids to their diacyl forms with comparable efficiency but excludes any exogenous monoacylated lipid species. This coupled remodeling enzyme tandem provides an effective means to examine substrate specificity of lipid regeneration and lysophospholipid transport per se across the membrane. The current chapter describes two distinct but complementary methods for the measurement of lysophospholipid transport across membranes using Escherichia coli spheroplasts., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Fission yeast can be used as a cell-based system for high-throughput drug screening. However, higher drug concentrations are often needed to achieve the same effect as in mammalian cells. Our goal here was to improve drug sensitivity so reduced drugs could be used. Three different methods affecting drug uptakes were tested using an FDA-approved HIV-1 protease inhibitor (PI) drug Darunavir (DRV). First, we tested whether spheroplasts without cell walls increase the drug sensitivity. Second, we examined whether electroporation could be used. Although small improvements were observed, neither of these two methods showed significant increase in the EC50 values of DRV compared with the traditional method. In contrast, when DRV was tested in a mutant strain PR836 that lacks key proteins regulating cellular efflux, a significant increase in the EC50 was observed. A comparison of nine FDA-approved HIV-1 PI drugs between the wild-type RE294 strain and the mutant PR836 strain showed marked enhancement of the drug sensitivities ranging from an increase of 0.56 log to 2.48 logs. Therefore, restricting cellular efflux through the adaption of the described fission yeast mutant strain enhances the drug sensitivity, reduces the amount of drug used, and increases the chance of success in future drug discovery. [ABSTRACT FROM AUTHOR]

Studies attempting to characterize the membrane translocation of antimicrobial and cell-penetrating peptides are frequently limited by the resolution of conventional light microscopy. This study shows that spheroplasts provide a valuable approach to overcome these limits. Spheroplasts produce less ambiguous images and allow for more systematic analyses of localization. Data collected with spheroplasts are consistent with studies using normal bacterial cells and imply that a particular peptide may not always follow the same mechanism of action.

Screening drug candidates for their affinity and selectivity for a certain binding site is a crucial step in developing targeted therapy. Here, we created a screening assay for receptor binding that can be easily scaled up and automated for the high throughput screening of Kv channel blockers. It is based on the expression of the KcsA-Kv1 hybrid channel tagged with a fluorescent protein in the E. coli membrane. In order to make this channel accessible for the soluble compounds, E. coli were transformed into spheroplasts by disruption of the cellular peptidoglycan envelope. The assay was evaluated using a hybrid KcsA-Kv1.3 potassium channel tagged with a red fluorescent protein (TagRFP). The binding of Kv1.3 channel blockers was measured by flow cytometry either by using their fluorescent conjugates or by determining the ability of unconjugated compounds to displace fluorescently labeled blockers with a known affinity. A fraction of the occupied receptor was calculated with a dedicated pipeline available as a Jupyter notebook. Measured binding constants for agitoxin-2, charybdotoxin and kaliotoxin were in firm agreement with the earlier published data. By using a mid-range flow cytometer with manual sample handling, we measured and analyzed up to ten titration curves (eight data points each) in one day. Finally, we considered possibilities for multiplexing, scaling and automation of the assay. [ABSTRACT FROM AUTHOR]

Fission yeast can be used as a cell-based system for high-throughput drug screening. However, higher drug concentrations are often needed to achieve the same effect as in mammalian cells. Our goal here was to improve drug sensitivity so reduced drugs could be used. Three different methods affecting drug uptakes were tested using an FDA-approved HIV-1 protease inhibitor (PI) drug Darunavir (DRV). First, we tested whether spheroplasts without cell walls increase the drug sensitivity. Second, we examined whether electroporation could be used. Although small improvements were observed, neither of these two methods showed significant increase in the EC50 values of DRV compared with the traditional method. In contrast, when DRV was tested in a mutant strain PR836 that lacks key proteins regulating cellular efflux, a significant increase in the EC50 was observed. A comparison of nine FDA-approved HIV-1 PI drugs between the wild-type RE294 strain and the mutant PR836 strain showed marked enhancement of the drug sensitivities ranging from an increase of 0.56 log to 2.48 logs. Therefore, restricting cellular efflux through the adaption of the described fission yeast mutant strain enhances the drug sensitivity, reduces the amount of drug used, and increases the chance of success in future drug discovery.

Vibrio cholerae, the agent of the deadly human disease cholera, propagates as a curved rod-shaped bacterium in warm waters. It is sensitive to cold but persists in cold waters in the form of viable but nondividing coccoidal-shaped cells. Additionally, V. cholerae is able to form nonproliferating spherical cells in response to cell wall damage. It was recently reported that L-arabinose, a component of the hemicellulose and pectin of terrestrial plants, stops the growth of V. cholerae. Here, we show that L-arabinose induces the formation of spheroplasts that lose the ability to divide and stop growing in volume over time. However, they remain viable, and upon removal of L-arabinose, they start expanding in volume, form branched structures, and give rise to cells with a normal morphology after a few divisions. We further show that WigKR, a histidine kinase/response regulator pair implicated in the induction of high-level expression of cell wall synthetic genes, prevents the lysis of the spheroplasts during growth restart. Finally, we show that the physiological perturbations result from the import and catabolic processing of L-arabinose by the V. cholerae homolog of the Escherichia coli galactose transport and catabolic system. Taken together, our results suggest that the formation of nongrowing spherical cells is a common response of vibrios exposed to detrimental conditions. They also permit us to define conditions preventing any physiological perturbation of V. cholerae when using L-arabinose to induce gene expression from the tightly regulated promoter of the Escherichia coli araBAD operon. [ABSTRACT FROM AUTHOR]

Vacuole generation occurs frequently during the enlargement of bacterial protoplasts and spheroplasts. Gram-positive Enterococcus faecalis protoplasts and gram-negative Lelliottia amnigena spheroplasts had large and small vacuoles inside the cytoplasm, respectively. Although no vacuoles were found at the early stage of cell enlargement, all enlarged cells used in the microinjection procedures had vacuoles. The plasma membrane of L. amnigena was more flexible than that of E. faecalis. In addition, E. faecalis protoplasts had unique discoidal structures as well as spherical structures in the cytoplasm. Our findings showed that the number of vacuoles increased as the L. amnigena plasma membrane expanded and that the size of vacuoles increased as the E. faecalis plasma membrane expanded, suggesting that bacterial cell enlargement involved vacuole generation. Thus, biosynthesis of the plasma and vacuolar membranes was synchronous with the bacterial cell enlargement. Differences in the plasma membrane flexibility might influence the different types of vacuole generation. [ABSTRACT FROM AUTHOR]

Screening drug candidates for their affinity and selectivity for a certain binding site is a crucial step in developing targeted therapy. Here, we created a screening assay for receptor binding that can be easily scaled up and automated for the high throughput screening of Kv channel blockers. It is based on the expression of the KcsA-Kv1 hybrid channel tagged with a fluorescent protein in the E. coli membrane. In order to make this channel accessible for the soluble compounds, E. coli were transformed into spheroplasts by disruption of the cellular peptidoglycan envelope. The assay was evaluated using a hybrid KcsA-Kv1.3 potassium channel tagged with a red fluorescent protein (TagRFP). The binding of Kv1.3 channel blockers was measured by flow cytometry either by using their fluorescent conjugates or by determining the ability of unconjugated compounds to displace fluorescently labeled blockers with a known affinity. A fraction of the occupied receptor was calculated with a dedicated pipeline available as a Jupyter notebook. Measured binding constants for agitoxin-2, charybdotoxin and kaliotoxin were in firm agreement with the earlier published data. By using a mid-range flow cytometer with manual sample handling, we measured and analyzed up to ten titration curves (eight data points each) in one day. Finally, we considered possibilities for multiplexing, scaling and automation of the assay.

Killer strains of S. cerevisiae harbor double-stranded RNA viruses and secrete protein toxins that kill virus-free cells. The K1 killer toxin acts on sensitive yeast cells to perturb potassium homeostasis and cause cell death. Here, the toxin is shown to activate the plasma membrane potassium channel of S. cerevisiae, TOK1. Genetic deletion of TOK1 confers toxin resistance; overexpression increases susceptibility. Cells expressing TOK1 exhibit toxin-induced potassium flux; those without the gene do not. K1 toxin acts in the absence of other viral or yeast products: toxin synthesized from a cDNA increases open probability of single TOK1 channels (via reversible destabilization of closed states) whether channels are studied in yeast cells or X. laevis oocytes.

Physiological conditions in living cells are strictly regulated to allow, optimize, and coordinate biological processes. The bacterial cell envelope is the compartment where the communication with the external environment takes place. This involves membrane proteins, key players in many biological processes that ensure bacterial survival. The biochemical characterization of membrane proteins, either integral, lipidated or peripheral is challenging due to their mixed protein‐lipid nature, making it difficult to purify and obtain considerable amounts of samples. In contrast to integral membrane proteins, lipidated proteins are usually purified as truncated soluble versions, neglecting the impact of the membrane environment. Here we report a simple and robust protocol to characterize bacterial lipidated proteins in spheroplasts from Escherichia coli using a β‐lactamase as a model. The Metallo‐β‐lactamase NDM‐1 is an enzyme anchored to the inner leaflet of the outer membrane of Gram‐negative bacteria. Kinetic parameters and stability of the lipidated NDM‐1 and the soluble unbound version (NDM‐1 C26A) were measured in spheroplasts and periplasm, respectively. These studies revealed that membrane anchoring increases the KM of the enzyme, consequently decreasing the catalytic efficiency, while not affecting its kinetic stability. This approach can be used to characterize lipidated proteins avoiding the purification step while mimicking its native environment. This approach also helps in filling the gap between in vitro and in vivo studies. [ABSTRACT FROM AUTHOR]

Introduction: It has already been reported that subinhibitory concentrations of β-lactam antibiotics can cause abnormal changes of bacterial forms, such as spheroplasts. Herein we report a case of Croatian male patient with Escherichia coli spheroplasts present in urine after treatment with tazobactam, on the tenth day of hospitalization. The aim of this report is to emphasize the inability of imaging based automated urine analysers to recognize some relatively uncommon forms of bacterial presentation in urine sediment. Materials and methods: During routine urine analysis, unusual particles were observed in patient urine. Urine sediment was examined by two urine analysers: Atellica 1500 (Siemens, Germany) and Iris iQ200 (Beckman Coulter, USA). Additionally, urine was sent for culture testing to Microbiology department. Results: Both urine analysers didn't indicate presence of bacteria in urine sediment. Unusual particles observed on the tenth day were classified as erythrocytes by both instruments. Dipstick test showed blood trace and microscopic analysis revealed bacteria in urine. Urine culture was positive for Escherichia coli. Careful examination of urine sediment has confirmed that shapes present in urine were abnormal bacterial forms called spheroplasts. Conclusions: Imaging based automated urine analysers are not able to recognize bacterial spheroplasts in urine sediment misclassifying it as erythrocytes. Microscopic examination remains the gold standard for urines with blood trace or negative blood, in which erythrocytes are reported by urine analyser in urine sediment. Failure to identify and follow up such cases may lead to inaccurate treatment decisions and puts patient safety at risk. [ABSTRACT FROM AUTHOR]

In two patients under treatment with various antibiotics, spheroplasts were detected with an automated urine sediment analyzer. Urinalysis was performed by an AutionMAX AX 4030-sediMAX platform. Spheroplasts can be easily misclassified as yeasts or erythrocytes, but when automated urine sediment analyzers are used by well-trained, and experienced operators they can be correctly identified and classified. Appropriate training of urine laboratory professionals in spheroplast detection and association with UTI, together with timely communication with the microbiologist and caring clinician, will provide prompt targeted treatment. [ABSTRACT FROM AUTHOR]

Mitochondria import hundreds of different precursor proteins from the cytosol and direct each of these to its specific mitochondrial subcompartment. The import routes and mechanisms by which precursors are transported into the outer membrane, the intermembrane space (IMS), the inner membrane and the matrix have been characterized in depth by use of very powerful in vitro assays. In the 'classical' import assays, radiolabeled precursor proteins are incubated with isolated mitochondria and the protein uptake is monitored by one or more of the following observations: intramitochondrial processing, resistance to externally added proteases, or the formation of disulfide bonds. In this chapter, we describe an alternative import assay which employs semi-intact yeast cells. This assay uses spheroplasts from which the cell wall had been removed by enzymatic digestion before the plasma membrane was partially permeabilized by a freeze-thawing step. Since the organellar architecture is largely maintained in semi-intact cells, this in vitro import assay allows to elucidate the targeting of precursor proteins from the cytoplasm to the mitochondrial surface. Thereby the contribution of other compartments such as the endoplasmic reticulum (ER) can be assessed. Here we describe how semi-intact cells are prepared and used in the in vitro import assay and discuss the pros and cons of this approach., (Copyright © 2024. Published by Elsevier Inc.)

Ion channels are tightly involved in various aspects of cell physiology, including cell signaling, proliferation, motility, endo- and exo-cytosis. They may be involved in toxin production and release by marine dinoflagellates, as well as harmful algal bloom proliferation. So far, the patch-clamp technique, which is the most powerful method to study the activity of ion channels, has not been applied to dinoflagellate cells, due to their complex cellulose-containing cell coverings. In this paper, we describe a new approach to overcome this problem, based on the preparation of spheroplasts from armored bloom-forming dinoflagellate Prorocentrum minimum. We treated the cells of P. minimum with a cellulose synthesis inhibitor, 2,6-dichlorobenzonitrile (DCB), and found out that it could also induce ecdysis and arrest cell shape maintenance in these microalgae. Treatment with 100–250 µM DCB led to an acceptable 10% yield of P. minimum spheroplasts and was independent of the incubation time in the range of 1–5 days. We show that such spheroplasts are suitable for patch-clamping in the cell-attached mode and can form 1–10 GOhm patch contact with a glass micropipette, allowing recording of ion channel activity. The first single-channel recordings of dinoflagellate ion channels are presented.

Antimicrobial peptides (AMPs) represent new alternatives to cope with the increasing number of multi-drug resistant microbial infections. Recently, a derivative of the frog-skin AMP esculentin-1a, Esc(1-21), was found to rapidly kill both the planktonic and biofilm forms of the Gram-negative bacterium Pseudomonas aeruginosa with a membrane-perturbing activity as a plausible mode of action. Lately, its diastereomer Esc(1-21)-1c containing two d -amino acids i.e. D Leu14 and D Ser17 revealed to be less cytotoxic, more stable to proteolytic degradation and more efficient in eradicating Pseudomonas biofilm. When tested in vitro against the free-living form of this pathogen, it displayed potent bactericidal activity, but this was weaker than that of the all- l peptide. To investigate the reason accounting for this difference, mechanistic studies were performed on Pseudomonas spheroplasts and anionic or zwitterionic membranes, mimicking the composition of microbial and mammalian membranes, respectively. Furthermore, structural studies by means of optical and nuclear magnetic resonance spectroscopies were carried out. Our results suggest that the different extent in the bactericidal activity between the two isomers is principally due to differences in their interaction with the bacterial cell wall components. Indeed, the lower ability in binding and perturbing anionic phospholipid bilayers for Esc(1-21)-1c contributes only in a small part to this difference, while the final effect of membrane thinning once the peptide is inserted into the membrane is identical to that provoked by Esc(1-21). In addition, the presence of two d -amino acids is sufficient to reduce the α-helical content of the peptide, in parallel with its lower cytotoxicity. [ABSTRACT FROM AUTHOR]

A feeding experiment was conducted in a closed recirculating system to evaluate the effects of freeze-dried spheroplasts prepared from Pyropia yezoensis (Ueda) on feed intake, growth and biochemical composition of sea cucumber, Apostichopus japonicus (Selenka). Pyropea spheroplasts ( PS) were prepared through enzymatic treatment to break down the complex mixture of polysaccharides cell walls that might be easier for growth energy partitioning. Sea cucumbers were fed-formulated diets with 10 (Diet 1), 30 (Diet 2) and 50 g/kg (Diet 3) inclusion level of PS. A diet without PS was used as a control (Diet 4). The experiment was conducted for 6 weeks maintaining water temperature 15 ± 1°C, photoperiod 18:06 hours (D:L). Feed was supplied ad-libitum at 16.00 h once in a day, and the remaining feed and faeces were removed in the next day. Results showed that the highest growth was observed in the 50 g/kg PS diet compared to other treatments. Total weight gain, mean weight gain, net yield, protein efficiency ratio ( PER) and protein gain (%) were significantly higher in the 50 g/kg PS diet ( p < .05 ). A significantly higher percentage of energy was allocated for growth in the 50 g/kg PS diet. The highest specific growth rate and feed conversion efficiency ( p < .05) were observed in the higher percentage of PS diet. Both the growth performance and biochemical analysis showed that superior growth was observed with increasing levels of PS in the diet. We infer that PS can be used as a new, cheaper feed ingredient in the formulated diet of A. japonicus. [ABSTRACT FROM AUTHOR]

The Schizosaccharomyces pombe shu1+ gene encodes a cellsurface protein required for assimilation of exogenous heme. In this study, shaving experiments showed that Shu1 is released from membrane preparations when spheroplast lysates are incubated with phosphoinositide-specific phospholipase C (PIPLC). Shu1 cleavability by PI-PLC and its predicted hydropathy profile strongly suggested that Shu1 is a glycosylphosphatidylinositol-anchored protein. When heme biosynthesis is selectively blocked in hem1Δ mutant cells, the heme analog zinc mesoporphyrin IX (ZnMP) first accumulates into vacuoles and then subsequently, within the cytoplasm in a rapid and Shu1-dependent manner. An HA4-tagged shu1+ allele that retained wild-type function localizes to the cell surface in response to low hemin concentrations, but under high hemin concentrations, Shu1-HA4 re-localizes to the vacuolar membrane. Inactivation of abc3+, encoding a vacuolar membrane transporter, results in hem1Δ abc3Δmutant cells being unable to grow in the presence of hemin as the sole iron source. In hem1Δ abc3Δ cells, ZnMP accumulates primarily in vacuoles and does not sequentially accumulate in the cytosol. Consistent with a role for Abc3 as vacuolar hemin exporter, results with hemin-agarose pulldown assays showed that Abc3 binds to hemin. In contrast, an Abc3 mutant in which an inverted Cys-Pro motif had been replaced with Ala residues fails to bind hemin with high affinity. Taken together, these results show that Shu1 undergoes rapid hemininduced internalization from the cell surface to the vacuolar membrane and that the transporter Abc3 participates in the mobilization of stored heme from the vacuole to the cytosol. [ABSTRACT FROM AUTHOR]

Background: The development of protective vaccines for Brucella spp. has been hampered by the difficulty in transformation of Brucella cells with foreign DNA for genetic manipulation. It seems that the formation of Brucella spheroplasts would increase the efficiency of transformation. The aim of this study was to devise an efficient method for the transformation of Brucella spp. Materials and Methods: At first, spheroplast of Brucella was prepared by glycine and ampicillin induction and transformed using optimized protocols of CaCl2, electroporation, and lipofection methods. Then, the efficacy of transformation was compared between the three-mentioned methods. Results: Ampicillin-induced spheroplasts from early-log phase culture of brucella when incubated in a medium-containing 0.2 M sucrose during cell recovery had higher transformation efficiency in three different methods. Comparison of the transformation efficiency of Brucella abortus RB51 using the CaCl2, lipofection, and electroporation methods revealed that the transformation efficiency with the lipofection method was significantly higher than with other two methods (P < 0.05). Conclusions: Lipofection method by lipofectamine 2000 on ampicillin-induced spheroplasts can be a suitable approach for Brucella transformation.

The formation of spherical aggregates during the growth of cell population has long been observed under various conditions. We observed the formation of such aggregates during proliferation of Huh-7.5 cells, a human hepatocarcinoma cell line, in a microfabricated low-adhesion microwell system (SpheroFilm; formed of mass-producible silicone elastomer) on the length scales up to 500 μm. The cell proliferation was also tracked with immunofluorescence staining of F-actin and cell proliferation marker Ki-67. Meanwhile, our complementary 3D Monte Carlo simulations, taking cell diffusion and division, cell-cell and cell-scaffold adhesion, and gravity into account, illustrate the role of these factors in the formation of spheroids. Taken together, our experimental and simulation results provide an integrative view of the process of spheroid formation for Huh-7.5 cells. [ABSTRACT FROM AUTHOR]

Recombination of antibody light and heavy chain libraries greatly increases the size of a two-chain paired antibody library, thus easing the construction of large antibody libraries. Here, light and heavy chain variable domains paired by a coiled coil were applied to a bacterial inner membrane display system. However, the probability of the correct pairing of light and heavy chains through random recombination after each round of flow cytometric sorting and cloning was very low in the presence of mostly unmatched light and heavy chain genes, resulting in inefficient enrichment; a target antibody clone in the ratio of 1:100,000 negative control spheroplasts was unable to be enriched by six rounds of sorting and cloning by a conventional sorting strategy (sorting the top 1 %). By just sorting the top 0.000025 % of spheroplasts, we succeeded in enriching the target antibody clone mixed with negative control spheroplasts in a ratio of 1:10 by just one round of sorting and cloning. Furthermore, using this gating strategy, we efficiently enriched for an antibody clone with an affinity slightly better than the parent antibody clone from mixed spheroplasts which were present in the ratio of 1 better affinity clone to 10 parent clones to 10 negative control clones after just two rounds of sorting and cloning, suggesting that this gating strategy is highly sensitive in distinguishing between clones with a small difference in affinity and also enriching for clones with a higher affinity. Taken together, the combination of the display of a two-chain paired antibody library and the use of stringent gating has significantly increased the efficiency of the antibody maturation system. [ABSTRACT FROM AUTHOR]

After losing their protective peptidoglycan, bacterial spheroplasts can resynthesize a cell wall to recreate their normal shape. In Escherichia coli, this process requires the Rcs response. In its absence, spheroplasts do not revert to rod shapes but instead form enlarged spheroids and lyse. Here, we investigated the reason for this Rcs requirement. Rcs-deficient spheroids exhibited breaks and bulges in their periplasmic spaces and failed to synthesize a complete peptidoglycan cell wall, indicating that the bacterial envelope was defective. To determine the Rcs-dependent gene(s) required for shape recovery, we tested spheroplasts lacking selected RcsB-regulated genes and found that colanic acid (CA) biosynthesis appeared to be involved. Surprisingly, though, extracellular CA was not required for recovery. Instead, lysis was caused by mutations that interrupted CA biosynthesis downstream of the initial glycosyl transferase, WcaJ. Deleting wcaJ prevented lysis of spheroplasts lacking ensuing steps in the pathway, and providing WcaJ in trans to a mutant lacking the entire CA operon triggered spheroplast enlargement and lysis. Thus, CA is not required for spheroplast recovery. Instead, CA intermediates accumulate as dead-end products which inhibit recovery of wall-less cells. The results strongly imply that CA may not be required for the survival E. coli L-forms. More broadly, these findings mandate that previous conclusions about the role of colanic acid in biofilm formation or virulence must be reevaluated. [ABSTRACT FROM AUTHOR]

This essay details a historical crossroad in biochemistry and microbiology in which penicillin was a co-agent. I narrate the trajectory of the bacterial cell wall as the precise target for antibiotic action. As a strategic object of research, the bacterial cell wall remained at the core of experimental practices, scientific narratives and research funding appeals throughout the antibiotic era. The research laboratory was dedicated to the search for new antibiotics while remaining the site at which the mode of action of this new substance was investigated. This combination of circumstances made the bacterial wall an ontology in transit. As invisible as the bacterial wall was for clinical purposes, in the biological laboratory, cellular meaning in regard to the action of penicillin made the bacterial wall visible within both microbiology and biochemistry. As a border to be crossed, some components of the bacterial cell wall and the biochemical destruction produced by penicillin became known during the 1950s and 1960s. The cell wall was constructed piece by piece in a transatlantic circulation of methods, names, and images of the shape of the wall itself. From 1955 onwards, microbiologists and biochemists mobilized new names and associated conceptual meanings. The composition of this thin and rigid layer would account for its shape, growth and destruction. This paper presents a history of biochemical morphology: a chemistry of shape - the shape of bacteria, as provided by its wall - that accounted for biology, for life itself. While penicillin was being established as an industrially-manufactured object, it remained a scientific tool within the research laboratory, contributing to the circulation of further scientific objects. [ABSTRACT FROM AUTHOR]

Lysophospholipid transporter (LplT) was previously found to be primarily involved in 2-acyl lysophosphatidylethanolamine (lyso-PE) recycling in Gram-negative bacteria. This work identifies the potent role of LplT in maintaining membrane stability and integrity in the Escherichia coli envelope. Here we demonstrate the involvement of LplT in the recycling of three major bacterial phospholipids using a combination of an in vitro lysophospholipid binding assay using purified protein and transport assays with E. coli spheroplasts. Our results show that lyso-PE and lysophosphatidylglycerol, but not lysophosphatidylcholine, are taken up by LplT for reacylation by acyltransferase/acyl-acyl carrier protein synthetase on the inner leaflet of the membrane. We also found a novel cardiolipin hydrolysis reaction by phospholipase A2 to form diacylated cardiolipin progressing to the completely deacylated headgroup. These two distinct cardiolipin derivatives were both translocated with comparable efficiency to generate triacylated cardiolipin by acyltransferase/acyl-acyl carrier protein synthetase, demonstrating the first evidence of cardiolipin remodeling in bacteria. These findings support that a fatty acid chain is not required for LplT transport. We found that LplT cannot transport lysophosphatidic acid, and its substrate binding was not inhibited by either orthophosphate or glycerol 3-phosphate, indicating that either a glycerol or ethanolamine headgroup is the chemical determinant for substrate recognition. Diacyl forms of PE, phosphatidylglycerol, or the tetra-acylated form of cardiolipin could not serve as a competitive inhibitor in vitro. Based on an evolutionary structural model, we propose a "sideways sliding" mechanism to explain how a conserved membrane-embedded α-helical interface excludes diacylphospholipids from the LplT binding site to facilitate efficient flipping of lysophospholipid across the cell membrane. [ABSTRACT FROM AUTHOR]

The mechanosensitive (MS) channel MscCG from the soil bacterium Corynebacterium glutamicum functions as a major glutamate exporter. MscCG belongs to a subfamily of the bacterial MscS-like channels, which play an important role in osmoregulation. To understand the structural and functional features of MscCG, we investigated the role of the carboxyl-terminal domain, whose relevance for the channel gating has been unknown. The chimeric channel MscS-(C-MscCG), which is a fusion protein between the carboxyl terminal domain of MscCG and the MscS channel, was examined by the patch clamp technique. We found that the chimeric channel exhibited MS channel activity in Escherichia coli spheroplasts characterized by a lower activation threshold and slow closing compared to MscS. The chimeric channel MscS-(C-MscCG) was successfully reconstituted into azolectin liposomes and exhibited gating hysteresis in a voltage-dependent manner, especially at high pipette voltages. Moreover, the channel remained open after releasing pipette pressure at membrane potentials physiologically relevant for C. glutamicum . This contribution to the gating hysteresis of the C-terminal domain of MscCG confers to the channel gating properties highly suitable for release of intracellular solutes. [ABSTRACT FROM AUTHOR]

Black leaf streak disease, also known as black Sigatoka, causes dramatic losses in production of banana and plantains fruits. The disease is caused by the pathogenic fungus Mycosphaerella fijiensis (anamorph Pseudocercospora fijiensis ; Mycosphaerellaceae). Genetic transformation of M. fijiensis would allow a better understanding of molecular basis of pathogenicity and design novel approaches to control the infection caused by this pathogen. However, transformation of this fungus has not been easy. We report here a protocol for genetic transformation of M. fijiensis employing underwater shock waves and intact conidia. The recombinant strains recovered showed genetic stability over > 10 generations. The frequency of transformation obtained was between 75 and 150 times higher than the efficiency reported in the only article published on transformation of M. fijiensis using spheroplasts. This improvement allowed the use of a thousand times less cells than the amount employed before, avoiding the need for cumbersome successive batch cultures. Our protocol is simple, highly efficient, fast and reproducible and together with the available genomes of M. fijiensis and Musa acuminata , it offers new possibilities to study the diverse mechanisms of pathogenesis of the fungus. [ABSTRACT FROM AUTHOR]

An investigation was made and a description given of the rheological properties of spheroplastics based on hollow glass microspheres and epoxy oligomers in generalised parameters of their particulate-filled structure. It is shown that the main generalised parameters of the particulate-filled structure, determining the viscosity and fluidity limit, are the parameters aav and Θ, which depend on the packing of particles and their content in the composite. Dependences of viscosity and fluidity limit of particulate-filled polymer composites in generalised parameter coordinates are given for the first time, and the spheroplastics are classified according to the structural principle. [ABSTRACT FROM AUTHOR]

Background: Antibiotics of the β-lactam group are able to alter the shape of the bacterial cell wall, e.g. filamentation or a spheroplast formation. Early determination of antimicrobial susceptibility may be complicated by filamentation of bacteria as this can be falsely interpreted as growth in systems relying on colorimetry or turbidometry (such as Vitek-2, Phoenix, MicroScan WalkAway). The objective was to examine an automated image analysis algorithm for quantification of filamentous bacteria using the 3D digital microscopy imaging system, oCelloScope. Results: Three E. coli strains displaying different resistant profiles and differences in filamentation kinetics were used to study a novel image analysis algorithm to quantify length of bacteria and bacterial filamentation. A total of 12 β-lactam antibiotics or β-lactam–β-lactamase inhibitor combinations were analyzed for their ability to induce filamentation. Filamentation peaked at approximately 120 min with an average cell length of 30 μm. Conclusion: The automated image analysis algorithm showed a clear ability to rapidly detect and quantify β-lactam-induced filamentation in E. coli. This rapid determination of β-lactam-mediated morphological alterations may facilitate future development of fast and accurate AST systems, which in turn will enable early targeted antimicrobial therapy. Therefore, rapid detection of β-lactam-mediated morphological changes may have important clinical implications. [ABSTRACT FROM AUTHOR]

Two meerkats (Suricata suricatta) housed in the same zoological garden in Japan died due to Yersinia pseudotuberculosis serotype 4b infection. Gross and microscopic lesions included necrotizing enteritis and enlargement of the spleen and liver with multifocal necrosis. Inflammatory cells, primarily neutrophils, and nuclear debris were associated with clusters of Gram-negative bacilli. Additionally, there were aberrant organism forms that were larger than bacilli and appeared as basophilic globular bodies. Immunohistochemical examination showed that the bacilli and globular bodies were strongly positive for Y. pseudotuberculosis O4 antigen. The globular bodies were considered a shape-changed form of Y. pseudotuberculosis, and these morphologically abnormal bacteria could present a diagnostic challenge. [ABSTRACT FROM AUTHOR]

Various strategies adapted to develop an efficient vaccine against foodborne pathogen, Listeria monocytogenes, have met with little success. Spheroplasts (bacterial cell devoid of cell wall) are likely to undergo membrane-membrane fusion, leading to the delivery of their content to the cytosol of antigen-presenting cells, thus facilitating MHC class I antigen processing and presentation. In this study, we evaluated the prophylactic potential of Listeria spheroplast-based vaccine against experimental murine listeriosis in comparison with heat-killed Listeria ( HKL) and archaeosome-entrapped Listeria whole-cell protein ( LWCP). Compared with HKL, the spheroplast-based vaccine was found to evoke better Th1 response as exhibited by the presence of type 1 cytokines in the host (interferon- γ and IL-12) and a high IgG2a/IgG1 ratio. Robust lympho-proliferative efficacy was apparent in both spheroplast-immunized and archaeosome-entrapped LWCP-immunized groups. The upregulation of costimulatory and effector memory markers upon immunization with spheroplasts was found to be at par with that evoked by archaeosome-entrapped LWCP-immunized group. Central memory response in gated CD8+ T cell was much higher in spheroplast-immunized animals when compared with archaeosome-entrapped LWCP group. The data presented here clearly demonstrate that spheroplasts evoked a robust immune response and offer better prophylactic potential against L. monocytogenes. [ABSTRACT FROM AUTHOR]

The article discusses a study related to integration and expression of heterologous genes in the oleaginous yeast, Lipomyces starkeyi. Topics discussed include development of a system for targeted recombinant DNA integration of multiple copies of a gene into Lipomyces starkeyi genome using spheroplast-polyethylene glycol transformation, importance of Lipomyces starkeyi as an excellent lipid producer and need for the establishment of methods for molecular breeding for Lipomyces starkeyi.

This report describes atypical cases of yersiniosis in squirrel monkeys in which aberrant forms of Yersinia pseudotuberculosis were seen. There were 2 outbreaks due to yersiniosis in squirrel monkeys in Japan. The monkeys had systemic necrotizing and hemorrhagic lesions with Gram-negative rod-shaped bacilli and microthromboembolism in the kidneys. Some lesions contained filaments, globular bodies, and other pleomorphic forms of bacteria. All forms were usually seen in the same lesions, and those with pleomorphic morphology appeared to be an intermediate form between the rod-shaped bacteria and the filaments or globular bodies. In addition, they had strong immunolabeling for Y. pseudotuberculosis, as did the rod-shaped bacteria. Therefore, the globular bodies, filaments, and others are strongly suspected to be shape-changed bacilli of Y. pseudotuberculosis. These morphologically altered bacteria could cause errors in diagnosis since they resemble fungi or protozoa, and special staining techniques, including immunohistochemistry, can be helpful in establishing the correct diagnosis. [ABSTRACT FROM PUBLISHER]

Using conventional approaches, the antigen-binding affinity of a novel format of bi-specific antibody (BsAb) cannot be determined until purified BsAb is obtained. Here, we show that new lipoprotein A (NlpA)-based bacteria display technology, combined with flow cytometry (FCM), can be used to detect antigen-binding affinity of BsAbs, in the absence of expression and purification work. Two formats of BsAb, scFv2-CH/CL and Diabody-CH/CL, specific for human interleukin 1β (hIL-1β) and human interleukin 17A (hIL-17A), were constructed and displayed in Escherichia coli using NlpA-based bacteria display technology. Conversion of these cells to spheroplasts, and their incubation with fluorescently conjugated antigens resulted in the selective labeling of spheroplasts expressing BsAb; enabling their antigen-binding affinity to be analyzed with FCM. The association and dissociation of BsAbs for binding to hIL-1β and hIL-17A were analyzed using FCM-based assays. The results showed that antigen-binding affinity of Diabody-CH/CL was significantly higher than that of scFv2-CH/CL. To confirm these results of FCM-based assays, BsAbs were expressed, purified and subjected to relative affinity measurements, in vitro and in vivo bioactivity analysis. The results showed that Diabody-CH/CL had greater relative affinities for both antigens, resulting in better blocking bioactivities on cellular level and effects on alleviating joint inflammation, and cartilage destruction and bone damage in collagen induced arthritis (CIA) mice model. These results indicate that BsAbs with good antigen-binding affinity can be identified by FCM-based assays without expression and purification work, and the indentified BsAb can serve as a lead compound for further drug development. [ABSTRACT FROM AUTHOR]

Gluconobacter oxydans ATCC 11894 produces dextran dextrinase (DDase, EC 2.4.1.2), which synthesizes dextran from the starch hydrolysate, dextrin and is known to cause ropy beer. G. oxydans ATCC 11894 was believed to possess both a secreted DDase (DD ext ) and an intracellular DDase (DD int ), expressed upon cultivation with dextrin and glucose, respectively. However, genomic Southern blot, peptide mass fingerprinting and reaction product-pattern analyses revealed that both DD ext and DD int were identical. The activity in the cell suspension and its liberation from the spheroplast cells indicated that DD int was localized on the cell surface. The localization of DDase was altered during the culture depending on the growth phase. During the early growth stage, DDase was exclusively liberated into the medium (DD ext ), and the cell-associated form (DD int ) appeared after depletion of glucose from the medium. [ABSTRACT FROM AUTHOR]