Correspondence to: Dennis H. Bamford, Viikki Biocenter, P.O. Box 56 (Viikinkaari 5), FIN-00014, University of Helsinki, Finland. Tel:358-9-191 59100 Fax:358-9-191 59098 E-mail:gen_phag{at}cc.helsinki.fi.
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Abstract |
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Studies on the viruscell interactions have proven valuable in elucidating vital cellular processes. Interestingly, certain virushost membrane interactions found in eukaryotic systems seem also to operate in prokaryotes (Bamford, D.H., M. Romantschuk, and P.J. Somerharju, 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:14671473; Romantschuk, M., V.M. Olkkonen, and D.H. Bamford. 1988. EMBO (Eur. Mol. Biol. Organ.) J. 7:18211829). 6 is an enveloped double-stranded RNA virus infecting a gram-negative bacterium. The viral entry is initiated by fusion between the virus membrane and host outer membrane, followed by delivery of the viral nucleocapsid (RNA polymerase complex covered with a protein shell) into the host cytosol via an endocytic-like route. In this study, we analyze the interaction of the nucleocapsid with the host plasma membrane and demonstrate a novel approach for dissecting the early events of the nucleocapsid entry process. The initial binding of the nucleocapsid to the plasma membrane is independent of membrane voltage (
) and the K+ and H+ gradients. However, the following internalization is dependent on plasma membrane voltage (
), but does not require a high ATP level or K+ and H+ gradients. Moreover, the nucleocapsid shell protein, P8, is the viral component mediating the membranenucleocapsid interaction.
Key Words: endocytosis, cell energetics, dsRNA virus entry, prokaryote, phi6
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Introduction |
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THE delivery of the viral nucleic acid into the host cell is a complex series of tightly regulated events, where the entering virus utilizes the host cell's machineries to reach its goal. Depending on the host cell and the virus type, different mechanisms have evolved. Classical bacterial virus work demonstrated that the viral DNA is delivered through the host cell envelope leaving the virus capsid outside, whereas animal viruses, in most cases, seem to internalize the viral particle or its subassemblies. Enveloped animal viruses gain access to the host cytosol using membrane fusion. The fusion reaction occurs either at the plasma membrane (PM),1 as exemplified by members of the families Paramyxoviridae and Retroviridae, or intracellularly upon endocytic uptake, as with viruses of the families Togaviridae and Orthomyxoviridae. Nonenveloped animal viruses, such as members of the families Picornaviridae and Adenoviridae, also mostly rely on endocytosis (
The best understood pathway for animal virus internalization occurs via receptor-mediated endocytosis involving coated vesicles and endosomes (
The endocytic uptake, via coated vesicle, relays on specific proteins that are activated upon ATP or GTP binding or hydrolysis. Both the recycling of the clathrin coat and the dynamin-directed pinching of the coated vesicle from the PM are ATP/GTP-dependent processes (p), rather than acidic milieu per se, drives this process.
Pseudomonas syringae enveloped bacteriophage 6 is a unique virus in the prokaryotic world. It has a segmented double-stranded (ds) RNA genome (
6, like other dsRNA viruses, has to deliver not only its genome, but also the viral polymerase particle (nucleocapsid core) into the cell to carry out the viral replication cycle. The polymerase complex is the innermost layer in the virion (see Figure 1, top) and it is composed of four protein species: a particle forming protein (P1) (
6 a model system for dsRNA virus genome packaging, replication, assembly, and maturation (
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The 6 host is a gram-negative bacterium. The viral lipid envelope, protein P8 shell, and the lytic enzyme P5 are required to deliver the viral core across two membranes and a peptidoglycan layer between them. The current view of the entry mechanism of
6 is illustrated in Figure 1, bottom. If purified NCs are added to host cell spheroplasts (cells with a removed outer membrane [OM] and peptidoglycan layer), they enter the cell by direct interaction with the exposed PM leading to production of mature infectious virus particles (
The entry of 6 is dependent on the energetic state of the host cell (
p) across the membrane. Membrane voltage (
, the transmembrane difference of electrical potential) is the major component of
p, the other component being the pH gradient (
pH) (
p or its components (
The 6 entry mechanism is unique among prokaryotes, but resembles the penetration mechanisms used by both enveloped (fusion with the OM) and nonenveloped (PM penetration by NC) animal viruses (see Figure 1, bottom). Here we describe, using spheroplast infection, an entry system that allows us to study the NC interactions with the PM and to dissect the early events in NC entry into two stages. The nature of the NCPM interaction is addressed by using specific agents interfering with the host
,
pH, proton pumping, or ATP stability. Moreover, we analyze the role of proteins P1, P2, P4, P7, and P8 in the NC entry.
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Materials and Methods |
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Materials
Wild-type 6 and its host P. syringae pathovar phaseolicola HB10Y (
Monensin (MN), nigericin (NG), valinomycin, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), polymyxin B (PMB) sulfate, polymyxin B nonapeptide, benzoic acid, apyrase (AP), N-ethylmaleimide, butylated hydroxytoluene (BHT), and Triton X-114 were purchased from Sigma Chemical Co. KCN, N,N'-dicyclohexylcarbodiimide (DCCD), NaF, and tetraphenylphosphonium (TPP+) chloride were products of Fluka Chemie AG. NaN3 was obtained from Riedel-deHaën AG, Na2HAsO4 from Merck Biochemica, and gramicidin D (GD) from Serva.
NC Isolation
For NC production, bacteriophage 6 was grown on HB10Y and purified as described previously (
Preparation of Host Cell Spheroplasts and Spheroplast Infection
The host cell spheroplasts were prepared as previously described (
Determination of , ATP Content, and Ion Fluxes
Extracellular concentrations of H+, K+, and TPP+ ions were monitored at 23°C using ion selective electrodes as previously described ( values were calculated using the Nernst equation (
Drug Treatments and Entry Assay
The effects of monovalent salts, ionophores, and energy-depleting agents on NC infection were studied by measuring the formation of plaques using the entry assay and simultaneously assaying the effects of the drugs on and ATP content of the spheroplasts. Spheroplasts were preincubated with drugs for 2 or 6 min at 23°C, and samples were withdrawn for
or ATP measurements and for the NC infection assay. In the standard entry assay, a 50-min infection was followed by an additional 10-min or 7.5-min treatment with NC-specific or nonspecific antiserum, respectively.
Transmission Electron Microscopy
For morphological analysis, NC-infected spheroplasts were subjected either to chemical fixation (CF) or to rapid freezing (RF) and freeze substitution (FS). Spheroplasts were infected with a multiplicity of ~200. Infected spheroplasts (spheroplasts in 30 mM sodium phosphate, pH 6, 3% [wt/vol] lactose, 2% [wt/vol] BSA, 100 mM NaCl, and 50% LB broth) were conventionally fixed with 3% (wt/vol) glutaraldehyde (in 50 mM sodium phosphate, pH 6, 3% [wt/vol] lactose, 2% [wt/vol] BSA) for 15 min at 23°C, followed by OsO4 after fixation. Before cryoprocessing (RF and FS), spheroplasts were collected (centrifuge 5415, 8,000 rpm, for 2 min, +4°C; Eppendorf-Netheler-Hinz GmbH) and resuspended in 20 mM potassium phosphate, pH 7.4, 3% (wt/vol) lactose, 8% (wt/vol) sucrose, 10% glycerol, 2% (wt/vol) BSA to a cell density of ~6 x 1010 cells/ml. Samples from the infection mixtures were rapidly frozen by slamming onto liquid nitrogenchilled copper blocks, and then freeze-substituted for 8 h at -90°C. Several substitution protocols were tested: (1) acetone and 12% (wt/vol) OsO4, (2) acetone and 12% (wt/vol) OsO4, followed by en-bloc staining with 0.5% (wt/vol) uranyl acetate (for 1 h, +4°C), (3) acetone and 3% (vol/vol) glutaraldehyde, and (4) methanol and 0.5% (wt/vol) uranyl acetate. All samples were embedded in Epon. Thin sections (4060 nm) were poststained with aqueous 0.5% (wt/vol) uranyl acetate (for 040 min at +2040°C) and lead citrate (for 5 min at +20°C) and viewed at 60100 kV with a JEOL 1200EX electron microscope.
Neutralization Assay
The NC neutralization tests were carried out with purified mAbs against 6 structural proteins P1, P4, P8, and P3, (
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Results |
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Spheroplasts Susceptible to NC Infection Are Energetically Active
Earlier studies with intact host cells have indicated that NC penetration through the host PM is an energy-dependent process ( values. The distribution of this ion between cells (or organelles) and the surrounding medium can be measured by selective electrodes that monitor the change of the TPP+ concentration in the incubation medium (
of intact spheroplasts was between 120 and 150 mV, and the cytosolic K+ level was ~40 mM. The acidification rate of the spheroplast media was ~2 x 103 H+ x spheroplast-1 x s-1, indicating that the proton pumps were actively extruding H+ ions through the PM. Furthermore, the intracellular ATP level of the spheroplasts was ~600 µM and the extracellular media contained ~3 µM ATP. Although the spheroplasts were rather well energized, their energetic state was not stable when incubated at 23°C with magnetic stirring, the electrochemical measurement conditions (Figure 2). In these conditions, the spheroplasts became depolarized and the intracellular K+ leaked out (Figure 2 b). Simultaneously, the ability of the spheroplasts to support NC infection was dramatically reduced (Figure 2 a, closed circles). However, storage of spheroplasts on ice without stirring for up to 90 min did not reduce their energetic level or capacity to produce mature viruses (Figure 2 a, open circles). The instability of the spheroplasts in conditions for electrochemical measurements was taken into account when the effects of different energy-depleting drugs on NCspheroplast infection were studied. Despite batch-to-batch variation in the ability of the spheroplasts to support NC infection, the trend of the results was consistent and allowed pooling of the data (as in Figure 5).
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During the systematic study of the NC infection, it also became apparent that the maximum virus yield was not affected by the temperature of the infection mixture (1030°C). However, the rate for achieving the maximum yield was temperature-dependent (not shown). The PM of P. syringae cells was permeable to TPP+ and the cells were sensitive to ionophoric antibiotics GD (a channel former) and NG (a K+ to H+ exchanger) even at +6°C, strongly indicating that the PM stays in a fluid state and, thus, is able to support the formation of invaginations even at low temperatures. Therefore, a low temperature could not be used to dissect the NC binding from the internalization.
The NC Entry Assay
The original NC infection assay (
The time course of IC formation on LB plates after different infection times in a test tube is depicted in Figure 3 a. The maximal yield is reached at ~45 min postinfection (p.i.). Treatment of the infection mixture with NC-specific polyclonal antiserum allows the dissection of the antibody-inhibitable NC particles (free and PM adsorbed) from those that have already reached an antibody-resistant environment (internalized particles). ICs formed by antibody-resistant NCs as a function of time are shown in Figure 3 b. In this case, the maximal level is reached at ~90 min. The total yield (Figure 3 a, closed and open circles) is the sum of the ICs formed by spheroplasts infected both by particles that are adsorbed (antibody inhibitable) and internalized (resistant to the antibody) at the time of plating.
We set up an NC entry assay based on the following three observations: (1) dilution-resistant NCs, inhibitable by antibodies (cell adsorbed particles) can form ICs on plates (Figure 3 a); (2) a 10-min treatment with NC-specific antiserum causes maximal inactivation of these particles, but still allows the antibody-resistant (internalized) NCs to form ICs (Figure 3 a, squares); and (3) treatment with the serum did not reduce the ability of spheroplasts to support the viral life cycle (Figure 3 a, open circles). Thus, in this investigation, NC entry was defined as a process where NCs become resistant to NC-specific antiserum. This was analyzed by comparing the IC counts obtained after a 10-min treatment with an NC-specific antiserum to those obtained after treatment with a nonspecific control antiserum.
The energy requirements of the NC internalization process were analyzed by using energy-depleting agents. Plaque assays, , and ATP level determinations were simultaneously carried out to monitor the cellular energy status and to correlate it to infection efficiency. As the different agents tested were diluted before plating the infected spheroplasts out, the entered or adsorbed NCs are able to produce progeny on the plates. When a drug inhibits the entry, the number of NCs in the antibody-resistant environment at the time of plating will be lower than in the control infection. However, if the drug does not affect the capability of spheroplasts to produce viruses, or the NC binding to spheroplasts, the antibody-inhibitable NCs (adsorbed to spheroplasts) can enter normally into the spheroplasts after dilution of the drug. Therefore, the number of ICs formed by these NCs will not differ from the control.
NC Internalization Is Dependent on , but Not on
pH
Extensive screening allowed us to find drug concentrations that had measurable effects on the energetic properties of the spheroplasts, but which did not affect IC production per se. On the basis of the effects on the NC entry, the drugs were categorized into three groups as shown in Figure 4. Compounds in the first category (Figure 4 a) did not affect the infection per se (Figure 4 a, black bars), but caused a distinct decrease in plaque numbers if NC-specific antiserum was used, implying an effect on entry (Figure 4 a, gray bars). This category includes uncouplers of oxidative phosphorylation, CCCP, and FCCP. These protonophores dissipate the p (
values (<80 mV) were observed at the concentrations used (25 µg/ml). In spite of the inhibitory effect on entry, the binding of the NC to the spheroplast surface was not reduced because of the decrease in
p (Figure 4 a, black bars).
In the second category (Figure 4 b), the drug treatment had no significant effect on the production of progeny either in the presence or absence of the NC-specific antiserum. MN and NG, the representatives of this category, reduce the existing H+ and K+ concentration gradients by exchanging extracellular H+ for intracellular K+ ions (pH is not involved (
PMB (a membrane-active antibiotic), N-ethylmaleimide (an inhibitor of the SH groupdependent processes), and NaN3 (an inhibitor of the respiratory chain and membrane H+-ATP synthase) are examples of drugs falling into the third category (PMB; Figure 4 c). Active concentrations of these drugs affected the IC formation both in the presence and absence of the NC-specific antiserum. The inhibition of the total IC production (Figure 4 c, black bars) indicates the following: (1) that incubation with these agents has irreversibly lowered the ability of spheroplasts to produce ICs, (2) the drug is irreversibly bound to membranes and cannot be removed by dilution, or (3) dilution-resistant adsorption of the NCs to spheroplasts has been affected in the presence of the agent. Since the distinction between these alternatives could not be made, these drugs were not used in further studies.
Increase of the Rescues the NC Internalization
Concentrations of KCl (Figure 5) or NaCl (not shown) up to 50 mM increased the total yield of ICs. At higher concentrations, reduced values were measured (Figure 6), and a decrease in the number of ICs after the NC-specific antiserum treatment was detected (Figure 5 and Figure 6 a). To ensure that the change in the medium osmolarity had not affected the internalization, the osmolarity of the infection mixture was increased with sucrose instead of salt. As shown in Figure 6 a, the sucrose containing infections did not differ from the control infections.
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The inhibitory effect of NaCl and KCl on the NC entry was reversed if MN or NG were added (Figure 6 a, NG and KCl). However, the electrogenic K+ carrier, valinomycin, could not rescue the effect of KCl (not shown). The measurements showed that NG also induced an increase in the
(Figure 6 a). Although NG works in an electroneutral manner, it can increase
if the respiratory chain is active (
in the presence of KCN nor was the NC entry rescued (Figure 6 a). Therefore, it appears that there is a threshold value in
between 95 and 120 mV (Figure 6 b) below which the NC internalization is strongly inhibited.
Reduction in Extra- or Intracellular ATP Content Does Not Affect NC Internalization
Different drug treatments were carried out to decrease the intra- and extracellular ATP content of the spheroplasts. To this end, we analyzed the effects of DCCD (an inhibitor of membrane ATP synthase), NaF (an inhibitor of ATP formation from glycolytic substrates), Na2HAsO4 (an ATP destabilizing agent), as well as AP (an ATP hydrolyzing enzyme). Some decrease in the ATP concentration was observed in the presence of these agents, but the most efficient reduction was achieved if the spheroplasts were washed before the drug treatments (Figure 7). The lowest extracellular ATP concentration measured was 75 nM (2.5% of the ATP level of unwashed spheroplasts) and the lowest intracellular ATP concentration was ~50 µM (8% of the ATP level of unwashed spheroplasts). Although some decrease in the total IC production was observed after these treatments, the infections treated with NC-specific antiserum remained at the control level, indicating that internalization of the NC was not affected at reduced ATP concentrations.
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Low pH at the Outer Surface of the PM Is Not Needed for Initial NC Adsorption or Internalization
A high concentration of H+ ions seems to be crucial for a number of virus entry processes ( as shown in Figure 6 a. The addition of 25 mM KCl or NaCl to the spheroplast suspension caused a clear pulse of acidification of the medium, indicating an exchange of H+ to K+ or Na+ ions at the outer surface of the spheroplasts (not shown). However, this did not considerably affect the IC formation (for KCl see Figure 5). Neither did the decrease in the surface charge after addition of high concentrations of the polycationic compound, PMB nonapeptide (300 µg/ml), reduce the IC counts (not shown). The density of H+ at the outer surface of the PM should also be reduced in the presence of NG, which changes the extracellular H+ to cytosolic K+, but no inhibition of NC internalization was detected (Figure 6 a). These results indicate that low pH at the outer surface of the PM is not crucial for the early stages in NC internalization.
EM Reveals NCs at Different Stages of Penetration into the Spheroplast
In a normal 6 infection, practically every cell is infected and the specific infectivity of the virus particles is close to one (
Previous morphological analyses of normal 6 infections using freeze-fracture EM showed enveloped NC sized particles in the cell interior in early infection (
The NCspheroplast infection system was used to further characterize the NCPM interactions. As a complement to conventional chemical fixation (CF) (Figure 8c and jm), which may induce artefactual membrane configurations (mesosomes; see
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We carried out the EM analysis at conditions optimized to trap the transient entry event. The infection was arrested by reducing either with CCCP or NaCl. As previously documented (
reduction, the spheroplasts constantly displayed PM invaginations and/or intracellular vesicles, indicating them to be intrinsic for spheroplasts, not artifacts of the fixation method (Figure 8, ac). The NC-infected spheroplasts showed NCPM interaction patterns similar to those observed earlier in energy-depleted normal infections (
, these transient events were difficult to capture, and indentations or invaginations were rare in all samples.
The NC Surface Protein P8 Mediates the NC Interaction with the PM
We also analyzed the role of different NC proteins in NC internalization. Previous studies have suggested the necessity of the protein P8 shell for the NC infectivity; uncoated NC particles devoid of P8 (the NC cores) were not infectious to host cell spheroplasts (
More detailed investigation using the panel of P8-specific mAbs revealed that most of them had a neutralizing effect on NC infectivity (Table 1). The nonneutralizing mAbs, 8J3, 8Q4, and 8Q7, did not precipitate NCs in an immunoprecipitation assay, indicating that their epitopes are not accessible on the NC surface (Table 1). To confirm that the inhibitory effect was not due to aggregation or disruption of NC particles, the sedimentation assay of NCs was carried out after treatment with several different P8-specific mAbs (Table 1). Although many of the neutralizing antibodies did appear to disrupt the NC particles, four nonaggregating and nondisrupting mAbs (8B1, 8D1, 8K4, and 8L1) still had neutralization activity, thus, confirming P8's role in the NC entry.
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Discussion |
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The endocytotic pathway commonly used by eukaryotic cells to internalize different types of molecules and molecular complexes is not thought to function in prokaryotes. However, phage 6 has to use an exceptional entry mechanism to deliver its polymerase complex particle (NC carrying the dsRNA genome) into the host cytosol without dissipating the
. We have shown morphological evidence here and in our previous studies (
The aim of the present study was to analyze the energy requirements of the 6 endocytic-like NC internalization. Romantschuk and co-workers (1988) showed that this process is dependent on the energetic state of the membrane. However, more detailed analysis of the energy requirements, during infection of an intact cell, is difficult as the initial association of the virus particle with the OM is also an energy-dependent process (
Specific ATP/GTP-dependent cytosolic proteins are needed to direct the normal (clathrin-dependent) endocytic uptake used by many animal viruses (6 NC to an antibody-resistant location was not dependent on high extra- or intracellular ATP levels (Figure 7). Therefore, it seems likely that there are no ATP-dependent cytosolic or PM-associated proteins involved, neither is the viral NTPase activity required.
p is commonly used in gram-negative bacteria to drive the transport of macromolecules into or across the membrane(s) (for reviews see
p-dependent fusion of the OM and PM at the site of phage adsorption (
-driven insertion of amphiphilic helices (
6 NC binding to the spheroplast membrane was not affected at reduced
p values (Figure 4 a). However, the subsequent NC transport to the antibody-resistant location was clearly dependent on
but not on
pH (Figure 4 and Figure 6).
Simultaneous measurements of and plaque formation allowed us to define a threshold value for the NC internalization (Figure 6, ~110 mV). Accordingly, the
p-dependent processes mentioned above, the T4 phage DNA entry, and the colicin insertion into PM, occur only when the
is above a threshold value (
dependence in NC entry might be associated to polypeptide chain translocation (probably of the NC shell protein P8) into the PM. Alternatively, the
might be required as the NC-containing invagination pinches off from the PM. In the bacterial PM, the negatively charged phospholipids, cardiolipin and phospatidylglycerol, are predominantly located in the outer leaflet and phosphatidylethanolamine in the inner leaflet (
p (
The NC surface protein P8 was shown to be crucial for the entry. Viral proteins involved in the entry are known to undergo large conformational changes (see for example production makes it difficult to dissect these effects (Figure 6). The NC adsorption and early steps in the formation of the PM invagination were not affected in conditions where the PM surface charge, and thus protonation, was reduced. We consider that the proton-dependent events are crucial later in the entry, in the process of the NC release from the entry vesicle.
6 entry can now be dissected into a number of stages (Figure 1, bottom) (
6 entry process interestingly highlights common universal mechanisms operating in both prokaryotic and eukaryotic cells, and points out that mechanisms that were thought to operate in eukaryotes only are also used in prokaryotes.
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Footnotes |
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P.M. Ojala's present address is Cell Cycle Laboratory, Haartman Institute, University of Helsinki, Finland.
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Acknowledgements |
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Sarah Butcher is acknowledged for critical reading of the manuscript. Marja-Leena Perälä (both from Institute of Biotechnology) is thanked for her skilful technical assistance and Arja Strandell (EM Unit, Institute of Biotechnology) for thin-sections.
This work has been supported by Finnish Academy of Science (grants 30562 and 37725) and EU grant B104-CT97-2364.
Submitted: 21 July 1998
Revised: 16 August 1999
Accepted: 21 September 1999
ble-stranded; DCCD, N,N'-dicyclohexylcarbodiimide; FCCP, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone; FS, freeze substitution; GD, gramicidin D; IC, infective center; MN, monensin; NC, nucleocapsid; NG, nigericin; OM, outer membrane; p.i., postinfection; PM, plasma membrane; PMB, polymyxin B; RF, rapid freezing; TPP+, tetraphenylphosphonium
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References |
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