Characterization of ion channels involved in the penetration of phage T4 DNA into Escherichia coli cells

The hypothesis of a channel-mediated transport of phage DNA into Escherichia coli cytoplasmic membrane has been formulated for a long time. In this paper, we present experimental evidence in favor of this proposal. We have analyzed the kinetics of the K+ efflux induced by T4 phage and ghosts (phage depleted of DNA) using a potassium selective electrode. We show that the K+ efflux is not catalyzed by the K+ transport systems. The Km of K+ efflux is the same for phage and ghosts. The rate of K+ efflux is linearly related to the multiplicity of infection. This suggests that phage and ghosts induce the formation of similar channels and that one channel is induced by one virion. The K+ efflux is associated with an influx of H+ and Na+ or Li+ which compete for entry through the channel. These ion fluxes may be responsible for the cell depolarization. The phage-induced channels allow the passage of DNA. They are only transiently opened, and their closing leads to cellular repolarization. The ghost-induced channels remain open. The insertion and conformation of the channels in the membrane depend on the temperature and their confirmation is voltage-dependent. We give an estimate of their size.     

Formation of ion channels in the Escherichia coli cytoplasmic membrane after exposure to bacteriophages T4 and lambda

The effects of phage T4 and lambda on the ion permeability of the E. coli cytoplasmic membrane were studied. It was shown that the phage-induced depolarization of the membrane is coupled with a simultaneous increase in a transmembrane pH gradient. Hence, the total value of the proton-motive force remains unchanged at moderate multiplicity of infection. The rise in the pH gradient occurs due to an increase in the activity of the redox H+-pump of the E. coli membrane. Analysis of the temperature dependence showed that the stimulating effect of the phage is observed at 6-8 degrees C. Apart from the phages, gramicidin is also capable of stimulating the H+-pump under these conditions, while the stimulating effect of valinomycin is diminished. These data suggest that the ion-permeable channels are formed in the membrane during the interaction of E. coli cells with the phages. The experimental results demonstrate that the channels are permeable to ions of monovalent metals. The phage can also increase the permeability of cell membranes to protons; however, the permeability to monovalent ions is higher when these ions are in excess.     

Interaction of the isolated DNA of lambda phage with spheroplasts of E. coli treated with sturine]

The method of centrifugation in sucrose density gradient (30-55%) of the spheroplast membrane preparations treated and untreated with sturine and infected with phage lambda DNA demonstrated that sturine, treatment increased the phage lambda DNA absorption three-fold. About 50% of the lambda DNA molecules adsorbed by spheroplasts are bound with the cytoplasmic membrane of spheroplasts treated with sturine; 50% of the lambda DNA molecules are bound with the cell wall membrane on the sturine-untreated spheroplasts. The data obtained allow to conclude that the stimulating effect of sturine in E. coli spheroplasts transfection by lambda DNA is connected with redistribution of phage DNA absorbed on spheroplasts from the cell wall to the cytoplasmic membrane facilitating the penetration of DNA and its fastening on the membrane.     

A trans-envelope protein complex needed for filamentous phage assembly and export

Assembly and export of filamentous phage requires four non-capsid proteins: the outer membrane protein, pIV; the inner membrane proteins, pI and pXI; and a cytoplasmic host factor, thioredoxin. Chemical cross-linking of intact cells demonstrates a trans-membrane complex containing pI and pIV. Formation of the complex protects pI from proteolytic cleavage by an endogenous protease. This protection also requires pXI, which is identical to the C-terminal portion of pI. This indicates that pXI, which is required for phage assembly in its own right, is also part of the complex. This complex forms in the absence of any other phage proteins or the DNA substrate; hence, it represents the first preinitiation step of phage morphogenesis. On the basis of protease protection data, we propose that the preinitiation complex is converted to an initiation complex by binding phage DNA, thioredoxin and the initiating minor coat protein(s).     

The TolQRA Proteins Are Required for Membrane Insertion of the Major Capsid Protein of the Filamentous Phage f1 during Infection

Infection of Escherichia coli by the filamentous bacteriophage f1 is initiated by interaction of the end of the phage particle containing the gene III protein with the tip of the F conjugative pilus. This is followed by the translocation of the phage DNA into the cytoplasm and the insertion of the major phage capsid protein, pVIII, into the cytoplasmic membrane. DNA transfer requires the chromosomally encoded TolA, TolQ, and TolR cytoplasmic membrane proteins. By using radiolabeled phages, it can be shown that no pVIII is inserted into the cytoplasmic membrane when the bacteria contain null mutations in tolQ, -R and -A. The rate of infection can be varied by using bacteria expressing various mutant TolA proteins. Analysis of the infection process in these strains demonstrates a direct correlation between the rate of infection and the incorporation of infecting bacteriophage pVIII into the cytoplasmic membrane.     

Permeability of the cell-to-cell membrane channel and its regulation in an insect cell junction

Summary Cells of organs and tissues commonly communicate directly with one another via permeable membrane junctions. Cell-to-cell channels, spanning the width of both membranes of a junction, are thought to provide the pathways between the cytoplasms of adjacent cells for the immediate exchange of ions and small molecules. We study these cell-to-cell channels in a cell model system, the salivary gland ofChironomus. Using intracellularly injected fluorescent labelled peptides and oligosaccharides of various molecular dimensions as channel permeability probes we find the channels to have a bore of about 2 nm. The channel permeability can be modulated and, in the extreme, the channels can be closed under various experimental conditions. With the aid of the Ca²⁺-sensitive photoprotein aequorin as monitor of cytoplasmic free Ca²⁺ concentration, we show that a determining factor in this modulation of channel permeability is the cytoplasmic free Ca²⁺ concentration. Moreover, results obtained by injection of different-sized and different-labelled channel permeability probes together with Ca²⁺ indicate that closure of the individual channels may occur in more than one step, i.e., by a graded reduction of channel bore. Presented in the symposium on Molecular and Morphological Aspects of Cell-Cell Communication at the 31st Annual Meeting of the Tissue Culture Association, St. Louis, Missouri, June 1–5, 1980. This symposium was supported, in part by Contract 263-MD-025754 from the National Cancer Institute and the Fogarty International Center. This work was supported by NH Grants 5P1GM23911-07 and 5T32-6M07403-04.     

The small viral membrane-associated protein P32 is involved in bacteriophage PRD1 DNA entry

The lipid-containing bacteriophage PRD1 infects a variety of gram-negative cells by injecting its linear double-stranded DNA genome into the host cell cytoplasm, while the protein capsid is left outside. The virus membrane and several structural proteins are involved in phage DNA entry. In this work we identified a new infectivity protein of PRD1. Disruption of gene XXXII resulted in a mutant phenotype defective in phage reproduction. The absence of the protein P32 did not compromise the particle assembly but led to a defect in phage DNA injection. In P32-deficient particles the phage membrane is unable to undergo a structural transformation from a spherical to a tubular form. Since P32(-) particles are able to increase the permeability of the host cell envelope to a degree comparable to that found with wild-type particles, we suggest that the tail-tube formation is needed to eject the DNA from the phage particle rather than to reach the host cell interior.     

Structure and assembly of filamentous bacteriophages

Filamentous bacteriophages are interesting paradigms in structural molecular biology, in part because of the unusual mechanism of filamentous phage assembly. During assembly, several thousand copies of an intracellular DNA-binding protein bind to each copy of the replicating phage DNA, and are then displaced by membrane-spanning phage coat proteins as the nascent phage is extruded through the bacterial plasma membrane. This complicated process takes place without killing the host bacterium.     

Host cell metabolic energy is not required for injection of bacteriophage T5 DNA

The addition of various metabolic inhibitors (uncouplers, cyanide, arsenate, ionophores) separately or together (for example, arsenate and an uncoupler) or even harsher methods of energy depletion did not prevent bacteriophage T5 from injecting its first-step-transfer DNA (a DNA segment 3 micron long) into the cytoplasm of host cells. The same indifference to metabolic energy was observed if first-step-transfer DNA was decapsidated and uncoiled before injection, thus precluding any energetic help from the phage capsid or from some tension stored in DNA tightly packed in the head. Penetration of the second-step-transfer DNA across the cytoplasmic membrane was studied by determining injection of superinfecting T5 A2- amber phages into Sup- bacteria containing proteins A1 and A2 previously encoded by the first-step-transfer DNA of a primary wild-type phage. The addition of various metabolic inhibitors after synthesis of proteins A1 and A2 but before superinfection did not prevent this penetration of second-step-transfer DNA. Thus, we conclude that traversal of the cytoplasmic membrane by the entire T5 DNA (a molecule 34 micron long) must occur by diffusion through protein channels.     

A study of the effect of ethanol on the synthesis of serine and the exchange of methyl groups in hepatocytes by NMR spectroscopy

The method of NMR spectroscopy was used to investigate the role of voltage-dependent anion channels in the outer mitochondrial membrane in the mechanism of ethanol hepatotoxicity using the synthesis of serine and exchange of methyl groups in hepatocytes metabolizing ¹³C-labeled glycine. Here we present and describe a methodological approach developed for the independent monitoring of the synthesis of serine in two intracellular compartments: the cytoplasm and mitochondria of intact hepatocytes, and quantification of different serine isotopomers synthesized in hepatocytes from ¹³C-labeled glycine. The data obtained indicate that the treatment of cells with ethanol as well as cysteamine (specific inhibitor of mitochondrial synthesis of serine) suppressed the level of mitochondrial but not cytoplasmic serine isotopomers. It is concluded that the decrease in the production of mitochondrial serine isotopomers in hepatocytes exposed to ethanol can be caused not only by decreased permeability of the outer mitochondrial membrane due to the closure of voltage-dependent anion channels and suppression of the exchange of substrates of serine synthesis in mitochondria but also by the reduction of the cytoplasmic and/or mitochondrial pool of pyridine nucleotides (NADH) during the oxidation of ethanol. Our work reveals a new mechanism of action of ethanol (alcohol intoxication) in hepatocytes through the regulation of glycine metabolism and opens new possibilities in the treatment of alcohol poisoning.     

Characterization of the Streptomyces lividans PspA response

Phage shock protein (Psp) is induced by extracytoplasmic stress that may reduce the energy status of the cell. It is encoded in Escherichia coli by the phage shock protein regulon consisting of pspABCDE and by pspF and pspG. The phage shock protein system is highly conserved among a large number of gram-negative bacteria. However, many bacterial genomes contain only a pspA homologue but no homologues of the other genes of the Psp system. This conservation indicates that PspA alone might play an important role in these bacteria. In Streptomyces lividans, a soil-borne gram-positive bacterium, the phage shock protein system consists only of the pspA gene. In this report, we showed that pspA encodes a 28-kDa protein that is present in both the cytoplasmic and the membrane fractions of the S. lividans mycelium. We demonstrated that the pspA gene is strongly induced under stress conditions that attack membrane integrity and that it is essential for growth and survival under most of these conditions. The data reported here clearly show that PspA plays an important role in S. lividans under stress conditions despite the absence of other psp homologues, suggesting that PspA may be more important in most bacteria than previously thought.     

Phage shock proteins B and C prevent lethal cytoplasmic membrane permeability in Yersinia enterocolitica

The bacterial phage shock protein (Psp) stress response system is activated by events affecting the cytoplasmic membrane. In response, Psp protein levels increase, including PspA, which has been implicated as the master effector of stress tolerance. Yersinia enterocolitica and related bacteria with a defective Psp system are highly sensitive to the mislocalization of pore-forming secretin proteins. However, why secretins are toxic to psp null strains, whereas some other Psp inducers are not, has not been explained. Furthermore, previous work has led to the confounding and disputable suggestion that PspA is not involved in mitigating secretin toxicity. Here we have established a correlation between the amount of secretin toxicity in a psp null strain and the extent of cytoplasmic membrane permeability to large molecules. This leads to a morphological change resembling cells undergoing plasmolysis. Furthermore, using novel strains with dis-regulated Psp proteins has allowed us to obtain unequivocal evidence that PspA is not required for secretin-stress tolerance. Together, our data suggest that the mechanism by which secretin multimers kill psp null cells is by causing a profound defect in the cytoplasmic membrane permeability barrier. This allows lethal molecular exchange with the environment, which the PspB and PspC proteins can prevent.     

The mode of action of vasopressin: membrane microstructure and biological transport

Vasopressin affects a variety of cell systems. This review is focused on permeability changes induced by vasopressin in tight epithelia such as the collecting duct of the mammalian kidney and the skin and the bladder of anurans. These vasopressin effects are discussed with reference to current concepts and models of the microstructure of the plasma membrane. The transport of three major chemical species--Na, urea and water--is analyzed. In each instance, the hormone appears to activate selective membrane pathways situated at the rat-limiting barrier of the epithelium, i.e., the apical membrane. Available data suggest that two intra-cellular messengers -- cAMP and calcium -- plan a key role in the coupling between stimulus (receptor occupancy) and biological effect (permeability change). The enhancement of Na transport (natriferic effect) depends on the opening and/or the insertion of Na channels, the biophysical and biochemical characteristics of which have been investigated by fluctuation analysis and by means of several chemical blockers of Na transport, particularly the amiloride molecule and its congeners. Likewise, the finding of inhibitors and activators of urea transport, which do not cause any appreciable change in Na or water permeability, led to the notion of selective urea channels or pores. Finally, the enhancement of water transport (hydrosmotic effect) possibly results from the insertion in the apical membrane of water channels already present in vesicular cytoplasmic structures. The restructuring of the apical membrane underlying the transition from a low to a higher state of water permeability is very likely related to the appearance of intramembrane particle aggregates detectable with the freeze-fracture technique in epithelia exposed to vasopressin. The putative water channels (or pores) appear to be so narrow that trans-apical water movement is constrained to single-file diffusion. Recent data also suggest that, in addition to cAMP, microtubules and microfilaments, the calmodulin-Ca complex is a major element in the hydrosmotic effect of vasopressin.     

Conversion of the FhuA transport protein into a diffusion channel through the outer membrane of Escherichia coli.

The FhuA receptor protein is involved in energy-coupled transport of Fe3+ via ferrichrome through the outer membrane of Escherichia coli. Since no energy source is known in the outer membrane it is assumed that energy is provided through the action of the TonB, ExbB and ExbD proteins, which are anchored to the cytoplasmic membrane. By deleting 34 amino acid residues of a putative cell surface exposed loop, FhuA was converted from a ligand specific transport protein into a TonB independent and nonspecific diffusion channel. The FhuA deletion derivative FhuA delta 322-355 formed stable channels in black lipid membranes, in contrast to wild-type FhuA which did not increase membrane conductance. The single-channel conductance of the FhuA mutant channels was at least three times larger than that of the general diffusion porins of E. coli outer membrane. It is proposed that the basic structure of FhuA in the outer membrane is a channel formed by beta-barrels. Since the loop extending from residue 316 to 356 is part of the active site of FhuA, it probably controls the permeability of the channel. The transport-active conformation of FhuA is mediated by a TonB-induced conformational change in response to the energized cytoplasmic membrane. The ferrichrome transport rate into cells expressing FhuA delta 322-355 increased linearly with increasing substrate concentration (from 0.5 to 20 microM), in contrast to FhuA wild-type cells, which displayed saturation at 5 microM. This implies that in wild-type cells ferrichrome transport through the outer membrane is the rate-limiting step and that TonB, ExbB and ExbD are only required for outer membrane transport.     

Permeability and channel-mediated transport of boric acid across membrane vesicles isolated from squash roots

Boron is an essential micronutrient for plant growth and the boron content of plants differs greatly, but the mechanism(s) of its uptake into cells is not known. Boron is present in the soil solution as boric acid and it is in this form that it enters the roots. We determined the boron permeability coefficient of purified plasma membrane vesicles obtained from squash (Cucurbita pepo) roots and found it to be 3 x 10(-7) +/-1.4 x 10(-8) cm s(-1), six times higher than the permeability of microsomal vesicles. Boric acid permeation of the plasma membrane vesicles was partially inhibited (30%-39%) by mercuric chloride and phloretin, a non-specific channel blocker. The inhibition by mercuric chloride was readily reversible by 2-mercaptoethanol. The energy of activation for boron transport into the plasma membrane vesicles was 10.2 kcal mol(-1). Together these data indicate that boron enters plant cells in part by passive diffusion through the lipid bilayer of the plasma membrane and in part through proteinaceous channels. Expression of the major intrinsic protein (MIP) PIP1 in Xenopus laevis oocytes resulted in a 30% increase in the boron permeability of the oocytes. Other MIPs tested (PIP3, MLM1, and GlpF) did not have this effect. We postulate that certain MIPs, like those that have recently been shown to transport small neutral solutes, may also be the channels through which boron enters plant cells.     

Effect of ethanol on synthesis of serine and exchange of methyl groups in hepatocytes by NMR spectroscopy

The method of NMR spectroscopy was used to investigate the role of voltage-dependent anion channels in the outer mitochondrial membrane in the mechanism of ethanol hepatotoxicity using the synthesis of serine and exchange of methyl groups in hepatocytes metabolizing 13C-labeled glycine. Here we present and describe a methodological approach developed for the independent monitoring of the synthesis of serine in two intracellular compartments: the cytoplasm and mitochondria of intact hepatocytes, and quantification of different serine isotopomers synthesized in hepatocytes from 13C-labeled glycine. The data obtained indicate that the treatment of cells with ethanol as well as cysteamine (specific inhibitor of mitochondrial synthesis of serine) suppressed the level of mitochondria but not cytoplasmic serine isotopomers. It is concluded that the decrease in the production of mitochondrial serine isotopomers in hepatocytes exposed to ethanol can be caused not only by decreased permeability of the outer mitochondrial membrane due to the closure of voltage-dependent anion channels and suppression of the exchange of substrates of serine synthesis in mitochondria but also by the restoration of the cytoplasmic and/or mitochondrial pool of pyridine nucleotides (NADH) during the oxidation of ethanol. Our work reveals a new mechanism of action of ethanol (alcohol intoxication) in hepatocytes through the regulation of glycine metabolism and opens new possibilities in the treatment of alcohol poisoning.     

SCAM analysis of Panx1 suggests a peculiar pore structure

Vertebrates express two families of gap junction proteins: the well-characterized connexins and the pannexins. In contrast to connexins, pannexins do not appear to form gap junction channels but instead function as unpaired membrane channels. Pannexins have no sequence homology to connexins but are distantly related to the invertebrate gap junction proteins, innexins. Despite the sequence diversity, pannexins and connexins form channels with similar permeability properties and exhibit similar membrane topology, with two extracellular loops, four transmembrane (TM) segments, and cytoplasmic localization of amino and carboxy termini. To test whether the similarities extend to the pore structure of the channels, pannexin 1 (Panx1) was subjected to analysis with the substituted cysteine accessibility method (SCAM). The thiol reagents maleimidobutyryl-biocytin and 2-trimethylammonioethyl-methanethiosulfonate reacted with several cysteines positioned in the external portion of the first TM segment (TM1) and the first extracellular loop. These data suggest that portions of TM1 and the first extracellular loop line the outer part of the pore of Panx1 channels. In this aspect, the pore structures of Panx1 and connexin channels are similar. However, although the inner part of the pore is lined by amino-terminal amino acids in connexin channels, thiol modification was detected in carboxyterminal amino acids in Panx1 channels by SCAM analysis. Thus, it appears that the inner portion of the pores of Panx1 and connexin channels may be distinct.     

Involvement of envelope-bound calcium in the transient depolarization of the Escherichia coli cytoplasmic membrane induced by bacteriophage T4 and T5 adsorption

We previously showed that adsorption of bacteriophages T4 and T5 to their respective outer membrane receptors induced a partial depolarization of the cytoplasmic membrane. As these membrane potential changes were independent of phage properties, we proposed that phage adsorption triggered the emission of a signal which must be transmitted between the two membranes. We show here that these two phages use different mechanisms of transmission of this stimulation signal. In the case of T4, but not of T5, a specific requirement for envelope-bound calcium was found. Indeed, addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prevented the membrane potential changes induced by T4. This envelope-bound calcium became accessible to the chelator only as a consequence of phage adsorption and remained in this state during the depolarization and repolarization. Membrane potential changes again occurred if calcium was added after the addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and phage. The same concentration (300 microM) of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prevented the T4-induced depolarization between multiplicities of infection of 6 and 30. This suggests that phage adsorption triggers both a conformational change of membrane components, the number of which reflects the number of stimuli (phages), and the liberation of a definite amount of calcium. This liberated calcium would, in turn, activate these modified membrane components to induce the depolarization. The fact that depolarization may be induced several times after a unique adsorption implies that these membrane components remain irreversibly modified.     

Mode of action of colicin ib: formation of ion-permeable membrane channels

Addition of purified colicin Ib to whole Escherichia coli cells or cytoplasmic membrane vesicles inhibits their subsequent ability to generate a membrane potential. In addition, this colicin is shown to bring about a voltage-dependent increase in the conductance of an artificial planar bilayer membrane prepared from soybean phospholipids. This results from the formation of ion-permeable channels. These data provide strong evidence that the depolarization of Escherichia coli cells by this colicin results from an Ib-induced increase in membrane permeability to ions.