Alteration of active transport after bacteriophage T5 infection.

Bacteriophage T5 absorption immediately followed by injection of the first-step-transfer DNA segment produces alterations in the bacterial membrane which reduce the uptake of amino acids and of o-nitrophenyl-beta-D-galactopyranoside. Concomitantly, intracellular ATP is hydrolyzed. The extent of inhibition of uptake and of ATP hydrolysis is cooperatively increased with the multiplicity of infection. Inhibition of active transport at a high multiplicity of infection (greater than 3) is also observed after the second step of DNA injection. In contrast, at low multiplicities of infection, phage proteins are able to enhance amino acid uptake. Infection of su- bacteria with amber mutants in the first-step-transfer DNA suggests that protein A1 is implicated in this enhancement.     

Evidence for heterogeneity in populations of T5 bacteriophage. II. Some particles are unable to inject their second-step-transfer DNA.

A new class of bacteriophage was characterized in purified T5 stocks. Regardless of the host cell, these phages were irreversibly blocked at the first-step-transfer stage under conditions in which whole DNA injection normally takes place. However, they expressed their first-step-transfer functions. These observations confirmed the previously established heterogeneity of T5 bacteriophage populations and provided a new way to define a phage function necessary to release the blocking of T5 DNA injection at the first-step-transfer stage.     

Identification of the gene controlling the synthesis of the major bacteriophage T5 membrane protein.

After infection of Escherichia coli B by bacteriophage T5, a major new protein species, as indicated by polyacrylamide gel electrophoresis, appears in the cells' membranes. Phage mutants with amber mutations in the first-step-transfer portion of their DNA have been tested for their ability to induce membrane protein synthesis after they infect E. coli B. We have found that phage with mutations in the Al gene of T5 do not induce the synthesis of the T5-specific major membrane protein, whereas phage that are mutant in the A2 gene do induce its synthesis. We conclude that gene Al must function normally for T5-specific membrane protein biosynthesis to occur and that only the first 8% (first-step-transfer piece) of the DNA need be present in the cell for synthesis to occur.     

Rescue of First-Step-Transfer Amber Mutants by “Second-Step-Transfer-Blocked” Bacteriophage T5 on an su− Strain

T5 st0 phages irreversibly blocked in the injection of their second-step-transfer DNA can produce active A1 and A2 proteins which complement first-step-transfer amber mutants infecting an su⁻ strain.     

The energetics of the injection process of bacteriophage lambda DNA and the role of the ptsM/pel-encoded protein

We have examined the nature of the role played in the process of phage lambda DNA injection by the bacterial protein coded by the ptsM/pel gene. Neither the specific inhibition of the activity of the PtsM protein, nor the addition of inhibitors of phosphotransferase system modified the efficiency of lambda DNA penetration. Thus, the PtsM/Pel protein does not seem to play a role through its transport function, although we have confirmed that it must be present for a successful lambda DNA injection. Moreover, the presence of various metabolic inhibitors (uncouplers, cyanide, arsenate) separately or together, or even harsher methods of energy depletion did not prevent lambda DNA penetration, suggesting that DNA is entering the cell cytoplasm by diffusion.     

Requirement for a fluid host cell membrane in injection of coliphage T5 DNA.

Injection of T5 first-step-transfer DNA was prevented at 29 degrees C, after adsorption to an unsaturated fatty acid mutant grown on elaidate (phase transition at 35 degrees C). Local anesthetics, which increase membrane fluidity, did not inhibit injection above transition temperature and could even reverse the inhibition observed at 29 degrees C on elaidate cells.     

Factors Influencing the Production of Intermediate Particles During Alkaline Degradation of Tobacco Mosaic Virus: Time, pH, Salt Concentration, and Temperature

When T5 bacteriophage infect a colicin Ib-containing host, a variety of membrane changes and inhibition of macromolecular synthesis occur. This work shows that all these changes also occur when a mutant of T5 that can only inject 8% of its DNA is used. This indicates that all the information necessary for the abortive infection is present on this 8% (first-step-transfer) DNA.     

DNA injection during bacteriophage T4 infection of Escherichia coli.

The process of phage T4 DNA injection into the host cell was studied under a fluorescent microscope, using 4',6-diamidino-2-phenylindole as a DNA-specific fluorochrome. The phage DNA injection was observed when spheroplasts were infected with the artificially contracted phage particles having a protruding core. The DNA injection was mediated by the interaction of the core tip with the cytoplasmic membrane of the spheroplast. A membrane potential was not required for the process of DNA injection. On the other hand, DNA injection upon infection by intact noncontracted phage of the intact host cell was inhibited by an energy poison. Based on these observations, together with results from previous work, a model for the T4 infection process is presented, and the role of the membrane potential in the infection process is discussed.     

Methionine transport in Yersinia pestis.

Yersinia pestis TJW, an avirulent wild-type strain, requires phenylalanine and methionine for growth. It was of interest to examine and define the methionine transport system because of this requirement. The methionine system showed saturation kinetics with a Km for transport of approximately 9 times 10(-7) M. After 8 s of methionine transport, essentially all of the methionine label appeared in S-adenosyl-L-methionine (SAM) as detected in ethanol extracts. Small amounts of free methionine was detected intracellularly after 1 min of transport. Addition of glucose increased significantly the amount of intracellular methionine at 1 min. A series of SAM metabolic products was detected after 90 s to 5 min of transport including: 5'-thiomethyladenosine, homoserine lactone, S-adenosyl homoserine, and a fluorescent methyl receptor compound. Results from assays for SAM synthetase in spheroplast fractions showed a small (16%) but significant portion of synthetase associated with the membrane. However, most of the enzyme activity was associated with the cytoplasmic fraction. Methionine transport was characterized by a high degree of stereospecificity. No competition occurred from structurally unrelated amino acids. Although uptake was inhibited by uncoupling and sulfhydryl reagents, no efflux was observed. Results using energy inhibitors on unstarved and starved cells showed that respiratory inhibitors such as potassium cyanide (KCN) and amytal were most effective, and that arsenate was least effective. KCN plus arsenate completely blocked utilization of energy derived from glucose, and KCN completely blocked utilization of energy deived from D-lactate. The data indicate that methionine transport in Y. pestis is linked to the trapping of methionine in SAM. The results further suggest that this transport system can be classified as a permease-bound system where transport is coupled to an energized membrane state and to respiration.     

Light-dependent rubidium uptake into isolated mesophyll protoplasts from leaves of Pisum sativum

A method is described for the isolation of large amounts of physiologically active protoplasts from leaves of Pisum sativum L. Rubidium uptake was determined after separation of the intact protoplasts from the loading medium by rapid centrifugation through a phthalate step gradient. In freshly isolated mesophyll protoplasts of Pisum sativum , rubidium uptake was carbonylcyanide- p -trifluoromethoxyphenylhydrazone reduced by metabolic inhibitors such as 5 μ M , 0.1 mW cyanide, 2 μ M DCMU and 5 m M arsenate and by dark incubation. Reduction of rubidium uptake by inhibition of aerobic respiration or the photosynthetic electron transport system demonstrates that both processes play a role in the energy supply for membrane transport in these protoplasts.     

Differential effects of metabolic inhibitors on cytoplasmic membrane associated and nuclear DNA

The cellular functions necessary for transport of cytoplasmic membrane associated DNA from nucleus to cytoplasm have been investigated utilizing inhibitors of macromolecular synthesis. Hydroxyurea, fluorodeoxyuridine, cytosine arabinoside, and ethidium bromide did not prevent transport of cytoplasmic membrane associated DNA to the cytoplasm. In contrast, rifampicin and N-demethyl rifampicin totally inhibited the appearance of newly synthesized DNA on cytoplasmic membranes, while dimethyl-benzyl-demethyl rifampicin was partially inhibitory.     

The role of ATP and membrane potential in the penetration of phage T17 DNA into the cell during infection

The role of ATP and membrane potential in phage T7 DNA injection into E. coli during infection has been studied. Entrance of phage T7 genes of class II and III was shown to be prevented by arsenate, indicating the requirement for phosphorylated macroergs in the phage DNA injection. The injection process was also inhibited by exposition of the cells to the uncoupler of oxidative phosphorylation. Dependence of the injection efficiency on the membrane-potential value has been shown to be sigmoidal, which suggests a regulatory role of the membrane potential in phage T7 DNA injection from the virion into the host cell.     

Phospholipase C–mediated hydrolysis of PIP2 releases ERM proteins from lymphocyte membrane

Mechanisms controlling the disassembly of ezrin/radixin/moesin (ERM) proteins, which link the cytoskeleton to the plasma membrane, are incompletely understood. In lymphocytes, chemokine (e.g., SDF-1) stimulation inactivates ERM proteins, causing their release from the plasma membrane and dephosphorylation. SDF-1–mediated inactivation of ERM proteins is blocked by phospholipase C (PLC) inhibitors. Conversely, reduction of phosphatidylinositol 4,5-bisphosphate (PIP2) levels by activation of PLC, expression of active PLC mutants, or acute targeting of phosphoinositide 5-phosphatase to the plasma membrane promotes release and dephosphorylation of moesin and ezrin. Although expression of phosphomimetic moesin (T558D) or ezrin (T567D) mutants enhances membrane association, activation of PLC still relocalizes them to the cytosol. Similarly, in vitro binding of ERM proteins to the cytoplasmic tail of CD44 is also dependent on PIP2. These results demonstrate a new role of PLCs in rapid cytoskeletal remodeling and an additional key role of PIP2 in ERM protein biology, namely hydrolysis-mediated ERM inactivation.     

Selective release of the Treponema pallidum outer membrane and associated polypeptides with Triton X-114.

The effects of the nonionic detergent Triton X-114 on the ultrastructure of Treponema pallidum subsp. pallidum are presented in this study. Treatment of Percoll-purified motile T. pallidum with a 1% concentration of Triton X-114 resulted in cell surface blebbing followed by lysis of blebs and a decrease in diameter from 0.25-0.35 micron to 0.1-0.15 micron. Examination of thin sections of untreated Percoll-purified T. pallidum showed integrity of outer and cytoplasmic membranes. In contrast, thin sections of Triton X-114-treated treponemes showed integrity of the cytoplasmic membrane but loss of the outer membrane. The cytoplasmic cylinders generated by detergent treatment retained their periplasmic flagella, as judged by electron microscopy and immunoblotting. Recently identified T. pallidum penicillin-binding proteins also remained associated with the cytoplasmic cylinders. Proteins released by Triton X-114 at 4 degrees C were divided into aqueous and hydrophobic phases after incubation at 37 degrees C. The hydrophobic phase had major polypeptide constituents of 57, 47, 38, 33-35, 23, 16, and 14 kilodaltons (kDa) which were reactive with syphilitic serum. The 47-kDa polypeptide was reactive with a monoclonal antibody which has been previously shown to identify a surface-associated T. pallidum antigen. The aqueous phase contained the 190-kDa ordered ring molecule, 4D, which has been associated with the surface of the organisms. Full release of the 47- and 190-kDa molecules was dependent on the presence of a reducing agent. These results indicate that 1% Triton X-114 selectively solubilizes the T. pallidum outer membrane and associated proteins of likely outer membrane location.     

Action of respiratory inhibitors on the electron transport system of Escherichia coli]

Bacteria of two strains of Escherichia coli (Q13 and MRE 600) were disintegrated by aluminium oxide. The influence of the respiratory inhibitors RF (a protein from reticulocytes), carboxin, Dexon (fungicides), thenoylftrifluoroacetone (TTFA), rotenone, antimycin A, myristic acid and monolaurin was tested on the succinate oxidase and the NADH oxidase system, respectively, of the membrane preparation obtained in this way as well as on the NADH oxidase activity of the cytosol. Among the inhibitors listed, only TTFA (5mM) inhibited the succinate oxidase system and Dexon (10 miconr), monolaurin (100 micron) and myristic acid (100 micron) inhibited the NADH oxidase system of the membranes. KCN (10 micron) inhibited both NADH oxidase systems. The inhibitory effects by monolaurin and myristic acid were prevent by human serum albumin and were markedly weaker than those on beef heart mitochondrial particles under similar conditions. The results argue for a divergent structure of the iron-sulphur proteins in the dehydrogenase regions of the electron transport system in comparison with animal and plant mitochondria and, moreover, confirm the specificity of RF and carboxin as well as the nature of Dexon as a group reagent on pyridine nucleotide dependent flavin enzymes.     

Ion channels are likely to be involved in the two steps of phage T5 DNA penetration into Escherichia coli cells.

Phage T5 injects its DNA into Escherichia coli cells in two steps; 8% of the chromosome is first injected, and then there is a pause during which proteins encoded by this DNA fragment are synthesized allowing the remaining DNA to be injected. Using a potassium-selective electrode, we show that the injection of the two DNA fragments is associated with an efflux in two steps, of cytoplasmic potassium. The rate of efflux is linearly related to the number of added phages suggesting that each phage induces the formation of at least one channel in the inner membrane. The first efflux occurs even in depolarized cells suggesting that the insertion and the opening of the channel can take place in the absence of the electrochemical gradient of protons (delta mu H+). The channel is in a closed configuration during the time required for the synthesis of the phage-encoded proteins; this closing and the second efflux are prevented by the depolarization of the cell. The insertion of the channel in the inner membrane requires a fluid membrane. The results obtained suggest that the function of this channel is to translocate phage T5 DNA.     

Energy requirement for pullulanase secretion by the main terminal branch of the general secretory pathway

The energy requirement for the second step in pullulanase secretion by the general secretory pathway was studied in Escherichia coli . In order to uncouple the two steps in the secretion pathway (across the cytoplasmic and outer membranes, respectively) and to facilitate kinetic analysis of secretion, a variant form of pullulanase lacking its N-terminal fatty acid membrane anchor was used. The transport of the periplasmic secretion intermediate form of this protein across the outer membrane was not inhibited by concentrations of sodium arsenate in excess of those required to reduce ATP levels to ≤10% of their normal value. Pullulanase secretion was inhibited by the protonophore carbonyl cyanide m -chlorophenyl hydrazone at concentrations which were similar to those reported by others to be required to prevent solute uptake or the export and processing of preproteins across the cytoplasmic membrane, but which were in excess of those required to fully dissipate the proton-motive force and to reduce lactose uptake to a significant extent.     

Real-time analysis of human immunodeficiency virus type 1 Env-mediated membrane fusion by fluorescence resonance energy transfer

Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env)-mediated membrane fusion occurs as a sequence of events that is triggered by CD4 binding to the Env gp120 subunit. In this study, we analyzed the dynamics of Env-mediated membrane fusion at the single-cell level using fluorescent fusion proteins and confocal laser fluorescent microscopy. Either enhanced cyan or yellow fluorescent protein (CFP and YFP, respectively) was fused to the end of the cytoplasmic regions of the HIV-1 receptors (CD4 and CCR5) and Env proteins. Real-time imaging of membrane fusion mediated by these recombinant proteins revealed that the kinetics of fusion in our system was faster than that previously reported. Analysis of the receptor interaction by fluorescence resonance energy transfer (FRET) at the single-cell level demonstrated a tendency for oligomerization of CD4-CD4, but not of CD4-CCR5, in the absence of Env-expressing cells. However, when Env-expressing cells attached to the receptor cells, FRET produced by CD4-CCR5 interaction was increased; the FRET intensity began to decline before the formation of the fusion pore. These changes in FRET may represent the temporal association of these receptors, triggered by gp120 binding, and their dissociation during the formation of the fusion pore. In addition, the FRET analysis of receptor interactions in the presence of fusion inhibitors showed that not only inhibitors acting on CCR5 but also the gp41-derived peptide T-20 interfered with CD4-CCR5 interaction during fusion. These data suggest that T-20 could affect the formation of Env-receptors complexes during the membrane fusion.     

FhuA-mediated phage genome transfer into liposomes: a cryo-electron tomography study

BACKGROUND: The transfer of phage genomes into host cells is a well established but only dimly understood process. Following the irreversible phage binding to a receptor in the bacterial outer membrane, the DNA is ejected from the viral capsid and transferred across the bacterial cell envelope. In Escherichia coli, the mere interaction of the phage T5 with its outer membrane receptor, the ferrichrome transporter FhuA, is sufficient to trigger the release of the DNA from the phage capsid. Although the structure of FhuA has been determined at atomic resolution, the understanding of the respective roles of phage and bacterial proteins in DNA channeling and the mechanisms by which the transfer of the DNA is mediated remains fragmentary. RESULTS: We report on the use of cryo-electron tomography to analyze, at a molecular level, the interactions of T5 phages bound to FhuA-containing proteoliposomes. The resolution of the three-dimensional reconstructions allowed us to visualize the phage-proteoliposome interaction before and after release of the genome into the vesicles. After binding to its receptor, the straight fiber of the phage T5 (the "tip" of the viral tail made of pb2 proteins) traverses the lipid bilayer, allowing the transfer of its double-stranded DNA (121,000 bp) into the proteoliposome. Concomitantly, the tip of the tail undergoes a major conformational change; it shrinks in length (from 50 to 23 nm), while its diameter increases (from 2 to 4 nm). CONCLUSIONS: Taking into account the crystal structure of FhuA, we conclude that FhuA is only used as a docking site for the phage. The tip of the phage tail acts like an "injection needle," creating a passageway at the periphery of FhuA, through which the DNA crosses the membrane. A possible mechanistic scenario for the transfer of the viral genome into bacteria is discussed.     

Hybridizable sequences between cytoplasmic ribosomal RNAs and 3 micron circular DNAs of Saccharomyces cerevisiae and Torulopsis glabrata.

We have shown that 2.8 and 3.1 micron circular DNA molecules, previously reported to be present in Saccharomyces cerevisiae and Torulopsis glabrata respectively, contain sequences hybridizing to cytoplasmic ribosomal RNAs. In S. cerevisiae the 2.8 micron circular DNA appears to be identical to the rDNA repeating unit from nuclear DNA, both in length (approximately 9000 base pairs) and in the location of the 25, 18 and 5.8S rRNA sequences on the large HindIII fragment (6500 bp) and the presence of the 5S rRNA sequence on the small HindIII fragment. The 3.1 micron molecule from T. glabrata is approximately 2000 base pairs longer than the S. cerevisiae molecule and in addition, one of the HindIII sites lies within the region hybridizing to 25, 18 and 5.8S rRNAs. In S. cerevisiae the 4-5 copies of the 2.8 micron circular DNA molecules per cell, which have an extra-nuclear location, do not appear to be essential for cell viability as in one strain they were undetectable.