Is the in vitro ejection of bacteriophage DNA quasistatic? A bulk to single virus study.

Bacteriophage T5 DNA ejection is a complex process that occurs on several timescales in vitro. By using a combination of bulk and single phage measurements, we quantitatively study the three steps of the ejection-binding to the host receptor, channel-opening, and DNA release. Each step is separately addressed and its kinetics parameters evaluated. We reconstruct the bulk kinetics from the distribution of single phage events by following individual DNA molecules with unprecedented time resolution. We show that, at the single phage level, the ejection kinetics of the DNA happens by rapid transient bursts that are not correlated to any genome sequence defects. We speculate that these transient pauses are due to local phase transitions of the DNA inside the capsid. We predict that such pauses should be seen for other phages with similar DNA packing ratios.

Bacteriophage T5 DNA ejection under pressure.

The transfer of the bacteriophage genome from the capsid into the host cell is a key step of the infectious process. In bacteriophage T5, DNA ejection can be triggered in vitro by simple binding of the phage to its purified Escherichia coli receptor FhuA. Using electrophoresis and cryo-electron microscopy, we measure the extent of DNA ejection as a function of the external osmotic pressure. In the high pressure range (7-16 atm), the amount of DNA ejected decreases with increasing pressure, as theoretically predicted and observed for lambda and SPP1 bacteriophages. In the low and moderate pressure range (2-7 atm), T5 exhibits an unexpected behavior. Instead of a unique ejected length, multiple populations coexist. Some phages eject their complete genome, whereas others stop at some nonrandom states that do not depend on the applied pressure. We show that contrarily to what is observed for the phages SPP1 and lambda, T5 ejection cannot be explained as resulting from a simple pressure equilibrium between the inside and outside of the capsid. Kinetics parameters and/or structural characteristics of the ejection machinery could play a determinant role in T5 DNA ejection.

Bacteriophage T5 structure reveals similarities with HK97 and T4 suggesting evolutionary relationships.

Evolutionary relationships between viruses may be obscure by protein sequence but unmasked by structure. Analysis of bacteriophage T5 by cryo-electron microscopy and protein sequence analysis reveals analogies with HK97 and T4 that suggest a mosaic of such connections. The T5 capsid is consistent with the HK97 capsid protein fold but has a different geometry, incorporating three additional hexamers on each icosahedral facet. Similarly to HK97, the T5 major capsid protein has an N-terminal extension, or Delta-domain that is missing in the mature capsid, and by analogy with HK97, may function as an assembly or scaffold domain. This Delta-domain is predicted to be largely coiled-coil, as for that of HK97, but is approximately 70% longer correlating with the larger capsid. Thus, capsid architecture appears likely to be specified by the Delta-domain. Unlike HK97, the T5 capsid binds a decoration protein in the center of each hexamer similarly to the "hoc" protein of phage T4, suggesting a common role for these molecules. The tail-tube has unusual trimeric symmetry that may aid in the unique two-stage DNA-ejection process, and joins the tail-tip at a disk where tail fibers attach. This intriguing mix of characteristics embodied by phage T5 offers insights into virus assembly, subunit function, and the evolutionary connections between related viruses.

Quantitative and qualitative hydrologic balance for a suburban watershed with a separate sewer system (Nantes, France).

A qualitative and quantitative budget at the outlet of the storm-water runoff system of a small suburban watershed is presented together with some data regarding waste-water. 445,000 m3 (34% of the rain-water volume) were drained by the storm-water runoff system and 40,879 m3 by the waste-water system from September 2002 to March 2004. Storm-water runoff is generally not heavily polluted with regard to trace metals but concentrations occasionally exceed the standards for surface water of good quality. On the contrary, pesticides (diuron and glyphosate) have very high concentrations especially in spring and autumn when their use is maximum. As the St Joseph storm-water runoff is finally discharged into the Erdre River, measures to reduce the use of these pollutants should be considered.

Characterization of a high-affinity complex between the bacterial outer membrane protein FhuA and the phage T5 protein pb5.

Binding of bacteriophage T5 to Escherichia coli cells is mediated by specific interactions between the receptor-binding protein pb5 (67.8 kDa) and the outer membrane iron-transporter FhuA. A histidine-tagged form of pb5 was overproduced and purified. Isolated pb5 is monomeric and organized mostly as beta-sheets (51%). pb5 functionality was attested in vivo by its ability to impair infection of E. coli cells by phage T5 and Phi80, and to prevent growth of bacteria on iron-ferrichrome as unique iron source. pb5 was functional in vitro, since addition of an equimolar concentration of pb5 to purified FhuA prevented DNA release from phage T5. However, pb5 alone was not sufficient for the conversion of FhuA into an open channel. Direct interaction of pb5 with FhuA was demonstrated by isolating a pb5/FhuA complex using size-exclusion chromatography. The stoichiometry, 1 mol of pb5/1 mol of FhuA, was deduced from its molecular mass, established by analytical ultracentrifugation after determination of the amount of bound detergent. SDS-PAGE and differential scanning calorimetry experiments highlighted the great stability of the complex: (i) it was not dissociated by 2% SDS even when the temperature was raised to 70 degrees C; (ii) thermal denaturation of the complex occurred at 85 degrees C, while pb5 and FhuA were denatured at 45 degrees C and 74 degrees C, respectively. The stability of the complex renders it suitable for high-resolution structural studies, allowing future analysis of conformational changes into both FhuA and pb5 upon adsorption of the virus to its host.

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.

Stability studies of FhuA, a two-domain outer membrane protein from Escherichia coli.

FhuA (MM 78.9 kDa) is an Escherichia coli outer membrane protein that transports iron coupled to ferrichrome and is the receptor for a number of bacteriophages and protein antibiotics. Its three-dimensional structure consists of a 22-stranded beta-barrel lodged in the membrane, extracellular hydrophilic loops, and a globular domain (the "cork") located within the beta-barrel and occluding it. This unexpected structure raises questions about the connectivity of the different domains and their respective roles in the different functions of the protein. To address these questions, we have compared the properties of the wild-type receptor to those of a mutated FhuA (FhuA Delta) missing a large part of the cork. Differential scanning calorimetry experiments on wild-type FhuA indicated that the cork and the beta-barrel behave as autonomous domains that unfold at 65 and 75 degrees C, respectively. Ferrichrome had a strong stabilizing effect on the loops and cork since it shifted the first transition to 71.4 degrees C. Removal of the cork destabilized the protein since a unique transition at 61.6 degrees C was observed even in the presence of ferrichrome. FhuA Delta showed an increased sensitivity to proteolysis and to denaturant agents and an impairment in phage T5 and ferrichrome binding.

Characterization of in vitro interactions between a truncated TonB protein from Escherichia coli and the outer membrane receptors FhuA and FepA.

High-affinity iron uptake in gram-negative bacteria depends upon TonB, a protein which couples the proton motive force in the cytoplasmic membrane to iron chelate receptors in the outer membrane. To advance studies on TonB structure and function, we expressed a recombinant form of Escherichia coli TonB lacking the N-terminal cytoplasmic membrane anchor. This protein (H(6)-'TonB; M(r), 24,880) was isolated in a soluble fraction of lysed cells and was purified by virtue of a hexahistidine tag located at its N terminus. Sedimentation experiments indicated that the H(6)-'TonB preparation was almost monodisperse and the protein was essentially monomeric. The value found for the Stokes radius (3.8 nm) is in good agreement with the value calculated by size exclusion chromatography. The frictional ratio (2.0) suggested that H(6)-'TonB adopts a highly asymmetrical form with an axial ratio of 15. H(6)-'TonB captured both the ferrichrome-iron receptor FhuA and the ferric enterobactin receptor FepA from detergent-solubilized outer membranes in vitro. Capture was enhanced by preincubation of the receptors with their cognate ligands. Cross-linking assays with the purified proteins in vitro demonstrated that there was preferential interaction between TonB and ligand-loaded FhuA. Purified H(6)-'TonB was found to be stable and thus shows promise for high-resolution structural studies.

High-conductance channel induced by the interaction of phage lambda with its receptor maltoporin.

Bacteriophage lambda that binds to liposomes bears its receptor maltoporin (LamB) and is able to inject its DNA into the internal space. During this process, the liposomes are permeabilized, suggesting that a transmembrane channel has formed (Roessner and Ihler (1986) J. Biol. Chem. 261, 386-390). This pore possibly constitutes the pathway used by lambda DNA to cross the membrane. We reconstituted purified LamB from Shigella in liposomes that were incubated with lambda phages. Addition of this mixture to a bilayer chamber resulted in the incorporation in planar bilayers of high-conductance channels whose conductance, kinetics and voltage dependence were totally different from those of maltoporin channels.

DNA delivery by phage as a strategy for encapsulating toroidal condensates of arbitrary size into liposomes.

We report a strategy for encapsulating and condensing DNA. When T5 phage binds to its membrane protein receptor, FhuA, its double stranded DNA (120,000 bp) is progressively released base pair after base pair in the surrounding medium. Using cryoelectron microscopy, we have visualized the structures formed after T5 phage DNA is released into neutral unilamellar proteoliposomes reconstituted with the receptor FhuA. In the presence of spermine, toroidal condensates of circumferentially wrapped DNA were formed. Most significantly, the sizes of these toroids were shown to vary, from 90 to 200 nm in their outer diameters, depending on the number of DNA stands transferred. We have also analyzed T5 DNA release in bulk solution containing the detergent-solubilized FhuA receptor. After DNA release in a spermine containing solution, huge DNA condensates with a diameter of about 300 nm were formed containing the DNAs from as many as 10-20 capsids. At alkaline pH, the condensates appeared as large hollow cylinders with a diameter of 200 nm and a height of 100-200 nm. Overall, the striking feature of our experiments is that, because of the progressive release of DNA from the phage capsid, the mechanism of toroid formation is fundamentally different from that in the classical studies in which highly dilute, "naked" DNA is condensed by direct addition of polyvalent cations; as a consequence, our method leads to toroids of arbitrary size.

Androctonin, a hydrophilic disulphide-bridged non-haemolytic anti-microbial peptide: a plausible mode of action.

Androctonin is a 25-residue non-haemolytic anti-microbial peptide isolated from the scorpion Androctonus australis and contains two disulphide bridges. Androctonin is different from known native anti-microbial peptides, being a relatively hydrophilic and non-amphipathic molecule. This raises the possibility that the target of androctonin might not be the bacterial membrane, shown to be a target for most amphipathic lytic peptides. To shed light on its mode of action on bacteria and its non-haemolytic activity, we synthesized androctonin, its fluorescent derivatives and its all-D-amino acid enantiomer. The enantiomer preserved high activity, suggesting a lipid-peptide interaction between androctonin and bacterial membranes. In Gram-positive and (at higher concentrations) Gram-negative bacteria, androctonin induced an immediate perturbation of the permeability properties of the cytoplasmic membrane of the bacterial energetic state, concomitant with perturbation of the morphology of the cell envelope as revealed by electron microscopy. Androctonin binds only to negatively charged lipid vesicles and induces the leakage of markers at high concentrations and with a slow kinetics, in contrast with amphipathic alpha-helical anti-microbial peptides that bind and permeate negatively charged vesicles, and to a smaller extent also zwitterionic ones. This might explain the selective lytic activity of androctonin towards bacteria but not red blood cells. Polarized attenuated total reflection-Fourier transform infrared spectroscopy revealed that androctonin adopts a beta-sheet structure in membranes and did not affect the lipid acyl chain order, which supports a detergent-like effect. The small size of androctonin, its hydrophilic character and its physicochemical properties are favourable features for its potential application as a replacement for commercially available antibiotics to which bacteria have developed resistance.

Phage DNA transport across membranes.

Phage nucleic acid transport is atypical in bacterial membrane transport: it is unidirectional and concerns a unique molecule the size of which may represent 50 times that of the bacterium. The rate of DNA transport, although it varies from one phage to another, can reach values as high as 3000 bp s(-1). This raises the following questions which will be discussed in this review. Is there a single mechanism of transport for all types of phages? Does the phage genome cross the outer and inner membranes by a unique mechanism? Is it transported as a free molecule or in association with proteins? How does it avoid periplasmic nucleases? Is such transport dependent on phage and/or host cell components? What is the driving force for transport? Recent cryoelectron microscopy experiments will be presented which show that it is possible to encapsulate a phage genome (121000 bp) into unilamellar liposomes. The interest of such a model system in gene delivery and in the study of the mechanisms of DNA compaction will be discussed.

An 8-A projected structure of FhuA, A "ligand-gated" channel of the Escherichia coli outer membrane.

The structure of FhuA, a siderophore and phage receptor in the outer membrane of Escherichia coli, has been investigated by electron crystallography. Bidimensional crystals of hexahistidine-tagged FhuA protein solubilized in N,N-dimethyldodecylamine-N-oxide were produced after detergent removal with polystyrene beads. Frozen-hydrated crystals (unit cell dimensions of a = 124 A, b = 98 A, gamma = 90 degrees ) exhibited a p22121 plane group symmetry. A projection map at 8 A resolution showed the presence of dimeric ring-like structures with an elliptical shape (48 x 40 A). Each monomer was composed of a ring of densities with a radial width of 8-10 A corresponding to a cylinder of beta sheets. Few densities are present inside the barrel, leaving a central channel approximately 25 A in diameter. A projection map of FhuA at 15 A resolution, which was calculated from negatively stained preparations, demonstrated that most of the central channel was masked by extramembrane domains. This map also revealed an asymmetric distribution of extramembrane domains in FhuA, with large domains located mainly on one side of the molecule. Comparison with density maps derived from recent atomic structure allowed further interpretation of the electron microscopy projection structures with regard to long hydrophilic loops governing the selectivity and opening of the channel.

FhuA, an Escherichia coli outer membrane protein with a dual function of transporter and channel which mediates the transport of phage DNA.

FhuA (M(r) = 78,900) is an Escherichia coli outer membrane protein which transports the ferric siderophore ferrichrome and is the receptor for phage T5, phi 80 and T1 and for colicin M. FhuA was purified chromatographically in non-ionic detergent (octyl glucoside). The circular dichroism spectrum indicates that FhuA is essentially organized in beta-strands like the majority of proteins of the outer membrane of Gram-negative bacteria. The structural parameters of FhuA were assessed from size exclusion chromatography, sedimention equilibrium and velocity experiments. FhuA is monomeric in solution and functional since binding of phage T5 causes the release of the phage genome, a double-stranded DNA of 121,000 base pairs, into the surrounding medium. Planar lipid bilayer experiments showed that the FhuA transporter is converted into a high conductance channel upon binding of phage T5. FhuA was reconstituted into large unilamellar vesicles (mean diameter 125 nm). Cryo-electron microscopy and fluorescence experiments, using a DNA intercalant YO-PRO 1, showed that binding of T5 to FhuA triggers the transfer of the phage genome into the proteoliposomes without altering their morphology. Two models can account for these observations, which apply both to in vitro and in vivo DNA transport. The simplest model supposes that the naked DNA is transported through the FhuA channel. Alternatively transfer of DNA might be mediated by pb2, the protein forming the phage straight fiber. pb2 would insert either directly in the membrane or inside the FhuA channel.

Mode of action of linenscin OC2 against Listeria innocua.

Linenscin OC2 is a small hydrophobic substance produced by the orange cheese coryneform bacterium Brevibacterium linens OC2. Linenscin OC2 inhibits growth of gram-negative bacteria with an altered outer membrane permeability and gram-positive bacteria. It is also able to lyse eucaryotic cells. The mode of action of linenscin OC2 on the Listeria innocua cytoplasmic membrane and the effects of environmental parameters were investigated. Addition of low doses of linenscin OC2 resulted in an immediate perturbation of the permeability properties of the cytoplasmic membrane and of the bacterial energetic state. Linenscin OC2 induced a loss of cytoplasmic potassium, depolarization of the cytoplasmic membrane, complete hydrolysis of internal ATP, efflux of inorganic phosphate, and transient increase in oxygen consumption. Potassium loss occurred in the absence of a proton motive force and was severely reduced at low temperatures, presumably as a result of increased ordering of the lipid hydrocarbon chains of the cytoplasmic membrane. We propose that linenscin OC2 interacts with the cytoplasmic membrane and that the permeability changes observed at low doses reflect the formation of pore-like structures in this membrane.

Protein-mediated DNA transfer into liposomes.

The transfer of a foreign genome into a bacterium by means of phage infection is a very efficient but poorly understood process. To analyse the mechanism of phage DNA transfer at a molecular level, we have reconstituted FhuA, the receptor for phage T5 in the outer membrane of Escherichia coli, into unilamellar vesicles made of natural phospholipids. Cryoelectron microscopy studies showed that the binding of the phage to FhuA triggered the transfer of its double-stranded DNA (121000 bp) into the proteoliposomes. DNA was entrapped within vesicles with a diameter ranging from 70 to 150 nm. The DNA appeared to be densely packed, but its presence did not alter the morphology of the liposomes, suggesting no DNA-lipid interactions. These liposomes represent an attractive model system for studying the mechanisms of DNA transport and condensation. They may also serve as alternative vehicles for the transfer of foreign genes into eukaryotic cells.

Reconstitution of FhuA, an Escherichia coli outer membrane protein, into liposomes. Binding of phage T5 to Fhua triggers the transfer of DNA into the proteoliposomes.

The Escherichia coli outer membrane protein FhuA catalyzes the transport of ferrichrome and is the receptor of bacteriophage T5. Purified FhuA was reconstituted into liposomes. The size of the proteoliposomes and the distribution of the proteins in the vesicles were determined by freeze fracture electron microscopy. Unilamellar vesicles with a diameter larger than 200 nm were observed frequently. FhuA was symetrically oriented in the proteoliposomes. Reconstituted FhuA was functional as binding of phage T5 induced the release of phage DNA and its transfer inside the vesicles.

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 < or = 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.

Studies on the energetics of proaerolysin secretion across the outer membrane of Aeromonas species. Evidence for a requirement for both the protonmotive force and ATP.

Aeromonas spp. secrete the channel-forming protein proaerolysin across their inner and outer membranes in separate steps using the general secretion pathway. Here we show that treating A. hydrophila or A. salmonicida with the protonophore carbonyl cyanide m-chorophenyl hydrazone blocks the second step in transport, secretion across the outer membrane from the periplasm, under conditions where the ATP levels in the cell are no different than the levels in control, secreting cells. A threshold for DeltaPsi was observed in the region of 120 mV, below which secretion by both species was inhibited. Treatment of cells with arsenate, which lowered ATP levels but did not affect DeltaPsi, also reduced secretion from the periplasm, an indication that there is an ATP requirement for this step independent of the requirement for DeltaPsi. Secretion across the outer membrane was also arrested by increasing the osmotic pressure of the medium, even though cellular ATP levels and DeltaPsi were not affected. This may be due to disruption of some necessary association between the inner and outer membranes.

Modeling ligand-gated receptor activity. FhuA-mediated ferrichrome efflux from lipid vesicles triggered by phage T5.

An in vitro assay of iron-ferrichrome translocation across the FhuA protein of outer membranes from Escherichia coli has been devised. Upon reconstitution into large lipid vesicles, bacteriophage T5 binds to this polyvalent receptor, triggering a conformational change that resulted in channel opening. This facilitates the translocation of an iron(III)-siderophore, without the complexities involved in the in vivo process. Efflux of 55Fe(III)-ferrichrome across FhuA channels was determined quantitatively by monitoring the release of trapped radioactivity. The assay is rapid, reliable, and specific, because other bacteriophages, such as Phi80, fail to trigger channel opening of the FhuA receptor.