A novel method to couple electrophysiological measurements and fluorescence imaging of suspended lipid membranes: the example of T5 bacteriophage DNA ejection.

We present an innovative method to couple electrophysiological measurements with fluorescence imaging of functionalized suspended bilayers. Our method combines several advantages: it is well suited to study transmembrane proteins that are difficult to incorporate in suspended bilayers, it allows single molecule resolution both in terms of electrophysiological measurements and fluorescence imaging, and it enables mechanical stimulations of the membrane. The approach comprises of two steps: first the reconstitution of membrane proteins in giant unilamellar vesicles; then the formation of a suspended bilayer spanning a 5 to 15 micron-wide aperture that can be visualized by high NA microscope objectives. We exemplified how the technique can be used to detect in real time the translocation of T5 DNA across the bilayer during its ejection from the bacteriophage capsid.

Bacteriophage-host interactions leading to genome internalization.

Bacteriophage infection is initiated by binding of the virion to a specific receptor located on the host surface. The genome is then released from the capsid and delivered to the host cytoplasm. Our knowledge of these early steps of infection has recently improved. The three-dimensional structure of numerous receptor binding proteins of tailed phages has been solved. Cryo-electron tomography has allowed characterization of the phage-host interactions in a cellular context and at nanometric resolution. The localization and motions of fluorescently labelled phages, receptors and viral DNA were monitored on individual bacteria. Altogether these approaches have revealed the intricacy of these early events and emphasize the link between infection and microbial architecture.

In vitro assembly of the T=13 procapsid of bacteriophage T5 with its scaffolding domain.

The Siphoviridae coliphage T5 differs from other members of this family by the size of its genome (121 kbp) and by its large icosahedral capsid (90 nm), which is organized with T=13 geometry. T5 does not encode a separate scaffolding protein, but its head protein, pb8, contains a 159-residue aminoterminal scaffolding domain (Delta domain) that is the mature capsid. We have deciphered the early events of T5 shell assembly starting from purified pb8 with its Delta domain (pb8p). The self assembly of pb8p is regulated by salt conditions and leads to structures with distinct morphologies. Expanded tubes are formed in the presence of NaCl, whereas Ca(2+) promotes the association of pb8p into contracted tubes and procapsids. Procapsids display an angular organization and 20-nm-long internal radial structures identified as the Delta domain. The T5 head maturation protease pb11 specifically cleaves the Delta domain of contracted and expanded tubes. Ca(2+) is not required for proteolytic activity but for the organization of the Delta domain. Taken together, these data indicate that pb8p carries all of the information in its primary sequence to assemble in vitro without the requirement of the portal and accessory proteins. Furthermore, Ca(2+) plays a key role in introducing the conformational diversity that permits the formation of a stable procapsid. Phage T5 is the first example of a viral capsid consisting of quasi-equivalent hexamers and pentamers whose assembly can be carried out in vitro, starting from the major head protein with its scaffolding domain, and whose endpoint is an icosahedral T=13 particle.

Quantitative time-resolved measurement of membrane protein-ligand interactions using microcantilever array sensors.

Membrane proteins are central to many biological processes, and the interactions between transmembrane protein receptors and their ligands are of fundamental importance in medical research. However, measuring and characterizing these interactions is challenging. Here we report that sensors based on arrays of resonating microcantilevers can measure such interactions under physiological conditions. A protein receptor--the FhuA receptor of Escherichia coli--is crystallized in liposomes, and the proteoliposomes then immobilized on the chemically activated gold-coated surface of the sensor by ink-jet spotting in a humid environment, thus keeping the receptors functional. Quantitative mass-binding measurements of the bacterial virus T5 at subpicomolar concentrations are performed. These experiments demonstrate the potential of resonating microcantilevers for the specific, label-free and time-resolved detection of membrane protein-ligand interactions in a micro-array format.

Phage T5 straight tail fiber is a multifunctional protein acting as a tape measure and carrying fusogenic and muralytic activities.

We report a bioinformatic and functional characterization of Pb2, a 121-kDa multimeric protein that forms phage T5 straight fiber and is implicated in DNA transfer into the host. Pb2 was predicted to consist of three domains. Region I (residues 1-1030) was mainly organized in coiled coil and shared features of tape measure proteins. Region II (residues 1030-1076) contained two alpha-helical transmembrane segments. Region III (residues 1135-1148) included a metallopeptidase motif. A truncated version of Pb2 (Pb2-Cterm, residues 964-1148) was expressed and purified. Pb2-Cterm shared common features with fusogenic membrane polypeptides. It formed oligomeric structures and inserted into liposomes triggering their fusion. Pb2-Cterm caused beta-galactosidase release from Escherichia coli cells and in vitro peptidoglycan hydrolysis. Based on these multifunctional properties, we propose that binding of phage T5 to its receptor triggers large conformational changes in Pb2. The coiled coil region would serve as a sensor for triggering the opening of the head-tail connector. The C-terminal region would gain access to the host envelope, permitting the local degradation of the peptidoglycan and the formation of the DNA pore by fusion of the two membranes.

The endonuclease domain of bacteriophage terminases belongs to the resolvase/integrase/ribonuclease H superfamily: a bioinformatics analysis validated by a functional study on bacteriophage T5.

Bacteriophage terminases are essential molecular motors involved in the encapsidation of viral DNA. They are hetero-multimers whose large subunit encodes both ATPase and endonuclease activities. Although the ATPase domain is well characterized from sequence and functional analysis, the C-terminal region remains poorly defined. We describe sequence-structure comparisons of the endonuclease region of various bacteriophages that revealed new sequence similarities shared by this region and the Holliday junction resolvase RuvC and to a lesser extent the HIV integrase and the ribonuclease H. Extensive sequence comparison and motif refinement led to a common signature of terminases and resolvases with three conserved acidic residues engaged in catalytic activity. Sequence analyses were validated by in vivo and in vitro functional assays showing that the nuclease activity of the endonuclease domain of bacteriophage T5 terminase was abolished by mutation of any of the three predicted catalytic aspartates. Overall, these data suggest that the endonuclease domains of terminases operate autonomously and that they adopt a fold similar to that of resolvases and share the same divalent cation-dependent enzymatic mechanism.

Encapsidation and transfer of phage DNA into host cells: from in vivo to single particles studies.

A remarkable property of bacteriophages is their capacity to encapsidate large amounts of DNA during morphogenesis and to maintain their genome in the capsid in a very stable form even under extreme conditions. Even as remarkable is the efficiency with which their genome is ejected from the phage particle and transferred into the host bacteria. Biophysical techniques have led to significant progresses in characterizing these mechanisms. The molecular motor of encapsidation of several phages as well as the organization of viral capsids have been described at atomic resolution. Cryo-electron microscopy and fluorescence microscopy have permitted to describe DNA ejection at the level of single phage particles. Theoretical models of encapsidation and ejection have been proposed that can be confronted to experimental data. This review will present the state of the art on the recent advances brought by biophysics in this field. Reference will be given to the work performed on double-stranded DNA phages and on one of its representative, phage T5, our working model.

The iron-siderophore transporter FhuA is the receptor for the antimicrobial peptide microcin J25: role of the microcin Val11-Pro16 beta-hairpin region in the recognition mechanism.

The role of the outer-membrane iron transporter FhuA as a potential receptor for the antimicrobial peptide MccJ25 (microcin J25) was studied through a series of in vivo and in vitro experiments. The requirement for both FhuA and the inner-membrane TonB-ExbB-ExbD complex was demonstrated by antibacterial assays using complementation of an fhuA(-) strain and by using isogenic strains mutated in genes encoding the protein complex respectively. In addition, MccJ25 was shown to block phage T5 infection of Escherichia coli, in vivo, by inhibiting phage adhesion, which suggested that MccJ25 prevents the interaction between the phage and its receptor FhuA. This in vivo activity was confirmed in vitro, as MccJ25 inhibited phage T5 DNA ejection triggered by purified FhuA. Direct interaction of MccJ25 with FhuA was demonstrated for the first time by size-exclusion chromatography and isothermal titration calorimetry. MccJ25 bound to FhuA with a 2:1 stoichiometry and a K(d) of 1.2 microM. Taken together, our results demonstrate that FhuA is the receptor for MccJ25 and that the ligand-receptor interaction may occur in the absence of other components of the bacterial membrane. Finally, both differential scanning calorimetry and antimicrobial assays showed that MccJ25 binding involves external loops of FhuA. Unlike native MccJ25, a thermolysin-cleaved MccJ25 variant was unable to bind to FhuA and failed to prevent phage T5 infection of E. coli. Therefore the Val11-Pro16 beta-hairpin region of MccJ25, which is disrupted upon cleavage by thermolysin, is required for microcin recognition.

Real-time imaging of DNA ejection from single phage particles.

Infection by tailed dsDNA phages is initiated by release of the viral DNA from the capsid and its polarized injection into the host. The driving force for the genome transport remains poorly defined. Among many hypothesis [1], it has been proposed that the internal pressure built up during packaging of the DNA in the capsid is responsible for its injection [2-4]. Whether the energy stored during packaging is sufficient to cause full DNA ejection or only to initiate the process was tested on phage T5 whose DNA (121,400 bp) can be released in vitro by mere interaction of the phage with its E. coli membrane receptor FhuA [5-7]. We present a fluorescence microscopy study investigating in real time the dynamics of DNA ejection from single T5 phages adsorbed onto a microfluidic cell. The ejected DNA was fluorescently stained, and its length was measured at different stages of the ejection after being stretched in a hydrodynamic flow. We conclude that DNA release is not an all-or-none process but occurs in a stepwise fashion and at a rate reaching 75,000 bp/sec. The relevance of this stepwise ejection to the in vivo DNA transfer is discussed.

DNA ejection from bacteriophage T5: analysis of the kinetics and energetics.

DNA ejection from bacteriophage T5 can be passively driven in vitro by the interaction with its specific host receptor. Light scattering was used to determine the physical parameters associated with this process. By studying the ejection kinetics at different temperatures, we demonstrate that an activation energy of the order of 70 k(B)T must be overcome to allow the complete DNA ejection. A complex shape of the kinetics was found whatever the temperature. This shape may be actually understood using a phenomenological model based on a multistep process. Passing from one stage to another requires the mentioned thermal activation of pressurized DNA inside the capsids. Both effects contribute to shorten or to lengthen the pause time between the different stages explaining why the T5 DNA ejection is so slow compared to other types of phage.

Main features on tailed phage, host recognition and DNA uptake.

Phage nucleic acid transport is atypical among membrane transport and thus poses a fascinating problem: transport is unidirectional; it concerns a unique molecule the size of which may represent 50 times that of the bacterium. The rate of DNA transport can reach values as high as 3 to 4 thousands base pairs/sec. This raises many questions, which will be addressed in this review. Is there a single mechanism of transport for all types of phages? How does the phage genome overcome the hydrophobic barrier of the host envelope? Is DNA transported as a free molecule or in association with proteins? Is such transport dependent on phage and/or host cell components? What is the driving force for transport? Data will be presented for a few selected tailed phages, which are the most common type of phages and for which DNA transport has been most extensively studied. Part of the review is devoted to recent in vitro data which have allowed to partly decipher the mechanism of phage T5 DNA transport.

Microcin E492 antibacterial activity: evidence for a TonB-dependent inner membrane permeabilization on Escherichia coli.

The mechanism of action of microcin E492 (MccE492) was investigated for the first time in live bacteria. MccE492 was expressed and purified to homogeneity through an optimized large-scale procedure. Highly purified MccE492 showed potent antibacterial activity at minimal inhibitory concentrations in the range of 0.02-1.2 microM. The microcin bactericidal spectrum of activity was found to be restricted to Enterobacteriaceae and specifically directed against Escherichia and Salmonella species. Isogenic bacteria that possessed mutations in membrane proteins, particularly of the TonB-ExbB-ExbD complex, were assayed. The microcin bactericidal activity was shown to be TonB- and energy-dependent, supporting the hypothesis that the mechanism of action is receptor mediated. In addition, MccE492 depolarized and permeabilized the E. coli cytoplasmic membrane. The membrane depolarization was TonB dependent. From this study, we propose that MccE492 is recognized by iron-siderophore receptors, including FepA, which promote its import across the outer membrane via a TonB- and energy-dependent pathway. MccE492 then inserts into the inner membrane, whereupon the potential becomes destabilized by pore formation. Because cytoplasmic membrane permeabilization of MccE492 occurs beneath the threshold of the bactericidal concentration and does not result in cell lysis, the cytoplasmic membrane is not hypothesized to be the sole target of MccE492.

Channeling phage DNA through membranes: from in vivo to in vitro.

We discuss current models of phage DNA transport through membranes. We present results that attempt to answer the following questions: is there a single mechanism of transport for all types of phage? is DNA transported as a free molecule or in association with proteins? what is the driving force for transport?

The biochemical and physiological characteristics of surface receptors of gram negative bacteria.

This review focuses on the properties of ferric iron surface receptors of Gram negative bacteria. We discuss the different strategies to acquire iron, and the fundamental role of these receptors in pathogenicity. The structure of some of these receptors, iron transport and regulation mechanisms are presented here.