Contractile injection systems of bacteriophages and related systems

Contractile tail bacteriophages, or myobacteriophages, use a sophisticated biomolecular structure to inject their genome into the bacterial host cell. This structure consists of a contractile sheath enveloping a rigid tube that is sharpened by a spike‐shaped protein complex at its tip. The spike complex forms the centerpiece of a baseplate complex that terminates the sheath and the tube. The baseplate anchors the tail to the target cell membrane with the help of fibrous proteins emanating from it and triggers contraction of the sheath. The contracting sheath drives the tube with its spiky tip through the target cell membrane. Subsequently, the bacteriophage genome is injected through the tube. The structural transformation of the bacteriophage T4 baseplate upon binding to the host cell has been recently described in near‐atomic detail. In this review we discuss structural elements and features of this mechanism that are likely to be conserved in all contractile injection systems (systems evolutionary and structurally related to contractile bacteriophage tails). These include the type VI secretion system (T6SS), which is used by bacteria to transfer effectors into other bacteria and into eukaryotic cells, and tailocins, a large family of contractile bacteriophage tail‐like compounds that includes the P. aeruginosa R‐type pyocins.     

Structural Study of the Serratia entomophila Antifeeding Prophage: Three-Dimensional Structure of the Helical Sheath

The sheath of the Serratia entomophila antifeeding prophage, which is pathogenic to the New Zealand grass grub Costelytra zealandica, is a 3-fold helix formed by a 4-fold symmetric repeating motif disposed around a helical inner tube. This structure, determined by electron microscopy and image processing, is distinct from that of the other known morphologically similar bacteriophage sheaths.The antifeeding prophage (Afp) of Serratia entomophila and Serratia proteamaculans is a naturally occurring virus tail-like structure which delivers a putative toxin molecule that leads to starvation of the New Zealand grass grub Costelytra zealandica (5). Afp is composed of 18 different gene products (molecular masses of 6.5 to 263 kDa). The first 16 open reading frames have orthologues (Photorhabdus virulence cassettes [PVC]) in the insecticidal bacterium Photorhabdus luminescens TTO1 genome (5). Afp and PVCs morphologically resemble a typical R-type bacteriocin (6, 12, 16) However, Afp is the only known phage tail-like protein complex that is not a bacteriocin-protein complex of considerable medical relevance that targets the same or closely related bacterial strains (1, 3, 8, 12). The major component of Afp is a contractile cylindrical outer sheath encasing an inner tube speculated to house the toxin molecule (6). A dome-shaped “head” defines one extremity of the tube, while the other end is attached to a “bell-shaped” structure with a base morphologically similar to the base plate of the T4 bacteriophage tail (9).Transmission electron micrographs of two-dimensional (2D) projections of negatively stained (Fig. ​(Fig.11 A) or frozen-hydrated and vitrified (Fig. ​(Fig.1B)1B) recombinant Afp particles (see Fig. S1 in the supplemental material) were used for computational image analysis. A globally averaged image of the Afp particle in the major configuration (called E here) (Fig. ​(Fig.1C),1C), generated using negatively stained specimens, clearly distinguished the morphologies of the various constituent structural parts. Thus, the cylindrical sheath appears to be formed by a periodic structure harboring a distinctive, inverted-V-shaped feature. A minor population of Afp particles displays an alternate configuration (called C here) where, concomitant with contraction of the sheath (averaged axial compression of ∼52% [see Table S1 in the supplemental material]), the inner tube, shorn off the bell-shaped structure, is revealed (Fig. ​(Fig.1A)1A) (6). Several other bacteriocins undergo such a high degree of compression, which has been characterized in detail for the tail sheath of bacteriophage T4 (9). We also generated individual global averages for the periodic sheath structure, for the bell-shaped structure, and for the inner tube (Fig. ​(Fig.1C)1C) which provide more accurate dimensions of these different sections (see Table S1 in the supplemental material) than those reported earlier (6).Open in a separate windowFIG. 1.(A) Electron micrograph of a negatively stained preparation of partially purified recombinant Afp particles. The gray, white, and black arrows point to an Afp particle in the extended (E) configuration, to an Afp particle with the sheath contracted, exposing the inner tube in the contracted (C) configuration, and to an inner tube with the bell-shaped structure attached at one end, respectively. (B) Cryoelectron micrograph recorded from a preparation similar to that shown in panel A. (C) Globally averaged images of Afp particles (3,026 images) in the E state and the three distinguishable parts visualized by negative staining. Bars, 200 Å. (D) Averaged power spectrum of the sheath of vitrified Afp particles. The black and white arrows indicate reflections delineating the axial rise (1/78.5 Å) and the helical pitch (1/118 Å), respectively. In panels A and C, lighter regions represent protein and the contrast is reversed in panel B. Global averages were created using classalign2 of the EMAN suite (10) and visualized in bshow (4).For a better insight, we determined the 3D structure of the central periodic section of the Afp particle in the E state. A global power spectrum derived from the cryoimages established the structure to be helical with a clear first meridional reflection at 1/78.5 Å and the first, strongest nonmeridional reflection at 1/118 Å (Fig. ​(Fig.1D).1D). These reflections correspond to the axial rise (Δx) and the pitch of the helix, respectively, and reflect a turn angle (Δψ) of about ±240° (3₂ helix) for the repeating motif. The correct sign, i.e., the hand, of the helix remains to be determined. Computationally excised overlapping segments of this helical section from images of vitrified and negatively stained Afp particles were subjected to 3D reconstruction using the iterative helical real-space reconstruction (IHRSR) algorithm (2) using the determined helical parameters (see Fig. S2 in the supplemental material). After a few iterations, the presence of an in-plane 4-fold symmetry (C4) was apparent in the density map (see Fig. S2 in the supplemental material), which was then imposed in the subsequent reconstruction exercises. However, no stable solution was forthcoming, even after many (e.g., 30) iterative cycles. This is generally indicative of the presence of heterogeneity in the form of variations in helix translation and/or twist angle (15) in the structure. As a first step, we focused our attention on the pitch value, and following classification (see Fig. S3 in the supplemental material), we found that the majority of the image segments correspond to a pitch of 120 Å. These segments were then selected out of the full data set and led to a stable and refined 3D reconstruction. We also obtained very comparable results for the helical section when images of negatively stained Afp sheath sections were used, thus supporting our computational approach (see Fig. S4 in the supplemental material) and general conclusions about the E state described below.Figure ​Figure22 A is an isosurface representation of the density map of the helical Afp sheath in the E state calculated at ∼21.5-Å resolution (see Fig. S5 in the supplemental material). To the best of our knowledge, a 4-fold rotational symmetry has not been seen for any other contractile T4 bacteriophage taillike structure, which points to the unique architecture of the Afp sheath. A power spectrum generated using the 2D projection from the final density map, compared to the experimental global power spectrum (Fig. ​(Fig.2D),2D), showed strong agreement, confirming the fidelity of the computational image analysis. The density map displays protein layers, ∼80 Å thick, that are stacked on each other in a periodic fashion. The uneven outer surface of the sheath is perforated and decorated with ∼35-Å protrusions. When rendered with a raised threshold, a characteristic feature of the map is a contiguous, high-electron-density region having an inverted-Y-shaped structure (Fig. 2C and E; see Fig. S4 in the supplemental material). At the modest ∼21.5-Å resolution, the boundary of the repeating subunit cannot be defined. A 25 ± 3-Å-wide central lumen is seen clearly when viewed along the helix axis (Fig. ​(Fig.2B)2B) and likely represents the pore of the inner tube (see also below). Using scanning transmission electron microscopy (STEM) (see Fig. S6 in the supplemental material), we estimated the averaged molecular mass of the central helical section of an Afp particle to be 9.8 ± 0.4 kDa/Å (Fig. ​(Fig.3)3) (14) and that of only the inner tube to be ∼2.5 kDa/Å, based on a relatively small pool of such images. These values translate to a mass contribution of approximately 145 kDa of the subunit whose periodic arrangement forms the outer component of the sheath (i.e., excluding the inner tube) (see Fig. S6 in the supplemental material). This value is not very different from the cumulative mass of the different proteins, i.e., homologous afp2, afp3, and afp4, thought to be involved in Afp sheath formation (5) (see Fig. S7 in the supplemental material).Open in a separate windowFIG. 2.Orthogonal isosurface rendering, at 1 σ (standard deviation) of the computed ∼21.5-Å density map of the helical sheath of the vitrified Afp particle viewed normal (A) and parallel (B) to the helix. The images were generated using the software package CHIMERA (13). The arrow indicates a surface perforation. (C) The Afp density map rendered at 3.5 σ to highlight the largest contiguous high-electron-density regions; one circumscribed by a black ellipse is computationally extracted and shown in panel E. (D) Comparison of the experimental, averaged power spectrum of the helical sheath of Afp (left) with that computed (right) from the 2D projection of the calculated density map. (F) Global average of the inner tube of the Afp particle and a plot of the surface density variation (scaled from 0 to 1) along the helical (y) axis. The dimension along the tube is plotted on the x axis.Open in a separate windowFIG. 3.(A) Dark-field micrograph of a freeze-dried, unstained preparation of Afp particles used in STEM measurements. An Afp particle in the E state, an Afp inner tube with the attached bell-shaped structure, and a tobacco mosaic virus particle, used as a calibration standard, are marked by the arrowhead and the gray and white arrows, respectively. (B) Histogram plot of the measured distributions of mass per unit length corresponding to the uniform periodic section of the Afp particles overlaid with a fitted Gaussian curve produced by using the ORIGIN6 software package.A paucity of images of the C state (∼5% of the complete data set) precluded a full, refined 3D reconstruction, but based on the available 2,774 overlapping image segments of the isolated inner tube, a global average was calculated. A plot of contrast variation (Fig. ​(Fig.2F)2F) indicates that the surface is characterized by ∼40-Å spaced elevated crests and invaginated grooves, in agreement with the calculated axial rise of ∼39 Å for the subunit (see Fig. S8 in the supplemental material) comprising the tube. Based on these preliminary results, it appears that the helical symmetry of the inner tube is markedly different from that of the sheath.Our observation that the pitch of the helix in the E state can vary by as much as ∼50 Å attests to the flexible nature of the sheath, which is required for compressibility and may be facilitated by the somewhat porous nature of the sheath (Fig. ​(Fig.2).2). Preliminary deductions (data not shown) based on a small pool of images of the C state suggest profound rearrangement of the elements of the sheath. How that translates to extrusion of the toxin remains to be revealed.      

The Type VI Secretion TssEFGK-VgrG Phage-Like Baseplate Is Recruited to the TssJLM Membrane Complex via Multiple Contacts and Serves As Assembly Platform for Tail Tube/Sheath Polymerization

The Type VI secretion system (T6SS) is a widespread weapon dedicated to the delivery of toxin proteins into eukaryotic and prokaryotic cells. The 13 T6SS subunits assemble a cytoplasmic contractile structure anchored to the cell envelope by a membrane-spanning complex. This structure is evolutionarily, structurally and functionally related to the tail of contractile bacteriophages. In bacteriophages, the tail assembles onto a protein complex, referred to as the baseplate, that not only serves as a platform during assembly of the tube and sheath, but also triggers the contraction of the sheath. Although progress has been made in understanding T6SS assembly and function, the composition of the T6SS baseplate remains mostly unknown. Here, we report that six T6SS proteins–TssA, TssE, TssF, TssG, TssK and VgrG–are required for proper assembly of the T6SS tail tube, and a complex between VgrG, TssE,-F and-G could be isolated. In addition, we demonstrate that TssF and TssG share limited sequence homologies with known phage components, and we report the interaction network between these subunits and other baseplate and tail components. In agreement with the baseplate being the assembly platform for the tail, fluorescence microscopy analyses of functional GFP-TssF and TssK-GFP fusion proteins show that these proteins assemble stable and static clusters on which the sheath polymerizes. Finally, we show that recruitment of the baseplate to the apparatus requires initial positioning of the membrane complex and contacts between TssG and the inner membrane TssM protein.     

Cryo‐EM reconstruction of Type VI secretion system baseplate and sheath distal end

The bacterial Type VI secretion system (T6SS) assembles from three major parts: a membrane complex that spans inner and outer membranes, a baseplate, and a sheath–tube polymer. The baseplate assembles around a tip complex with associated effectors and connects to the membrane complex by TssK. The baseplate assembly initiates sheath–tube polymerization, which in some organisms requires TssA. Here, we analyzed both ends of isolated non‐contractile Vibrio cholerae sheaths by cryo‐electron microscopy. Our analysis suggests that the baseplate, solved to an average 8.0 Å resolution, is composed of six subunits of TssE/F₂/G and the baseplate periphery is decorated by six TssK trimers. The VgrG/PAAR tip complex in the center of the baseplate is surrounded by a cavity, which may accommodate up to ~450 kDa of effector proteins. The distal end of the sheath, resolved to an average 7.5 Å resolution, shows sixfold symmetry; however, its protein composition is unclear. Our structures provide an important step toward an atomic model of the complete T6SS assembly.     

In vivo structures of an intact type VI secretion system revealed by electron cryotomography

The type VI secretion system (T6SS) is a versatile molecular weapon used by many bacteria against eukaryotic hosts or prokaryotic competitors. It consists of a cytoplasmic bacteriophage tail‐like structure anchored in the bacterial cell envelope via a cytoplasmic baseplate and a periplasmic membrane complex. Rapid contraction of the sheath in the bacteriophage tail‐like structure propels an inner tube/spike complex through the target cell envelope to deliver effectors. While structures of purified contracted sheath and purified membrane complex have been solved, because sheaths contract upon cell lysis and purification, no structure is available for the extended sheath. Structural information about the baseplate is also lacking. Here, we use electron cryotomography to directly visualize intact T6SS structures inside Myxococcus xanthus cells. Using sub‐tomogram averaging, we resolve the structure of the extended sheath and membrane‐associated components including the baseplate. Moreover, we identify novel extracellular bacteriophage tail fiber‐like antennae. These results provide new structural insights into how the extended sheath prevents premature disassembly and how this sophisticated machine may recognize targets.     

The gp27-like Hub of VgrG Serves as Adaptor to Promote Hcp Tube Assembly

Contractile injection systems are multiprotein complexes that use a spring-like mechanism to deliver effectors into target cells. In addition to using a conserved mechanism, these complexes share a common core known as the tail. The tail comprises an inner tube tipped by a spike, wrapped by a contractile sheath, and assembled onto a baseplate. Here, using the type VI secretion system (T6SS) as a model of contractile injection systems, we provide molecular details on the interaction between the inner tube and the spike. Reconstitution into the Escherichia coli heterologous host in the absence of other T6SS components and in vitro experiments demonstrated that the Hcp tube component and the VgrG spike interact directly. VgrG deletion studies coupled to functional assays showed that the N-terminal domain of VgrG is sufficient to interact with Hcp, to initiate proper Hcp tube polymerization, and to promote sheath dynamics and Hcp release. The interaction interface between Hcp and VgrG was then mapped using docking simulations, mutagenesis, and cysteine-mediated cross-links. Based on these results, we propose a model in which the VgrG base serves as adaptor to recruit the first Hcp hexamer and initiates inner tube polymerization.     

The HsiB1C1 (TssB-TssC) Complex of the Pseudomonas aeruginosa Type VI Secretion System Forms a Bacteriophage Tail Sheathlike Structure

Protein secretion systems in Gram-negative bacteria evolved into a variety of molecular nanomachines. They are related to cell envelope complexes, which are involved in assembly of surface appendages or transport of solutes. They are classified as types, the most recent addition being the type VI secretion system (T6SS). The T6SS displays similarities to bacteriophage tail, which drives DNA injection into bacteria. The Hcp protein is related to the T4 bacteriophage tail tube protein gp19, whereas VgrG proteins structurally resemble the gp27/gp5 puncturing device of the phage. The tube and spike of the phage are pushed through the bacterial envelope upon contraction of a tail sheath composed of gp18. In Vibrio cholerae it was proposed that VipA and VipB assemble into a tail sheathlike structure. Here we confirm these previous data by showing that HsiB1 and HsiC1 of the Pseudomonas aeruginosa H1-T6SS assemble into tubules resulting from stacking of cogwheel-like structures showing predominantly 12-fold symmetry. The internal diameter of the cogwheels is ∼100 Å, which is large enough to accommodate an Hcp tube whose external diameter has been reported to be 85 Å. The N-terminal 212 residues of HsiC1 are sufficient to form a stable complex with HsiB1, but the C terminus of HsiC1 is essential for the formation of the tubelike structure. Bioinformatics analysis suggests that HsiC1 displays similarities to gp18-like proteins in its C-terminal region. In conclusion, we provide further structural and mechanistic insights into the T6SS and show that a phage sheathlike structure is likely to be a conserved element across all T6SSs.     

Caulobacter crescentus bacteriophage phiCbK: structure and in vitro self-assembly of the tail

Electron micrographs of negatively stained and platinum-shadowed bacteriophage φCbK have been analyzed by optical diffraction and computer Fourier transformation. The results show that the phage tail is a helical “stacked disc” structure with an annular repeat of about 38 Å and with 3-fold rotational symmetry about the helix axis. Phage tails exhibited lateral and rotational flexibility and were found to possess variable helical parameters. The smaller angle of rotation about the helix axis between equivalent asymmetric units on adjacent discs measured from a number of tail images was found to have an average value of 41.5±0.9 °. Cross-sectional views of short tail fragments were obtained after sonication at 0 °C. These views confirmed the 3-fold symmetry of the 38 Å annular unit, which most probably consists of three identical subunits of the major tail protein. Formation of extended tail polymers, both linear and circular, was found to take place spontaneously in vitro after sonication. On the basis of these results, a low-resolution model for the tail helix is presented. The questions of head-tail symmetry mismatch in the phage and of tail length regulation are discussed.     

Structural and Functional Studies of gpX of Escherichia coli Phage P2 Reveal a Widespread Role for LysM Domains in the Baseplates of Contractile-Tailed Phages

A variety of bacterial pathogenicity determinants, including the type VI secretion system and the virulence cassettes from Photorhabdus and Serratia, share an evolutionary origin with contractile-tailed myophages. The well-characterized Escherichia coli phage P2 provides an excellent system for studies related to these systems, as its protein composition appears to represent the “minimal” myophage tail. In this study, we used nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of gpX, a 68-residue tail baseplate protein. Although the sequence and structure of gpX are similar to those of LysM domains, which are a large family associated with peptidoglycan binding, we did not detect a peptidoglycan-binding activity for gpX. However, bioinformatic analysis revealed that half of all myophages, including all that possess phage T4-like baseplates, encode a tail protein with a LysM-like domain, emphasizing a widespread role for this domain in baseplate function. While phage P2 gpX comprises only a single LysM domain, many myophages display LysM domain fusions with other tail proteins, such as the DNA circulation protein found in Mu-like phages and gp53 of T4-like phages. Electron microscopy of P2 phage particles with an incorporated gpX-maltose binding protein fusion revealed that gpX is located at the top of the baseplate, near the junction of the baseplate and tail tube. gpW, the orthologue of phage T4 gp25, was also found to localize to this region. A general colocalization of LysM-like domains and gpW homologues in diverse phages is supported by our bioinformatic analysis.     

Molecular assembly and structure of the bacteriophage T4 tail

The tail of bacteriophage T4 undergoes large structural changes upon infection while delivering the phage genome into the host cell. The baseplate is located at the distal end of the contractile tail and plays a central role in transmitting the signal to the tail sheath that the tailfibers have been adsorbed by a host bacterium. This then triggers the sheath contraction. In order to understand the mechanism of assembly and conformational changes of the baseplate upon infection, we have determined the structure of an in vitro assembled baseplate through the three-dimensional reconstruction of cryo-electron microscopy images to a resolution of 3.8 Å from electron micrographs. The atomic structure was fitted to the baseplate structure before and after sheath contraction in order to elucidate the conformational changes that occur after bacteriophage T4 has attached itself to a cell surface. The structure was also used to investigate the protease digestion of the assembly intermediates and the mutation sites of the tail genes, resulting in a number of phenotypes.     

Structure–Function Analysis of the C-Terminal Domain of the Type VI Secretion TssB Tail Sheath Subunit

The type VI secretion system (T6SS) is a specialized macromolecular complex dedicated to the delivery of protein effectors into both eukaryotic and bacterial cells. The general mechanism of action of the T6SS is similar to the injection of DNA by contractile bacteriophages. The cytoplasmic portion of the T6SS is evolutionarily, structurally and functionally related to the phage tail complex. It is composed of an inner tube made of stacked Hcp hexameric rings, engulfed within a sheath and built on a baseplate. This sheath undergoes cycles of extension and contraction, and the current model proposes that the sheath contraction propels the inner tube toward the target cell for effector delivery. The sheath comprises two subunits: TssB and TssC that polymerize under an extended conformation. Here, we show that isolated TssB forms trimers, and we report the crystal structure of a C-terminal fragment of TssB. This fragment comprises a long helix followed by a helical hairpin that presents surface-exposed charged residues. Site-directed mutagenesis coupled to functional assay further showed that these charges are required for proper assembly of the sheath. Positioning of these residues in the extended T6SS sheath structure suggests that they may mediate contacts with the baseplate.     

Stretching the arms of the type VI secretion sheath protein

The bacterial type VI secretion system (T6SS) is a multicomponent complex responsible for the translocation of effector proteins into the external milieu. The T6SS consists of an external sheath, an internal rigid tube, a baseplate, and a T6SS‐specific membrane complex. Secretion is accomplished by the contraction of the sheath, which expels the effector‐loaded tube. In this issue of EMBO reports, Brackmann et al 1 show how modifications of the sheath subunits can lock the T6SS assembly in the extended state. These findings allowed Wang et al 2 and Nazarov et al 3 to purify the T6SS sheath–tube–baseplate complex in the extended pre‐secretion state and to analyze its structure using cryo‐electron microscopy (cryoEM).     

Type VI secretion system sheath inter‐subunit interactions modulate its contraction

Secretion systems are essential for bacteria to survive and manipulate their environment. The bacterial type VI secretion system (T6SS) generates the force needed for protein translocation by the contraction of a long polymer called sheath. The sheath is a six‐start helical assembly of interconnected VipA/VipB subunits. The mechanism of T6SS sheath contraction is unknown. Here, we show that elongating the N‐terminal VipA linker or eliminating charge of a specific VipB residue abolishes sheath contraction and delivery of effectors into target cells. Mass spectrometry analysis identified the inner tube protein Hcp, spike protein VgrG, and other components of the T6SS baseplate significantly enriched in samples of the stable non‐contractile sheaths. The ability to lock the T6SS in the pre‐firing state opens new possibilities for understanding its mode of action.     

The tail structure of bacteriophage T4 and its mechanism of contraction

Bacteriophage T4 and related viruses have a contractile tail that serves as an efficient mechanical device for infecting bacteria. A three-dimensional cryo-EM reconstruction of the mature T4 tail assembly at 15-A resolution shows the hexagonal dome-shaped baseplate, the extended contractile sheath, the long tail fibers attached to the baseplate and the collar formed by six whiskers that interact with the long tail fibers. Comparison with the structure of the contracted tail shows that tail contraction is associated with a substantial rearrangement of the domains within the sheath protein and results in shortening of the sheath to about one-third of its original length. During contraction, the tail tube extends beneath the baseplate by about one-half of its total length and rotates by 345 degrees , allowing it to cross the host's periplasmic space.     

Afp14 is involved in regulating the length of Anti‐feeding prophage (Afp)

The anti‐feeding prophage (Afp), a phage‐tail‐like particle that causes cessation of feeding in the New Zealand grass grub, Costelytra zealandica, is encoded by 18 open reading frames (afp1–18). C‐terminal truncations of afp14 resulted in shortened Afp particles, suggesting that Afp14 is involved in Afp length determination. We constructed an Afp assembly system (afp1–18), wherein Afp14 was truncated after the N‐terminal 88 residues. This construct, when expressed in trans in Escherichia coli expressing a N‐terminal 98‐amino acid Afp14 construct, yielded fully assembled Afp but no assembled Afp was detected in the case of a N‐terminal 96‐amino acid Afp14 construct. These results suggested that the 98 N‐terminal, amino acid residues of Afp14 is crucial for the initiation of Afp assembly via baseplate formation. Trans‐based expression of wild‐type afp14 resulted in Afp particles of varying lengths, all of which were shorter than the wild‐type Afp particle. On the other hand, similar expression of Afp14 harboring a C‐terminal extension (KLLEH₆) resulted in elongated Afp particles. This information, combined with bioinformatics data, allowed us to propose a model delineating the mechanism and role of Afp14 in the maturation of the Afp particle.     

The bacteriophage T4 DNA injection machine

The tail of bacteriophage T4 consists of a contractile sheath surrounding a rigid tube and terminating in a multiprotein baseplate, to which the long and short tail fibers of the phage are attached. Upon binding of the fibers to their cell receptors, the baseplate undergoes a large conformational switch, which initiates sheath contraction and culminates in transfer of the phage DNA from the capsid into the host cell through the tail tube. The baseplate has a dome-shaped sixfold-symmetric structure, which is stabilized by a garland of six short tail fibers, running around the periphery of the dome. In the center of the dome, there is a membrane-puncturing device, containing three lysozyme domains, which disrupts the intermembrane peptidoglycan layer during infection.     

Ultrastructure and Host Specificity of Bacteriophages of Streptococcus cremoris, Streptococcus lactis subsp. diacetylactis, and Leuconostoc cremoris from Finnish Fermented Milk “Viili”

“Viili,” a fermented milk product, has a firm but viscous consistency. It is produced with traditional mesophilic mixed-strain starters, which have various stabilities in dairy practice. Thirteen morphologically different types of phages were found in 90 viili samples studied by electron microscopy. Ten of the phage types had isometric heads with long, noncontractile tails, two had elongated heads with long, noncontractile tails, and one had a unique, very long elongated head with a short tail. Further morphological differences were found in the tail size and in the presence or absence of a collar, a baseplate, and a tail fiber. To find hosts for the industrially significant phages, we examined the sensitivities of 500 bacterial isolates from starters of the viili. Seven of the phages attacked Streptococcus cremoris strains, three attacked S. lactis subsp. diacetylactis strains, and four attacked Leuconostoc cremoris strains. Some phages differed only in their host specificity. Hosts were not found for 4 of the 13 morphological types of phages.     

The Periplasmic Protein TolB as a Potential Drug Target in Pseudomonas aeruginosa

The Gram-negative bacterium Pseudomonas aeruginosa is one of the most dreaded pathogens in the hospital setting, and represents a prototype of multi-drug resistant “superbug” for which effective therapeutic options are very limited. The identification and characterization of new cellular functions that are essential for P. aeruginosa viability and/or virulence could drive the development of anti-Pseudomonas compounds with novel mechanisms of action. In this study we investigated whether TolB, the periplasmic component of the Tol-Pal trans-envelope protein complex of Gram-negative bacteria, represents a potential drug target in P. aeruginosa. By combining conditional mutagenesis with the analysis of specific pathogenicity-related phenotypes, we demonstrated that TolB is essential for P. aeruginosa growth, both in laboratory and clinical strains, and that TolB-depleted P. aeruginosa cells are strongly defective in cell-envelope integrity, resistance to human serum and several antibiotics, as well as in the ability to cause infection and persist in an insect model of P. aeruginosa infection. The essentiality of TolB for P. aeruginosa growth, resistance and pathogenicity highlights the potential of TolB as a novel molecular target for anti-P. aeruginosa drug discovery.     

Structure of the head-tail connector of bacteriophage φ29

The head-to-tail connecting region of bacteriophage φ29 has been studied by isolating neck-tail complexes from disrupted phage. These complexes can be isolated with appendages (from wild-type phage) or without appendages (from phage mutant sus12). Treatment of the neck-tail complex without appendages with urea or guanidinium hydrochloride releases the tail protein (p9) from the neck complex (proteins p10 and p11). Electron micrographs of φ29 necks show that they are composed of two collars and a thin axial tube. There is an internal hole along the longitudinal axis, from the upper collar to the thin tube.Image-processing analysis of electron micrographs of two-dimensional crystals of necks shows that the neck of phage φ29 consists of 12 external units and an internal area of apparent 6-fold symmetry, with a hole in the centre.