ABSTRACT
The bacterial virus lambda (λ) is a temperate bacteriophage that can lysogenize host Escherichia coli (E. coli) cells. Lysogeny requires λ repressor, the cI gene product, which shuts off transcription of the phage genome. The λ N protein, in contrast, is a transcriptional antiterminator, required for expression of the terminator-distal genes, and thus, λ N mutants are growth-defective. When E. coli is infected with a λ double mutant that is defective in both N and cI (i.e., λN-cI-), at high multiplicities of 50 or more, it forms polylysogens that contain 20-30 copies of the λN-cI- genome integrated in the E. coli chromosome. Early studies revealed that the polylysogens underwent "conversion" to long filamentous cells that form tiny colonies on agar. Here, we report a large set of altered biochemical properties associated with this conversion, documenting an overall degeneration of the bacterial envelope. These properties reverted back to those of nonlysogenic E. coli as the metastable polylysogen spontaneously lost the λN-cI- genomes, suggesting that conversion is a direct result of the multiple copies of the prophage. Preliminary attempts to identify lambda genes that may be responsible for conversion ruled out several candidates, implicating a potentially novel lambda function that awaits further studies.
Subject(s)
Bacteriophage lambda/growth & development , Lysogeny/physiology , Prophages/growth & development , Bacteriophage lambda/drug effects , Bacteriophage lambda/genetics , Bacteriophage lambda/ultrastructure , Cytoplasm/drug effects , Cytoplasm/metabolism , Dactinomycin/pharmacology , Escherichia coli/virology , Genes, Viral , Lysogeny/drug effects , Membrane Proteins/metabolism , Models, Biological , Nalidixic Acid/pharmacology , Peptidoglycan/metabolism , Prophages/drug effects , Prophages/ultrastructure , Viral Proteins/metabolismABSTRACT
The long flexible tail tube of bacteriophage lambda connects its capsid to the tail tip. On infection, a DNA ejection signal is passed from the tip, along the tube to the capsid that triggers passage of the DNA down the tube and into the host bacterium. The tail tube is built from repeating units of the major tail protein, gpV, which has two distinctive domains. Its N-terminal domain has the same fold as proteins that form the rigid inner tubes of contractile tail phages, such as T4, and its C-terminal domain adopt an Ig-like fold of unknown function. We determined structures of the lambda tail tube in free tails and in virions before and after DNA ejection using cryoelectron microscopy. Modeling of the density maps reveals how electrostatic interactions and a mobile loop participate in assembly and also impart flexibility to the tube while maintaining its integrity. We also demonstrate how a common protein fold produces rigid tubes in some phages but flexible tubes in others.
Subject(s)
Bacteriophage lambda/ultrastructure , Capsid Proteins/ultrastructure , Siphoviridae/ultrastructure , Viral Tail Proteins/ultrastructure , Amino Acid Sequence/genetics , Bacteriophage lambda/genetics , Capsid/chemistry , Capsid/ultrastructure , Capsid Proteins/genetics , Cryoelectron Microscopy , Models, Molecular , Siphoviridae/genetics , Static Electricity , Viral Tail Proteins/genetics , Virion/genetics , Virion/ultrastructureABSTRACT
Complex double-stranded DNA viruses utilize a terminase enzyme to package their genomes into a preassembled procapsid shell. DNA packaging triggers a major conformational change in the proteins assembled into the shell and most often subsequent addition of a decoration protein that is required to stabilize the structure. In bacteriophage λ, DNA packaging drives a procapsid expansion transition to afford a larger but fragile shell. The gpD decoration protein adds to the expanded shell as trimeric spikes at each of the 140 three-fold axes. The spikes provide mechanical strength to the shell such that it can withstand the tremendous internal forces generated by the packaged DNA in addition to environmental insults. Hydrophobic, electrostatic, and aromatic-proline noncovalent interactions have been proposed to mediate gpD trimer spike assembly at the expanded shell surface. Here, we directly examine each of these interactions and demonstrate that hydrophobic interactions play the dominant role. In the course of this study, we unexpectedly found that Trp308 in the λ major capsid protein (gpE) plays a critical role in shell assembly. The gpE-W308A mutation affords a soluble, natively folded protein that does not further assemble into a procapsid shell, despite the fact that it retains binding interactions with the scaffolding protein, the shell assembly chaparone protein. The data support a model in which the λ procapsid shell assembles via cooperative interaction of monomeric capsid proteins, as observed in the herpesviruses and phages such as P22. The significance of the results with respect to capsid assembly, maturation, and stability is discussed.
Subject(s)
Bacteriophage lambda/chemistry , Capsid Proteins/chemistry , DNA, Viral/chemistry , Glycoproteins/chemistry , Protein Precursors/chemistry , Virus Assembly/genetics , Bacteriophage lambda/genetics , Bacteriophage lambda/metabolism , Bacteriophage lambda/ultrastructure , Biomechanical Phenomena , Capsid Proteins/genetics , Capsid Proteins/metabolism , DNA Packaging , DNA, Viral/genetics , DNA, Viral/metabolism , Gene Expression , Glycoproteins/genetics , Glycoproteins/metabolism , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Mutation , Protein Domains , Protein Folding , Protein Multimerization , Protein Precursors/genetics , Protein Precursors/metabolism , Protein Structure, Secondary , Static ElectricityABSTRACT
We compared four bacteriophage species, T5, λ, T7, and Φ29, to explore the possibilities of DNA reorganization in the capsid where the chain is highly concentrated and confined. First, we did not detect any change in DNA organization as a function of temperature between 20 to 40 °C. Second, the presence of spermine (4+) induces a significant enlargement of the typical size of the hexagonal domains in all phages. We interpret these changes as a reorganization of DNA by slight movements of defects in the structure, triggered by a partial screening of repulsive interactions. We did not detect any signal characteristic of a long-range chiral organization of the encapsidated DNA in the presence and in the absence of spermine.
Subject(s)
Bacteriophage T7/chemistry , Bacteriophage lambda/chemistry , Capsid/chemistry , DNA, Viral/chemistry , Siphoviridae/chemistry , Spermine/chemistry , Bacteriophage T7/ultrastructure , Bacteriophage lambda/ultrastructure , Capsid/ultrastructure , Cryoelectron Microscopy , DNA Packaging , DNA, Viral/ultrastructure , Nucleic Acid Conformation , Siphoviridae/ultrastructure , Species Specificity , Temperature , ThermodynamicsABSTRACT
Releasing the packaged viral DNA into the host cell is an essential process to initiate viral infection. In many double-stranded DNA bacterial viruses and herpesviruses, the tightly packaged genome is hexagonally ordered and stressed in the protein shell, called the capsid. DNA condensed in this state inside viral capsids has been shown to be trapped in a glassy state, with restricted molecular motion in vitro. This limited intracapsid DNA mobility is caused by the sliding friction between closely packaged DNA strands, as a result of the repulsive interactions between the negative charges on the DNA helices. It had been unclear how this rigid crystalline structure of the viral genome rapidly ejects from the capsid, reaching rates of 60,000 bp/s. Through a combination of single-molecule and bulk techniques, we determined how the structure and energy of the encapsidated DNA in phage λ regulates the mobility required for its ejection. Our data show that packaged λ-DNA undergoes a solid-to-fluid-like disordering transition as a function of temperature, resulting locally in less densely packed DNA, reducing DNA-DNA repulsions. This process leads to a significant increase in genome mobility or fluidity, which facilitates genome release at temperatures close to that of viral infection (37 °C), suggesting a remarkable physical adaptation of bacterial viruses to the environment of Escherichia coli cells in a human host.
Subject(s)
Bacteriophage lambda/chemistry , DNA, Viral/chemistry , Phase Transition , Virus Diseases/virology , Bacteriophage lambda/ultrastructure , Capsid/chemistry , Cryoelectron Microscopy , DNA, Viral/ultrastructure , Escherichia coli/virology , Fluorescence , Humans , Kinetics , Microscopy, Atomic Force , ThermodynamicsABSTRACT
Long DNA molecules remain difficult to image by atomic force microscopy (AFM) because of their tendency to entanglement and spontaneous formation of networks. We present a comparison of two different DNA deposition methods operating at room temperature and humidity conditions, aimed at reproducible imaging of isolated and relaxed λ DNA conformations by AFM in air. We first demonstrate that a standard deposition procedure, consisting in adsorption of DNA in the presence of divalent cations followed by washing and air-drying steps, yields a coexistence of different types of λ DNA networks with a only a few isolated DNA chains. In contrast, deposition using a spin-coating-based technique results in reproducible coverage of a significant fraction of the substrate area by isolated and relaxed λ DNA molecules, with the added benefit of a reduction in the effect of a residual layer that normally embeds DNA strands and leads to an apparent DNA height closer to the expected value. Furthermore, we show that deposition by spin-coating is also well-suited to visualize DNA-protein complexes. These results indicate that spin-coating is a simple, powerful alternative for reproducible sample preparation for AFM imaging.
Subject(s)
Aluminum Silicates/chemistry , Bacteriophage lambda/genetics , Bacteriophage lambda/ultrastructure , DNA, Viral/chemistry , DNA, Viral/ultrastructure , Microscopy, Atomic Force/methods , AdsorptionABSTRACT
Herpes simplex type 1 virus (HSV-1) and bacteriophage λ capsids undergo considerable structural changes during self-assembly and DNA packaging. The initial steps of viral capsid self-assembly require weak, non-covalent interactions between the capsid subunits to ensure free energy minimization and error-free assembly. In the final stages of DNA packaging, however, the internal genome pressure dramatically increases, requiring significant capsid strength to withstand high internal genome pressures of tens of atmospheres. Our data reveal that the loosely formed capsid structure is reinforced post-assembly by the minor capsid protein UL25 in HSV-1 and gpD in bacteriophage λ. Using atomic force microscopy nano-indentation analysis, we show that the capsid becomes stiffer upon binding of UL25 and gpD due to increased structural stability. At the same time the force required to break the capsid increases by â¼70% for both herpes and phage. This demonstrates a universal and evolutionarily conserved function of the minor capsid protein: facilitating the retention of the pressurized viral genome in the capsid. Since all eight human herpesviruses have UL25 orthologs, this discovery offers new opportunities to interfere with herpes replication by disrupting the precise force balance between the encapsidated DNA and the capsid proteins crucial for viral replication.
Subject(s)
Bacteriophage lambda/physiology , Capsid Proteins/metabolism , Capsid/ultrastructure , Glycoproteins/metabolism , Herpesvirus 1, Human/physiology , Viral Proteins/metabolism , Virus Assembly , Animals , Bacteriophage lambda/metabolism , Bacteriophage lambda/ultrastructure , Capsid/metabolism , Chlorocebus aethiops , Herpesvirus 1, Human/metabolism , Herpesvirus 1, Human/ultrastructure , Vero CellsABSTRACT
In bacteriophage λ, the overlapping open reading frames G and T are expressed by a programmed translational frameshift similar to that of the gag-pol genes of many retroviruses to produce the proteins gpG and gpGT. An analogous frameshift is widely conserved among other dsDNA tailed phages in their corresponding "G" and "GT" tail genes even in the absence of detectable sequence homology. The longer protein gpGT is known to be essential for tail assembly, but the requirement for the shorter gpG remained unclear because mutations in gene G affect both proteins. A plasmid system that can direct the efficient synthesis of tails was created and used to show that gpG and gpGT are both essential for correct tail assembly. Phage complementation assays under conditions where levels of plasmid-expressed gpG or gpGT could be altered independently revealed that the correct molar ratio of these two related proteins, normally determined by the efficiency of the frameshift, is also crucial for efficient assembly of functional tails. Finally, the physical connection between the G and T domains of gpGT, a consequence of the frameshift mechanism of protein expression, appears to be important for efficient tail assembly.
Subject(s)
Bacteriophage lambda/genetics , Bacteriophage lambda/physiology , Viral Proteins/genetics , Viral Tail Proteins/metabolism , Virus Assembly/genetics , Amino Acid Sequence , Bacteriophage lambda/metabolism , Bacteriophage lambda/ultrastructure , Base Sequence , Codon, Terminator/genetics , Codon, Terminator/physiology , Frameshift Mutation/genetics , Frameshift Mutation/physiology , Genes, Viral/physiology , Glycoproteins/genetics , Glycoproteins/metabolism , Glycoproteins/physiology , Models, Biological , Molecular Sequence Data , Open Reading Frames/genetics , Viral Proteins/metabolism , Viral Tail Proteins/genetics , Viral Tail Proteins/physiologyABSTRACT
Bacteriophage lambda is one of the most exhaustively studied of the double-stranded DNA viruses. Its assembly pathway is highly conserved among the herpesviruses and many of the bacteriophages, making it an excellent model system. Despite extensive genetic and biophysical characterization of many of the lambda proteins and the assembly pathways in which they are implicated, there is a relative dearth of structural information on many of the most critical proteins involved in lambda assembly and maturation, including that of the lambda major capsid protein. Toward this end, we have utilized a combination of chemical cross-linking/mass spectrometry and computational modeling to construct a pseudo-atomic model of the lambda major capsid protein as a monomer, as well as in the context of the assembled procapsid shell. The approach described here is generalizable and can be used to provide structural models for any biological complex of interest. The procapsid structural model is in good agreement with published biochemical data indicating that procapsid expansion exposes hydrophobic surface area and that this serves to nucleate assembly of capsid decoration protein, gpD. The model further implicates additional molecular interactions that may be critical to the assembly of the capsid shell and for the stabilization of the structure by the gpD decoration protein.
Subject(s)
Bacteriophage lambda/physiology , Capsid Proteins/chemistry , Capsid Proteins/metabolism , Capsid/chemistry , Models, Molecular , Amino Acid Sequence , Bacteriophage lambda/chemistry , Bacteriophage lambda/drug effects , Bacteriophage lambda/ultrastructure , Capsid/drug effects , Capsid/metabolism , Capsid/ultrastructure , Capsid Proteins/drug effects , Cross-Linking Reagents/pharmacology , Glycoproteins/chemistry , Glycoproteins/metabolism , Mass Spectrometry/methods , Models, Biological , Molecular Dynamics Simulation , Molecular Sequence Data , Protein Multimerization/physiology , Protein Stability/drug effects , Protein Structure, Quaternary , Validation Studies as Topic , Virus Assembly/drug effects , Virus Assembly/physiologyABSTRACT
The DNA structure in phage capsids is determined by DNA-DNA interactions and bending energy. The effects of repulsive interactions on DNA interaxial distance were previously investigated, but not the effect of DNA bending on its structure in viral capsids. By varying packaged DNA length and through addition of spermine ions, we transform the interaction energy from net repulsive to net attractive. This allowed us to isolate the effect of bending on the resulting DNA structure. We used single particle cryo-electron microscopy reconstruction analysis to determine the interstrand spacing of double-stranded DNA encapsidated in phage λ capsids. The data reveal that stress and packing defects, both resulting from DNA bending in the capsid, are able to induce a long-range phase transition in the encapsidated DNA genome from a hexagonal to a cholesteric packing structure. This structural observation suggests significant changes in genome fluidity as a result of a phase transition affecting the rates of viral DNA ejection and packaging.
Subject(s)
Bacteriophage lambda/genetics , Bacteriophage lambda/ultrastructure , Capsid/ultrastructure , DNA, Viral/ultrastructure , Cryoelectron Microscopy , DNA Packaging , DNA, Viral/chemistry , Genome, Viral , Nucleic Acid Conformation , Virus AssemblyABSTRACT
When the process of cell-fate determination is examined at single-cell resolution, it is often observed that individual cells undergo different fates even when subject to identical conditions. This "noisy" phenotype is usually attributed to the inherent stochasticity of chemical reactions in the cell. Here we demonstrate how the observed single-cell heterogeneity can be explained by a cascade of decisions occurring at the subcellular level. We follow the postinfection decision in bacteriophage lambda at single-virus resolution, and show that a choice between lysis and lysogeny is first made at the level of the individual virus. The decisions by all viruses infecting a single cell are then integrated in a precise (noise-free) way, such that only a unanimous vote by all viruses leads to the establishment of lysogeny. By detecting and integrating over the subcellular "hidden variables," we are able to predict the level of noise measured at the single-cell level.
Subject(s)
Bacteriolysis , Bacteriophage lambda/physiology , Escherichia coli/virology , Lysogeny , Bacteriological Techniques , Bacteriophage lambda/ultrastructureABSTRACT
Cryo-electron microscopy produces 3D density maps of molecular machines, which consist of various molecular components such as proteins and RNA. Segmentation of individual components in such maps is a challenging task, and is mostly accomplished interactively. We present an approach based on the immersive watershed method and grouping of the resulting regions using progressively smoothed maps. The method requires only three parameters: the segmentation threshold, a smoothing step size, and the number of smoothing steps. We first apply the method to maps generated from molecular structures and use a quantitative metric to measure the segmentation accuracy. The method does not attain perfect accuracy, however it produces single or small groups of regions that roughly match individual proteins or subunits. We also present two methods for fitting of structures into density maps, based on aligning the structures with single regions or small groups of regions. The first method aligns centers and principal axes, whereas the second aligns centers and then rotates the structure to find the best fit. We describe both interactive and automated ways of using these two methods. Finally, we show segmentation and fitting results for several experimentally-obtained density maps.
Subject(s)
Cryoelectron Microscopy/statistics & numerical data , Models, Molecular , Molecular Conformation , Algorithms , Bacteriophage lambda/chemistry , Bacteriophage lambda/ultrastructure , Chaperonin 10/chemistry , Chaperonin 10/ultrastructure , Chaperonin 60/chemistry , Chaperonin 60/ultrastructure , Computer Simulation , Protein Conformation , Protein Subunits , Reoviridae/chemistry , Reoviridae/ultrastructure , Ribosomes/chemistry , Ribosomes/ultrastructure , Static Electricity , Structural Homology, ProteinABSTRACT
The use of sub-nanometer resolution electron density as spatial constraints for de novo and ab initio structure prediction requires knowledge of protein boundaries to accurately segment the electron density for the prediction algorithms. Here we present a procedure where even poorly segmented density can be used to determine the fold of the protein. The method is automated, fast, capable of searching for multiple copies of a protein fold, and accessible to densities encompassing more than a thousand residues. The automation is particularly powerful as it allows the procedure to take full advantage of the expanding repository in the Protein Data Bank. We have tested the method on nine segmented sub-nanometer image reconstruction electron densities. The method successfully identifies the correct fold for the six densities for which an atomic structure is known, identifies a fold that agrees with prior structural data, a fold that agrees with predictions from the Fold & Function Assignment server, and a fold that correlates with secondary structure prediction. The identified folds in the last three examples can be used as templates for comparative modeling of the bacteriophage P22 tail-machine (a 3MDa complex composed of 39 protein subunits).
Subject(s)
Protein Folding , Proteins/chemistry , Proteins/ultrastructure , Algorithms , Animals , Automation , Bacterial Proteins/chemistry , Bacterial Proteins/ultrastructure , Bacteriophage P22/chemistry , Bacteriophage P22/ultrastructure , Bacteriophage lambda/chemistry , Bacteriophage lambda/ultrastructure , Cattle , Chaperonin 60/chemistry , Chaperonin 60/ultrastructure , Cryoelectron Microscopy , Crystallography, X-Ray , Databases, Protein , Image Processing, Computer-Assisted/statistics & numerical data , Models, Molecular , Reoviridae/chemistry , Reoviridae/ultrastructure , Rhodopsin/chemistry , Rhodopsin/ultrastructure , Software Design , Static ElectricityABSTRACT
Large, cooperative assemblies of proteins that wrap and/or loop genomic DNA may "epigenetically" shift configurational equilibria that determine developmental pathways. Such is the case of the lambda bacteriophage which may exhibit virulent (lytic) or quiescent (lysogenic) growth. The lysogenic state of lambda prophages is maintained by the lambda repressor (CI), which binds to tripartite operator sites in each of the O(L) and O(R) control regions located about 2.3 kbp apart on the phage genome and represses lytic promoters. Dodd and collaborators have suggested that an initial loop formed by interaction between CI bound at O(R) and O(L) provides the proper scaffold for additional CI binding to attenuate the P(RM) promoter and avoid over production of CI. Recently, the looping equilibrium as a function of CI concentration was measured using tethered particle motion analysis, but the oligomerization of CI in looped states could not be determined. Scanning force microscopy has now been used to probe these details directly. An equilibrium distribution of looped and unlooped molecules confined to a plane was found to be commensurate to that for tethered molecules in solution, and the occupancies of specific operator sites for several looped and unlooped conformations were determined. Some loops appeared to be sealed by oligomers of 6-8, most by oligomers of 10-12, and a few by oligomers of 14-16.
Subject(s)
Bacteriophage lambda/chemistry , DNA, Viral/chemistry , Repressor Proteins/chemistry , Viral Regulatory and Accessory Proteins/chemistry , Bacteriophage lambda/ultrastructure , Binding Sites , Calibration , DNA Fragmentation , DNA, Viral/ultrastructure , Microscopy, Atomic Force , Molecular Weight , Nucleic Acid Conformation , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The nanoindentation response of empty viral capsids is modeled using three-dimensional finite element analysis. Simulation with two different geometries, spherical and icosahedral, is performed using the finite element code Abaqus. The capsids are modeled as nonlinear Hookean elastic, and both small and large deformation analysis is performed. The Young's modulus is determined by calibrating the force-indentation curve to data from atomic force microscopy (AFM) experiments. Force-indentation curves for three different viral capsids are directly compared to experimental data. Predictions are made for two additional viral capsids. The results from the simulation showed a good agreement with AFM data. The paper demonstrates that over the entire range of virus sizes (or Foppl-von Karman numbers) spherical and icosahedral models yield different force responses. In particular, it is shown that capsids with dominantly spherical shape (for low Foppl-von Karman numbers) exhibit nearly linear relationship between force and indentation, which has been experimentally observed on the viral shell studies so far. However, we predict that capsids with significant faceting (for large Foppl-von Karman numbers) and thus more pronounced icosahedral shape will exhibit rather nonlinear deformation behavior.
Subject(s)
Bacteriophages/chemistry , Bacteriophages/ultrastructure , Capsid Proteins/chemistry , Capsid/chemistry , Capsid/ultrastructure , Bacteriophage lambda/chemistry , Bacteriophage lambda/ultrastructure , Calibration , Computer Simulation , Finite Element Analysis , Linear Models , Microscopy, Atomic Force , Models, Molecular , Molecular Conformation , Nonlinear DynamicsABSTRACT
We report the cryo-EM structure of bacteriophage lambda and the mechanism for stabilizing the 20-A-thick capsid containing the dsDNA genome. The crystal structure of the HK97 bacteriophage capsid fits most of the T = 7 lambda particle density with only minor adjustment. A prominent surface feature at the 3-fold axes corresponds to the cementing protein gpD, which is necessary for stabilization of the capsid shell. Its position coincides with the location of the covalent cross-link formed in the docked HK97 crystal structure, suggesting an evolutionary replacement of this gene product in lambda by autocatalytic chemistry in HK97. The crystal structure of the trimeric gpD, in which the 14 N-terminal residues required for capsid binding are disordered, fits precisely into the corresponding EM density. The N-terminal residues of gpD are well ordered in the cryo-EM density, adding a strand to a beta-sheet formed by the capsid proteins and explaining the mechanism of particle stabilization.
Subject(s)
Bacteriophage lambda/metabolism , Bacteriophage lambda/ultrastructure , Capsid Proteins/chemistry , Capsid Proteins/metabolism , Cryoelectron Microscopy , Glycoproteins/chemistry , Glycoproteins/metabolism , Bacteriophage lambda/physiology , Capsid/chemistry , Capsid/physiology , Capsid/ultrastructure , Capsid Proteins/physiology , Glycoproteins/physiology , Models, Molecular , Protein Binding , Protein Structure, Quaternary , Time Factors , Virus Assembly/physiologyABSTRACT
Recent in vitro experiments have shown that DNA ejection from bacteriophage can be partially stopped by surrounding osmotic pressure when ejected DNA is digested by DNase I in the course of ejection. In this work, we argue by a combination of experimental techniques (osmotic suppression without DNase I monitored by UV absorbance, pulse-field electrophoresis, and cryo-transmission electron microscopy visualization) and simple scaling modeling that intact genome (i.e., undigested) ejection in a crowded environment is, on the contrary, enhanced or eventually complete with the help of a pulling force resulting from DNA condensation induced by the osmotic stress itself. This demonstrates that in vivo, the osmotically stressed cell cytoplasm will promote phage DNA ejection rather than resist it. The further addition of DNA-binding proteins under crowding conditions is shown to enhance the extent of ejection. We also found some optimal crowding conditions for which DNA content remaining in the capsid upon ejection is maximum, which correlates well with the optimal conditions of maximum DNA packaging efficiency into viral capsids observed almost 20 years ago. Biological consequences of this finding are discussed.
Subject(s)
Bacteriophages/genetics , DNA, Viral/metabolism , Bacteriophage lambda/genetics , Bacteriophage lambda/ultrastructure , Bacteriophages/ultrastructure , Cryoelectron Microscopy , DNA, Viral/chemistry , DNA, Viral/ultrastructure , Dextrans/chemistry , Electrophoresis, Gel, Pulsed-Field , HMGB1 Protein/chemistry , Osmotic Pressure , Polyethylene Glycols/chemistry , Spectrophotometry, Ultraviolet , Spermine/chemistryABSTRACT
Terminase enzymes are common to complex double-stranded DNA viruses and function to package viral DNA into the capsid. We recently demonstrated that the bacteriophage lambda terminase gpA and gpNu1 proteins assemble into a stable heterotrimer with a molar ratio gpA1/gpNu1(2). This terminase protomer possesses DNA maturation and packaging activities that are dependent on the E. coli integration host factor protein (IHF). Here, we show that the protomer further assembles into a homogeneous tetramer of protomers of composition (gpA1/gpNu1(2))4. Electron microscopy shows that the tetramer forms a ring structure large enough to encircle duplex DNA. In contrast to the heterotrimer, the ring tetramer can mature and package viral DNA in the absence of IHF. We propose that IHF induced bending of viral DNA facilitates the assembly of four terminase protomers into a ring tetramer that represents the catalytically competent DNA maturation and packaging complex in vivo. This work provides, for the first time, insight into the functional assembly state of a viral DNA packaging motor.
Subject(s)
Bacteriophage lambda/enzymology , DNA Replication/genetics , DNA, Viral/biosynthesis , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/metabolism , Molecular Motor Proteins/chemistry , Molecular Motor Proteins/metabolism , Virus Assembly/physiology , Bacteriophage lambda/genetics , Bacteriophage lambda/physiology , Bacteriophage lambda/ultrastructure , Catalysis , DNA, Viral/chemistry , DNA, Viral/ultrastructure , Endodeoxyribonucleases/ultrastructure , Molecular Motor Proteins/ultrastructure , Molecular Weight , Promoter Regions, Genetic , UltracentrifugationABSTRACT
Advanced techniques for observing protein localization in live bacteria show that the distributions are dynamic. For technical reasons, most such techniques have not been applied to outer membrane proteins in Gram-negative bacteria. We have developed two novel live-cell imaging techniques to observe the surface distribution of LamB, an abundant integral outer membrane protein in Escherichia coli responsible for maltose uptake and for attachment of bacteriophage lambda. Using fluorescently labelled bacteriophage lambda tails, we quantitatively described the spatial distribution and dynamic movement of LamB in the outer membrane. LamB accumulated in spiral patterns. The distribution depended on cell length and changed rapidly. The majority of the protein diffused along spirals extending across the cell body. Tracking single particles, we found that there are two populations of LamB--one shows very restricted diffusion and the other shows greater mobility. The presence of two populations recalls the partitioning of eukaryotic membrane proteins between 'mobile' and 'immobile' populations. In this study, we have demonstrated that LamB moves along the bacterial surface and that these movements are restricted by an underlying dynamic spiral pattern.