Preparation of Plasmid λdv from Bacteriophage λ: Role of Promoter-Operator in the Plasmid Replicon

A technique has been described for selection of bacteria carrying plasmid lambdadv. With this technique, the effects of mutations in the promoter-operators were compared on the production and perpetuation of the plasmid. It was found that "left" promoter-operator that controls leftward gene expressions can be deleted from the plasmid genome. Some mutations of "right" promoter-operator (pRoR) that controls expression of genes tof, O, and P affect the stability of the plasmid. However, the plasmid genome accomodates a variety of pRoR mutations within a reasonable but different degree of constitutivity. Some new promoter mutations that allow bypass of the pRoR cannot be carried in the plasmid genome. From these findings it was proposed that the plasmid replicon has one indispensable promoter-operator that controls expression of all the genes related to its own replication, although a variety of constitutive mutations can be accommodated in the pRorR.     

Ionic switch controls the DNA state in phage λ

We have recently found that DNA packaged in phage λ undergoes a disordering transition triggered by temperature, which results in increased genome mobility. This solid-to-fluid like DNA transition markedly increases the number of infectious λ particles facilitating infection. However, the structural transition strongly depends on temperature and ionic conditions in the surrounding medium. Using titration microcalorimetry combined with solution X-ray scattering, we mapped both energetic and structural changes associated with transition of the encapsidated λ-DNA. Packaged DNA needs to reach a critical stress level in order for transition to occur. We varied the stress on DNA in the capsid by changing the temperature, packaged DNA length and ionic conditions. We found striking evidence that the intracapsid DNA transition is ‘switched on’ at the ionic conditions mimicking those in vivo and also at the physiologic temperature of infection at 37°C. This ion regulated on-off switch of packaged DNA mobility in turn affects viral replication. These results suggest a remarkable adaptation of phage λ to the environment of its host bacteria in the human gut. The metastable DNA state in the capsid provides a new paradigm for the physical evolution of viruses.     

On the Clustered Exchanges of the Recbcd Pathway Operating on Phage λ

Lytic cycle crosses of Red(-) Gam(-) phage λ were conducted in rec(+) Escherichia coli carrying one or another plasmid with homology to λ. λ X λ recombinants and λ X plasmid recombinants were formed by RecBCD-mediated recombination. We showed previously that the act of recombining with a plasmid alters the disposition of selected λ X λ exchanges. This work reports that the relationships between the λ X plasmid and the λ X λ exchanges is unaltered by the removal from one λ parent of the homology shared with the plasmid. This result supports our view that a reciprocal exchange, allowing for cointegrate formation, is associated with but mechanistically separable from a (presumably) nonreciprocal λ X λ exchange. The nature of this relationship is independent of λ's Rap function, which is shown to alter the ratio of cointegrate formation (splices) to marker pick-up (patches) in λ X plasmid recombination mediated by the RecBCD pathway.     

Role of the tof gene in the production and perpetuation of the λdv plasmid

Molecular Genetics and Genomics - A series of λ derivatives carrying tof mutations were tested for their ability to give rise to plasmid λ dv. Phages carrying tof mutations that distorted...     

Chi-Stimulated Recombination between Phage λ and the Plasmid λdv

Chi promotes Rec-mediated recombination between phage lambda DNA and the homologous plasmid lambda dv. In the absence of Chi, some of the interactions splice lambda dv into lambda, whereas others patch information from lambda dv into lambda. When Chi is in the phage DNA, splices and patches are increased in frequency by the same factor. This result strengthens the analogy between Chi and recombination-promoting elements in fungi. It also rules out one model for the previously reported orientation dependence of Chi phenotype.     

Structure–Function Analysis of the DNA Translocating Portal of the Bacteriophage T4 Packaging Machine

Tailed bacteriophages and herpesviruses consist of a structurally well conserved dodecameric portal at a special 5-fold vertex of the capsid. The portal plays critical roles in head assembly, genome packaging, neck/tail attachment, and genome ejection. Although the structures of portals from phages φ29, SPP1, and P22 have been determined, their mechanistic roles have not been well understood. Structural analysis of phage T4 portal (gp20) has been hampered because of its unusual interaction with the Escherichia coli inner membrane. Here, we predict atomic models for the T4 portal monomer and dodecamer, and we fit the dodecamer into the cryo-electron microscopy density of the phage portal vertex. The core structure, like that from other phages, is cone shaped with the wider end containing the “wing” and “crown” domains inside the phage head. A long “stem” encloses a central channel, and a narrow “stalk” protrudes outside the capsid. A biochemical approach was developed to analyze portal function by incorporating plasmid-expressed portal protein into phage heads and determining the effect of mutations on head assembly, DNA translocation, and virion production. We found that the protruding loops of the stalk domain are involved in assembling the DNA packaging motor. A loop that connects the stalk to the channel might be required for communication between the motor and the portal. The “tunnel” loops that project into the channel are essential for sealing the packaged head. These studies established that the portal is required throughout the DNA packaging process, with different domains participating at different stages of genome packaging.     

Revisiting the NMR structure of the ultrafast downhill folding protein gpW from bacteriophage λ

GpW is a 68-residue protein from bacteriophage λ that participates in virus head morphogenesis. Previous NMR studies revealed a novel α+β fold for this protein. Recent experiments have shown that gpW folds in microseconds by crossing a marginal free energy barrier (i.e., downhill folding). These features make gpW a highly desirable target for further experimental and computational folding studies. As a step in that direction, we have re-determined the high-resolution structure of gpW by multidimensional NMR on a construct that eliminates the purification tags and unstructured C-terminal tail present in the prior study. In contrast to the previous work, we have obtained a full manual assignment and calculated the structure using only unambiguous distance restraints. This new structure confirms the α+β topology, but reveals important differences in tertiary packing. Namely, the two α-helices are rotated along their main axis to form a leucine zipper. The β-hairpin is orthogonal to the helical interface rather than parallel, displaying most tertiary contacts through strand 1. There also are differences in secondary structure: longer and less curved helices and a hairpin that now shows the typical right-hand twist. Molecular dynamics simulations starting from both gpW structures, and calculations with CS-Rosetta, all converge to our gpW structure. This confirms that the original structure has strange tertiary packing and strained secondary structure. A comparison of NMR datasets suggests that the problems were mainly caused by incomplete chemical shift assignments, mistakes in NOE assignment and the inclusion of ambiguous distance restraints during the automated procedure used in the original study. The new gpW corrects these problems, providing the appropriate structural reference for future work. Furthermore, our results are a cautionary tale against the inclusion of ambiguous experimental information in the determination of protein structures.     

Isolation of a λdv Plasmid Carrying the Bacterial gal Operon

A lambdadvgal plasmid carrying genes for controlled plasmid replication from phage lambda and the bacterial gal operon was isolated as a deletion mutant of phage lambdagalq4, which carries the gal operon between lambda genes P and Q. The plasmid DNA was found in cell extracts as covalently closed circular molecules. The plasmid was characterized by using genetic crosses, digestion with the specific endonuclease EcoRI, sucrose gradient centrifugation, and electron microscopy. In one clone analyzed, the plasmid was a complete dimer (O(lambda)P(lambda)galO(lambda)P(lambda)gal); in a subclone derived from it, the plasmid was a partial dimer with only one copy of gal (O(lambda)P(lambda)O(lambda)P(lambda)gal). The partial dimer may be a recombination product of the complete dimer, since test crosses show that the gal and lambda sequences in the plasmid can be separated by recombination. Analyses of the EcoRI digests of plasmid DNAs indicated one cleavage site per lambda gene sequence and none in the gal operon. A lambdadvgal monomer was approximately 6.7 x 10(6) daltons and the lambda gene and gal components were 3.9 x 10(6) and 2.8 x 10(6) daltons, respectively. The lambdadvgal plasmid can be introduced into a new bacterial host by transfection at an efficiency of 10(-6) per DNA molecule.     

Polar Mutations in the Left Arm of Bacteriophage λi434

By studying complementation between frameshift and nonsense mutants located in the structural genes for the head of bacteriophage lambdai434, we found mutations in gene B which are polar on genes C and D and one mutation in gene E which is polar on gene F.     

Probing the viral metallome: searching for metalloproteins in bacteriophage λ-- the hunt begins

Although the proteome and genome of bacteriophages are well developed, there is little knowledge about metals and their interactions with the phages, even though metals have been observed in stabilizing phage particles. With expanding studies of phage display and its promising applications, metalloprotein investigations in the bacteriophage areas are necessary to understand whether or not metalloproteins are included in the viral coat proteome. Since these virus studies are still in their infancy, lambda phage was chosen due to its high metal-binding potential as suggested by the cysteine/methionine rich proteins in the viral coat. After large-scale preparation and further purification of lambda phage according to standard protocols, state-of-the-art metallomics techniques via combinations of chromatographies and mass spectrometries were utilized for screening metal-associated species in lambda phage. The lambda phage sample was first separated using non-denaturing size exclusion chromatography with selective metal detection by ICPMS for screening associated metals and generating size distribution fractions for the various metal species, some of which include metalloproteins. Various molecular size distribution patterns were exhibited for the metals detected, Mn, Fe, Co, Ni, Cu and Zn, at different molecular weight ranges. On the other hand numerous other metals were not associated with the coat proteins, as they were not detected in the different molecular weight fractions. Further identification for putative metallopeptides and metalloproteins was accomplished by collecting various metal species' fractions offline and subsequently analyzing tryptically-digested fractions via nanoLC-Chip-ESI-MS. By searching appropriate MS databases with both Spectrum Mill and MASCOT search engines, the main capsid protein, gpE, a capsid decoration protein, gpD, and main tail component protein, gpV, were found and are known for associations with the detected transition metals. These findings will likely provide valuable information for lambda phage engineered applications.     

Interaction between the Sbcc Gene of Escherichia Coli and the Gam Gene of Phage λ

gam mutants of phage lambda carrying long palindromes fail to form plaques on wild-type Escherichia coli but do grow on strains that are mutant in the sbcC gene. gam + lambda carrying the same palindrome grow on both hosts and on a host deleted for the recB, C and D genes. These results suggest that the Gam protein of lambda, known to interact also with E. coli's recBCD protein, can interact with the product of the sbcC gene.     

The λ-components of the “intermediate” fractions of the carrageenan form Iridaea undulosa

The “intermediate” fractions of the carrageenan of Iridaea undulosa, with are precipitated at 1.20–1.25m, 1.35–1.40m, and 1.55–1.65m concentrations of potassium chloride, are mixtures of μ/ν-carrageenans and λ-carrageenans. The λ-carrageenans contain mainly 3-linked, 2-sulfated (33.5–39.0%) and 2,6-disulfated (5.6–11.8%) galactose units, together with 4-linked, 2-sulfated (1.8–5.9%), 6-sulfated and/or 2,6-disulfated (39.0–40.1%), and other 2,6-disubstituted (3.7–4.3%) galactose residues. The presence of this last unit suggests that some C-6 atoms must be either branching points or blocked by glycosidic linkages. When the compositions of these gel-forming λ-carrageenans are compared with those of the “soluble” λ-carrageenans from the same seaweed, it is clear that in the latter ones there is a higher diversification of the structural units and more “solubility-promoting” residues.     

Double-chain-cut sites are recombination hotspots in the red pathway of phage λ

The Red recombination pathway of phage λ is shown to target recombination to doublechain ends of DNA. A double-chain cut, delivered in vivo to only one of two parents participating in a λ lytic cross by a type II restriction endonuclease, increases the proportion of crossing over in the interval containing the cut compared with other intervals. The stimulating effect of a cut is evident whether replication is inhibited or permitted. Cut stimulation can move away from the initial cut-site, presumably by doublechain degradation. Movement of the stimulating effect of a cut is dependent on the Escherichia coli gene recA when the cross is carried out under conditions that inhibit phage replication. When replication is permitted, all aspects of cut-stimulated recombination are independent of recA. Evidence is presented to show that the reaction that is stimulated by cutting is often non-reciprocal at the molecular level.     

Structural and Functional Characterization of the Redβ Recombinase from Bacteriophage λ

The Red system of bacteriophage λ is responsible for the genetic rearrangements that contribute to its rapid evolution and has been successfully harnessed as a research tool for genome manipulation. The key recombination component is Redβ, a ring-shaped protein that facilitates annealing of complementary DNA strands. Redβ shares functional similarities with the human Rad52 single-stranded DNA (ssDNA) annealing protein although their evolutionary relatedness is not well established. Alignment of Rad52 and Redβ sequences shows an overall low level of homology, with 15% identity in the N-terminal core domains as well as important similarities with the Rad52 homolog Sak from phage ul36. Key conserved residues were chosen for mutagenesis and their impact on oligomer formation, ssDNA binding and annealing was probed. Two conserved regions were identified as sites important for binding ssDNA; a surface basic cluster and an intersubunit hydrophobic patch, consistent with findings for Rad52. Surprisingly, mutation of Redβ residues in the basic cluster that in Rad52 are involved in ssDNA binding disrupted both oligomer formation and ssDNA binding. Mutations in the equivalent of the intersubunit hydrophobic patch in Rad52 did not affect Redβ oligomerization but did impair DNA binding and annealing. We also identified a single amino acid substitution which had little effect on oligomerization and DNA binding but which inhibited DNA annealing, indicating that these two functions of Redβ can be separated. Taken together, the results provide fresh insights into the structural basis for Redβ function and the important role of quaternary structure.