A duplex structure involving two non-complementary DNA strands can be formed and stabilized by M13 phage proteins

M13 phage is a long, thin nucleoprotein filament containing a single-stranded DNA loop. Exposing the filaments to a chloroform/water interface at 20 °C causes them to contract into hollow spherical particles (spheroids), while exposure at low temperatures yields short, thick rods (I-forms). All of the DNA remains within the I-forms while a specific third remains within the spheroids. Here, a photo-crosslinking reagent, psoralen, has been used to probe secondary structure of the DNA in situ in these chloroform-relaxed phage forms. Following photo-crosslinking, the DNA that had been held within the spheroids appeared to be a duplex rod when seen by electron microscopy, while the DNA extruded from the spheroids was an open single-stranded DNA loop. Photo-crosslinking of the DNA in the I-forms yielded linear duplex DNA rods close to the length of M13 phage filaments. Similar observations derived from experiments with deletion and insertion mutant phage showed that the stability of the duplex rods did not depend on the sequence homology between the two opposing strands. These results showthat two non-homologous strands of DNA can exist in an apparently duplex structure and suggest that this is directed by proteins, possibly involving interaction at a membrane.     

Minor coat protein composition and location of the A protein in bacteriophage f1 spheroids and I-forms.

The filamentous bacteriophage f1 can be transformed into a spherical particle (spheroid) or an intermediate shortened filament with a flared end (I-forms) by exposure to a chloroform-water interface at 22 or 4 degrees C, respectively. The protein composition of bacteriophage f1 spheroids and I-forms was examined by separating the proteins from the purified. [35S]cysteine-labeled particles by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis. Quantitation of the radioactivity on the gels showed that I-forms and spheroids contain the same complement of minor coat proteins as do untreated f1 phage. This composition is unchanged after removal of the DNA, either by digestion with micrococcal nuclease or by centrifugation of the particles through CsCl density gradients, indicating that none of the minor coat proteins is held in the particles solely through an interaction with the DNA. We also examined the location of the A protein in I-forms by decoration with ferritin-conjugated antibodies and examination under the electron microscope and found that the A protein is located specifically at the flared end of the I-form particle, through which the DNA is extruded and at which contraction into spheroids begins. The implications of these results with regard to the orientation of the DNA within the capsid and the process of infection are discussed.     

Coat protein conformation in M13 filaments,I-forms and spheroids

Circular dichroism studies of the filamentous coliphage M13 were carried out to determine conformational changes in the major capsid protein (the B protein) that occur during contraction of the filaments to I-forms and spheroids. The α-helicity of the B protein is somewhat lower in the I-forms than in filaments and much lower in spheroids. This conformational change may explain the increased detergent and lipid solubility of both I-forms and spheroids relative to filaments.     

Filamentous bacteriophage contract into hollow spherical particles upon exposure to a chloroform-water interface

The bacteriophage M13 is a 1 μm long filament consisting of a circular single-stranded DNA loop firmly held within a tubular protein capsid. We report here that exposure to a chloroform-water interface initiates a 20 fold contraction of each filament into a hollow protein sphere. In these 0.04 μm diameter particles, termed M13 “spheroids,” two thirds of the DNA is apparently extruded through a hole in the wall of the spheroid; the portion of DNA remaining inside the shell centers about the origins of M13 DNA replication. These results suggest that the filament, upon exposure to a membrane environment, undergoes an ordered change whereby the DNA is released into the cell and the coat protein is changed to a form more easily solubilized by the membrane lipids.     

Isolation of chloroform-resistant mutants of filamentous phage: localization in models of phage structure

Interaction of fd or M13 filamentous phage with a chloroform/water interface induces morphological change, contracting the filaments sequentially into shortened rods (I-forms), and then into spheroidal particles (S-forms). To further investigate this phage contraction, 34 and 26 chloroform-resistant isolates of fd and M13, respectively, were selected after chloroform treatment of wild-type phages at pH 8. 2 and 4 degrees C. DNA sequencing of gene VIII of the 34 fd isolates revealed five different mutants: these were D5H, M28L, V31L, I37T, and S50T. All 26 M13 isolates were I37T. These mutants exhibited variable sensitivity to chloroform, but all contracted much more slowly than wild-type phage during treatment at 4 degrees C. They all contracted like wild-type phage at 37 degrees C. Site-directed mutagenesis showed that the indicated single mutations carried the chloroform resistance. In structural models of the phage, the D5H locus is on the outside and the S50T locus is on the inside. The M28L and I37T loci are buried in a mostly hydrophobic region in the middle. Although these four mutants are spread out radially, they are localized in the axial direction into a thin disk in the model. The last mutant locus, V31L, is out of this disk, but this locus is proximal to the M28L and I37T loci and also in contact with the surface via a deep hydrophobic hole or depression. These five mutants, their locations, and their variable affects on contraction suggest that chloroform-induced contraction involves a specific mechanism rather than a generalized solvent-induced denaturation and that the critical structural changes occur in a localized level in the phage. These results add weight to suggestions that the sequential contraction of filaments-->I-forms-->S-forms mimic corresponding steps in phage penetration, and, in the reverse order, for phage assembly.     

Association of M13 I-forms and spheroids with lipid vesicles

Electron microscopy and density gradient centrifugation were used to demonstrate that the coat protein of M13 I-forms and spheroids, but not of filaments, can form some type of association with lipid vesicles in vitro. The association was detected only when the phage particles were incubated with dilauroylphosphatidylcholine (DLPC) or dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicles (SUV) above the phase transition temperature of the lipid. Under these conditions the I-form coat protein was resistant to proteolytic digestion, and the viral DNA was also associated with the vesicles.     

A model for fd phage penetration and assembly

Below 15 degrees C, chloroform causes fd phage to contract to I-forms, which are compact structures about 1/3 as long as the original phage. Above 15 degrees C, chloroform causes I-forms to contract to even more compact spheroidal S-forms. Here we show that the coat protein structure in I-forms is the same as the protein structure in the phage and the protein structure in S-forms is the same as the protein structure in bilayers. The conversions from fd----I-forms----S-forms are therefore suggested to mimic steps in fd penetration. The same conversions, in reverse order, are suggested to mimic steps in fd assembly.     

Proposed molten globule intermediates in fd phage penetration and assembly

The fd filamentous phage can be contracted to short rods called I-forms and to spheroidal particles called S-forms. The conversions from fd----I-forms----S-forms were previously suggested to mimic steps in fd penetration. The same conversions, in reverse order, were suggested to mimic steps in fd assembly. The I-forms and S-forms bind the hydrophobic probe, 1-anilino-napthalene-8-sulfonate (ANS); under the same conditions, fd binds this probe very poorly. Rigidly packed side chains in fd and nonrigidly packed side chains in I-forms and S-forms would explain the differences in ANS binding. A compilation of the properties of I-forms and S-forms indicate that: (i) they have compact structures; (ii) they have secondary structures of the same type as native phage; (iii) they have non-native morphologies; and (iv) they may have nonrigid side chain packing. These are the properties of molten globules.     

Filamentous phage are released from the bacterial membrane by a two-step mechanism involving a short C-terminal fragment of pIII.

Filamentous phage assemble at the membrane of infected cells. The phage filament is released from the membrane at the end of assembly, after four to five copies of the minor proteins, pIII and pVI, have been added to the end of the virion. In the absence of pIII or pVI, phage filaments are not released, but remain associated with the cells. The C-terminal portion of pIII, termed the "C" domain, is required for the release of stable virions.With the use of pIII C-terminal fragments of increasing size, termination of assembly can be divided into various steps. An 83-residue fragment leads to the incorporation of pVI into the assembling phage, but does not release it from the membrane. A slightly longer fragment (93 residues) is sufficient to release the particle into the culture supernatant. However, these released particles are unstable in the detergent, sarkosyl, which does not disrupt wild-type phage. A fragment of >121 residues is needed for the particle to become detergent resistant. Thus, the C-domain can be divided into two subdomains: C2, sufficient for release, and C1, required for virion stability.A model for termination of phage assembly is proposed in which pIII and pVI dock to the membrane-associated filament and form a pre- termination complex. Then, a conformational change involving the C2 domain of pIII disrupts the hydrophobic interactions with the inner membrane, releasing the phage from the cells. The pIII-mediated release of phage from the membranes points to one possible mechanism for excision of membrane-anchored protein complexes from lipid bilayers.     

Phage tf-1: a filamentous bacteriophage specific for bacteria harbouring the IncT plasmid pIN25

Phage tf-1 is a filamentous phage which is about 800 nm in length, 10 nm in width and has slightly tapered ends. The phage was isolated from sewage and formed plaques or propagated only on Escherichia coli, Salmonella typhimurium and Klebsiella oxytoca strains harbouring the IncT plasmid pIN25 at 30 degrees C. It adsorbed in large numbers to pIN25-encoded long thick flexible conjugative pili formed at 30 degrees C and also to the short form of these pili synthesized at 37 degrees C. The reason for the failure to form plaques at 37 degrees C is not known. The adsorption site is a short length of the pilus shaft extending 100-200 nm back from the distal tip. Efficient phage tf-1 adsorption to the same site was found for pili determined by other IncT plasmids in spite of the fact that phage tf-1 did not plate or propagate on strains harbouring them. However, areas of specific partial clearing on lawns of these plasmid-containing bacteria were produced by phage in high concentrations. Lack of plaque-formation could be due to inefficient intracellular assembly coupled to avid adsorption of any liberated phage to pili. The phage differs from all but one other filamentous phage by being sensitive to diethyl ether.     

Morphological Features of a Filamentous Phage of Vibrio cholerae O139 Bengal

A filamentous phage was isolated from carrier strain AI-1841 of Vibrio cholerae O139 Bengal and thus was termed fs phage. The phage was measured to be approximately 1 μm in length and 6 nm in width. One end of the phage was slightly tapered and had a fibrous appendage. The plaques developed on strain AI-4450 of V. cholerae O139 were small and turbid. The phage grew in strain AI-4450 and reached a size of 10⁸ to 10⁹ pfu/ml at 5 hr after infection without inducing any lysis of the host bacteria. The group of phages attached on rod-shaped materials like fimbriae of this bacteria, with their fibrous appendages at the pointed end, were often found in the phage-infected culture. The anti-fimbrial serum effectively inhibited the infection of fs phage to the host strain AI-4450. We thus concluded that the phage can be adsorbed on fimbriae with a fibrous appendage on the pointed end of the phage filament.     

Crystal structure of the two N-terminal domains of g3p from filamentous phage fd at 1.9 A: evidence for conformational lability.

Infection of Escherichia coli by filamentous bacteriophages is mediated by the minor phage coat protein g3p and involves two distinct cellular receptors, the F' pilus and the periplasmic protein TolA. Recently we have shown that the two receptors are contacted in a sequential manner, such that binding of TolA by the N-terminal domain g3p-D1 is conditional on a primary interaction of the second g3p domain D2 with the F' pilus. In order to better understand this process, we have solved the crystal structure of the g3p-D1D2 fragment (residues 2-217) from filamentous phage fd to 1.9 A resolution and compared it to the recently published structure of the same fragment from the related Ff phage M13. While the structure of individual domains D1 and D2 of the two phages are very similar (rms<0.7 A), there is comparatively poor agreement for the overall D1D2 structure (rms>1.2 A). This is due to an apparent movement of domain D2 with respect to D1, which results in a widening of the inter-domain groove compared to the structure of the homologous M13 protein. The movement of D2 can be described as a rigid-body rotation around a hinge located at the end of a short anti-parallel beta-sheet connecting domains D1 and D2. Structural flexibility of at least parts of the D1D2 structure was also suggested by studying the thermal unfolding of g3p: the TolA binding site on D1, while fully blocked by D2 at 37 degrees C, becomes accessible after incubation at temperatures as low as 45 degrees C. Our results support a model for the early steps of phage infection whereby exposure of the coreceptor binding site on D1 is facilitated by a conformational change in the D1D2 structure, which in vivo is induced by binding to the F' pilus on the host cell and which can be mimicked in vitro by thermal unfolding.     

The effect of combined heat and ultrasound on multicellular tumour spheroids

The effect of combined ultrasound and heat treatments on Chinese hamster multicellular spheroids of varying size was investigated using growth rate, single cell survival and ultrastructural damage as endpoints. Ultrasonic irradiation at 37 degrees C had no effect on the growth rate of 200-730 microns spheroids. Similarly there was no effect on the growth rate of 350 microns spheroids when irradiated during a 60 min exposure to 41.5 degrees C. However, spheroids of 200-700 mm diameter showed growth delay when held at 43 degrees C for 1 h. The effect was enhanced with concomitant ultrasound irradiation but was not dependent on spheroid size. When 200 and 400 microns spheroids held at 43 degrees C for 60 min were irradiated with different ultrasonic intensities a dose-dependent decrease in surviving fraction and a dose-dependent increase in growth delay was obtained. When surviving fraction was plotted as a function of growth delay a good correlation was obtained, suggesting that the combination of heat and ultrasound irradiation does not produce cytostasis in the surviving cells of either 200 or 400 microns spheroids. At the ultrastructural level increased cytoplasmic vacuolation was the only result of ultrasonic irradiation at 37 degrees C. Exposure to 43 degrees C for 60 min was required to elicit thermal damage. This took the form of membrane evagination at the spheroid surface, vacuolation of the cytoplasm, grouping of organelles around the periphery of the nucleus, and fragmentation of the nucleolus. These effects were enhanced with concomitant ultrasonic irradiation but other features were also noted, viz. disaggregation of polyribosomes, dilation of the rough endoplasmic reticulum and blebbing of the nuclear membrane. Damage was independent of spheroid size. These results are in agreement with previous data obtained from single-cell studies. Indicating that there is a non-thermal, non-cavitational component to the cell killing in multicellular spheroids resulting from combined heat and ultrasound treatment.     

The isolation and characterization of a temperate phage, Y46/(E2), from Erwinia herbicola Y40.

A temperate phage was induced from exponential phase cells of Erwinia herbicola Y46 by treatment with mitomycin C. The phage was purified by single plaque isolation, and produced in bulk by successive cultivation in young cultures of E. herbicola Y 178. Phages were concentrated from culture filtrates by rate zonal centrifugation and resuspension in 0.02 M Tris buffer, pH 7.2, twice, yielding suspensions of about 5 times 10(11) PFU/ml. Purification was achieved by centrifugation in buffered sucrose solutions. The band at the 30/40% sucrose interface yielded intact particles having regular hexagonal heads and lonb contractile tails, with base plates. Fibers were not seen. The mean dimensions were head, 51 nm; neck length, 11 nm; overall tail length, extended, 98 nm and contracted, 75 nm; diameter of tail sheath, 24 nm. The phage was stable from pH 4.0 to 11.0, but unstable at pH 3.0, the response being independent of the suspending medium used. At pH 3.0, a survival curve having biphasic appearance was observed, which was not due to a mixed population of phages. Stability to heat was good up to 45 degrees C, above which a logarithmic decline with temperature increase occurred. The average inactivation rate constant at 50 degrees C and pH 6.8 was 0.15 min-1. Adsorption to E. herbicola Y 178 cells exhibited first-order kinetics, the adsorption rate constant being 2.5 times 10(-10) ml/min. One-step growth-curve experiments indicated a burst size of 35-40, and a minimum latent period of 80 min. Probit analysis gave a mean latent period of 140 min (SD 25). The phage caused lysis of only E. herbicola strains Y178 and Y186.     

Two regions of M13 phage genome hybridizing with human DNA are similar to several keratin genes.

DNA from two regions of the phage M13 genome hybridizes with DNA restriction fragments from genomes of various species including man [15, 20]. As the pattern of hybridization is individual-specific, this phage M13 probe can be used for DNA fingerprinting. We demonstrate here that the regions of many keratin genes coding for glycine-rich parts of C and N end domains are very similar to the phage M13 probe, and this similarity may be responsible for hybridization.     

F-Actin-depolymerizing activity of human serum.

Non-heated human and animal sera contain a factor which exhibited an inhibiting activity on the staining of actin-containing structures by anti-actin antibodies in indirect immunofluorescence experiments. The presence of this factor lowered the viscosity of F-actin preparations and caused, as studied by electron-microscopy, a depolymerization of F-actin filaments as well as inhibition of filament formation of G-actin. The factor was, after its reaction with F-actin, liberated seemingly unaffected, indicating an enzymatic activity. The factor tentatively termed 'F-actin depolymerizing factor' was heat-sensitive and trypsin sensitive but resisted reduction. It was Ca2+ dependent and the staining inhibiting reaction was faster at 30 degrees C and 37 degrees C than at lower temperatures. Gel filtration experiments on Sephadex G-200 suggested a molecular size of the actin depolymerizing factor slightly higher than that of albumin. The electrophoretic mobility was that of gamma 2 globulin. The physiological role of the factor might be to prevent the presence of F-actin filaments within the circulation.     

SOS induction in Escherichia coli by infection with mutant filamentous phage that are defective in initiation of complementary-strand DNA synthesis.

We report that the SOS response is induced in Escherichia coli by infection with mutant filamentous phage that are defective in initiation of the complementary (minus)-strand synthesis. One such mutant, R377, which lacks the entire region of the minus-strand origin, failed to synthesize any detectable amount of primer RNA for minus-strand synthesis. In addition, the rate of conversion of parental single-stranded DNA of the mutant to the double-stranded replicative form in infected cells was extremely slow. Upon infection, R377 induced the SOS response in the cell, whereas the wild-type phage did not. The SOS induction was monitored by (i) induction of beta-galactosidase in a strain carrying a dinD::lacZ fusion and (ii) increased levels of RecA protein. In addition, cells infected with R377 formed filaments. Another deletion mutant of the minus-strand origin, M13 delta E101 (M. H. Kim, J. C. Hines, and D. S. Ray, Proc. Natl. Acad. Sci. USA 78:6784-6788, 1981), also induced the SOS response in E. coli. M13Gori101 (D. S. Ray, J. C. Hines, M. H. Kim, R. Imber, and N. Nomura, Gene 18:231-238, 1982), which is a derivative of M13 delta E101 carrying the primase-dependent minus-strand origin of phage G4, did not induce the SOS response. These observations indicate that single-stranded DNA by itself induces the SOS response in vivo.     

Effects of high temperature on Escherichia coli F pili.

The effects of high temperatures (46 to 50 degrees C) on the production of F pili by Escherichia coli were studied by electron microscopy. Attached F pili rapidly disappeared at 48 and 50 degrees C but not at 46 degrees C. Free pili were not denatured at these temperatures. The pili that disappeared from the cells at 50 degrees C did not appear as free pili in the culture supernatant fluid, indicating that the pili had retracted to the cell surface or into the cell. The adsorption of either R17 phage or F pili antibody to the sides of pili prevented retraction. The disappearance of pili was accompanied by a loss in the ability to adsorb R17 phage but not M13 phage, suggesting that the tip of a pilus remains exposed after retraction.     

Isolation, ultrastructure, and partial characterization of collagen from the perineurium of the Florida lobster, Panulirus argus.

Highly concentrated extracellular filaments in the perineurium of the Florida spiny lobster, Panulirus argus, were isolated using ultracentrifugation and linear sucrose gradients. The pellet obtained was highly enriched for the filaments as observed by transmission electron microscopy. Fibril diameter and axial periodicity measurements were obtained from filaments positively and negatively stained with uranyl acetate. A period between 14.0 and 25.0 nm and an average fibril diameter of 15.0 nm were observed. The filaments proved resistant to solubilization by most conventional agents and by several collagenases. NaOH (0.1 M at 100 degrees C) safely dissolved the filaments for measurements of protein content by the Lowry method and carbohydrate content with anthrone reagent. These tests revealed a protein content of approximately 84% and a high carbohydrate content of approximately 15%. Polyacrylamide electrophoresis of an acid-pepsin filament extract revealed a highly concentrated band (approximately 100,000) corresponding to the alpha-1 and alpha-2 bands of vertebrate type I collagen. Wide angle X-ray diffraction yielded meridional reflections that confirmed the filaments as collagen when compared with mammalian collagen X-ray diffraction. The amino acid composition was determined with a computer-assisted Beckman amino acid analyzer, which showed a glycine content of 279 residues/1000. Hydroxylysine and hydroxyproline were present in lower concentrations than expected.