Original Paper Intervirology 2007;50:264–272 DOI: 10.1159/000102469
Received: August 10, 2006 Accepted: February 15, 2007 Published online: May 8, 2007
Physicochemical Characterization of El Tor Vibriophage S20 Moumita Dutta Amar N. Ghosh Division of Electron Microscopy, National Institute of Cholera and Enteric Diseases, Kolkata, India
Abstract Objective: To characterize Vibrio cholerae El Tor typing phage S20 (ATCC No. 51352-B3). Methods and Results: The phage has a hexagonal head and a short tail. Cryo-electron microscopy and three-dimensional image reconstruction showed that the phage head has icosahedral symmetry. The phage has two major structural polypeptides of 50 and 42 kDa. Adsorption of the phage to its host followed a biphasic kinetics and its intracellular growth is characterized by a latent period of 12 min and a burst size of around 60 particles per infected cell. The phage was found to be stable at a pH range 5.0–9.0 and moderately thermotolerant and highly UV sensitive. Phage genome comprises a 40.7 8 1.5-kb linear DNA molecule with random circular permutation and terminal redundancy. The restriction endonucleases AccI, HpaII, HaeIII, HindIII, EcoRV, HincII, DraI and XmnI cut vibriophage S20 DNA. Conclusion: Vibriophage S20, which belongs to Podoviridae, has an icosahedral head and the genome, which is double-stranded linear DNA, has random circular permutation and terminal redundancy. Copyright © 2007 S. Karger AG, Basel
© 2007 S. Karger AG, Basel 0300–5526/07/0504–0264$23.50/0 Fax +41 61 306 12 34 E-Mail
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Introduction
Cholera is the most serious global health problem among all diarrheal diseases for more than a century. The causative agent of such an acute infectious disease is the highly pathogenic Gram-negative, comma-shaped, single polar flagellated bacterium Vibrio cholerae. Vibrios are broadly classified into two serotypes: O1 and non-O1 [1]. The serogroup O1 strain of V. cholerae is again subdivided into two biotypes: classical and El Tor [1]. Since 1961, the El Tor biotype slowly replaced the classical strain in global epidemics and after 1966, all cholera epidemics were due to El Tor strains [1]. Vibriophages, also known as choleraphages, are capable of infecting and lysing the bacterium V. cholerae. Phage typing of V. cholerae is a widely accepted method for intraspecies classification and has immense practical value for tracking down cholera epidemics [1]. In 1968, a new phage typing scheme was developed for V. cholerae biotype El Tor by Basu and Mukerjee [2] using a set of five bacteriophages, namely group I–V. Later this scheme was found to be inadequate for epidemiological purposes as most strains are clustered into two phage types [3]. To overcome those limitations a new phage typing scheme has been developed by Chattopadhyay et al. [4] including five new phages, namely N4, S5, S20, M4 and D10 besides the five phages of Basu and Mukerjee.
Amar N. Ghosh Division of Electron Microscopy National Institute of Cholera and Enteric Diseases P-33, C.I.T. Road, Scheme-XM, Beleghata, Kolkata 700010 (India) Tel. +91 33 2370 0448, Fax +91 33 2370 5066, E-Mail
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Key Words Vibriophage Choleraphage DNA Partial denaturation mapping Cryo-electron microscopy
Media and Buffers The nutrient broth (Himedia) used as the bacterial growth medium contained peptic digest of animal tissue 5 g, NaCl 5 g, beef extract 1.5 g, yeast extract 1.5 g in a liter with final pH 7.4. Nutrient agar plates (solid bottom agar) contained 15 g agar in nutrient broth (Himedia) in a liter with final pH 7.4. The phage titer was found to be higher in the soft agar overlay method than liquid nutrient broth culture. The soft top agar for overlay plating contained 1% agar in nutrient broth (Himedia). Phages were kept as suspensions in 50 mM Tris-HCl pH 7.4–10 mM MgCl2 (TM buffer).
Chemicals As a gel standard, a protein mixture (PMW-M) containing phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), soyabean trypsin inhibitor (20.1 kDa) and lysozyme (14.3 kDa) was used from Bangalore Genei Pvt. Ltd. (India). Formamide was used from Sigma Chemical Co. (St. Louis, Mo., USA) and cytochrome C was used from Polaron equipment limited (Division of BioRad, Watford, UK).
Phage Techniques Plate Stock Preparation. Phage lysate of S20 was prepared from single plaque isolates using V. cholerae O1 biotype El Tor MAK757 as the propagating strain by the soft agar overlay method. Concentration of Vibriophage S20 Solutions. The phage lysate was centrifuged in a Sorvall ultracentrifuge with a fixed-angle 13094 rotor at 35,000 rpm at 4 ° C for 1.5 h. The pellet was re-suspended in 1.5 ml of TM buffer, pH 7.4 to concentrate the phage solution and stored at 4 ° C. Purification of Phage Stock. The phage was found to be unstable in CsCl. The concentrated phage particles were purified by a preformed sucrose step gradient (10–40%) ultracentrifugation in a Sorvall ultracentrifuge swing-out TH-660 rotor at 35,000 rpm at 4 ° C for 1.5 h. Purified phage solution was re-suspended in 1 ml TM buffer and dialyzed at 4 ° C overnight against TM buffer with three changes and stored at 4 ° C. Adsorption Kinetics of Phage S20. This experiment was done as described for bacteriophage T4 [13]. Briefly, 4 ! 108 exponentially growing V. cholerae cells per ml at 37 ° C were infected with a multiplicity of infection (m.o.i.) of 0.1. After infection, every 2 min over a period of 20 min, the concentration of nonadsorbed phages was determined by diluting the solution and plating it. One Step Growth Curve of S20. The one step growth parameters were studied as described by Stent [13]. About 4 ! 108 exponentially growing V. cholerae cells per ml at 37 ° C were infected with an m.o.i. of 0.1. Five minutes after infection an aliquot was withdrawn and titrated for free phages. Eight minutes after infection the culture was diluted and samples were titrated at various time points and plated. pH Inactivation. Aliquots of nutrient broth medium adjusted to pH 3–13 with 1 N HCl or 1 N NaOH with enough phage to give an initial titer of 107 pfu/ml were incubated at 37 ° C. After 1 h incubation samples were withdrawn, diluted and assayed for surviving phages. The survival at any pH was expressed as percentage of the maximum survival [14]. Thermal Inactivation. The thermal inactivation kinetics of the phage S20 was studied in nutrient broth medium at 50, 60 and 70 ° C [14]. UV Inactivation. Vibriophage S20 suspension (108 pfu/ml) in TM buffer was exposed for irradiation at room temperature (26– 28 ° C) in the dark with constant agitation. UV source was a 15watt Philips germicidal lamp emitting primarily at 254 nm. The distance of sample from source was 55 cm. At different time intervals 0.1 ml sample was removed and assayed for pfu by agar overlay method [14]. SDS-Polyacrylamide Gel Electrophoresis. Bacteriophage protein samples were analyzed under denaturing conditions by electrophoresis in SDS-polyacrylamide gel following the method as described by Laemmli [15] except that a 12% slab gel apparatus
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Methods Bacterial Strain and Bacteriophage MAK757, the universal strain for propagating V. cholerae O1 biotype El Tor phages was used in this study for the growth of vibriophage S20 [ATCC (The American Type Culture Collection) No. 51352-B3].
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Characterization of vibriophages are important for the phage taxonomy [5]. Phages have also been used as therapeutic agent but could not be clearly established as a preventive measure against cholera [6]. However, emergence of multi-antibiotic resistant bacteria renewed interest in the possibilities of bacteriophage therapy. Vibriophages have also been used to construct potential vaccine strains against the deadly disease cholera using temperate phages VcA-1 and a temperature-sensitive derivative of VcA-2 deleting their cholera toxin gene [5]. Presence of vibriophages is found to be inversely proportional with the V. cholerae strains present in the aquatic environment which in turn negatively influence the seasonal outbreak of cholera cases [7]. Amongst lytic El Tor vibriophages till now e5 [8], e4 [9], N4 [10], N5 [11] and D10 [12] were characterized at their molecular level by different physicochemical methods and room temperature electron microscopy. Cryoelectron microscopy, which is a better choice for visualization of closer-to-native structure, has not been used for deducing the morphology of vibriophages. The unstained, frozen-hydrated phage particles are examined by cryo-electron microscopy. The present study deals with the characterization of vibriophage S20 which infect and lyse V. cholerae El Tor biotypes. Parameters such as latent period, burst size, adsorption kinetics, UV tolerance, thermotolerance were determined. The phage genome was characterized using electron microscopy and other biochemical techniques. Three-dimensional (3D) image reconstruction was done by cryo-electron microscopy and single particle analysis method. To our knowledge this is the first report of 3D image reconstruction of any vibriophage using cryo-electron microscopy.
DNA Methods Isolation of DNA. Phage S20 DNA was isolated using the phenol-chloroform extraction method as described for bacteriophage lambda [16]. The phage DNA was dialyzed against 20 mM NaCl-5 mM EDTA buffer, pH 7.4 for 48 h at 4 ° C and stored at –20 ° C. Restriction Endonuclease Digestion and Agarose Gel Electrophoresis of DNA. The digestions were performed according to the instructions of the manufacturer (Bangalore Genei Pvt. Ltd., India). The generated DNA fragments were separated by agarose gel electrophoresis. Electron Microscopy Negative Staining. Carbon-coated copper grids were made hydrophilic by subjecting them to glow-discharge in a Jeol HDT 400 hydrophilic treatment device before use. A drop of S20 phage was placed on a glow-discharged grid for 1 min. The excess liquid was withdrawn with filter paper and 2 drops of 2% aqueous uranyl acetate was applied to the grid for 30 s. The excess stain was drawn off and the grid was air dried. Specimen Preparation and Cryo-Electron Microscopy of the Phages. For cryo-electron microscopy holey carbon grids were used. 1.5 l of purified phage suspension (1011 pfu/ml) was put on a holey carbon grid that was made hydrophilic on a glow-discharge unit immediately before use. After blotting nearly to dry with a piece of filter paper, the grid was subjected to ultra-rapid freezing by plunging it quickly into liquid ethane at –180 ° C and a thin layer of vitreous ice was formed in the holes of the support films. The procedure was carried out using a Leica EM CPC instrument. For visualization, the grid was placed on a GATAN 626DH cryo-holder and was inserted into the electron microscope (FEI Tecnai 12 BioTwin fitted with a SIS MegaView III CCD camera). Electron micrographs of frozen, hydrated phages were taken using low-dose software at a defocus value of –1 m. Image Processing and Visualization. The 3D reconstruction of the phage was performed using EMAN 1.61 software [17] operating on Linux Red Hat 8 platform. Images of individual DNA-filled virus particles were selected, centered and manually boxed out with the boxer program of the EMAN package. For all data sets, rotationally averaged power spectra were calculated from all of the boxed out particles, noise parameters of contrast transfer function (CTF) were manually adjusted to fit the power spectra, all images were individually phase-flipped and CTF parameters were saved using the ctfit program of the EMAN package. 3D model was visualized using Chimera software [18]. For determination of final resolution eotest was performed using EMAN. Radial density plot was done with bradial program of Bsoft software [19]. DNA Spreading. DNA was spread by the basic protein monolayer technique of Kleinschmidt et al. [20] with the modification described by Inman [21]. The hyperphase or spreading solution contained S20 DNA (0.1 g/ml), pBR322 marker DNA (0.025 g/ ml), 1 mM Na 2CO3, 1 mM EDTA, 50% formamide, 0.01% cyto-
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chrome C, at pH 7.4. The hypophase was double distilled water. Carbon-coated copper grid was used to pick up the DNA and stained with alcoholic uranyl acetate. Partial Denaturation Mapping of S20 DNA. The phage DNA was exposed to a high pH buffer at which the DNA molecule was partially denatured (preferentially AT-rich regions) and formaldehyde was used so that it could react with the single-stranded DNA to prevent subsequent re-annealing [21]. The DNA solution (7 l) was mixed with the 3 l high pH buffer containing 0.0678 M Na 2CO3, 0.0107 M EDTA and 34% formaldehyde and incubated at 37 8 1 ° C for 10–15 min. Formamide and cytochrome C to a final concentration of 50 and 0.01%, respectively, were added and chilled on ice for 5 min and spread on double-distilled water [10, 12]. From the electron micrographs of partially denatured molecules position of all single-stranded loops and length of DNAs were measured. All molecules were individually normalized to the contour length of the native DNA length and alignments were done with respect to the denaturation pattern rather than the physical ends. Evidence for Terminal Redundancy in Phage Genome. Relation between terminal redundancy and circular permutation was detected by a complete denaturation-self-reannealing experiment in permuted DNA molecules [23, 24]. The presence of circular permutation is confirmed if circular homoduplex DNA with singlestranded protrusions is observed in such renatured molecules and the circular path should represent the DNA length that does not include the redundancy. The length of the single-stranded protrusions is a measure of the terminal redundancy in the circularly permuted DNA. The condition for homoduplex formation was standardized. 8 l of phage DNA was mixed gently with 2 l of 10! Tris-EDTA (pH 8.0) and formamide with 35% final concentration. The mixture was heated in a water bath at 85 ° C for 7 min to denature and 0.6 vol of 5! SSC was added and re-annealed at 4 ° C for 6 days. Homoduplexed DNA was spread on double-distilled water, stained with alcoholic uranyl acetate. Rotary Shadowing. All grids with spread DNA were rotary shadowed with evaporated heavy metals (Pt-Pd) or Pt (100%) at a low angle in a JEOL JEE 400 high vacuum evaporator to increase the contrast of DNA before visualization [22]. Electron Microscope. FEI Tecnai 12 BioTwin (cryo) and Philips 420T transmission electron microscopes were used to inspect all specimens in this study.
Results
Host Specificity and Plaque Morphology Phage S20 lysates prepared by infecting V. cholerae cells with a multiplicity of infection (m.o.i) of 0.1 yielded a titer of about 1010–1011 pfu/ml. Plaques on 1.5% agar plates revealed a big hollow zone with slightly wrinkled margin (average diameter 5.5 8 1.0 mm) after 20 h incubation at 37 ° C. The host range of phage S20 was determined from plating efficiency on different V. cholerae strains. It was found that phage S20 could not produce plaque on non-O1 V. cholerae SG24 strain, non-O1 non-O139 V. cholerae VC141 and V. cholerae classical strain 569B Inaba. Dutta/Ghosh
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was used. The high titre phage lysate (1011 pfu/ml) was dissociated in a sample loading buffer (5!) containing 60 mM Tris-HCl (pH 6.8), 25% (v/v) glycerol, 2% (w/v) SDS, 14.4 mM -mercaptoethanol, 0.1% (w/v) bromophenol blue and the sample was heated on a boiling water bath at 100 ° C for 5 min, cooled at room temperature and after a brief centrifugation the samples were applied to the casted gel. The protein bands were stained with Coomassie brilliant blue.
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Host Adsorption and One Step Growth Characteristics The adsorption of vibriophage S20 to V. cholerae El Tor MAK757 strain followed a biphasic kinetics at 37 ° C. This was characterized by a very rapid initial phase with a rate constant 4.3 ! 10 –9 ml–1 min–1; thereafter, the process continued at a slower rate with an adsorption constant 3.3 ! 10 –10 ml–1 min–1. Growth of S20 within its host was characterized by a latent period of 12 min, a rise period of 10 min and a burst size of around 60 8 15 plaqueforming units (pfu) per cell. pH Stability It is important to study the pH stability of vibriophages for microbiological interest, for example, their survival in the pH environment of gut and also for practical purposes related to the possible therapeutic uses. Vibriophages are usually found to be more stable in the alkaline pH than in the acidic pH. At pH 10.0 survival percentage was around 70. Below pH 5.0, the phage has survival percentage of around 10 and above pH 9.0 this phage was gradually inactivated in the three pH ranges 9–10, 10–11 and 11–13 (in nutrient broth medium). The phage S20 El Tor Vibriophage S20
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was found to be stable (more than 90% survival after 1 h) in the pH range 5.0–9.0. Thermal Inactivation The thermal death points of vibriophages are important parameters to be determined for their identification and classification. For example, phages with a high degree of thermostability have better chance of survival in organic composts, in which temperature may exceed 70 ° C. Figure 1a shows the kinetics of thermal inactivation of the phage S20 at different temperatures. In respect of heat sensitivity, the homogeneity of phage population was indicated by the fact that at all temperatures inactivation obeyed the first-order reaction kinetics. At 50–60 ° C the phages were inactivated rather slowly but above 60 ° C this phage was inactivated rapidly. The half-life of thermal inactivation of S20 at 60 ° C was found to be 4.2 min. UV Inactivation Ultraviolet light is one of the most extensively used agents for inactivation of phages. Besides inactivation of Intervirology 2007;50:264–272
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Fig. 1. a Kinetics of thermal inactivation of the phage S20 at 50 ° C (I), 60 ° C (y) and 70 ° C (o). b UV inactivation of phage S20.
Protein Profile Structural proteins of the phage S20 were analyzed by disrupting the phage particles and separating the proteins by electrophoresis on 12% polyacrylamide gel under denaturing condition (SDS-PAGE). The results show 16 structural polypeptides ranging in molecular weights from 15 to 90 kDa with two major components of 50 and 42 kDa. Morphology of the Phage with Negative Staining Negatively stained phage S20 particles had a hexagonal head of 58.6 8 1.7 nm diameter with a short tail of 11.5 8 1.8 nm length. Measurements were done on negatives manually with a magnifier fitted with a graticule and also by using analySIS software. Based on its morphology, phage S20 can be placed under the Podoviridae family according to the Universal Virus Database of the International Committee on Taxonomy of Viruses (ICTVdB). Negatively stained catalase crystals having alternate lattice plane spacings of 8.75 and 6.85 nm were used for magnification calibration. Three-Dimensional Image Reconstruction of Vibriophage S20 3D reconstruction of viruses from cryo-electron microscopy has contributed greatly to the understanding of the viral structural biology. Different orientations of isometric capsids in the vitrified buffer yielded hexagonal particles. Most of them contained DNA within their cores and the remaining appeared to be empty. Here we present results of a 3D reconstruction of just the head of the DNAfilled phage particles using EMAN (Electron Micrograph ANalysis) software, because the phage has got a very small featureless tail that probably attracts no interest in its detailed study. The 3D image reconstruction of single particle vibriophage S20 head is shown in figure 2a–c. It can be seen from the colored images of the phage head 268
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that the major capsid proteins are arranged as capsomers (more dense blue than the rest of the surface). The capsomers along a 2-fold axis have a hole in the middle like the P4 head and the capsomers have a large hole in the middle when viewed along a 5-fold axis. The 5-fold capsomers protrude more from the capsid surface than the other capsomers. When the image is viewed along the 3fold axes, one icosahedral face can be seen as represented by the triangle in figure 2b. The vertices of the triangle are formed by three capsomers of 5-fold positions and there are three other elongated shaped capsomers in between them. Although the subunits are not recognizable at this resolution of the reconstruction, from the triangular structure it can be inferred that the phage is likely to have a triangulation number T = 4. From SDS-PAGE study a major protein of around 42 kDa was found which is very similar to the gpN (42 kDa) major capsid protein of P4 phage [25]. When the phage head is viewed from inside along a 5fold axis, grooves are seen in the region corresponding to the capsomers protruding from the outside surface (fig. 2d). There are weaker densities (light green color) that extend inward along the groove of the capsomers. The color-coded surface represents the inward densities as less blue and the densities outward as more blue which agrees well with the outside view of the colored phage head. The spatial frequency at which the FSC drops below 0.5 is a measure of the resolution and in the present reconstruction is 52 Å. DNA Packaging Packaging of the DNA inside the phage head was studied with the help of a radial density plot using the bradial program of Bsoft software [19]. Here we have tried to interpret the internal packaging from the radial density plot (fig. 2e). Radial density profiles of S20 vibriophage show the highest density peak at the right end of the radial plot corresponding to the outer capsid protein shell. The other six peaks in the density distribution of filled S20 particles (at 4, 8, 12, 15, 19 and 23 nm positions) are almost equally spaced (4 nm) and are presumably due to the DNA present in the phage head. The radial density closer to the center is almost the same as the outer protein shell and may be due to the presence of a proteinaceous core. Such a core has been reported to be present in coliphage T7 particles [26]. Phage S20 Genome Electron microscopy shows that phage S20 DNA is linear as the two free ends are clearly visible and the contour Dutta/Ghosh
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the phage particles, it also produces important physiological and genetic effects such as the ability to stimulate genetic recombination and exert mutagenic action. It has also been used to study intracellular phage development. The survival percentage of UV-irradiated S20 phage was obtained by infecting V. cholerae MAK757, as shown in figure 1b. UV inactivation was found to obey the firstorder reaction kinetics. Times required for the 37% (D37) and 10% (D10) survival of the S20 phage at a distance of 55 cm from a 15-watt UV lamp were 7 and 16 s, respectively. Around 80% phage particles were inactivated within 10 s of exposure.
length of the DNA was measured manually with a finepointed divider, also by using analySIS software. The length of S20 DNA was computed to be 40.7 8 1.5 kb. The internal calibration standard was double-stranded circular plasmid pBR322 DNA whose length was assumed to be 4.36 kb. The molecular mass of phage S20 DNA was calculated to be 27.1 8 1.0 ! 106 Da.
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and SfiI enzymes. Circularly, permuted DNA generally shows submolar fragments on digestion with restriction enzymes. The temperate phage A118 of Listeria monocytogenes has DNA with circular permutation and terminal redundancy, but restriction endonuclease digestion of its genome did not show any submolar fragment [27]. Phage S20 DNA also failed to reveal a submolar fragment like A118 phage DNA with restriction digestion.
Restriction Endonuclease Digestion of S20 DNA Vibriophage S20 DNA was treated with different restriction endonucleases and the 1% agarose gel electrophoresis result showed that double-stranded DNA-specific restriction endonucleases AccI, HpaII, HaeIII, HindIII, EcoRV, HincII, DraI and XmnI cut S20 DNA into distinct fragments confirmed that the phage S20 genome consists of double-stranded DNA. Hind III restriction fragments of bacteriophage DNA were used as size standards. Phage DNA was found to be resistant to SmaI, NotI, StuI, AssI, XbaI, AvaI, PvuI, ClaI, HpaI, NsiI, NheI, EcoRI, PstI, SalI, BamHI, KpnI, BglII, BstEII, BasI, BglI, PvuII, NruI,
Partial Denaturation Mapping of S20 DNA The partially denatured S20 DNA molecules were manipulated (by reversal or by shifting to the left or right) such that the denatured sites could be aligned. Here all the DNA molecules had a characteristic AT-rich region and all the molecules were aligned with respect to that site. But the ends of the molecules do not align to any particular position. The denaturation pattern delineates the end position of the molecules. Alignment of the maps with respect to the denaturation sites in figure 3a shows that S20 DNA has a circular permutation. Figure 3b shows
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Fig. 2. Shaded surface representation of vibriophage S20. (a) 2-fold, (b) 3-fold (the facet triangle is shown in white color) and (c) 5-fold view of the 3D image of vibriophage S20. d Inside view of phage head along the 5-fold axis. e Radial density plot of filled S20 particle.
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Fig. 3. a Partial denaturation maps of S20 DNA molecules. Here each horizontal line represents a molecule and black rectangles show the measured position of denatured sites arising from ATrich regions. But the sites are not in the same place in every molecule. All molecules are aligned with respect to the denaturation pattern in a 0–80-kb scale to include all the molecules. b Histogram showing the position of AT-rich regions for the individual maps shown in (a). The vertical scale 0–1 shows the fraction of molecules denatured, and peak numbers are discussed in the text. c Histogram of the position of the right ends of the molecules with respect to the denaturation maps which shows a random distribution of ends.
phage of El Tor V. cholerae, at its genomic level. The adsorption of phage S20 to its host MAK 757 follows biphasic kinetics at 37 ° C. But the burst size was very low compared to another El Tor typing phage e5 (100 pfu per cell). The half-life of thermal inactivation of e5 phage was 12.5 min compared to 4.2 min of S20 phage at 60 ° C. SDSPAGE of purified S20 capsid has revealed two major structural polypeptides (50 and 42 kDa) in approximately equimolar amounts. Most likely, these correspond to the phage S20 major capsid proteins. Here also this phage resembles phage e5 and another phage e4 as they have a major structural polypeptide of 50 kDa. From the study of partial denaturation mapping, it has been shown that the phage DNA represents a set of terminally redundant and circularly permuted linear DNA molecules. Homoduplex analysis, by electron microscopy, confirmed the presence of terminal redundancy in the DNA molecules by showing double-stranded circles with single-stranded tails. Taking out the amount of terminal repetition from the native DNA length, the unit genome size can be determined which is 36.9 8 0.6 kb. There is evidence of other phages where the presence of circular permutation and terminal redundancy were confirmed by the homoduplex structures, namely, coliphage 15 [28] and T2 [29], salmonella phage P22 [30], group H streptococcal phage Ф42 [31], group A streptococcal bacteriophage SP24 [23] and the Listeria monocytogenes phage A118 [27]. Single particle 3D computerized image reconstruction technique in addition to cryo-electron microscopy developing over the years shows strong potential to give usable information for solving the structure of viruses. Cryoelectron microscopy provides much structural information with the help of the radial density profile, whereas negative staining gives us only surface details. Here the spherical density distribution of filled particles as a function of radius shows six peaks revealing probably the DNA packaging pattern inside. Also, a peak near the center may be due to a proteinaceous core that may be used as an anchor by the DNA for the packaging. The inner surface when viewed along 5-fold symmetry shows a weak density region protruding inward. It is interesting to note that phage S20 is unusual in many respects. Although negatively stained phage S20 particles look very much like coliphage T7 and their genome size is almost the same, their triangulation numbers are found to be different. In this respect S20 phage being a member of the Podoviridae family resembles coliphage P4, which belongs to the Siphoviridae family. Also, the 11.6-kb length of the P4 DNA [32] is very short compared to the S20 DNA. This apparent paradox could not be resolved as no
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Homoduplex Analysis of Phage S20 DNA Partial denaturation mapping showed that the S20 genome has random circular permutation. This was further confirmed and the extent of terminal redundancy was determined by the homoduplex experiment of the same population of matured phage DNA. When this experiment was performed, circular DNA was indeed observed with single-stranded protrusions or tails confirming that the mature phage DNA exists as a population of permuted and terminally redundant molecules. The circumference of circular molecules was 36.9 kb, about 9.4% smaller than the linear phage molecule. The single-stranded tails protruding from the duplex circles were equal in size to the terminal redundancy and were measured. The contour length of the tails was 3.8 kb or about 9.24% of the circular DNA molecule. Therefore, the length of the tails agrees well with the amount of the redundancy.
Discussion
The host-virus (V. cholerae and its bacteriophage) structure-function relation can be better explored if its phage is characterized in more detail at the molecular level. Here we have studied vibriophage S20, a typing
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the histogram of the aligned partial denaturation maps and the major peaks were numbered from the left (1–5) and the peaks were repeated after peak 5. It can be seen that the positions of the peaks were repeated after the DNA length without the amount of redundancy. Figure 3c shows a histogram of the right end of the DNA molecules with respect to the aligned denaturation map. The distribution of the ends reveals that the ends were not restricted in a short region but randomly distributed. Partial denaturation mapping clearly showed a high degree of permutation was present among the set of S20 DNAs.
data are available on the morphogenetic genes especially the genes coding the capsid proteins. Finally, characterization of the novel bacteriophage S20 using physicochemical methods, conventional transmission electron microscopy, cryo-electron microscopy and 3D image reconstruction studies has expanded our knowledge and understanding of the different physiological, morphological and physicochemical parameters of a vibriophage structure. Higher-resolution cryo-electron microscopic studies in future will help resolve the many questions which need to be answered to understand viral life cycles in more detail.
Acknowledgements This work was supported by the Department of Science and Technology, Government of India (Grant No. SP/SO/B-38/99). We are grateful to Dr. S.K. Bhattacharya, Director of this Institute, for his interest and encouragement in the present work. We would like to thank Dr. Steve Ludtke for his support with valuable suggestions regarding the EMAN software package and Dr. J. Bernard Heymann for the use of his B-Soft software. We would also like to thank the Computer Graphics Laboratory, University of California, San Francisco, Calif., USA for providing the UCSF Chimera software with free downloading.
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