Influence of the Chitosan Oligomer on the Phage Particles and Reproduction of Phage 1-97A in the Culture of Bacillus thuringiensis

The causes of bacteriophage 1-97A inactivation by the chitosan oligomer with a polymerization degree of 15 and the influence of the oligomer on the phage reproduction in the culture of Bacillus thuringiensissubsp. galleriae, strain 1-97, were studied. The study of the inactivation kinetics showed that, in 1 h, virtually all chitosan was bound to the phage particles, causing, as evidenced by electron microscopy, DNA release from the phage head, destruction of the phage particles, and agglutination of the phage particles or of their tails in the region of the basal plate. High-polymeric chitosan caused more pronounced destruction of the phage particles than the oligomer. It was established that chitosan prevented the production of complete phage particles. One of the mechanisms of such an influence may be the production in the presence of chitosan of phage particles devoid of DNA.     

Effect of chitosan derivatives on the development of phage infection in cultured Bacillus thuringiensis

The influence of chitosan fragments with different degrees of polymerization and some chemical chitosan derivatives on the infection of Bacillus thuringiensis by phage 1-97A was studied. It was shown that chitosan inhibits phage infection and inactivates phage particles. The extent of inhibition of phage infection inversely depended on the degree of polymerization of chitosan fragments. On the contrary, the extent of inactivation of phage virulence was proportional to the degree of polymerization. Chitosan derivatives did not inhibit the growth of bacilli. Deaminated chitosan derivatives at a concentration of 100 mg/ml efficiently inhibited phage reproduction, exhibiting no correlation between the degree of deamination and antiviral activity. The anionic derivative chitosan sulfate and N-succinate-6-O-sulfate did not inactivate phage, did not influence bacterial growth, and did not inhibit the process of viral infection.     

Influence of chitosan derivatives on the development of phage infection in theBacillus thuringiensis culture

The influence of chitosan fragments with different degrees of polymerization and some chemical chitosan derivatives on the infectionof Bacillus thuringiensis by phage 1–97 A was studied. It was shown that chitosan inhibits phage infection and inactivates phage particles. The extent of inhibition of phage infection inversely depended on the degree of polymerization of chitosan fragments. On the contrary, the extent of inactivation of phage virulence was proportional to the degree of polymerization. Chitosan derivatives did not inhibit the growth of bacilli. Deaminated chitosan derivatives at a concentration of 100 μg/ml efficiently inhibited phage reproduction, exhibiting no correlation between the degree of deamination and antiviral activity. The anionic derivative chitosan sulfate andN-succinate-6-O-sulfate did not inactivate the phage, did not influence bacterial growth, and did not inhibit the process of viral infection.     

Two types of structural organization of double-stranded DNA in phage particles

Structural arrangement of linear unmodified duplex DNA in phage particles FI5 and SB1 was studied by the techniques of absorption spectrum, CD, scanning microcalorimetry. Hyperchromism is not registered for SB1 DNA in situ at 260 nm, in contrast to FI5 and other phages, but is visible at 260-290 nm. Phage SB1 had an unusual CD spectrum: intensity of the positive band at 280 nm was, practically, identical to the free SB1 DNA intensity. However, the character of SB1 DNA in situ melting in 1.5% HCHO corresponds to FI5 (and other phages) melting in situ in the presence of HCHO. Analysis of these spectral studies and calorimetry permitted one to identify the peaks on the thermograms of phages FI5 and SB1. Variability of heat capacity in the zone of phage particles destruction is supposed to be connected with DNA rearrangements.     

The production of generalized transducing phage by bacteriophage lambda

Generalized tranduction has for about 30 years been a major tool in the genetic manipulation of bacterial chromosomes. However, throughout that time little progress has been made in understanding how generalized transducing particles are produced. The experiments presented in this paper use phage λ to assess some of the factors that affect that process. The results of those experiments indicate: (1) the production of generalized transducing particles by bacteriophage λ is inhibited by the phage λ exonuclease (Exo). Also inhibited by λ Exo is the production of λdocR particles, a class of particles whose packaging is initiated in bacterial DNA and terminated at the normal phage packaging site, cos. In contrast, the production of λdocL particles, a class of particles whose packaging is initiated at cos and terminated in bacterial DNA, is unaffected by λ Exo; (2) λ-generalized transducing particles are not detected in induced lysis-defective (S⁻) λ lysogens until about 60–90 min after prophage induction. Since wild-type λ would normally lyse cells by 60 min, the production of λ-generalized transducing particles depends on the phage being lysis-defective; (3) if transducing lysates are prepared by phage infection then the frequency of generalized transduction for different bacterial markers varies over a 10–20-fold range. In contrast, if transducing lysates are prepared by the induction of a λ lysogen containing an excision-defective prophage, then the variation in transduction frequency is much greater, and markers adjacent to, and on both sides of, the prophage are transduced with much higher frequencies than are other markers ; (4) if the prophage is replication-defective then the increased transduction of prophage-proximal markers is eliminated; (5) measurements of total DNA in induced lysogens indicate that part of the increase in transduction frequency following prophage induction can be accounted for by an increase in the amount of prophage-proximal bacterial DNA in the cell. Measurements of DNA in transducing particles indicate that the rest of the increase is probably due to the preferential packaging of the prophage-proximal bacterial DNA.

These results are most easily interpreted in terms of a model for the initiation of bacterial DNA packaging by λ, in which the proteins involved (Ter) do not recognize any particular sequence in bacterial DNA but rather     

Characterization of Natronobacterium magadii phage ΦCh1, a unique archaeal phage containing DNA and RNA

A novel archaeal bacteriophage, ΦCh1, was isolated from a haloalkalophilic archaeon Natronobacterium magadii upon spontaneous lysis. The phage-cured strain N. magadii (L13) was used to demonstrate infectivity of phage ΦCh1. The turbid-plaque morphology and the fact that N. magadii cells isolated from plaques were able to produce phage indicated that ΦCh1 is a temperate phage. The phage morphology resembles other members of Myoviridae -infecting Halobacterium species. In solution below 2 M NaCl, the phage lost its morphological stability and infectivity. One- and two-dimensional SDS–PAGE of phage particles revealed at least four major and five minor proteins with molecular masses ranging from 15 to 80 kDa and acidic isoelectric points. Southern blot analysis of chromosomal DNA of a lysogenic N. magadii strain showed that ΦCh1 exists as a chromosomally integrated prophage. The phage particles contain both double-stranded, linear DNA (approx. 55 kbp) as well as several RNA species (80–700 nucleotides). Hybridization of labelled RNA fragments to total DNA from N. magadii and ΦCh1 showed that the virion-associated RNA is host encoded. Part of the phage DNA population is modified and restriction analysis revealed evidence for adenine methylation. Phage ΦCh1 is the first virus described for the genus Natronobacterium , and the first phage containing DNA and RNA in mature phage particles.     

Packaging of ColE1 DNA having a lambda phage cohesive end site.

The mechanism of lambda phage-mediated transduction of hybrid colicin E1 DNAs of various lengths was studied, and factors influencing the formation of these transducing particles were investigated. The results were as follows: 1. The presence of a cohesive end site of lambda phage (coslambda) on colicin E1 DNA was essential for packaging of the DNA. 2. Packaging of colicin E1 DNAs, which carry coslambda with molecular sizes corresponding to 68% of that of lambda phage DNA, was observed in the absence of all known recombination functions of E. coli K-12 and of lambda phage. 3. Hybrid colicin E1 DNAs having coslambda with molecular sizes corresponding to 28% of that of lambda phage DNA were packaged within lambda phage particles as trimers; hybrid DNAs with coslambda of 40 and 47% of the length of lambda phage DNA were packaged as dimers; and those with molecular sizes of 68% of that of lambda phage DNA were packaged mostly as monomers. These results demonstrated that two factors are essential for the packaging of DNAs within lambda phage particles; the presence of coslambda on the DNA molecule and an appropriate size of DNA.     

Cleavage of DNA from 2 phages of Bacillus thuringiensis by EcoRI and HindIII endonucleases

The DNA of two Bacillus thuringiensis phages was restricted by endonucleases EcoRI and HindIII and the electrophoretic distribution of the fragments in agarose gel was studied. EcoRI was shown to restrict the DNA of phage 1-97A into 8 fragments and the DNA of phage 1-97B into 12 fragments. Restriction with HindIII results in the formation of 22 and 9 fragments for phage 1-97A and phage 1-97B, respectively. The molecular mass of the DNAs determined by summing up EcoRI restricts is 80.87 MDa for phage 1-97A and 32.45 MDa for phage 1-97B.     

Inactivation of coliphages by chitosan derivatives

The effect of chitosan fragments with different degrees of polymerization and the chemical derivatives of chitosan differing in the number of amino groups and total molecule charge on phages T2, T4, and T7 was studied. The interaction of chitosan with bacteriophage particles inactivated them to the extent dependent on the chemical properties of chitosan and its concentration. Phage T2 was found to be most susceptible to inactivation by chitosan. The polycationic nature of chitosan plays an important role in the inactivation of phages. It is assumed that the abnormal rearrangement of the basal plate of phages, the loss of long tail fibers, and, probably, modification of the receptor-recognizing phage proteins may be responsible for the inactivation of coliphages by chitosan.     

Inactivation and reactivation of B. megatherium phage

Preparation of Reversibly Inactivated (R.I.) Phage.- If B. megatherium phage (of any type, or in any stage of purification) is suspended in dilute salt solutions at pH 5-6, it is completely inactivated; i.e., it does not form plaques, or give rise to more phage when mixed with a sensitive organism (Northrop, 1954). The inactivation occurs when the phage is added to the dilute salt solution. If a suspension of the inactive phage in pH 7 peptone is titrated to pH 5 and allowed to stand, the activity gradually returns. The inactivation is therefore reversible. Properties of R.I. Phage.- The R.I. phage is adsorbed by sensitive cells at about the same rate as the active phage. It kills the cells, but no active phage is produced. The R.I. phage therefore has the properties of phage "ghosts" (Herriott, 1951) or of colicines (Gratia, 1925), or phage inactivated by ultraviolet light (Luria, 1947). The R.I. phage is sedimented in the centrifuge at the same rate as active phage. It is therefore about the same size as the active phage. The R.I. phage is most stable in pH 7, 5 per cent peptone, and may be kept in this solution for weeks at 0 degrees C. The rate of digestion of R.I. phage by trypsin, chymotrypsin, or desoxyribonuclease is about the same as that of active phage (Northrop, 1955 a). Effect of Various Substances on the Formation of R.I. Phage.- There is an equilibrium between R.I. phage and active phage. The R.I. form is the stable one in dilute salt solution, pH 5 to 6.5 and at low temperature (<20 degrees C.). At pH >6.5, in dilute salt solution, the R.I. phage changes to the active form. The cycle, active right harpoon over left harpoon inactive phage, may be repeated many times at 0 degrees C. by changing the pH of the solution back and forth between pH 7 and pH 6. Irreversible inactivation is caused by distilled water, some heavy metals, concentrated urea or quanidine solutions, and by l-arginine. Reversible inactivation is prevented by all salts tested (except those causing irreversible inactivation, above). The concentration required to prevent R.I. is lower, the higher the valency of either the anion or cation. There are great differences, however, between salts of the same valency, so that the chemical nature as well as the valency is important. Peptone, urea, and the amino acids, tryptophan, leucine, isoleucine, methionine, asparagine, dl-cystine, valine, and phenylalanine, stabilize the system at pH 7, so that no change occurs if a mixture of R.I. and active phage is added to such solutions. The active phage remains active and the R.I. phage remains inactive. The R.I. phage in pH 7 peptone becomes active if the pH is changed to 5.0. This does not occur in solutions of urea or the amino acids which stabilize at pH 7.0. Kinetics of Reversible Inactivation.- The inactivation is too rapid, even at 0 degrees to allow the determination of an accurate time-inactivation curve. The rate is independent of the phage concentration and is complete in a few seconds, even in very dilute suspensions containing <1 x 10(4) particles/ml. This result rules out any type of bimolecular reaction, or any precipitation or agglutination mechanism, since the minimum theoretical time for precipitation (or agglutination) of a suspension of particles in a concentration of only 1 x 10(4) per ml. would be about 300 days even though every collision were effective. Mechanism of Salt Reactivation.- Addition of varying concentrations of MgSO(4) (or many other salts) to a suspension of either active or R.I. phage in 0.01 M, pH 6 acetate buffer results in the establishment of an equilibrium ratio for active/R.I. phage. The higher the concentration of salt, the larger proportion of the phage is active. The results, with MgSO(4), are in quantitative agreement with the following reaction: See PDF for Equation Effect of Temperature.- The rate of inactivation is too rapid to be measured with any accuracy, even at 0 degrees C. The rate of reactivation in pH 5 peptone, at 0 and 10 degrees , was measured and found to have a temperature coefficient Q(10) = 1.5 corresponding to a value of E (Arrhenius' constant) of 6500 cal. mole(-1). This agrees very well with the temperature coefficient for the reactivation of denatured soy bean trypsin inhibitor (Kunitz, 1948). The equilibrium between R.I. and active phage is shifted toward the active side by lowering the temperature. The ratio R.I.P./AP is 4.7 at 15 degrees and 2.8 at 2 degrees . This corresponds to a change in free energy of -600 cal. mole(-1) and a heat of reaction of 11,000. These values are much lower than the comparative one for trypsin (Anson and Mirsky, 1934 a) or soy bean trypsin inhibitor (Kunitz, 1948). Neither the inactivation nor the reactivation reactions are affected by light. The results in general indicate that there is an equilibrium between active and R.I. phage. The R.I. phage is probably an intermediate step in the formation of inactive phage. The equilibrium is shifted to the active side by lowering the temperature, adjusting the pH to 7-8 (except in the presence of high concentrations of peptone), raising the salt concentration, or increasing the valency of the ions present. The reaction may be represented by the following: See PDF for Equation The assumption that the active/R.I. phage equilibrium represents an example of native/denatured protein equilibrium predicts all the results qualitatively. Quantitatively, however, it fails to predict the relative rate of digestion of the two forms by trypsin or chymotrypsin, and also the effect of temperature on the equilibrium.     

Coliphages inactivation using chitosan derivatives

The effect of chitosan fragments with different degrees of polymerization and the chemical derivatives of chitosan differing in the number of amino groups and total molecule charge on phages T2, T4, and T7 was studied. The interaction of chitosan with bacteriophage particles inactivated them to the extent dependent on the chemical properties of chitosan and its concentration. Phage T2 was found to be most susceptible to inactivation by chitosan. The polycationic nature of chitosan plays an important role in the inactivation of phages. It is assumed that the abnormal rearrangement of the basal plate of phages, the loss of long tail fibers, and probably, modification of the receptor-recognizing phage proteins may be responsible for the inactivation of coliphages by chitosan.     

Fate of transforming bacteriophage HP1 deoxyribonucleic acid in Haemophilus influenzae lysogens.

The biological fate of temperate phage HP1 deoxyribonucleic acid (DNA) was followed after uptake by defectively lysogenic competent Haemophilus influenzae cultures. The similar inactivation kinetics of three single phage genetic markers and of their triple combination indicated a complete rather than partial destruction of about half of the adsorbed DNA molecules. Intracellular DNA breakdown products were tentatively identified by hydroxyapatite column chromatography as short single strands and extensively damaged short double strands. Integrated donor DNA (after single-strand insertion?) was still highly efficient for triple-marker co-transformation. This suggests that whole or nearly whole donor DNA molecules were integrated. Some donor DNA was never integrated but remained largely unaltered. This DNA fraction did not contain significant amounts of recipient prophage marker activity. It is concluded that it had not participated in some kind of reciprocal recombination event involving the recipient chromosome. Since very similar phage DNA marker inactivation rates were observed after adsorption by competent nonlysogenic recipients (transfection), the relationship between biological inactivation of adsorbed donor phage DNA and its integration in lysogenic recipients is not clear.     

Eliminating helper phage from phage display

Phage display technology involves the display of proteins or peptides, as coat protein fusions, on the surface of a phage or phagemid particles. Using standard technology, helper phage are essential for the replication and assembly of phagemid particles, during library production and biopanning. We have eliminated the need to add helper phage by using 'bacterial packaging cell lines' that provide the same functions. These cell lines contain M13-based helper plasmids that express phage packaging proteins which assemble phagemid particles as efficiently as helper phage, but without helper phage contamination. This results in genetically pure phagemid particle preparations. Furthermore, by using constructs differing in the form of gene 3 that they contain, we have shown that the display, from a single library, can be modulated between monovalent (phagemid-like) and multivalent display (phage-like) without any further engineering. These packaging cells eliminate the use of helper phage from phagemid-based selection protocols; reducing the amount of technical preparation, facilitating automation, optimizing selections by matching display levels to diversity, and effectively using the packaged phagemid particles as means to transfer genetic information at an efficiency approaching 100%.     

A simple and efficient procedure for the isolation of high-quality phage lambda DNA using a DEAE-cellulose column

A simple and rapid procedure for purifying large quantities of bacteriophage lambda particles and DNA is described. The procedure involves DEAE-cellulose column chromatography of the phage particles and elution of the phage particles from the column with a low-ionic-strength buffer. The resulting phage were well separated from RNA, DNA, and proteins derived from Escherichia coli host cells. The lambda DNA was prepared from the purified phage particles by the conventional method of phenol extraction and ethanol precipitation. This procedure did not use nucleases, proteases, detergents, or CsCl density gradient centrifugation. The lambda DNA obtained by this method was equivalent in purity to the material prepared by CsCl density gradient centrifugation and amenable to restriction enzyme digestion, ligation, radiolabeling, and double-stranded DNA sequencing. A detailed protocol is described for obtaining 0.5 to 1.0 mg DNA from a 1-liter liquid lysate in less than 5 h. This procedure is simple, inexpensive, and timesaving, and is particularly suitable for large-scale isolation of lambda DNA.     

Photosensitized inactivation of T7 phage as surrogate of non-enveloped DNA viruses: efficiency and mechanism of action

We investigated the efficiency and the mechanism of action of a tetraphenyl porphyrin derivative in its photoreaction with T7 phage as surrogate of non-enveloped DNA viruses. TPFP was able to sensitize the photoinactivation of T7 phage in spite of the lack of its binding to the nucleoprotein complex. The efficiency of TPFP photosensitization was limited by the aggregation and by the photobleaching of porphyrin molecules. Addition of sodium azide or 1,3-dimethyl-2-thiourea (DMTU) to the reaction mixture moderated T7 inactivation, however, neither of them inhibited T7 inactivation completely. This result suggests that both Type I and Type II reaction play a role in the virus inactivation. Optical melting studies revealed structural changes in the protein part but not in the DNA of the photochemically treated nucleoprotein complex. Polymerase chain reaction (PCR) also failed to demonstrate any DNA damage. Circular dichroism (CD) spectra of photosensitized nucleoprotein complex indicated changes in the secondary structure of both the DNA and proteins. We suggest that damages in the protein capsid and/or loosening of protein-DNA interaction can be responsible for the photodynamic inactivation of T7 phage. The alterations in DNA secondary structure might be the result of photochemical damage in phage capsid proteins.     

The net hydration of phage lambda

The banding density of phage lambda varies with the activity of water when the phage particles are banded in a series of different cesium salts. The results are comparable to those Hearst and Vinograd for free DNA. Lambda phage ghosts show less net hydration than the phage particles and band in a fairly narrow range of densities in these cesium salts. The phage banding density may be predicted to a first approximation by a simple additive approximation: the total net hydration of the phage is approximately equal to the net hydrations of free λ DNA λ hosts, all measured at the same water activity. The simple additive approximation is not adequate, however, to explain the banding density differences between a deletion mutant and phage lambda in the different cesium salts. The density differences evidently are sensitive to second-order effects: they apparently are affected by a restriction of DNA hydration inside the phage head, which depends both on water activity and on DNA length (or free volume inside the phage head). This becomes a striking effect in Cs₂SO₄ solutions where the net DNA hydration is large. Changing the phage banding density by substituting 5-bromouracil for thymine, which increases the DNA mass while leaving the DNA volume relatively unchanged, gives results consistent with a restriction of the net DNA hydration that depends on the DNA volume. Data on the sedimentation velocity behavior that λ and λb₂ in diferrent salts are presented and discussed. It appears possible to estimate the size of a DNA deletion from the phage sedimentation coefficient.     

Pore-forming properties of the adsorption protein of filamentous phage fd

The gene 3-encoded adsorption protein (g3p) of filamentous phage fd has been purified to homogeneity by using high-performance liquid chromatography. Removal of SDS from the SDS-solubilized g3p results in spontaneous oligomerization of the g3p. Reconstitution into artificial lipid bilayer membranes shows that the oligomer forms large aqueous pores that remain open for seconds and are insensitive to changes in membrane potential. The estimated diameter of the pores suggest that they are large enough to allow passage of phage single-stranded DNA. The implications of these findings for phage infection are discussed.     

Weigle reactivation of diploid M13 phage

Summary The limited ability of ultraviolet (UV)-irradiated E. coli cells to W-reactivate UV-irradiated, single-stranded DNA phages fd and M13 was investigated. The kinetics of induction for W-reactivation of UV-irradiated fd phage are different from that for other SOS functions. W-reactivation of UV-irradiated M13 phage was studied using phage particles that contain at least two single-stranded DNA genomes. No effect on the extent of W-reactivation of diploid phage was observed, compared to that of normal haploid phage, indicating that the mechanism of W-reactivation of single-stranded DNA phages does not involve recombination between partially replicated genomes.     

T7噬菌体转运真核表达载体入胞表达平台的构建

[目的]构建携带锚定序列的真核表达载体,研究T7噬菌体识别、包裹和转运真核表达载体进入细胞实现蛋白表达的可行性,为DNA疫苗研发建立新的技术平台.[方法]本研究通过重叠延伸PCR方法获得候选锚定序列并插入真核表达载体;建立荧光定量PCR方法比较T7噬菌体识别、包裹真核表达载体的效率;激光共聚焦显微镜观察T7噬菌体转运真...     

Lysis from without of S. aureus K1 by the combined action of phage and virolysin

Lysis from without (LFW) occurs in two steps: (1) sensitization of cells by phage, which renders the cells susceptible to (2) destruction of an essential cell structure by an extracellular lytic enzyme. Virolysin, from phage-infected cells, was used in these studies. Normal cell autolysin is also effective. Evidence is presented that: 1. Neither phage nor lysin alone causes LFW. 2. Sensitization requires phage adsorption. 3. It can be caused by non-infectious particles. This establishes a new biological activity of the particle. 4. Heat, U.V., detergents, penicillin, and other damaging agents also sensitize cells. 5. Sensitization involves a non-lethal, reversible reaction. 6. Sensitization by phage prevents virus synthesis. Following adsorption, a cell can undergo sensitization or infection but not simultaneously. When only a few particles are adsorbed, infection can occur; when sufficient particles are adsorbed, sensitization takes place. 7. Quantitative aspects of LFW are described. Lysis proceeds logarithmically. The lysis end-point depends upon the phage concentration but is independent of the enzyme concentration.