Transient electric birefringence studies of T2 bacteriophage and T2 ghost

The transient electric birefringence behavior of bacteriophage T2 and the T2 ghost or protein coal was studied. The field free relaxation measurements show both the intact virus and its ghost to have two rotary diffusion coefficients. These coefficients have values of 555 ± 54 and 111 ± 22 sec.^?1 for the intact virus and 688 ± 89 and 161 ± 29 sec.^?l for the ghost. The equivalent ellipsoids for the fast and slow relaxation coefficients were obtained by use of Perrin's equation and were related to the bacteriophage structure in terms of a possible extension of the tail fibers or an enlargement of the head structure. The saturation of the specific birefringence of the phage and the ghost when compared with the specific birefringence of the free nucleic acid gave an average optical orientation of 10 to 18% of the nucleic acid parallel to the main axis of the phage. The analysis of the birefringence versus applied field strength in the Kerr region gave the following values for the anisotropy of the polarixability. α_e,33 – α_e,11 and intrinsic dipole, μ, of both phage and ghost : for T2 phage α_e,33 – α_e,11 = 5.0 × 10^?14 cm.³ and μ = 64,400 Debyes; for T2 ghost α_e,33 – α_e,11 = 7.9 × 10^?14cm.³ and μ = 57,200 Debyes. The high intrinsic dipole for phage and ghost is interpreted as to be associated with the mechanisms of the virus for attachment, to the host cell wall.     

Release of respiratory control in Escherichia coli after bacteriophage adsorption: process independent of DNA injection.

Adsorption of phages T4, T5, and BF23 to previously starved Escherichia coli cells triggered the immediate release of respiratory control. A similar stimulation of respiration was induced after T4 ghost attachment, showing that this process was independent of the mechanism of DNA injection. Rather, this change in the respiratory rate was related to the transient depolarization of the cytoplasmic membrane also induced after phage and ghost adsorption. Both processes were suppressed by addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid in the case of T4 (phage and ghosts) but not of T5 and BF23. The increase in respiratory rate observed after phage adsorption was of a magnitude similar to that induced by protonophores. Since other treatments that depolarize the membrane without a massive proton influx did not increase the rate of respiration of starved bacteria with the same efficiency, these results suggest that phage adsorption induced an entry of protons into the cell cytoplasm.     

Purification of B. megatherium Phage G and Evidence for a Muralytic Enzyme as an Integral Part of the Phage

The Purification Of B. megatherium G phage is described and it is shown that DEAE cellulose chromatography combined with conventional methods gave a phage preparation which was at least 95 per cent pure, and contained 2.16 µg nitrogen/10¹¹ infective particles. The phage particle weight in molecular weight units was 91 x 10⁶. The small amount of contaminating material appeared to represent phage "ghosts." An essentially 1:1 ratio of particles to infective units was found when data from electron microscopic counts or data from chemical analysis were related to phage infectivity. Comparison, by several methods, of the G phage and coliphage T2 shows that T2 is 2.6 times larger than G phage. The specific activity of the muralytic component obtained by disintegration of phage preparations with urea was unchanged by the purification indicating that the phage-"bound" muralytic activity is an integral part of the phage structure.     

Physical and genetical analysis of bacteriophage T4 generalized transduction

This report describes a comparison of the efficiency of transduction of genes in E. coli by the generalized transducing bacteriophages T4GT7 and P1CM. Both phages are capable of transducing many genetic markers in E. coli although the frequency of transduction for particular genes varies over a wide range. The frequency of transduction for most genes depends on which transducing phage is used as well as on the donor and recipient bacterial strains. Analysis of T4GT7 phage lysates by cesium chloride density gradient centrifugation shows that transducing phage particles contain primarily bacterial DNA and carry little, if any, phage DNA. In this regard transducing phages P1CM and T4GT7 are similar; both phages package either bacterial or phage DNA but not both DNAs into the same particle.     

Inactivation of bacteriophages by decay of incorporated radioactive phosphorus

The inactivation of the phages T1, T2, T3, T5, T7, and lambda by decay of incorporated P(32) has been studied. It was found that these phages fall into two classes of sensitivity to P(32) decay: at the same specific activity of P(32) in their deoxyribonucleic acid (DNA), T2 and T5 are inactivated three times as rapidly as T1, T3, T7, and lambda. Since the strains of the first class were found to contain about three times as much total phosphorus per phage particle as those of the second) it appears that the fraction of all P(32) disintegrations which are lethal is very nearly the same in all the strains. This fraction alpha depends on the temperature at which decay is allowed to proceed, being 0.05 at -196 degrees C., 0.1 at +4 degrees C., and 0.3 at 65 degrees C. Decay of P(32) taking place only after the penetration of the DNA of a radioactive phage particle into the interior of the bacterial cell can still prevent the reproduction of the parental phage, albeit inactivation now proceeds at a slightly reduced rate. T2 phages inactivated by decay of P(32) can be cross-reactivated; i.e., donate some of their genetic characters to the progeny of a mixed infection with a non-radioactive phage. They do not, however, exhibit any multiplicity reactivation or photoreactivation. The fact that at low temperatures less than one-tenth of the P(32) disintegrations are lethal to the phage particle and the dependence of the fraction of lethal disintegrations on temperature can be accounted for by the double stranded structure of the DNA macromolecule.     

Phospholipase activity in bacteriophage-infected Escherichia. II. Activation of phospholipase by T4 ghost infection.

The release of free fatty acids from the phospholipids of Escherichia coli is initiated immediately after the attachment of T4 ghosts. A similar accumulation of free fatty acids is observed if the cells are infected with T4 phage in the presence of chloramphenicol or puromycin. An early accumulation of free fatty acids, however, is not observed in T4 infections in which chloramphenicol or puromycin are not present, nor does it occur if the E. coli are infected with T4 phage before ghost infection, suggesting that phage products can prevent the phospholipid deacylation. If E. coli is infected with T4 ghosts before T4 phage infection, the accumulation of free fatty acids is not suppressed. When phospholipase-deficient E, coli are infected with T4 ghosts the appearance of free fatty acids is not observed, suggesting that T4 ghost attachment can activate the phospholipase of wild-type E. coli. Although the formation of free fatty acid apparently is a consequence of activation of the detergent-resistant phospholipase of the outer membrane, it is not observed in mutants deficient in the detergent-sensitive phospholipase.     

Rescue of abortive T7 gene 2 mutant phage infection by rifampin.

Infection of Escherichia coli with T7 gene 2 mutant phage was abortive; concatemeric phage DNA was synthesized but was not packaged into the phage head, resulting in an accumulation of DNA species shorter in size than the phage genome, concomitant with an accumulation of phage head-related structures. Appearance of concatemeric T7 DNA in gene 2 mutant phage infection during onset of T7 DNA replication indicates that the product of gene 2 was required for proper processing or packaging of concatemer DNA rather than for the synthesis of T7 progeny DNA or concatemer formation. This abortive infection by gene 2 mutant phage could be rescued by rifampin. If rifampin was added at the onset of T7 DNA replication, concatemeric DNA molecules were properly packaged into phage heads, as evidenced by the production of infectious progeny phage. Since the gene 2 product acts as a specific inhibitor of E. coli RNA polymerase by preventing the enzyme from binding T7 DNA, uninhibited E. coli RNA polymerase in gene 2 mutant phage-infected cells interacts with concatemeric T7 DNA and perturbs proper DNA processing unless another inhibitor of the enzyme (rifampin) was added. Therefore, the involvement of gene 2 protein in T7 DNA processing may be due to its single function as the specific inhibitor of the host E. coli RNA polymerase.     

Inducible reactivation of bacteriophage T7 damaged by methyl methanesulfonate or UV light.

We examined the effects of host mutations affecting "SOS"-mediated UV light reactivation on the survival of bacteriophage T7 damaged by UV light or methyl methanesulfonate (MMS). Survival of T7 alkylated with MMS was not affected by the presence of plasmid pKM101 or by a umuC mutation in the host. The survival of UV light-irradiated T7 was similar in umuC+ and umuC strains but was slightly enhanced by the presence of pKM101. When phage survival was determined on host cells preirradiated with a single inducing dose of UV light, these same strains permitted higher survival than that seen with noninduced cells for both UV light- and MMS-damaged phage. The extent of T7 reactivation was approximately proportional to the UV light inducing dose inflicted upon each bacterial strain and was dependent upon phage DNA damage. Enhanced survival of T7 after exposure to UV light or MMS was also observed after thermal induction of a dnaB mutant. Thus, lethal lesions introduced by UV light or MMS are apparently repaired more efficiently when host cells are induced for the SOS cascade, and this inducible reactivation of T7 is umuC+ independent.     

Green fluorescent protein (GFP)-dependent separation of bacterial ghosts from intact cells by FACS

BACKGROUND: E. coli and Salmonella ghost preparations, produced by applying the PhiX174 protein E-mediated lysis system, contain nonlysed bacteria at a very low percentage. To use the ghosts as vaccines, additional methods have to be identified to remove any viable cell, to end up in totally inactivated ghost fractions. Materials and Methods To increase the purity of ghost fractions, we established a green fluorescent protein (GFP)-dependent "in vivo staining" method to be combined with the E-mediated lysis system. Several gfp expression vectors were constructed, and the corresponding cellular fluorescence was analyzed. Bacterial fluorescence, exclusively preserved in nonlysed cells, was utilized to separate these cells from ghost preparations via flow cytometric sorting. RESULTS: High-level production of GFP prior to induction of the lysis system did not affect bacterial growth rates and caused no inhibitory effects on the subsequent protein E-mediated lysis of the cells. The population of reproductive or inactivated but nonlysed cells was highly fluorescent at mean intensities 215-fold higher than ghosts, which exhibited fluorescence at background level. Fluorescent cells could effectively be separated from ghost preparations via flow cytometric sorting. Cell sorting subsequent to protein E-mediated lysis reduced the number of viable cells within ghost preparations by a factor of 3 x 10(5). CONCLUSIONS: The presented procedure is compatible with the protein E-mediated lysis system, is highly effective in separation of nonlysed fluorescent cells, and may serve as a prototype for ghost-purification in applications where only a minimum number of viable cells within ghost preparations can be tolerated.     

利用荧光标记的T7噬菌体研究配体/受体的相互作用

将鸡传染性法氏囊病病毒(IBDV)衣壳蛋白VP2展示到T7噬菌体表面,以FITC标记纯化的重组噬菌体,通过荧光显微镜观察与流式细胞仪检测,研究标记噬菌体与病毒受体细胞--法氏囊B细胞的相互作用.结果展示有IBDV VP2蛋白的噬菌体经FITC标记后仍然具有与受体细胞结合的特性,荧光显微镜下可见绿色荧光,流式数据显示其平均荧光强度明显高于阴性对照,且IBDV疫苗株TAD可明显阻断其结合.由此得出结论,FITC标记与噬菌体展示技术相结合,可进行配体/受体间相互作用的研究.     

Effect of super high magnetic field on lysogenic lambda phage with a temperature sensitive repressor

Summary Escherichia coli where bacteriophage was lysogenic was grown under the super high magnetic field (11.7 Tesla) and the effect of the field on the transition from lysogenic to lytic process of the phase was investigated. The occurrence of phage particles due to induction of phage was stimulated under 11.7 T in comparison with that in geomagnetic field by raising temperature from 30 to 45°C. Especially at 35°C, the phase titer was tenfold larger. No significant effect of the field on the phase particleper se was observed. A potential application of high magnetic strength as a controlling factor ofin vivo switching was implied.     

Synthesis of an S-Adenosylmethionine-cleaving Enzyme in T3-infected Escherichia coli and Its Disturbance by Co-infection with Enzymatically Incompetent Bacteriophage

Synthesis of an S-adenosylmethionine-cleaving enzyme evoked by infection of Escherichia coli with phage T3 was independent of the multiplicity of infection with the wild type, T3 sam(+). It was depressed, however, by mixed infection with related phages genetically incapable of directing enzyme production, such as T3 sam(-), or phage T7. The depressor effect of enzymatically incompetent genomes depended on their proportion among the input phage and not on their absolute multiplicity. The effect was more pronounced with homologous, enzymatically incompetent phage (T3 sam(-)) than with heterologous phage (T7). After ultraviolet irradiation, enzymatically incompetent genomes lost their depressing power; at a survival level of 10(-7), no depression by either homologous or heterologous phage upon T3 sam(+)-directed enzyme synthesis was detected.     

UNG-dependent correction of molecular heteroduplexes of M13 phage DNA in Escherichia coli cells

Correction of heteroduplex DNA obtained by hybridization of uracil-containing single-stranded M13mp18 phage DNA and "mutant" synthetic oligonucleotide with deletion of cytosine in SalGI site was studied in ung+ and ung- E. coli strains. Uracil-containing DNA was prepared after growth of phage in an E. coli strain dut- ung-. The DNA was hybridized with "mutant" oligonucleotide then complementary DNA chain was synthesized by T4 DNA polymerase. Ung+ and ung- E. coli cells were transformed by DNA. In all experiments mutation frequency in ung+ was higher than in ung- cells (approximately 6-fold) and reached 11-50%. Absolute number of mutants was higher in ung+ cells. The results indicate that high level of mutagenesis depends on uracil repair system polarizing the correction of heteroduplex DNA.     

Regulatory Properties of Acetokinase from Veillonella alcalescens

Ghosts of T4 bacteriophage inhibit the uptake of thiomethyl-beta-galactoside (TMG), alpha-methylglucoside, glucose-6-phosphate, and glycerol in Escherichia coli B. The transport of orthonitrophenyl-beta-galactoside (ONPG) is also inhibited to a lesser degree and without alteration of the apparent K(m) of transport. These effects of ghosts parallel those of energy poisons on these systems. However, no one energy poison can produce such pronounced inhibitory effects in all these systems. The effect of the intact phage in these systems was either absent or very slight relative to the ghost. The effect of ghosts on the uptake of TMG was not immediate; at 10 C, no effect of the ghosts was apparent for at least 2 min. This suggests that a step, more temperature dependent than the attachment of the ghost, is necessary for the inhibitory action. The intracellular level of adenosine triphosphate (ATP) in the ghost-infected cells fell to less than 25% of the control value, and the ATP lost from the cell appeared in extracellular medium. Phage, on the other hand, caused no decrease in the intracellular ATP level. This loss of ATP from the cells after ghost infection suggests an alteration of the barrier properties of the membrane so that ATP can leave the cell; however, the accessibility of extracellular ONPG to intracellular beta-galactosidase does not increase. The dissimilarity of the actions of phage and ghosts on all properties examined does not support the model that the initial events in their infections are identical but that the intact phage, unlike the ghost, can provide information for the repair of its effects.     

Genes 1.2 and 10 of bacteriophages T3 and T7 determine the permeability lesions observed in infected cells of Escherichia coli expressing the F plasmid gene pifA.

Infections of F plasmid-containing strains of Escherichia coli by bacteriophage T7 result in membrane damage that allows nucleotides to exude from the infected cell into the culture medium. Only pifA of the F pif operon is necessary for "leakiness" of the T7-infected cell. Expression of either T7 gene 1.2 or gene 10 is sufficient to cause leakiness, since infections by phage containing null mutations in both of these genes do not result in permeability changes of the F-containing cell. Even in the absence of phage infection, expression from plasmids of either gene 1.2 or 10 can cause permeability changes, particularly of F plasmid-containing cells. In contrast, gene 1.2 of the related bacteriophage T3 prevents leakiness of the infected cell. In the absence of T3 gene 1.2 function, expression of gene 10 causes membrane damage that allows nucleotides to leak from the cell. Genes 1.2 and 10 of both T3 and T7 are the two genes involved in determining resistance or sensitivity to F exclusion; F exclusion and leakiness of the phage-infected cell are therefore closely related phenomena. However, since leakiness of the infected cell does not necessarily result in phage exclusion, it cannot be used as a predictor of an abortive infection.     

Effect of Bacteriophage Ghost Infection on Protein Synthesis in Escherichia coli

The rate of protein synthesis by Escherichia coli markedly decreased within 1 min after phage T4 infection, whereas a complete cessation of protein synthesis was observed within at least 25 sec after T4 ghost infection. The cellular level of amino acids and aminoacyl-transfer ribonucleic acid (tRNA) did not change drastically upon infection with ghosts, indicating that the inhibition of protein synthesis took place at a step(s) beyond aminoacyl-tRNA formation. The host messenger RNA remained intact and still bound to ribosomes shortly after ghost infection. Kinetic studies of the effect of ghosts on host protein synthesis revealed that nascent peptide chains on ribosomes were not released upon ghost infection.     

Dual Functions of Bacteriophage T4D Gene 28 Product: Structural Component of the Viral Tail Baseplate Central Plug and Cleavage Enzyme for Folyl Polyglutamates II. Folate Metabolism and Polyglutamate Cleavage Activity of Uninfected and Infected Escherichia coli Cells and Bacteriophage Particles

We investigated the role of the T4D bacteriophage gene 28 product in folate metabolism in infected Escherichia coli cells by using antifolate drugs and a newly devised assay for folyl polyglutamate cleavage activity. Preincubation of host E. coli cells with various sulfa drugs inhibited phage production by decreasing the burst size when the phage particles produced an altered gene 28 product (i.e., after infection under permissive conditions with T4D 28^ts or T4D am28). In addition, we found that another folate analog, pyrimethamine, also inhibited T4D 28^ts production and T4D 28am production, but this analog did not inhibit wild-type T4D production. A temperature-resistant revertant of T4D 28^ts was not sensitive to either sulfa drugs or pyrimethamine. We developed an assay to measure the enzymatic cleavage of folyl polyglutamates. The high-molecular-weight folyl polyglutamate substrate was isolated from E. coli B cells infected with T4D am28 in the presence of labeled glutamic acid and was characterized as a folate compound containing 12 to 14 labeled glutamate residues. Extracts of uninfected bacteria liberated glutamate residues from this substrate with a pH optimum of 8.4 to 8.5. Extracts of bacteriophage T4D-infected E. coli B cells exhibited an additional new folyl polyglutamate cleavage activity with a pH optimum of about 6.4 to 6.5, which was clearly distinguished from the preexisting activity in the uninfected host cells. This new activity was induced in E. coli B cells by infection with wild-type T4D and T4D amber mutants 29⁻, 26⁻, 27⁻, 51⁻, and 10⁻, but it was not induced under nonpermissive conditions by T4D am28 or by T4D 28^ts. Mutations in gene 28 affected the properties of the induced cleavage enzyme. Wild-type T4D-induced cleavage activity was not inhibited by pyrimethamine, whereas the T4D 28^ts activity induced at a permissive temperature was inhibited by this folate analog. Folyl polyglutamate cleavage activity characteristic of the activity induced in host cells by wild-type T4D or by T4D gene 28 mutants was also found in highly purified preparations of these phage ghost particles. The T4D-induced cleavage activity could be inhibited by antiserum prepared against highly purified phage baseplates. We concluded that T4D infection induced the formation of a new folyl polyglutamate cleavage enzyme and that this enzyme was coded for by T4D gene 28. Furthermore, since this gene product was a baseplate tail plug component which had both its antigenic sites and its catalytic sites exposed on the phage particle, it was apparent that this enzyme formed part of the distal surface of the phage baseplate central tail plug.     

Distribution of "minor" nicks in bacteriophage T5 DNA.

Bacteriophage T5 DNA can be released from the phage particle in such a way that one end of 5 to 10% of the DNA molecules remains attached to either the phage head or tail. Under partial denaturation conditions, the DNA preferentially denatures in the vicinity of a nick so that the nicks can be located relative to the end that remains attached to the phage head or tail. Two classes of nicks were found. "Major" nicks were those found in more than 20% of the molecules and were located at the same points along the DNA molecule as reported by others. "Minor" nicks were found in 5 to 10% of the molecules and often occurred at specific locations near a "major" nick.     

Unusual occurrence of EcoP1 and EcoP15 recognition sites and counterselection of type II methylation and restriction sequences in bacteriophage T7 DNA

Selected and counterselected oligodeoxynucleotide sequences were identified in the total sequence of bacteriophage T7 DNA using a statistical criterion derived for a probability model of the Markov chain type. All extremely rare tetra- and pentadeoxynucleotides are (or contain) recognition sequences for the Escherichia coli DNA methylases dam or dcm. Most of the 37 hexadeoxynucleotides absent from T7 DNA are recognition sequences for type II modification/restriction enzymes of E. coli or related species. In contrast to most restriction sites counterselected during evolution, the EcoP1 site GGTCT occurs 126 times in the T7 genome, and phage T7 replication is severely repressed in P1-lysogenic host cells. We demonstrate that the frequency of the EcoP1 site is determined by that of the overlapping recognition sites for T7 primase, an essential phage enzyme. The recognition site of a type III enzyme, EcoP15, is also not counterselected. In T7 DNA all 36 EcoP15 sites are arranged in such a manner that the sequence CAGCAG is confined to the H strand, the complementary sequence CTGCTG to the L strand. This "strand bias" is highly significant and, therefore, very probably selected. A functional relation between this strand bias and the refractive behaviour of phage T7 to EcoP15 restriction is suspected.